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UNIWERSYTET MIKOŁAJA KOPERNIKA w TORUNIU<br />
COLLEGIUM MEDICUM im. LUDWIKA RYDYGIERA<br />
W BYDGOSZCZY<br />
MEDICAL<br />
AND BIOLOGICAL<br />
SCIENCES<br />
(dawniej ANNALES ACADEMIAE MEDICAE BYDGOSTIENSIS)<br />
TOM XXVII/2 kwiecień – czerwiec ROCZNIK 2013
R E D A K T O R N A C Z E L N Y<br />
E d i t o r - i n - C h i e f<br />
Beata Augustyńska<br />
Z A S T Ę P C A R E D A K T O R A N A C Z E L N E G O<br />
Co- e d i t o r<br />
Jacek Manitius<br />
K O M I T E T R E D A K C Y J N Y<br />
E d i t o r i a l B o a r d<br />
Aleksander Araszkiewicz, Beata Augustyńska, Michał Caputa, Stanisław Dąbrowiecki, Gerard Drewa, Eugenia Gospodarek,<br />
Bronisław Grzegorzewski, Waldemar Halota, Olga Haus, Marek Jackowski, Henryk Kaźmierczak, Alicja Kędzia,<br />
Michał Komoszyński, Wiesław Kozak, Konrad Misiura, Ryszard Oliński, Danuta Rość, Karol Śliwka, Eugenia Tęgowska,<br />
Bogdana Wilczyńska, Zbigniew Wolski, Zdzisława Wrzosek, Mariusz Wysocki<br />
K O M I T E T D O R A D C Z Y<br />
A d v i s o r y B o a r d<br />
Gerd Buntkowsky (Berlin, Germany), Giovanni Gambaro (Padova, Italy), Edward Johns (Cork, Ireland),<br />
Massimo Morandi (Chicago, USA), Vladimir Palička (Praha, Czech Republic)<br />
A d r e s r e d a k c j i<br />
A d d r e s s o f E d i t o r i a l O f f i c e<br />
Redakcja Medical and Biological Sciences<br />
ul. Powstańców Wielkopolskich 44/22, 85-090 Bydgoszcz<br />
Polska – Poland<br />
e-mail: medical@cm.umk.pl, annales@cm.umk.pl<br />
tel. 52 585 33 26<br />
www.medical.cm.umk.pl<br />
Informacje w sprawie prenumeraty: tel. 52 585 33 26<br />
e-mail: medical@cm.umk.pl, annales@cm.umk.pl<br />
ISSN 1734-591X<br />
UNIWERSYTET MIKOŁAJA KOPERNIKA W TORUNIU<br />
COLLEGIUM MEDICUM im. LUDWIKA RYDYGIERA<br />
BYDGOSZCZ 2013
Medical and Biological Sciences, 2013, 27/2<br />
CONTENTS<br />
p.<br />
REVIEWS<br />
D o r o t a R o g a l a , D o r o t a J a c h i m o w i c z - W o ł o s z y n e k , Ż a n e t a S k i n d e r ,<br />
M a ł g o r z a t a G i e r s z e w s k a – The use of information systems in health care practice . . . . . 5<br />
P a w e ł S u t k o w y , A l i n a W o ź n i a k , C e l e s t y n a M i l a - K i e r z e n k o w s k a<br />
– Positive effect of generation of reactive oxygen species on the human organism . . . . . . . . . . . . . . . 13<br />
ORIGINAL ARTICLES<br />
M a c i e j G a g a t , K o r y n a K r ó l i k o w s k a , A n n a K l i m a s z e w s k a - W i ś n i e w s k a ,<br />
M a g d a l e n a I z d e b s k a , A l i n a G r z a n k a – The effect of mild hyperthermia<br />
on morphology, ultrastructure and F-actin organization in HL-60 cell line . . . . . . . . . . . . . . . . . . . . . . 19<br />
W o j c i e c h P a w ł o w i c z – Model study of epithelial motility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27<br />
Z u z a n n a P i e k o r z , I r e n a B u ł a t o w i c z , A g n i e s z k a R a d z i m i ń s k a , A n d r z e j<br />
L e w a n d o w s k i , S z y m o n P i e k o r z , G r z e g o r z G r a b a r c z y k , M o n i k a<br />
Cies i e l s k a – The influence of hatha yoga exercise on arterial pressure and pulse . . . . . . . . . . . . . . 33<br />
A d r i a n R e ś l i ń s k i , S t a n i s ł a w D ą b r o w i e c k i , K a t a r z y n a G ł o w a c k a , J a k u b<br />
S z m y t k o w s k i – The influence of octenidine dihydrochloride on bacterial biofilm<br />
on the surface of a polypropylene mesh . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41<br />
E w a J o a n n a S z y m e l f e j n i k , A n n a C h i b a – The interdependence of nutritional status<br />
and blood pressure in female students . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
Medical and Biological Sciences, 2013, 27/2<br />
SPIS TREŚCI<br />
str.<br />
PRACE POGLĄDOWE<br />
D o r o t a R o g a l a , D o r o t a J a c h i m o w i c z - W o ł o s z y n e k , Ż a n e t a S k i n d e r ,<br />
M a ł g o r z a t a G i e r s z e w s k a – Wykorzystanie systemów informacyjnych w praktyce<br />
jednostek opieki zdrowotnej . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5<br />
P a w e ł S u t k o w y , A l i n a W o ź n i a k , C e l e s t y n a M i l a - K i e r z e n k o w s k a<br />
– Pozytywne aspekty generowania reaktywnych form tlenu w organizmie człowieka . . . . . . . . . . . . . . 13<br />
PRACE ORYGINALNE<br />
M a c i e j G a g a t , K o r y n a K r ó l i k o w s k a , A n n a K l i m a s z e w s k a - W i ś n i e w s k a ,<br />
M a g d a l e n a I z d e b s k a , A l i n a G r z a n k a – Wpływ łagodnej hipertermii na morfologię,<br />
ultrastrukturę i organizację F-aktyny w komórkach linii HL-60 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19<br />
W o j c i e c h P a w ł o w i c z – Modelowe badania aktywności ruchowej nabłonka . . . . . . . . . . . . . . . . . . . . 27<br />
Z u z a n n a P i e k o r z , I r e n a B u ł a t o w i c z , A g n i e s z k a R a d z i m i ń s k a , A n d r z e j<br />
L e w a n d o w s k i , S z y m o n P i e k o r z , G r z e g o r z G r a b a r c z y k , M o n i k a<br />
Cies i e l s k a – Wpływ treningu hatha jogi na wartości ciśnienia tętniczego i częstość tętna . . . . . . 33<br />
A d r i a n R e ś l i ń s k i , S t a n i s ł a w D ą b r o w i e c k i , K a t a r z y n a G ł o w a c k a , J a k u b<br />
S z m y t k o w s k i – Wpływ roztworu dichlorowodorku oktenidyny na biofilm wytworzony<br />
na powierzchni siatki polipropylenowej . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41<br />
E w a J o a n n a S z y m e l f e j n i k , A n n a C h i b a – Współzależność między stanem odżywienia<br />
a ciśnieniem tętniczym u studentek . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49<br />
Regulamin ogłaszania prac w Medical and Biological Sciences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57
Medical and Biological Sciences, 2013, 27/2, 5-11<br />
REVIEW / PRACA POGLĄDOWA<br />
Dorota Rogala¹, Dorota Jachimowicz-Wołoszynek¹, Żaneta Skinder¹, Małgorzata Gierszewska²<br />
THE USE OF INFORMATION SYSTEMS IN HEALTH CARE PRACTICE<br />
WYKORZYSTANIE SYSTEMÓW INFORMACYJNYCH<br />
W PRAKTYCE JEDNOSTEK OPIEKI ZDROWOTNEJ<br />
1 Department of Organization and Management in Health Care UMK in Torun, Collegium Medicum<br />
of L. Rydygiera in Bydgoszcz<br />
Leader: Dorothy Jachimowicz-Wołoszynek, PhD<br />
2 Department of Fundamentals of Obstetric Care in Torun, Nicolaus Copernicus University,<br />
Collegium Medicum of L. Rydygiera in Bydgoszcz<br />
Leader: Margaret Gierszewska, PhD<br />
S u m m a r y<br />
Information systems are now the basis of the operation of<br />
each unit in health care. This paper describes the objectives<br />
and benefits of the introduction of new technologies in<br />
information systems and examples of practical use of the<br />
systems in Poland and worldwide. In the paper chosen<br />
programs used to monitor patient's health status and their<br />
usefulness to professionals have been described. The inherent<br />
savings, which are brought by the computerization of<br />
healthcare facilities, have been pointed out as well as the<br />
contribution of European Union funds in financing the<br />
programs.<br />
S t r e s z c z e n i e<br />
Systemy informacyjne stanowią obecnie podstawę<br />
funkcjonowania każdej jednostki w ochronie zdrowia. W<br />
pracy opisano cele i korzyści z wprowadzania nowych<br />
technologii w systemach informacyjnych oraz przykłady<br />
praktycznego wykorzystanie systemów w Polsce i na<br />
świecie. Opisano wybrane programy monitorujące stan<br />
zdrowia pacjenta oraz ich przydatność dla profesjonalistów.<br />
Wskazano na oszczędności, jakie niesie ze sobą<br />
informatyzacja placówek ochrony zdrowia oraz udział<br />
środków z Unii Europejskiej w finansowaniu programów.<br />
Key words: information systems, telemedicine, computerization<br />
Słowa kluczowe: systemy informacyjne, telemedycyna, informatyzacja<br />
INTRODUCTION<br />
Decision-making and management of healthcare<br />
units are largely determined by the information<br />
collected. The group of procedures consisting of<br />
collecting, processing, storing and transmitting<br />
information to support and facilitate the management<br />
can be defined as an information system [1]. Such a<br />
system is essential to normal/correct functioning of all<br />
organizations and companies, including facilities<br />
providing healthcare services. Today, healthcare<br />
institutions intending to gain a strong position in the<br />
evolving market of medical services are forced to<br />
carefully choose specific activities relevant to the<br />
management mechanisms of the information resources,<br />
so that they are appropriately used in order to increase
6<br />
Dorota Rogala et al.<br />
its chance to gain and maintain a permanent<br />
competitive predominance in the market environment.<br />
In healthcare, the strategic and universal purpose of<br />
the development of information systems is to develop<br />
and improve the efficient use of existing resources of<br />
the information system, through the computerization of<br />
its various parts, standardization of structures,<br />
dictionaries, and communication protocols;<br />
additionally by improving the quality and credibility of<br />
information as well as the utilization of generated<br />
information [2].<br />
The basic objectives of information systems in<br />
terms of the interests of health care are:<br />
- providing the highest quality of services,<br />
- efficient management of the unit in a competitive<br />
environment,<br />
- facilitating the work of medical staff [1].<br />
The modern health care system requires a high<br />
degree of computerization in institutions operating in<br />
the healthcare market, implementing of which has been<br />
greatly accelerated in recent years, for example, due to<br />
the necessity of carrying out settlements with the<br />
(tax)payer in the reformed health insurance system.<br />
The compulsion to collect and transmit large<br />
amounts of data enforced the introduction of measures<br />
aimed at increasing a degree of computerization and it<br />
has improved information systems of these units [3,4].<br />
The main disadvantage is the fact that the use of paper<br />
documents is time-consuming, and there are difficulties<br />
in obtaining quick access to data. The existence of<br />
electronic information flow enables to save time, and<br />
this, in turn, allows taking the effort to improve quality<br />
and productivity.<br />
Experience shows, that the use of IT tools in the<br />
information process also removes the source of<br />
frustration and reduces the overall workload, which<br />
prevents the generation of conflict and stress reactions<br />
in employees [5].<br />
The benefits of the implementation of information<br />
systems are multidimensional. The primary effects of<br />
computerization include:<br />
- improving the effectiveness of personnel, through<br />
the rapid flow of information and better access to it,<br />
- improving the quality of services, by providing<br />
tools to support medical staff in carrying out treatment<br />
and broadening the scope of information used in the<br />
treatment,<br />
- greater control of sources of costs in the hospital,<br />
- assistance in the management at higher levels, by<br />
allowing analysis of the data collected,<br />
- reduction of costs associated with processing<br />
large amounts of information appearing in the<br />
treatment process [1].<br />
COMPUTERIZATION IN HEALTH CARE<br />
Every implemented system clearly defines the area<br />
of its influence. It should also be clearly aimed at one<br />
of the spheres of the covered project or to meet the<br />
selected need. The United States and Germany began<br />
work on a system dealing with financial issues. United<br />
Kingdom, Sweden, France and Japan realized it<br />
primarily on the patient. These are two different<br />
approaches, which at some point need to take account<br />
of and complement each other. Only equitable<br />
introduction of patient medical data correlated with<br />
their financial information allows looking at the overall<br />
hospital system [1]. Hospitals and medical centers are<br />
implementing systems, which are supportive for the<br />
management of so-called grey- administrative sphere<br />
and medical, so-called white.<br />
Computerization can be complex or run in stages.<br />
In the event of a complex computerization in the<br />
hospital, systems and specialized software are<br />
integrated. An example of complex actions of<br />
computerization was the Oncology Centre in<br />
Bydgoszcz, where on the request of the Center,<br />
MEDINFO system was implemented to support part of<br />
the "white", some of the "grey" sphere and the<br />
settlement of the National Health Fund (NFZ). The<br />
system took almost all spheres of activities of the<br />
Centre, reducing the time of registration of patients and<br />
improving the quality of the service [6].<br />
Computerization was carried out in stages in<br />
Kujawsko-Pomorskie Pulmonology Center in<br />
Bydgoszcz. In 2000, administrative system SKID (now<br />
Xpertis) was introduced, containing the following<br />
modules: HR - Payroll, Finance - Accountant, Cashier,<br />
Inventory, Fixed Assets, Homebanking. The system<br />
allows the preparation of financial statements, data<br />
analysis, planning and forecasting. In 2001, the<br />
medical computer system was implemented, which<br />
included most of the functionality of the medical<br />
aspects of hospital activities such as: Patient's<br />
movement (Admissions) (Admissions modules, Ward,<br />
Statistics), Pharmacy and Medication on the ward,<br />
(Medical, Clinical)Analytical Laboratory, Diagnostic<br />
Labs, the module of Bacteriology. Later, some new<br />
modules of the system were introduced: Pathology,<br />
Rehabilitation, Endoscopy, Outpatients Clinic and the
The use of information systems in health care practice 7<br />
module, which was not integrated with the system,<br />
supporting the Laboratory of Respiratory Disorders<br />
during sleep. In 2007, the medical system was<br />
supplemented by a digital radiology system within the<br />
TELEMEDICINE program. The system allows the<br />
distribution of diagnostic images throughout the<br />
hospital's computer network at the indicated computer<br />
stations. [7] The current system Xpertis provides multivariant<br />
reports with numerous levels of summation,<br />
simulation analysis. It allows comparing actual and<br />
forecasted results. Additionally, it gives a direct and<br />
immediate access to data collected in other modules of<br />
the system [8].<br />
Already completed systems are naturally slower to<br />
respond to organizational and legal changes. Their<br />
implementation requires a large amount of time, and<br />
sometimes, it happens that quickly there is a need to<br />
make major modifications, replacement or introduction<br />
of a new system. In private clinics, information<br />
systems must take into account the specificity of their<br />
activities. Since the beginning of foundation (since the<br />
late 90), the Family Medicine Company SA has been<br />
creating on their own computer system. The work does<br />
a team of company analysts and programmers. The<br />
company has got 16 own medical clinics and includes<br />
5 institutional clinics. In each of these, there is<br />
independent software operating on the principle client<br />
– server with central data synchronization on - line.<br />
The synchronization method is an own solution of IT<br />
specialists as well as analysts, and ensures sharing of<br />
information between providers in actual time.<br />
Currently, the system supports several areas of the<br />
company: managing the database of patients and their<br />
rights to services, customer management (contractors),<br />
including contracts with the NFZ, employers and<br />
insurers, work time management – the module of<br />
planning doctors' and other staff rota, booking patients<br />
visits, keeping records of services and electronic<br />
medical records. The system also has analytic<br />
functions, reporting modules, which provide the<br />
information management; therefore, it is possible to<br />
improve efficiency and service quality. The system is<br />
constantly being developed and implemented with new<br />
modules, including the run of treatment rooms,<br />
promotion of cooperation with suppliers of medical<br />
services (diagnostic imaging facilities, laboratories)<br />
and the stock management. This structure allows the<br />
system to quickly adjust to the constantly changing<br />
external and internal conditions. The company's<br />
experience shows that this kind of own system can<br />
successfully handle the smaller health care facilities<br />
[9].<br />
Computerization of healthcare facilities is an<br />
expensive process. Introduction of modern solutions in<br />
many hospitals is possible thanks to funds acquired<br />
from the European Union.<br />
By the end of 2011, the Regional Specialist<br />
Children's Hospital in Olsztyn is likely to complete a<br />
complex computerization, funded by the European<br />
Regional Development Fund (cost of the project was<br />
3.9 million PLN). The project gives the patient the<br />
opportunity to register online and access to patient<br />
records at designated hospitals and clinics [10].<br />
The county Hospital in Kedzierzyn-Koźle received<br />
6.3 million PLN, funded by the European Union, to<br />
implement an integrated information system. After the<br />
introduction of the new system, the hospital predicts<br />
the growth of market position and the opportunity to<br />
compete with other commercial entities and to achieve<br />
benefits associated with obtaining savings in the<br />
operation by eliminating unnecessary expenditures<br />
incurred by operation activities [11].<br />
In a similar manner, one of the largest psychiatric<br />
hospitals in Poland, Wolski Hospital in Warsaw, was<br />
computerized. The cost of the project, entitled<br />
'E - Hospital - the creation of a digital information<br />
system of telemedicine, data collection, processing and<br />
archiving data for the Wolski Hospital SP Hospital in<br />
Warsaw', was approximately 4.2 million PLN. Half of<br />
the amount came from European Union funds within<br />
the Integrated Regional Operational Development<br />
Programme. The great importance to this institution<br />
was the introduction of electronic patient information<br />
flow - from admission to discharge, all the information<br />
are recorded at the so-called electronic patient record<br />
[12].<br />
The introduction of information systems also<br />
translates into real savings. The University Hospital<br />
Aachen (Germany) is a hospital which employs over<br />
5700 staff members and treats approximately 179 000<br />
patients in the year. With the development of the<br />
facility, the list of used operating systems has grown.<br />
Integration and analysis of large amounts of data from<br />
different systems caused many difficulties. In early<br />
2003, the hospital decided to implement SAS 9<br />
software company SAS Institute across the<br />
organization. The implementation included such<br />
elements as: data integration, business intelligence<br />
(software for budgeting and analysis, supporting the<br />
decision-making processes), information portal, as well
8<br />
Dorota Rogala et al.<br />
as solutions for managing results. The biggest<br />
challenge before the implementation of SAS 9 was the<br />
integration of data from various systems including:<br />
Medico - Hospital Information System (3.4 million<br />
records are updated every day), Swisslab - Laboratory<br />
Information System (10 million records generated each<br />
year), ERP system used for hospital resources<br />
management. The benefits of the new system included<br />
increased income from reimbursement of medical<br />
costs, reduced operating time for the exchange of<br />
information within the organization and reduced costs<br />
of external services. Five years after the<br />
implementation of SAS 9, the return on investment<br />
after tax was estimated at 569% [13].<br />
Norwegian Rikshospitalet University Hospital<br />
estimates that by introducing a comprehensive and<br />
modern information system, it reached annual savings<br />
of 66 million dollars, which represents 7% of the<br />
budget of the hospital [14].<br />
THE USE OF INFORMATION SYSTEMS<br />
Achievements in the development of modern<br />
medical technology have influenced the development<br />
of telemedicine, using technology for diagnosis,<br />
treatment, consultation and monitoring of the health<br />
status of patients. It gives a chance to reduce costs by<br />
allowing the treatment and care at the patient's home.<br />
Televisits are particularly valued by chronically ill<br />
patients and those, who because of a long<br />
convalescence / stay at home for a long period of time.<br />
The audio-visual contact with a doctor or nurse<br />
improves patient's psychological comfort, despite the<br />
actual distance. Statistical data from the state of New<br />
York in the U.S. show that the use of telemedicine in<br />
home care resulted in reduction of the frequency of<br />
ambulance and emergency visits in the hospital among<br />
patients over 65 the age by approximately 70%. In the<br />
Netherlands, modern telemedicine devices (based on<br />
the model so-called Smart home) were used in seniors’<br />
home (nursing home/residential home) in Eindhoven,<br />
and in Finland, a project is being created to equip one<br />
of the villages with the home telecare system. In<br />
France, a project PROSAFE designed for people with<br />
memory loss including Alzheimer's disease was<br />
developed It monitors the health of patients in addition<br />
to their behavior, the cases of disappearance from the<br />
residence, falls, etc.<br />
Telemedicine is also used to monitor the health<br />
status of patients in various types of diseases. MyCare<br />
program operating at Georgetown University Medical<br />
Center in Washington sends glucose measurements to<br />
the monitoring center from the miniature meters, which<br />
are provided for diabetic patients. At the same time,<br />
patients receive test results with recommendations on<br />
how to proceed. Thus, it is possible to prevent serious<br />
consequences resulting from the delayed<br />
implementation of therapy. At Vanderbilt University,<br />
USA, telemedicine is used to study the image of the<br />
eye of patients with diabetes. Also, the information is<br />
sent via the Internet to the monitoring center, where the<br />
information is analyzed for the diagnosis of<br />
complications associated with diabetes. This is<br />
particularly convenient for patients living in remote<br />
villages, because it eliminates the need for personal<br />
visits for diagnostic purposes. In the UK, the health of<br />
patients with asthma is monitored. The patient has a<br />
small mobile phone – computer that senses the<br />
deterioration of the patient’s condition. The<br />
information is passed on automatically to the doctor,<br />
who takes the necessary actions. Similar pilot<br />
programs are applied in remote monitoring of patients<br />
with hypertension, suffering from cardiac arrhythmia.<br />
In Brazil, remote diagnoses were introduced in<br />
dermatology patients with suspicious skin changes<br />
[15].<br />
In Poland, telemedicine ‘crawls’. So far, there has<br />
been only a few interactive educational and training<br />
videoconferencing - such as the tripartite; one from<br />
Washington to Warsaw and Bielsko Biala or heart<br />
surgery at Children's Health Center in Warsaw. Local<br />
telemedicine networks are formed around large<br />
medical centers. They focus mainly on developing and<br />
implementation of systems to transmit ECG signals by<br />
telephone, and X-ray images, ultrasound, CT via the<br />
Internet and using the Intranet for consultation.<br />
Although, all Polish hospitals have access to the<br />
Internet, according to the report ‘The study of the<br />
needs and potential opportunities in telemedicine in<br />
county hospitals,’ which was developed in cooperation<br />
with the Department of Medical Informatics and<br />
Telemedicine Medical Academy in Warsaw and the<br />
Association of Polish Counties, only a few facilities<br />
have digital equipment to record test results.<br />
Ambulances, equipped with equipment that enables the<br />
electronic transfer of ECG results to the cardiac center<br />
on call, function only in 16% of hospitals. Even more<br />
rarely, consultations are carried out during the<br />
transport of the patient (11%). The telemedicine<br />
system in ambulances has been operating for several
The use of information systems in health care practice 9<br />
years in Bydgoszcz. 16 rescue teams, including 5<br />
specialist teams, are included in this system.<br />
In the field of emergency medical service, Personal<br />
Digital Assistant (PDA) or personal digital assistant<br />
gives great opportunities to take advantage of. Already<br />
in 2007, the system was tested in the emergency<br />
stations in Katowice and Mikołów. The use of PDAs<br />
by procedures is following:<br />
- the doctor gets a request on the PDA along with a<br />
map of directions to the patient,<br />
- the software has also the option to fill the<br />
departure card,<br />
- in module 'interview, examination, medical<br />
procedure " a description of the patient symptoms is<br />
filled out (in the electronic version of the card, a doctor<br />
does not have to enter it manually, but to record),<br />
- the drug used does not have to be manually<br />
entered, but can be selected from the available database<br />
(this allows a statistical analysis of the drugs<br />
economy),<br />
- when the crew returns to base, the doctor sends<br />
the contents of the PDAs to the server and signs the<br />
printed document [16].<br />
The PDA is also used in telemedicine. Since 2004,<br />
one of U.S. companies has promoted its device, which,<br />
when connected to the PDA, allows the patient to<br />
observe own heartbeat. The data obtained can be sent<br />
to the doctor. At The College of Medicine in Texas use<br />
of information technology PDA in diabetes<br />
management by patients with type 2 diabetes in the<br />
ambulatory setting has been shown to improve selfcare<br />
and glycemic control [17].<br />
One of the pioneering activities of the PDA<br />
technology in Poland is the study of bronchial asthma<br />
and chronic obstructive pulmonary disease (COPD)<br />
within the COMPASS program, implemented by<br />
pulmonologists and clinical internists of Medical<br />
College in Krakow. The program allows registration of<br />
data of the disease, its course and treatment by<br />
hundreds of specialists from approximately 14 000<br />
patients with COPD and asthma. Another research<br />
program, which uses the PDA, is OPTIMO, or ‘optimal<br />
care of patients with diabetes in Poland.’ This program<br />
is focused on populations with diabetes under the care<br />
of a specialist diabetes clinic. It provides information<br />
about the characteristics of the research population<br />
including diagnostic and therapeutic methods<br />
depending on the duration of illness, severity of<br />
complications or comorbidities. It is planned to use<br />
PDAs in the daily work of doctors. Using an electronic<br />
questionnaire, the results of the study, complications<br />
and treatment data will be entered. This will not only<br />
enable safe collection of the information of patients<br />
(without being able to identify the members of the<br />
public), but also will help physicians to care for<br />
patients through the use of clinical decision support<br />
system (CDSS - Clinical Decision Support System),<br />
developed on the basis of current Polish Diabetes<br />
Association guidelines. The program will remind to<br />
carry out tests provide information on current methods<br />
of action, criteria, definitions, classifications of<br />
complications, and the expected health effects in<br />
diabetes treatment. With OPTIMO a doctor will be<br />
able to quickly view the information about the methods<br />
used in the patient's treatment, the results of the<br />
patient's tests and the possible complications.<br />
Moreover, the program plays an educational role,<br />
because every month participating doctors will receive<br />
multimedia information materials as well as sets of<br />
questions, and educational points will be awarded for<br />
correct answers. The program OPTIMO undergoes<br />
further changes, because the incoming comments and<br />
suggestions from the physicians participating in the<br />
program are included in the modification of the<br />
electronic questionnaire and the clinical decision<br />
support system.<br />
The PDA is also used in ECG and hearing<br />
screening tests, using objective and audiometric<br />
methods. Data is collected for ‘Program of care for<br />
people with hearing impairment in Poland’[16].<br />
The studies in Sweden revealed a positive attitude<br />
towards the PDA. The PDA seems to be a valuable<br />
tool for personnel and students in health care but there<br />
is a need for further intervention studies, randomized<br />
controlled trials and studies with various health care<br />
groups in order to identify its appropriate functions and<br />
software applications [18].<br />
AUTOMATIC IDENTIFICATION OF PATIENT<br />
In most European countries, the extensive use of<br />
automatic identification in healthcare is already a<br />
norm. In Poland, this solution is not yet widespread.<br />
For example, identification of the patient can be done<br />
by putting bands with a printed barcode on the wrist of<br />
a registered person.<br />
In the U.S. there are even more advanced<br />
technologies, such as radio frequency identification,<br />
known as RFID tags. Patient information can be<br />
obtained using the identification numbers allocated to
10<br />
Dorota Rogala et al.<br />
them. The band put on the patient, contains both chip<br />
signal on a specific radio frequency and the barcode.<br />
By using such solutions the hospital staff has secure<br />
and virtually unlimited access to all data, including<br />
those from the laboratory and pharmaceutical<br />
information center. The system eliminated the need for<br />
paper documentation. The fact that the information is<br />
instantly updated in each place also reduced the<br />
number of errors in prescribing medicines. This system<br />
is particularly convenient when using shift work<br />
system, where the patient is looked after by more than<br />
one person and the instant sharing of information is<br />
more difficult. In addition, physicians and nurses using<br />
the wireless platform right at the bedside can order any<br />
necessary lab tests, write notes about patient care or<br />
update the order of the administration of medication.<br />
The extension of the functionality of the RFID for<br />
tracking blood for transfusion and monitoring of<br />
surgical instruments has been introduced [19].<br />
An assessment RFID-enabled blood transfusion<br />
was conducted for two hospitals: the University of<br />
Iowa Hospital and Clinics (UIHC) and Mississippi<br />
Baptist Health System (MBHS). The study estimated<br />
that RFID technology could reduce morbidity and<br />
mortality effects substantially among patients receiving<br />
transfusions [20].<br />
The RFID system has been also introduced in the<br />
University Hospital of Jena in Germany and designed<br />
to improve patient safety. A significant increase in the<br />
quality of medical services and virtually complete<br />
elimination of the risk of medication dispensing errors<br />
was observed. In addition to improved quality of<br />
treatment, the RFID infrastructure is helpful in the<br />
optimization of logistic processes. It allows<br />
management of drug supplies depending on demand,<br />
which reduces the amount of capital tied up in the<br />
stock of hospital pharmacies [21].<br />
It is worth noting that for the security of<br />
distribution of blood, identification systems use mobile<br />
devices for printing labels on a regular basis. Such<br />
activities are aimed at reducing wrong-labeled samples<br />
of blood which, according to some studies, may be<br />
account for up to 5.8% of all containers [22].<br />
In-patient and outpatient facilities systems are even<br />
less common.<br />
RFID applications have the potential to increase the<br />
reliability of healthcare environments. RFID<br />
technology not only offers tracking capability to locate<br />
equipment, supplies and people in real time, but also<br />
provides efficient and accurate access to medical data<br />
for health professionals. However, the reality of RFID<br />
adoption in healthcare is far behind earlier<br />
expectations. Major barriers to adopt this technology<br />
include technological limitations, lack of global<br />
standards and secure authentication systems. In<br />
healthcare environments remains as a challenge the<br />
further use of this technology [23, 24, 25, 26].<br />
SUMMARY<br />
The examples of practical applications of modern<br />
systems confirm not only their multidimensional<br />
significance for the patient, but also for the<br />
management of health care institutions. Effective use<br />
of the system means not only improvement of<br />
administrative standards, but it provides savings in the<br />
form of reduced overall cost of treatment and care in<br />
our country.<br />
REFERENCES<br />
1. Trąbka W., Komnata W., Stalmach L., Kozierkiewicz A.<br />
Szpitalne systemy informatyczne, wyd. 2.<br />
Uniwersyteckie Wydawnictwo Medyczne „Vesalius”,<br />
Kraków 1999; 9-26,<br />
2. Sala D., Kozierkiewicz A., Potrzeby informacyjne dla<br />
zarządzania w opiece zdrowotnej, Antidotum, 1998, 2,<br />
39-49,<br />
3. Kozierkiewicz A., Strug A., Informatyka w ochronie<br />
zdrowia, [w:]: Transformacja systemu ochrony zdrowia<br />
w Polsce, Dokument MZiOS, Warszawa, 1998, 275-282,<br />
4. Bęben A., Czy pamiętasz znachora?, Rynek Zdrowia,<br />
2007, 10, 44-45,<br />
5. Pilch-Kowalczyk M., Zarządzanie zakładem diagnostyki<br />
obrazowej, Pasaż Medyczny, 2008, 7, 16-18,<br />
6. Koziński M., Czy RUM uzdrowi służbę zdrowia?,<br />
Teleinfo, 2006, 19, 13-15,<br />
7. Informacja dotycząca zabezpieczenia informatycznego<br />
oraz teleinformatycznego w Kujawsko-Pomorskim<br />
Centrum Pulmonologii w Bydgoszczy, Kujawsko-<br />
Pomorskie Centrum Pulmonologii, Bydgoszcz, 2007,<br />
8. Informacja finansowa dotycząca dostarczenia i<br />
uruchomienia rozwiązania XPERTIS dla Kujawsko-<br />
Pomorskiego Centrum Pulmonologii w Bydgoszczy,<br />
Kujawsko-Pomorskie Centrum Pulmonologii,<br />
Bydgoszcz, 2008,<br />
9. Brzozowski A., Aplikacje zdobywają oddziały, Rynek<br />
Zdrowia, 2006, 1, 75-76,<br />
10. Informatyzacja to szybkość działania, Medinfo, 2011, 8,<br />
38,<br />
11. Wydatki stają się racjonalne, Medinfo, 2011, 8, 38,<br />
12. Marianek M., Operacje E-szpital, Pasaż Medyczny, 2008,<br />
2, 33-34,<br />
13. Brzozowski A., U nas za wcześnie?, Rynek Zdrowia,<br />
2006, 11, 44-46,
The use of information systems in health care practice 11<br />
14. Jakubiak L., Elektroniczna perełka, Rynek Zdrowia,<br />
2007, 10, 42-44,<br />
15. Braniecki M., Telemedycyna, dostępne on-line<br />
2005.10.27,<br />
www.oil.org.pl/xml/oil/oil68/gazeta/numery/n2003/2003<br />
06/.<br />
16. Bęben A., Kieszonkowe biuro, Rynek Zdrowia, 2007, 9,<br />
35-37,<br />
17. Forjuoh SN., Reis MD., Couchman GR., et al, Improving<br />
diabetes self-care with a PDA in ambulatory care,<br />
Telemed J E Health., 2008, 14, 3, 273-279,<br />
18. Lindquist AM., Johansson PE., Petersson GI., et al., The<br />
use of the Personal Digital Assistant (PDA) among<br />
personnel and students in health care: a review, J Med<br />
Internet Res. 2008, 28, 10, 4,<br />
19. Klepczarek P., Bezpieczna droga pacjenta –<br />
automatyczna identyfikacja w służbie zdrowia, Pasaż<br />
Medyczny, 2008, 3, 31-34,<br />
20. Briggs L., Davis R., Gutierrez A., et al., RFID in the<br />
blood supply chain--increasing productivity, quality and<br />
patient safety, J Healthc Inf Manag., 2009, 23, 4, 54-63,<br />
21. Brzozowski A., Z komputerem w obchód, Rynek<br />
Zdrowia, 2006, 11, 47- 49,<br />
22. Lippi G., Phlebotomy Issues and Quality Improvement in<br />
Results of Laboratory Testing, Clin. Lab., 2006, 5,<br />
23. Wu ZY., Chen L., Wu JC., A reliable RFID mutual<br />
authentication scheme for healthcare environments, J<br />
Med Syst., 2013, 37, 2,<br />
24. Yao W., Chu CH., Li Z., The adoption and<br />
implementation of RFID technologies in healthcare: a<br />
literature review, J Med Syst., 2012, 36, 6, 3507-3525,<br />
25. Ting SL., Kwok SK., Tsang AH., et al., Critical elements<br />
and lessons learnt from the implementation of an RFIDenabled<br />
healthcare management system in a medical<br />
organization, J Med Syst., 2011, 35, 4, 657-669,<br />
26. Briggs L., Davis R., Gutierrez A., et al., RFID in the<br />
blood supply chain--increasing productivity, quality and<br />
patient safety, J Healthc Inf Manag., 2009, 23, 4, 54-63.<br />
Address for correspondence:<br />
Dorothy Rogala<br />
85-830 Bydgoszcz<br />
ul. Sandomierska 16<br />
e-mail-dorotarogala@op.pl<br />
telephone: 880 44 55 91<br />
Received: 12.04.2012<br />
Accepted for publication: 13.11.2012
Medical and Biological Sciences, 2013, 27/2, 13-17<br />
REVIEW / PRACA POGLĄDOWA<br />
Paweł Sutkowy, Alina Woźniak, Celestyna Mila-Kierzenkowska<br />
POSITIVE EFFECT OF GENERATION OF REACTIVE OXYGEN SPECIES<br />
ON THE HUMAN ORGANISM<br />
POZYTYWNE ASPEKTY GENEROWANIA REAKTYWNYCH FORM TLENU<br />
W ORGANIZMIE CZŁOWIEKA<br />
The Chair of Medical Biology, Nicolaus Copernicus University,<br />
Ludwik Rydygier Collegium Medicum in Bydgoszcz<br />
Head: dr hab. Alina Woźniak, prof. UMK<br />
S u m m a r y<br />
For aerobes including human, the oxygen is an<br />
indispensable element for life, but simultaneously at too high<br />
concentration this gas becomes toxic. On the physiological<br />
level, the appropriate concentration of oxygen which is the<br />
source of reactive oxygen species (ROS), is necessary for the<br />
proper functioning of the organism. O 2 and its derivatives as<br />
free radicals influence the maintenance of homeostasis of the<br />
human organism by acting at a cellular level on repair,<br />
protective, and energetic mechanisms, and at an intercellular<br />
level on communication between neighbouring cells and<br />
tissues. In turn, excess or impaired removal of ROS lead to<br />
so-called ‘oxidative stress’ that is associated with the<br />
pathogenesis of many diseases – from cancers to<br />
neurodegenerative, autoimmune and infectious diseases. In<br />
the paper the significance of only physiological level of<br />
reactive oxygen species in the human organism was<br />
presented.<br />
S t r e s z c z e n i e<br />
Dla organizmów aerobowych, w tym człowieka, tlen jest<br />
pierwiastkiem niezbędnym do życia, ale równocześnie<br />
w zbyt wysokich jego stężeniach staje się toksyczny. Na<br />
poziomie fizjologicznym, odpowiednie stężenie tlenu<br />
i reaktywnych form tlenu (RFT), których jest źródłem, jest<br />
niezbędne do prawidłowego funkcjonowania organizmu. O 2<br />
i jego wolnorodnikowe pochodne wpływając na mechanizmy<br />
naprawcze, ochronne i energetyczne komórek oraz na<br />
komunikację między sąsiadującymi komórkami i tkankami,<br />
biorą udział w utrzymaniu homeostazy organizmu człowieka.<br />
Nadmiar RFT lub zaburzenia w ich usuwaniu prowadzą<br />
z kolei do tzw. stresu oksydacyjnego, który związany jest<br />
z patogenezą wielu chorób – od nowotworów, po choroby<br />
neurodegeneracyjne, autoimmunologiczne i infekcyjne.<br />
W pracy przedstawiono znaczenie wyłącznie fizjologicznego<br />
poziomu reaktywnych form tlenu w organizmie człowieka.<br />
Key words: reactive oxygen species, oxidant-antioxidant balance, human physiology<br />
Słowa kluczowe: reaktywne formy tlenu, równowaga oksydacyjno-antyoksydacyjna, fizjologia człowieka<br />
INTRODUCTION<br />
Nowadays, there is clear that oxygen is a kind of<br />
double-edged sword. It allows aerobic organisms to<br />
obtain significantly more energy than anaerobic<br />
organisms in fermentation processes; moreover, the<br />
oxygen is an essential element that allows them to live.<br />
On the other hand, aerobic organisms did not feel
14<br />
Paweł Sutkowy et al.<br />
better at higher concentration of oxygen than<br />
atmospheric – then the oxygen is toxic to them.<br />
Depending on exposure time and oxygen<br />
concentration, toxic effects range from being worse to<br />
diseases (neurodegenerative, autoimmune, infectious,<br />
cancerous) and permanent organs damage [1-3]. In the<br />
paper only physiological importance of oxygen<br />
derivatives in the human organism was presented.<br />
SOURCES OF REACTIVE OXYGEN SPECIES<br />
IN THE HUMAN ORGANISM<br />
Reactive oxygen species (ROS), including oxygen<br />
free radicals (OFR), can be formed in the human<br />
organism by an action of external physical factors such<br />
as ionizing and ultraviolet radiation, ultrasounds, as<br />
well as one of the mildest method of treatment of<br />
biological material – lyophilisation. ROS can be also<br />
produced as a result of an action of air ionizers, even<br />
though producers and dealers ensure that they have a<br />
positive effect on our health and ward off allergies [1].<br />
The oxygen is 7-8 times more soluble in organic<br />
solvents than in water. This property of oxygen is<br />
crucial for the human organism because physical<br />
properties of lipid layer of biological membrane are<br />
similar to properties of organic solvents. Therefore, the<br />
most important sources of ROS in the human organism<br />
are endogenous sources. There are many reactions<br />
during which intracellular ROS are generated: redox<br />
cycling of xenobiotics, respiratory proteins oxidation,<br />
reactions inside peroxisomes, oxidation of reduced<br />
forms of low-molecular cell components,<br />
photoreduction/photooxidation reactions and reactions<br />
of some specific enzymes. Nevertheless, a main source<br />
of ROS in the human organism is the respiratory<br />
electron transport chain [1, 4].<br />
CELLULAR RESPIRATION – THE ELECTRON<br />
TRANSPORT CHAIN (ETC)<br />
Aerobic respiration is based on a reduction of<br />
molecules of oxygen (O 2 ) to molecules of water,<br />
thereupon the energy is obtained:<br />
C 6 H 12 O 6 (glucose) + 6O 2 → 6CO 2 + 6H 2 O + 36ATP<br />
In fact, the molecular oxygen absorbed from the air<br />
is metabolised in the respiratory chain that is located at<br />
the inner membrane of mitochondria. Four electrons<br />
and four protons with cooperation of many enzymes<br />
and coenzymes are required to complete reducing by<br />
cell the oxygen to the water (fig. 1) [1].<br />
O 2 reduction may also be incomplete by premature<br />
electron leakage to the oxygen that occurs. Then, there<br />
is usually the one-electron reduction, thus being one of<br />
the main source of superoxide radical anion (O •− 2 ) – a<br />
harmful OFR that gives next ROS – fig. 1, 2. The<br />
oxygen reduction may also be two- or/and threeelectron,<br />
giving other reactive oxygen species as the<br />
end products – figure 1 [5].<br />
Fig. 1. Reduction of oxygen in the respiratory chain<br />
Ryc. 1. Redukcja tlenu w łańcuchu oddechowym<br />
Fig. 2. Superoxide radical anion as a source of other reactive<br />
oxygen species<br />
Ryc. 2. Anionorodnik ponadtlenkowy jako źródło innych<br />
reaktywnych form tlenu<br />
SELECTED EXAMPLES OF MOST IMPORTANT<br />
RADICALS<br />
The most abundant and important radicals in the<br />
human organism are those located on carbon, oxygen,<br />
sulfur, and nitrogen atoms. The carbon-centered<br />
radicals such alkyl (RC • HR’), hydroxyalkyl<br />
(RC • HOH), acyl (RC • =O), α-(alkylthio) alkyl<br />
(RSC • HR’) radicals are formed mostly due to hydrogen<br />
atom elimination from organic molecules. Especially<br />
significant among the oxygen-centered radicals are:<br />
hydroxyl ( • OH), peroxyl (ROO • ), alkoxyl (RO • ),<br />
phenoxyl (ArO • ), and semiquinone (HO-ArO • ) radicals<br />
and a superoxide radical anion (O 2 •− ). While the • OH is<br />
the most reactive among the O-centered radicals in<br />
biological systems, ROO • is probably the most
Positive effect of generation of reactive oxygen species on the human organism 15<br />
numerous and, except for O •−<br />
2 , these radicals are<br />
oxidants. The representative of S-centered radicals is<br />
the thiyl radical (RS • ) which is an intermediate in the<br />
one-electron reduction of thiols (RSH) to disulfides<br />
(RSSR). Some sulfur radical cations (R2S •+ ,<br />
(R2SSR2) •+ , (RSSR) •+ , Ar2S •+ ) have also attracted<br />
interest for their application in biochemical synthesis<br />
as intermediates in biological redox systems. N-<br />
centered radicals (the most common: • NO, • NO 2 and<br />
• NO 3 ) have great importance in the field of<br />
physiological functions of the human organism,<br />
especially nitric oxide ( • NO). This molecule may have<br />
•−<br />
antioxidant or oxidant properties depending on O 2<br />
concentration. Superoxide radical anion gives in<br />
reaction with nitric oxide peroxynitrite (ONOO – ), as<br />
strong oxidant [1, 2].<br />
MOLECULAR IMPORTANCE OF ROS<br />
ROS are the most numerous oxidants in the cell,<br />
while reducers (antioxidants) are a group of their<br />
scavengers. The ratio of the one to the other id est the<br />
redox potential of the cell determines whether, a cell<br />
will divide, differentiate or die [6, 7] – figure 3.<br />
Fig. 3. Redox regulation of the cell activity; SOD –<br />
superoxide dismutase, GPx – glutathione<br />
peroxidase, CAT – catalase, GSSG – oxidized<br />
glutathione, GSH – reduced glutathione [7]; own<br />
modification<br />
Ryc. 3. Regulacja reakcji utleniania i redukcji w komórce;<br />
SOD – dysmutaza ponadtlenkowa, GPx –<br />
peroksydaza glutationowa, CAT – katalaza, GSSG –<br />
glutation utleniony, GSH – glutation zredukowany<br />
[7]; w modyfikacji własnej<br />
A temporary increase of ROS concentrations due to<br />
the release of various growth factors during a<br />
stimulation of tissue, such as cytokines and hormones,<br />
including insulin, is accompanied. This is not a side<br />
effect, but necessary factor for the correct response of<br />
the cell to these ligands actions. ROS are necessary in<br />
mitogenic signal transduction, gene expression,<br />
metabolic regulation and repair processes within the<br />
cells [7-15].<br />
In 1995, it was demonstrated that growth factor<br />
obtained from platelet (platelet-derived growth factor –<br />
PDGF) for its effect requires the transient increase of<br />
concentration of the hydrogen peroxide (H 2 O 2 ). The<br />
use of catalase or N-acetylcysteine, which is the<br />
precursor of glutathione, inhibits an action of this agent<br />
[16]. Metabolic pathways which lead to the increase of<br />
ROS levels in cells exposed to growth factors of<br />
tyrosine receptors have been described and<br />
characterized [17]. Furthermore, the insulin action is<br />
regulated by mechanism dependent on redox balance.<br />
The short-lived increase of ROS concentrations is<br />
essential for the regulation of phosphatases activities<br />
[18], which together with kinases control the process of<br />
reversible phosphorylation of proteins of the insulin<br />
signal conduction [19]. As early as in 1970s it was<br />
found that stimulation of adipocytes by insulin is<br />
accompanied by elevated ROS concentrations. The<br />
necessity of ROS was confirmed in blocking<br />
translocation of glucose transfer protein (GLUT4) by<br />
the insulin after the supply of catalase to adipocytes<br />
[20]. Significance of ROS for the insulin signal<br />
conduction was also confirmed by results of the studies<br />
where Nox, siRNA and DPI inhibitors mutations were<br />
used [21]. The action of angiotensin II [22],<br />
5-hydroxytryptamine [23] and 17β-estradiol [24] cause<br />
also the transient increase of ROS concentration within<br />
cells.<br />
Moreover, it is very possible that interactions<br />
between cells are modulated by changes in<br />
concentrations of ROS [7, 8]. Reactive oxygen species<br />
act as a second messenger in transmission of<br />
intracellular information and can induce sanitation and<br />
apoptosis processes, thus they have anti-tumor<br />
functions [12, 13]. These compounds also play a key<br />
role in inflammation through the activation of<br />
transcription factors NF-kappaB [25, 26] and AP-1,<br />
and the acetylation/deacetylation of nuclear histone. In<br />
vitro studies it was shown that many substances like<br />
polyphenols, for example curcumin and resveratrol
16<br />
Paweł Sutkowy et al.<br />
present in a diet, have anti-inflammatory properties<br />
[27].<br />
High-reactivity molecules such as ROS have also<br />
crucial importance for correct functioning of locomotor<br />
system. During a rest, free radicals are produced into<br />
muscles as a consequence of activity of the<br />
mitochondrial electron transport. Inside of working<br />
muscles, capillary endothelial enzymes (xanthine<br />
oxidase, nitric oxide synthase – NOS), phospholipase<br />
A2 and in the myocardium NADH oxidoreductase are<br />
responsible for their additional production. This way,<br />
generated reactive oxygen species such as:<br />
• NO,<br />
ONOO – , O •− 2 and • OH, significantly contribute to the<br />
achievement of maximum force of the muscle<br />
contraction and increase strength of tetanic<br />
contractions of the muscle [4].<br />
In the human organism nitric oxide and other N-<br />
centered radicals which create reactive nitrogen species<br />
group (RNS) are particularly important [2]. • NO was<br />
described as endothelium-derived relaxing factor<br />
(EDRF) that plays an important role in the regulation<br />
of blood pressure [28]. It is also significant<br />
neurotransmitter [2] and takes part in regulation of the<br />
activity of NF-kappaB [25, 26]. Moreover, the nitric<br />
oxide-derived radicals may nitrosate and oxidize<br />
tyrosine residues of proteins and affect their<br />
phosphorylation, thus they can modulate activities of<br />
enzymatic proteins [29]. The next important function<br />
of RNS is to destroy pathogens in the process named<br />
the respiratory burst of phagocytes (neutrophils,<br />
macrophages and monocytes) [30]. The respiratory<br />
burst is initiated by creation of active complex of an<br />
enzyme – the NADPH oxidase. This enzyme is<br />
activated due to numerous mediators which include<br />
cytokines, which react with a suitable receptor on the<br />
surface of the cell. It allows production of superoxide<br />
anion (O •− 2 ) as a result of displacement of an electron<br />
•−<br />
from NADPH to molecular oxygen (O 2 ). Created O 2<br />
undergoes a dismutation to hydrogen peroxide (H 2 O 2 ).<br />
The dismutation reaction is catalyzed by superoxide<br />
dismutase (SOD) but may also be spontaneous. In the<br />
presence of ferrous ions hydroxyl radicals ( • OH) are<br />
produced and due to an action of myeloperoxidase a<br />
hypochlorous acid (HOCl) is formed that can react<br />
with amines to form chloramines. The high activity of<br />
inducible nitric oxide synthase (iNOS) inside of<br />
activated phagocytes is another source of antibacterial<br />
agents. Initially, a created nitric oxide (NO) reacts with<br />
•−<br />
O 2 and a highly bactericidal peroxynitrite (ONOO – )<br />
is produced. The nitric oxide in high concentrations has<br />
also antiseptic properties [28-30].<br />
Reactive oxygen species (ROS), including oxygen<br />
free radicals (OFR), play a significant role in a<br />
regulation of proper functioning of human organism.<br />
Still more facts show that inside of the human body<br />
ROS are produced continuously and their<br />
concentrations are tightly controlled by intra- and<br />
extracellular antioxidants.<br />
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2. Bobrowski K. Free radicals in chemistry, biology and<br />
medicine: contribution of radiation chemistry.<br />
Nukleonika 2005; 50(3): 67-76.<br />
3. Ziemlański Ś, Wartanowicz M. Rola antyoksydantów w<br />
stanie zdrowia i choroby. Pediatr. Współcz.<br />
Gastroenterol. Hepatol. Żywienie Dziecka 1999; 1(2/3):<br />
97-105.<br />
4. Clanton et al. Evidence for ROS production in skeletal<br />
muscle. P.S.E.B.M. 1999; 222: 253-262.<br />
5. Sutkowy P. Wpływ jednorazowej kriostymulacji<br />
ogólnoustrojowej na stężenie dialdehydu malonowego<br />
(MDA) i drobnocząsteczkowych antyoksydantów we<br />
krwi osób amatorsko uprawiających sport. Uniwersytet<br />
Mikołaja Kopernika, Collegium Medicum im Ludwika<br />
Rydygiera w Bydgoszczy. Praca magisterska, Toruń<br />
2010.<br />
6. Schafer FQ, Buettner GR. Redox environment of the cell<br />
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disulfide/glutathione couple. Free Radic Biol Med 2001;<br />
30(11): 1191-212.<br />
7. Burdon RH. Superoxide and hydrogen peroxide in<br />
relation to mammalian cell proliferation. Free Radic Biol<br />
Med 1995; 18(4): 775-794.<br />
8. Suzuki YJ, Forman HJ, Sevanian A. Oxidants as<br />
stimulators of signal transduction. Free Radic Biol Med<br />
1997; 22(1-2): 269-285.<br />
9. Sun Y, Oberley LW. Redox regulation of transcriptional<br />
activators. Free Radic Biol Med 1996; 21(3): 335-348.<br />
10. Allen RG, Tresini M. Oxidative stress and gene<br />
regulation. Free Radic Biol Med 2000; 28(3): 463-499.<br />
11. Arrigo AP. Gene expression and the thiol redox state.<br />
Free Radic Biol Med 1999; 27(9-10): 936-944.<br />
12. Chandra J, Samali A, Orrenius S. Triggering and<br />
modulation of apoptosis by oxidative stress. Free Radic<br />
Biol Med 2000; 29(3-4): 323-333.<br />
13. Gardner AM et al. Apoptotic vs. nonapoptotic<br />
cytotoxicity induced by hydrogen peroxide. Free Radic<br />
Biol Med 1997; 22(1-2): 73-83.<br />
14. Vogt W. Oxidation of methionyl residues in proteins:<br />
tools, targets, and reversal. Free Radic Biol Med 1995;<br />
18(1): 93-105.<br />
15. Monteiro HP, Stern A. Redox modulation of tyrosine<br />
phosphorylation-dependent signal transduction pathways.<br />
Free Radic Biol Med 1996; 21(3): 323-333.
Positive effect of generation of reactive oxygen species on the human organism 17<br />
16. Sundaresan M et al. Requirement for generation of H 2 O 2<br />
for platelet-derived growth factor signal transduction.<br />
Science 1995; 270: 296-299.<br />
17. Kamata H et al. Epidermal growth factor receptor is<br />
modulated by redox through multiple mechanisms:<br />
effects of reductants and H 2 O 2 . Eur J Biochem 2000;<br />
267: 1933-1944.<br />
18. Tonks NK. Redox redux: revisiting PTPs and the control<br />
of cell signaling. Cell 2005; 121: 667-670.<br />
19. Van der Wijk T, Blanchetot C, den Hertog J. Regulation<br />
of receptor protein-tyrosine phosphatase dimerization.<br />
Methods 2005; 35: 73-79.<br />
20. Czech MP, Fain JN. Cu ++ - dependent thiol stimulation of<br />
glucose metabolism in white fat cells. J Biol Chem 1972;<br />
247: 6218-6223.<br />
21. Mahadev K et al. The NAD(P)H oxidase homolog Nox4<br />
modulates insulin-stimulated generation of H2O2 and<br />
plays an integral role in insulin signal transduction. Mol<br />
Cell Biol 2004; 24: 1844-1854.<br />
22. Ushio-Fukai M et al. Reactive oxygen species mediate<br />
the activation of Akt/protein kinase B by angiotensin II in<br />
vascular smooth muscle cells. J Biol Chem 1999; 274:<br />
699-704.<br />
23. Mukhin YV. 5-Hydroxytryptamine 1A<br />
receptor/gibetagamma stimulates mitogen-activated<br />
protein kinase via NAD(P)H oxidase and reactive oxygen<br />
species upstream of src in Chinese hamster ovary<br />
fibroblasts. Biochem J 2000; 347: 61-67.<br />
24. Felty Q et al. Estrogen-induced mitochondrial reactive<br />
oxygen species as<br />
1. signal-transducing messengers. Biochemistry 2005; 44:<br />
6900-6909.<br />
25. Flohé L et al. Redox regulation of NF-kappa B<br />
activation. Free Radic Biol Med 1997; 22(6): 1115-1126.<br />
26. Janssen-Heininger YM, Poynter ME, Baeuerle PA.<br />
Recent advances towards understanding redox<br />
mechanisms in the activation of nuclear factor kappaB.<br />
Free Radic Biol Med 2000; 28(9): 1317-1327.<br />
27. Rahman I, Biswas SK, Kirkham PA. Regulation of<br />
inflammation and redox signaling by dietary<br />
polyphenols. Biochem Pharmacol 2006; 72: 1439-1452.<br />
28. Kowalczyk E i wsp. Tlenek azotu – oksydant czy<br />
antyoksydant? Wiad Lek 2005; 58(9–10): 540–542.<br />
29. Squadrito GL, Pryor WA. Oxidative chemistry of nitric<br />
oxide: the roles of superoxide, peroxynitrite, and carbon<br />
dioxide. Free Radic Biol Med 1998; 25(4-5): 392-403.<br />
30. Klebanoff SJ. Reactive nitrogen intermediates and<br />
antimicrobial activity: role of nitrite. Free Radic Biol<br />
Med 1993; 14(4): 351-360.<br />
Address for correspondence:<br />
mgr Paweł Sutkowy<br />
Katedra Biologii Medycznej<br />
Uniwersytet Mikołaja Kopernika<br />
Collegium Medicum w Bydgoszczy<br />
ul. Karłowicza 24<br />
85-092 Bydgoszcz<br />
tel.: 52 585 37 37<br />
tel./fax: 52 585 37 42<br />
e-mail: pawel2337@wp.pl<br />
Received: 13.11.2012<br />
Accepted for publication: 5.02.2013
Medical and Biological Sciences, 2013, 27/2, 19-25<br />
ORIGINAL ARTICLE / PRACA ORYGINALNA<br />
Maciej Gagat, Koryna Królikowska, Anna Klimaszewska-Wiśniewska, Magdalena Izdebska, Alina Grzanka<br />
THE EFFECT OF MILD HYPERTHERMIA ON MORPHOLOGY, ULTRASTRUCTURE<br />
AND F-ACTIN ORGANIZATION IN HL-60 CELL LINE<br />
WPŁYW ŁAGODNEJ HIPERTERMII NA MORFOLOGIĘ, ULTRASTRUKTURĘ I ORGANIZACJĘ<br />
F-AKTYNY W KOMÓRKACH LINII HL-60<br />
Department of Histology and Embryology, Nicolaus Copernicus University in Toruń, Collegium Medicum in<br />
Bydgoszcz, Karłowicza 24, 85-092 Bydgoszcz, Poland<br />
Head: Prof. Alina Grzanka, Ph.D.<br />
S u m m a r y<br />
I n t r o d u c t i o n . Hyperthermia is a well-established<br />
physical stimulus, which is applied as an adjunctive therapy<br />
with various cancer treatments, such as radiotherapy and<br />
chemotherapy. However, the precise mechanism of heat<br />
action at the cellular level remains to be elucidated, and<br />
appears to be multi-dimensional. The purpose of the current<br />
study was to determine the effect of mild hyperthermia on the<br />
actin cytoskeleton in the HL-60 cell line. In addition, the<br />
morphological and ultrastructural approaches were used to<br />
determine the type of hyperthermia-induced cell death.<br />
M a t e r i a l a n d m e t h o d s . All studies were<br />
performed using human promyelocytic leukemia cell line<br />
(HL-60). Actin filaments were visualized with phalloidin<br />
conjugated to Alexa Fluor® 488 using fluorescence<br />
microscopy. Morphological and ultrastructural changes in the<br />
HL-60 cells were analysed by light and electron microscopy,<br />
respectively.<br />
R e s u l t s . Exposure of HL-60 cells to mild<br />
hyperthermia resulted in the reorganization of the actin<br />
cytoskeleton and the appearance of characteristic apoptotic<br />
features, including cell shrinkage, chromatin condensation<br />
and margination. In addition, swollen mitochondria were<br />
observed. The morphological and ultrastructural changes<br />
increased in severity with an increase in recovery time.<br />
Similarly, actin filament remodeling was observed<br />
immediately after the heat shock and was more evident 3 and<br />
6 hrs after the treatment. These effects were mainly reflected<br />
by a higher definition of the dense cortical F-actin ring as<br />
well as the appearance of brightly fluorescent F-actin dots<br />
and networks scattered throughout the cytoplasm.<br />
C o n c l u s i o n s . Presented data suggest that actin<br />
filament reorganization is involved in the process of<br />
apoptosis initiated by mild hyperthermia. Furthermore, the<br />
results of our studies showed that the severity of<br />
hyperthermia-induced morphological and ultrastructural<br />
changes as well as alterations in actin organization depend<br />
not only on the temperature treatment but also on the<br />
duration of post heat shock recovery.<br />
S t r e s z c z e n i e<br />
W s t ę p . Hipertermia jest dynamicznie rozwijającą się<br />
metodą lecznia nowotworów, stosowaną w skojarzeniu<br />
z radio- i/lub chemioterapią. Precyzyjny mechanizm<br />
działania hipertermii na poziomie komórkowym nie został,<br />
jak dotąd w pełni poznany i wydaje się on wielowymiarowy.<br />
Celem niniejszej pracy była analiza wpływu łagodnej<br />
hipertermii na organizację filamentów aktynowych<br />
w komórkach linii HL-60. Za zasadną uznano także ocenę<br />
zmian morfologicznych i ultrastrukturalnych wywołanych<br />
przez hipertermię, celem określenia rodzaju uruchamianej<br />
śmierci zachodzącej w badanej linii.<br />
M a t e r i a ł y i m e t o d y . Badania przeprowadzono<br />
na komórkach linii białaczki promielocytowej HL-60.<br />
Filameny aktynowe wyznakowano falloidyną skoniugowaną<br />
z Alexa Fluor® 488 i oglądano w mikroskopie<br />
fluorescencyjnym. Morfologiczne i ultrastrukturalne zmiany<br />
w komórkach oceniono odpowiednio, przy użyciu<br />
mikroskopu świetlnego oraz transmisyjnego mikroskopu<br />
elektronowego.
20<br />
Maciej Gagat et al.<br />
W y n i k i . W wyniku ekspozycji komórek HL-60 na<br />
podwyższoną temperaturę obserwowano reorganizację<br />
cytoszkieletu aktynowego oraz pojawienie się komórek o<br />
cechach charakterystycznych dla procesu apoptozy, takich<br />
jak obkurcznie cyto- i nukleoplazmy, kondensacja i<br />
marginalizacja chromatyny czy obrzmienie mitochondriów.<br />
W wyniku rearanżacji, F-aktyna lokalizowała sie głównie w<br />
części korowej komórki w formie pierścienia lub w<br />
cytoplazmie w postaci wyraźnie wyznakowanych sieci i<br />
skupień. Stopień nasilenia zmian w komórkach wzrastał wraz<br />
ze wzrostem czasu regeneracji komórek.<br />
W n i o s k i . Uzyskane wyniki pozwalają stwierdzić, że<br />
cytoszkielet aktynowy zaangażowany jest w realizację<br />
procesu apoptozy indukowanej przez łagodną hipertermię.<br />
Ponadto sugerują one, że na wystąpienie zmian w organizacji<br />
filamentów aktynowych, jak również zmian w morfologii i<br />
ultrastrukturze komórek ma wpływ, nie tylko zastosowany<br />
profil temperaturowy, ale również czas regeneracji komórek.<br />
Key words: hyperthermia, actin filaments, HL-60 cell line<br />
Słowa kluczowe: hipertermia, filamenty aktynowe, linia komórkowa HL-60<br />
INTRODUCTION<br />
The term ‘hyperthermia’ refers to raising the<br />
temperature of a part of the body or of the whole body<br />
above the threshold temperature set at a particular<br />
moment by the thermoregulatory system of the<br />
organism [1,2]. Hyperthermia has been used for its<br />
medical properties since ancient times. Presumably, the<br />
oldest written medical report with references to<br />
hyperthermia was found in the Egyptian Edwin Smith<br />
surgical papyrus, dated around 3000 BC [3]; whereas,<br />
the use of hyperthermia for cancer therapy was first<br />
reported by Hypocrites in the treatment of breast tumor<br />
[4]. Currently, hyperthermia is a well-established<br />
physical modality, which is used as an adjunctive<br />
therapy with various established cancer treatments,<br />
such as radiotherapy and chemotherapy [5].<br />
The scientific rationales for using hyperthermia in<br />
cancer treatment are based on the data from in vitro,<br />
animal and preclinical studies [3,6]. It has been shown<br />
that the alterations in plasma membrane permeability<br />
caused by hyperthermia lead to better infiltration and<br />
drug absorption by the tumor [7]. It has been also<br />
suggested that hyperthermia treatment activates the<br />
immune system against the tumor [8]. Moreover,<br />
cancer cells are selectively sensitive to heat shock<br />
treatment, while the same conditions rarely affect the<br />
growth of normal cells. Additionally, an increase in<br />
thermosensitivity of tumor cells results in the reduction<br />
of tumor blood flow which makes their environment<br />
more hypoxic and acidic. Moreover, several studies<br />
have shown that hypoxia and particularly acidity<br />
enhance the cytotoxicity of hyperthermia [9,10].<br />
The mechanisms of hyperthermia cytotoxicity<br />
involve denaturation of cellular proteins and their<br />
subsequent aggregation as well as plasma membrane<br />
damage, inhibition of DNA repair, and changes in<br />
intracellular calcium homeostasis. The heat-induced<br />
alterations in cell structure and function lead to cell<br />
cycle arrest and cell death [11].<br />
Among the most noteworthy alterations in heatshocked<br />
cells are the induction of heat shock proteins<br />
(HSPs) and reorganization of the cytoskeleton [12].<br />
The actin cytoskeleton is involved in the regulation of<br />
fundamental cellular processes such as motility,<br />
cytokinesis, adhesion and endocytosis [13].<br />
Furthermore, it also participates in signal transduction<br />
regulating cell growth, survival, and death. Therefore,<br />
the actin cytoskeleton may represent an attractive<br />
target for hyperthermic therapy against different types<br />
of cancer [13, 14]. However, the mechanism of<br />
structural and functional actin remodeling in response<br />
to heat shock is not fully understood.<br />
The aim of this study was to determine the effect of<br />
mild hyperthermia on actin cytoskeleton organization<br />
in the HL-60 cell line, in the context of involvement in<br />
cell death process.<br />
MATHERIAL AND METHODS<br />
Cell culture and treatment<br />
The human promyelocytic leukemia cell line<br />
HL-60 was purchased from the American Type Culture<br />
Collection (ATCC, Manassas, VA; CCL-240). The<br />
cells were cultured in RPMI 1640 medium (Lonza)<br />
supplemented with 10% fetal bovine serum (FBS,<br />
PAA) and 10 mg/ml gentamicin (Lonza), at 37ºC in a<br />
humidified 5% CO 2 atmosphere. After 48 hr cell<br />
culture, HL-60 cells were subjected to heat shock<br />
(40ºC, 2 hrs), followed by post-heat shock recovery at<br />
37ºC for 0, 3 and 6 hrs. Control cells were cultured<br />
identically without exposure to heat treatment. Cell<br />
viability was assessed by the trypan blue dye exclusion<br />
method.
The effect of mild hyperthermia on morphology, ultrastructure and F-actin organization in HL-60 cell line 21<br />
Light microscopy studies<br />
For the morphological analysis, HL-60 cells were<br />
fixed in 4% paraformaldehyde. After fixation, the cells<br />
were incubated with 0.1M glycine solution (Roth) and<br />
then the cell suspension was centrifuged onto glass<br />
slides. Thereafter, the cells were stained with Mayer's<br />
hematoxylin and rinsed under running tap water and<br />
dehydrated in a graded series of alcohols and xylenes.<br />
The preparations were observed using an Eclipse E800<br />
microscope (Nikon) with NIS-Elements ver. 3.30<br />
image analysis system (Nikon) and CCD camera<br />
(DS-5Mc-U1; Nikon).<br />
Transmission electron microscopy studies<br />
For ultrastructural analysis, the cells were fixed<br />
with 3.6% glutaraldehyde and then postfixed with 1%<br />
osmium tetroxide, dehydrated with graded series of<br />
alcohol and acetone, and embedded in Epon 812. The<br />
polymerization of the resin was performed at 37°C for<br />
24 hrs, and then at 65°C for 120 hrs. Selected parts of<br />
material were cut into ultra-thin sections by using an<br />
OmU3 ultramicrotome (Reichert) and then<br />
counterstained with uranyl acetate and lead citrate. The<br />
material was examined using JEM 100 CX electron<br />
microscope (JEOL).<br />
Fluorescence microscopy studies<br />
For fluorescence labeling of actin, the cells were<br />
fixed with 4% paraformaldehyde. After fixation, the<br />
cells were incubated with 0.1M glycine solution (Roth)<br />
and then the cell suspension was centrifuged onto glass<br />
slides. The cells were then permeabilized with 0.1%<br />
Triton X-100 (Serva Feinbiochemica). To enable<br />
visualization of F-actin, the cells were incubated with<br />
phalloidin conjugated to Alexa Fluor 488 (Invitrogen,<br />
diluted 1:40). The nuclei of the cells were labeled with<br />
4’,6-diamidino-2-phenyloindole (DAPI; Sigma-<br />
Aldrich). Slides were mounted in Aqua-Poly/Mount<br />
(Polysciences) and analysed by using an Eclipse E800<br />
microscope with a Y-FL fluorescence attachment<br />
(Nikon), NIS-Elements ver. 3.30 image analysis<br />
system (Nikon) and CCD camera (DS-5Mc-U1;<br />
Nikon).<br />
Statistical analysis<br />
The non-parametric Mann-Whitney U test was<br />
performed to compare the differences between<br />
experimental groups. The results were considered<br />
statistically significant at p
22<br />
Maciej Gagat et al.<br />
The effect of mild hyperthermia on the morphology<br />
and ultrastructure of HL-60 cells<br />
The evaluation of changes in the morphology and<br />
ultrastructure of heat-shocked HL-60 cells was<br />
performed. Control HL-60 cells were circular or oval<br />
in shape and contained intact nuclei. Only a few cells<br />
were morphologically changed (Fig. 2A).<br />
The appearance of HL-60 cells fixed immediately<br />
after a mild heat shock (40°C/2hrs) was not<br />
significantly different from that of the control (Fig.<br />
2B). However, with increasing recovery time more<br />
advanced changes in cell morphology and<br />
ultrastructure occurred in the heat-treated cultures.<br />
After 3 and 6 hrs of recovery, the shrunken cells with<br />
irregular and undulating surface as well as chromatin<br />
condensation and marginalization were observed (Fig.<br />
2C,D,F, 3C). The cells with fragmented nuclei were<br />
also noticed (Fig. 2D). Additional changes, including<br />
swollen mitochondria (Fig. 3C) and vacuolization of<br />
the cytoplasm (Fig. 3D) were also detected. Moreover,<br />
a few cells with the morphological appearance of<br />
mono-nucleated giant cells were seen (Fig. 2C,F).<br />
Fig. 2. The effect of mild hyperthermia on morphology of<br />
HL-60 cells, stained with Mayer's hematoxylin; Nontreated<br />
cells (A); The HL-60 cells were heat shocked<br />
at 40ºC for 2 hrs (B) and returned to 37ºC for: 3 hrs<br />
(C,D) or 6 hrs (E,F); The enlarged cells with one big<br />
nucleus are seen (C,F arrows I); The shrunken cells<br />
with fragmented nuclei (D arrows II) and/or<br />
chromatin condensation (B, C, D, F arrows III) can<br />
be also observed; The swollen cell with fragmented<br />
nucleus is seen (D, E arrows IV)<br />
Ryc. 2. Wpływ łagodnej hipertermii na morfologię komórek<br />
linii HL-60, barwionych hematoksyliną Mayera;<br />
Komórki kontrolne (A); Komórki HL-60 poddano<br />
szokowi cieplnemu w temperaturze 40ºC przez 2h<br />
(B), po czym ponownie umieszczono w 37ºC na okres<br />
3 (C,D) lub 6h (E,F); Widoczne powiększone komórki<br />
z jednym dużym jądrem (C, F strzałki I); Widoczne<br />
obkurczone komórki z fragmentację jądra (D strzałki<br />
II) i/lub kondensacją chromatyny (B,C,D,F strzałki<br />
III) Widoczne również obrzmiałe komórki z<br />
fragmentacją jądra (D,E strzałki IV)<br />
Fig. 3. The<br />
effect mild<br />
hyperthermia<br />
on the<br />
ultrastructure<br />
of HL-60<br />
cells; Nontreated<br />
cells<br />
(A); The HL-<br />
60 cells were<br />
heat shocked<br />
at 40ºC for 2<br />
hrs (B) and<br />
returned to<br />
37ºC for: 3 hrs<br />
(C) or 6 hrs<br />
(D); Visible<br />
swollen<br />
mitochondria<br />
(C arrows I);<br />
Cytoplasmic<br />
vacuolization<br />
is seen (C,D<br />
arrows II)<br />
Ruc. 3. Wpływ<br />
łagodnej<br />
hipertermii na<br />
ultrastrukturę<br />
komórek HL-<br />
60; Komórki<br />
kontrolne (A);<br />
Komórki HL-<br />
60 poddano<br />
szokowi<br />
cieplnemu w temperaturze 40ºC przez 2h (B), po czym<br />
ponownie umieszczono w 37ºC na okres 3 (C) lub 6h (D);<br />
Widoczne obrzmiałe mitochondria (C strzałki I) oraz<br />
wakuolizacja cytoplazmy (C,D strzałki II)
The effect of mild hyperthermia on morphology, ultrastructure and F-actin organization in HL-60 cell line 23<br />
The effect of mild hyperthermia on F-actin<br />
organization<br />
Alexa Fluor 488-phalloidin staining was applied for<br />
visualization of changes in filamentous actin<br />
organization after treatment of HL-60 cells with mild<br />
hyperthermia. Control HL-60 cells displayed a spread<br />
F-actin cytoskeleton with well-defined peripheral<br />
limits and intact nuclei. Only a few nuclei were<br />
morphologically changed (Fig. 4A,A').<br />
dense cortical F-actin ring as well as the appearance of<br />
bright F-actin clusters and network scattered<br />
throughout the cytoplasm (Fig. 4C,D). Moreover,<br />
directly after heating, the cells with fragmented nuclei<br />
and degradation of the F-actin cytoskeleton were often<br />
observed (Fig. 4B,B'). These alterations were also<br />
noticed following a recovery time of 3 and 6 hrs (Fig.<br />
4C,C',D,D'). After the recovery period, actin filaments<br />
were arranged circumferentially in dense, ring-like<br />
structures or in the form of densifications or<br />
aggregations under the plasma membrane (Fig. 4D).<br />
Furthermore, F-actin became organized in dots and<br />
networks scattered throughout the cytoplasm (Fig. 4C).<br />
Fig. 4. The effect of mild hyperthermia on the F-actin<br />
organization in the HL-60 cells; F-actin was stained<br />
with Alexa Fluor 488 phalloidin, nuclei were<br />
counterstained with DAPI; Non-treated cells (A,A');<br />
The HL-60 cells were heat shocked at 40ºC for 2 hrs<br />
(B,B') and returned to 37ºC for: 3 hrs (C,C') or 6 hrs<br />
(D,D')<br />
Ryc. 4. Wpływ łagodnej hipertermii na organizację F-aktyny<br />
w komórkach linii HL-60; F-aktynę wyznakowano<br />
falloidyną skoniugowaną z Alexa Fluor 488, jądra<br />
wybarwiono DAPI; Komórki kontrolne (A,A');<br />
Komórki HL-60 poddano szokowi cieplnemu w<br />
temperaturze 40ºC przez 2h (B,B'), po czym<br />
ponownie umieszczono w 37ºC na okres 3 (C,C') lub<br />
6h (D,D')<br />
The structural remodeling of actin was observed<br />
immediately after heat shock treatment but was more<br />
evident 3 and 6 hrs after the exposure. These effects<br />
were reflected mainly by a higher definition of the<br />
Discussion<br />
Hyperthermia is a type of medical therapy in<br />
which body tissues are exposed to slightly<br />
higher temperatures to damage or make cancer cells<br />
more sensitive to other chemical or physical factors.<br />
Heat-shock destroys enzyme complexes present in the<br />
cytoplasm and mitochondria membrane but also causes<br />
alterations in enzyme system cycles, which is involved<br />
with changes in chromatin organization, regulation of<br />
gene expression and ion homeostasis [12, 15]. Our<br />
previous studies showed the effect of 41 and 44.5ºC on<br />
the morphology, ultrastructure and actin cytoskeleton<br />
of CHO AA8 cells [16, 17]. We also investigated the<br />
influence of hyperthermia (43.5 and 45ºC) on H1299<br />
lung cancer cells [18]. In our studies, we noticed shape<br />
changes and characteristic, apoptotic features in heattreated<br />
CHO AA8 and H1299 cells [16-18].<br />
Furthermore, H1299 cells treated with 43.5 and 45°C<br />
heat stress showed similar changes in cell shape and<br />
membrane structure, which were probably the<br />
consequence of loss or reduction of integrins at the cell<br />
surface [18]. Hyperthermia also induces changes in<br />
actin organization, especially in switching from<br />
filamentous to monomeric state [19]. In CHO AA8 and<br />
H1299 reorganization of actin cytoskeleton was also<br />
observed [16-18]. In this paper, analyses were<br />
performed using a non-adherent human promyelocytic<br />
leukemia cell line (HL-60) and, as previously, we<br />
observed changes in the morphology and ultrastructure<br />
after heat shock treatment. It is known that mild<br />
hyperthermia promotes cell viability and proliferation<br />
[20]. In this study, we did not find any differences in<br />
viability between the control and heat-shocked cells.<br />
Following a recovery period of 3 and 6 hrs, a slight<br />
reduction in cell viability was noticed, but these data<br />
were statistically insignificant. 3 and 6 hrs after
24<br />
Maciej Gagat et al.<br />
heating, the shrunken cells with condensed and<br />
marginated chromatin were present. The cells with<br />
fragmented nuclei were also noticed. The same results<br />
were reported by Luchetti et al. (2002), who used 1 hr<br />
hyperthermia (43ºC) to induce apoptotic response of<br />
HL-60 cells, followed by a recovery time (6 hrs, 37°C)<br />
[21]. Moreover, at the ultrustructural level, the swollen<br />
mitochondria were detected. Cole et al. (1988) and<br />
Wheatley et al. (1989) observed the mitochondrial<br />
damage in cells exposed to hyperthermia [22,23]. The<br />
43 and 45°C hyperthermia induced a decrease in pH<br />
and mitochondrial matrix density. Many researchers<br />
revealed that the effect of heat shock on the<br />
mitochondrial membrane potential is associated with a<br />
change in the cellular redox status of cells.<br />
Depolarization of mitochondrial membrane results in<br />
the reactive oxygen species (ROS) outburst [22,23]. In<br />
our previous studies with CHO AA8 or H1299 cells,<br />
apart from apoptotic cells, we observed giant cells with<br />
one enlarged nucleus or with the features of<br />
micronucleation. These morphological changes are<br />
characteristic for mitotic catastrophe [16-18]. The same<br />
results were presented by Nakahata et al. (2002), who<br />
observed tumour cells with multiple micronuclei<br />
whose number increased with the post-treatment<br />
recovery time [24]. In the present study, a few mononucleated<br />
giant cells were noted, but we cannot<br />
identify them as cells with the mitotic catastrophe<br />
phenotype.<br />
Additionally, in this paper, we showed<br />
hyperthermia-induced reorganization of actin<br />
filaments. It is known that actin plays an important role<br />
not only in cell motility, membrane ruffing and<br />
formation of lamellipodia in adherent cells, but it is<br />
also involved in proliferation, differentiation, and<br />
apoptosis [13,14]. In 2003, Grzanka et al. presented a<br />
correlation between actin reorganization and apoptotic<br />
body formation during apoptosis [25]. The same<br />
scientists noticed that F-actin was seen in the nuclei of<br />
apoptotic cells [26]. Proapoptotic effect of<br />
hyperthermia has been demonstrated in various cell<br />
lines. It is known that heat shock induces changes not<br />
only in the actin cytoskeleton but also in the<br />
organization of microtubules and intermediate<br />
filaments, e.g. vimentin polymers [18]. In the present<br />
study, we observed F-actin especially at the cell<br />
periphery. We did not reveal any significant<br />
differences immediately after mild heat shock<br />
treatment (40°C/2hrs). 3 and 6 hrs after heating, we<br />
observed dense actin filaments arranged<br />
circumferentially in the ring-like structures or in the<br />
form of densifications or aggregations under the<br />
plasma membrane. We also found F-actin in form of<br />
dots and networks scattered in the cytoplasm of HL-60<br />
cells. Our previous studies showed similar effect of<br />
cytostatic drugs (arsenic trioxide, doxorubicin, and<br />
taxol) on actin organization in HL-60 cells and<br />
confirmed that actin reorganization is associated with<br />
cell death process [25-27]. After treatment with<br />
cytostatic drugs, the actin network in the cytoplasm<br />
undergoes reorganization to form aggregates, which<br />
are necessary for the formation of apoptotic bodies.<br />
Additionally, Luchetti et al. (2002) suggested that actin<br />
could be directly involved in chromatin rearrangement<br />
occurring during apoptotic cell death [21].<br />
In conclusion, our studies suggest that actin<br />
filament reorganization is involved in the process of<br />
apoptosis initiated by mild hyperthermia. Furthermore,<br />
we also showed that the severity of hyperthermiainduced<br />
morphological and ultrastructural changes as<br />
well as alterations in actin organization depend not<br />
only on the temperature treatment but also on the<br />
duration of post-heat shock recovery period.<br />
REFERENCES<br />
1. Habash R.W.Y., Bansal R., Krewski D. et al.: Thermal<br />
Therapy, Part 2: Hyperthermia Techniques. Crit Rev<br />
Biomed Eng, 2006; 34: 491-542<br />
2. Chicheł A., Skowronek J., Kubaszewska M. et al.:<br />
Hyperthermia – description of a method and a review of<br />
clinical applications. Rep Pract Oncol Radiother, 2007;<br />
12: 267-275<br />
3. Zee J.: Heating the patient: a promising approach? Ann<br />
Oncol, 2002; 13: 1173-1184<br />
4. Fiorentini G., Szasz A: Hyperthermia today: Electric<br />
energy, a new opportunity in cancer treatment. J Cancer<br />
Res Ther, 2006; 2: 41-46<br />
5. Wust P., Hildebrandt B., Sreenivasa G., Rau B.,<br />
Gellermann J., Riess H., Felix R., Schlag P.M:<br />
Hyperthermia in combined treatment of cancer. THE<br />
LANCEL Oncol., 2002; 3: 487-497<br />
6. Hildebrandt B., Wust P.: The biologic rationale of<br />
hyperthermia. Cancer Treat Res. 2007; 134: 171-84<br />
7. Kong G., Anyarambhatla G., Petros W.B. et al.: Efficacy<br />
of liposomes and hyperthermia in a human tumor<br />
xenograft model: importance of triggered drug release.<br />
Cancer Res, 2000; 60: 6950-6957<br />
8. Milani V., Noessner E., Ghose S. et al.: Heat shock<br />
protein 70: role in antigen presentation and immune<br />
stimulation. Int J Hyperther, 2002; 18: 563-575<br />
9. Song Ch.W., Lyons J.C., Griffin R.J. et al.: Increase in<br />
Thermosensitivity of tumor cells by lowering<br />
intracellular pH. Cancer Res, 1993; 53: 1599-1601
The effect of mild hyperthermia on morphology, ultrastructure and F-actin organization in HL-60 cell line 25<br />
10. Sakaguchi Y., Maehara Y., Baba H. et al.: Flavone acetic<br />
acid increases the antitumor effect of hyperthermia in<br />
mice. Cancer Res, 1992; 52: 3306-3309<br />
11. Hildebrandt B., Wust P., Ahlers O. et al.: The cellular<br />
and molecular basis of hyperthermia. Crit Rev<br />
Oncol/Hematol, 2002; 43: 33-56<br />
12. Coss R.A., Linnemans W.A.: The effects of hyperthermia<br />
on the cytoskeleton. Int J Hyprther, 1996; 12: 173-196<br />
13. Reisler E., Egelman E.H.: Actin structure and function:<br />
What we still do not understand. J Biol Chem, 2007; 282:<br />
36133-36137<br />
14. Desouza M., Gunning P.W., Stehn J.R.: The actin<br />
cytoskeleton as a sensor and mediator of apoptosis.<br />
BioArchitecture, 2012; 2: 75-87<br />
15. Streffer C. : Metabolic changes during and after<br />
hyperthermia. Int J Hyperthermia, 1985; 1: 305-319<br />
16. Grzanka D., Stepien A., Grzanka A. et al.: Hyperthermiainduced<br />
reorganization of microtubulrs and<br />
microfilaments and cel killing in CHO AA8 cell line.<br />
Neoplasma, 2008; 55: 409-415<br />
17. Gagat M., Grzanka A.A., Grzanka A.: Evaluation of the<br />
effect of mile hyperthermia on morphology in CHO AA8<br />
cell line. Med Biol Scie, 2010; 24: 25-32<br />
18. Pawlik A., Nowak J.M., Grzanka D. et al.:<br />
Hyperthermia induces cytoskeletal alterations and mitotic<br />
catastrophe in p53-deficient H1299 lung cancer cells.<br />
Acta Histochem, 2012<br />
19. Luchetti F., Mannello F., Canonico B. et al.: Integrin and<br />
cytoskeleton behaviour in human neuroblastoma cells<br />
during hyperthermia-related apoptosis. Apoptosis, 2004;<br />
9: 635-648<br />
20. Shui C., Scutt A.: Mild heat shock induces proliferation,<br />
alkaline phosphatase activity, and mineralization in<br />
human bone marrow stromal cells and Mg-63 cell in<br />
vitro. J Bone Miner Res, 2001; 16: 731-741<br />
21. Luchetti F., Burattini S., Ferri P., et al.: Actin<br />
involvement in apoptotic chromatin changes of<br />
hemopoietic cells undergoing hyperthermia. Apoptosis,<br />
2002; 7: 143-152.<br />
22. Cole A., Armour E.P.: Ultrastructural study of<br />
mitochondrial damage in CHO cells exposed to<br />
hyperthermia. Radiat Res, 1988; 115: 421-435<br />
23. Wheatley D.N., Kerr C., Gregory D.W.: Heat-induced<br />
damage to HeLa-S3 cells: correlation of viability,<br />
permeability, osmosensitivity, phase-contrast light-,<br />
scanning electron- and transmission electronmicroscopical<br />
findings. Int J Hyperthermia, 1989; 5: 145-<br />
162<br />
24. Nakahata K., Miyakoda M., Suzuki K., Kodama S.,<br />
Watanabe M.: Heat shock induces centrosomal<br />
dysfunction, and causes non-apoptotic mitotic<br />
catastrophe in human tumour cells. Int J Hyperthermia,<br />
2002; 18: 332-43<br />
25. Grzanka A., Grzanka D., Orlikowska M.: Cytoskeletal<br />
reorganization during process of apoptosis induced by<br />
cytostatic drugs in K-562 and HL-60 leukemia cell lines.<br />
Biochem Pharmacol, 2003; 66: 1611-1617<br />
26. Grzanka A., Grzanka D., Orlikowska M.: Fluorescence<br />
and ultrastructural localization of actin distribution<br />
patterns in the nucleus of HL-60 and K-562 cell lines<br />
treated with cytostatic drugs. Oncol Rep, 2004; 11: 765-<br />
70<br />
27. Izdebska M., Grzanka D., Gackowska L., Żuryń A.,<br />
Grzanka A.: The influence of Trisenox on actin<br />
organization in HL-60 cells. Cent Eur J Biol, 2009; 4:<br />
351-361<br />
Address for correspondence:<br />
Prof. Alina Grzanka, Ph.D.<br />
Nicolaus Copernicus University in Toruń<br />
Collegium Medicum in Bydgoszcz<br />
Department of Histology and Embryology<br />
24 Karłowicza St., 85-092 Bydgoszcz, Poland<br />
tel.: +48525853725; fax: +48525853734<br />
e-mail: agrzanka@cm.umk.pl<br />
Received: 13.11.2012<br />
Accepted for publication: 26.03.2013
Medical and Biological Sciences, 2013, 27/2, 27-31<br />
ORIGINAL ARTICLE / PRACA ORYGINALNA<br />
Wojciech Pawłowicz<br />
MODEL STUDY OF EPITHELIAL MOTILITY<br />
MODELOWE BADANIA AKTYWNOŚCI RUCHOWEJ NABŁONKA<br />
Department of Pathobiochemistry and Clinical Chemistry, Collegium Medicum, Nicolaus Copernicus University,<br />
M. Skłodowskiej-Curie 9, 85-094 Bydgoszcz, Poland<br />
Head of Department: Professor Tomasz Tyrakowski<br />
S u m m a r y<br />
One of the oldest achievements of human thought is the<br />
use of plants and plant extracts in therapeutics. Drugs of<br />
plant origin are characterized by multi-effects. In recent<br />
years, much interest in medicinal plants containing a mixture<br />
of biologically active substances with antimicrobial<br />
properties has increased. In medicine, extracts from plants<br />
and their secondary metabolites and plant extracts have been<br />
used for many years, but now by the development of organic<br />
chemistry, pharmacology and medicine, we can determine<br />
which biologically active substances produced by these<br />
plants are useful. Antimicrobial activity described selected<br />
groups of plant secondary metabolites, which potentially<br />
would allow their use as antimicrobial substances in<br />
medicine. These substances can be complementary to the<br />
basic medical treatment because their main advantage is the<br />
lower incidence of side effects. This paper presents an<br />
overview of research on the antimicrobial properties of<br />
alkaloids, coumarins, flavonoids, essential oils, phytosterols,<br />
and phenolic acids. Natural substances that inhibit the growth<br />
of microorganisms are becoming an alternative to synthetic<br />
compounds, as confirmed by this literature review.<br />
S t r e s z c z e n i e<br />
C e l e m b a d a n i a jest odnalezienie substancji<br />
mogących wpływać na aktywność ruchową nabłonka<br />
w modelu eksperymentalnym poruszającego się ślimaka<br />
Achatina achatina. Ze względu na fizjologiczne,<br />
biochemiczne i fizyczne podobieństwa pomiędzy nabłonkami<br />
różnych gatunków, wyniki mogą mieć znaczenie dla<br />
mechanizmów fizjologicznego oddziaływania tych<br />
substancji.<br />
M a t e r i a ł i m e t o d a . Sfilmowano proces ruchu<br />
ślimaków Achatina achatina zarówno spontaniczny jak i po<br />
wstrzyknięciu w stopę wybranych wcześniej neuroprzekaźników<br />
(serotoniny, kompleksu serotoninowokreatyninowego<br />
adrenaliny, noradrenaliny i dopaminy) oraz<br />
ambroksolu. Parametry mierzono wykorzystując filmy<br />
nagrane od spodu przez szklaną płytę po której poruszały się<br />
zwierzęta. Fale aktywności ruchowej nabłonka występowały<br />
na powierzchni stopy podczas ruchu.<br />
W y n i k i i d y s k u s j a . Każda z badanych<br />
substancji miała wpływ na ruch ślimaków. Wstrzyknięcie<br />
serotoniny, kompleksu serotoninowo-kreatyninowego,<br />
noradrenaliny i ambroksolu spowodowało wzmożenie<br />
aktywności ruchowej ślimaków, z kolei adrenalina<br />
i dopamina nie powodowały takich zmian, lub też działały<br />
hamująco. Ważne parametry, których zmiany<br />
zaobserwowano, to długość fali skurczu i odległości między<br />
nimi, częstotliwość fali skurczu, prędkość ślimaka,<br />
przesunięcie ciała na jedną falę skurczu i wydajność skurczu.<br />
W n i o s k i . Wyniki wskazują na możliwość<br />
zastosowania ślimaka A. achatina jako biologicznego<br />
modelu, ponieważ w czasie ruchu ślimaka można<br />
zaobserwować aktywność motoryczną nabłonka sterowaną<br />
przez układ nerwowy i wywoływaną skurczami tkanki<br />
mięśniowej, podobnie jak w ludzkich tkankach.<br />
Wstrzyknięcie serotoniny, kompleksu serotoninowo-kreatyninowego<br />
adrenaliny, noradrenaliny i dopaminy<br />
w organizmie modelowym A. achatina zmienia fale skurczu<br />
nabłonka na stopie. Postuluje się, że podobny proces<br />
zachodzi w innych nabłonkach, w tym ludzkich.<br />
Key words: epithelium, Achatina achatina, motility, pedal wave<br />
Słowa kluczowe: nabłonek, Achatina achatina, aktywność ruchowa, fale skurczu
28<br />
Wojciech Pawłowicz<br />
INTRODUCTION<br />
While general functions and physiology of<br />
epithelial tissue have been thoroughly recognized, less<br />
attention is focused on its motility, particularly the one<br />
related to movements of different substrates on<br />
epithelial surface. Various types of epithelium show<br />
different patterns of movement, which serves specific<br />
purposes, but the nature and exact mechanisms of its<br />
regulation are not fully known. To better understand<br />
the nature of these movements, a biological model<br />
could be helpful, aside from traditional methods which,<br />
like the Ussing apparatus, involve using tissue samples.<br />
In search for such a model, a set of similarities<br />
between snail pedal epithelium and other epithelia has<br />
been evaluated. As the animal moves forward series of<br />
repeated epithelial darkening areas called pedal waves<br />
are observed, which origin at the back of the foot and<br />
move forward towards the head as the animal moves<br />
forward [1]. Because snail movements are similar in<br />
physical nature to bowel movements, snails have the<br />
potential to be used as biological models. The wave<br />
progression and how it relates to overall mollusk<br />
adhesive locomotion in normal, unmodified conditions<br />
has already been studied [2,3,4]. The results showed<br />
that both pedal wave frequency and pedal wave length<br />
could play a key role in regulation of locomotion;<br />
however, these studies have so far proved inconclusive<br />
[5].<br />
Fig. 1. Side view of a moving snail illustrating the pedal<br />
waves where the epithelium tissue is lifted and<br />
contracted. Different vector lengths illustrate the<br />
variety of individual waves<br />
Ryc. 1. Widok z boku na poruszającego się ślimaka<br />
ilustrujący fale na stopie, gdzie nabłonek ulega<br />
skurczeniu i podniesieniu. Różnice w wektorach<br />
ilustrują zróżnicowanie poszczególnych fal<br />
Another important factor is a thin layer of mucus<br />
produced by snail, on which it slides, and which<br />
alternately serves as adhesive material. Probably, the<br />
pedal waves are the way to regulate friction between<br />
the sole of the foot and the surface. The mucus, being a<br />
non-newtonian fluid, is efficient in transferring<br />
movement energy from the sole of the foot to the<br />
surface [6, 7, 8]. By its prosperities, the animal is also<br />
able to adapt to various conditions and use it more<br />
efficiently [9]. Mammalian epithelia, such as these<br />
covering the airways or gastrointernal tract, are also<br />
known to produce mucus that plays a key role in<br />
moving foreign substrates on their surfaces. In this<br />
study, the problem whether snails respond to<br />
pharmacological modification of their movement<br />
pattern was examined. Vulnerability to<br />
neurotransmitters, drugs and other chemical substances<br />
are a key aspect in considering the use of snails as a<br />
potential biological model of epithelium movement.<br />
MATERIAL AND METHODS<br />
The study used control and study groups of 10<br />
Achatina achatina snails between 16 and 23 grams of<br />
body mass and 40 to 50mm of shell length. The snails<br />
were kept in a moist terrarium with a peat surface, at<br />
room temperature, and were fed with cabbage with<br />
addition of egg shells. During the study the snails were<br />
placed on a transparent glass surface, and their<br />
movement was then recorded with a digital camera<br />
CCD DFK 41 AV02.AS with a CCTV 5-50 mm<br />
F/1.8 lens, using IC Capture.AS 2.0 software. The<br />
recordings were then processed and examined using<br />
VirtualDub 1.9.9 and image analysis software.<br />
Recordings were all conducted starting at 9 a.m., at<br />
room temperature, and were approximately 30<br />
seconds long per animal.<br />
The following variables were used in the study:<br />
length of single pedal waves [mm], length of single<br />
intervals between pedal waves [mm], speed of<br />
snail’s head [mm/s], frequency of pedal waves [1/s],<br />
total distance covered per pedal wave [mm] and<br />
epithelium fold coefficient. The last value was<br />
calculated by the following equation and shows how<br />
the distance covered by the animal corresponding<br />
with the pedal wave length:<br />
u – epithelium fold coefficient [%]<br />
shw – distance covered per pedal wave [mm]<br />
lw – single pedal wave length [mm]<br />
During the study the following neurotransmitters<br />
were tested: epinephrine, norepinephrine, dopamine,<br />
serotonin (both pure and in a serotonin-creatinine<br />
complex). Additionally, ambroxol – a secretolytic<br />
agent and an inhibitor of Na+ that decreases mucus
Model study of epithelial motility 29<br />
density – was used to check the role of mucus. The<br />
studied neurotransmitters and ambroxol were<br />
applied in a form of an injection, dissolved in a<br />
physiological equivalent of Ringer’s solution set<br />
specifically for the snails (80mmol/l NaCl, 4mmol/l<br />
KCl, 8mmol/l CaCl2, 5mmol/l MgCl2, 5mmol/l<br />
Tris, pH 7.8) [10]. The same solution was used in<br />
negative control groups. In each case the substance’s<br />
dosage was 2 μg per gram of body mass. All<br />
variables were measured in a zero and negative<br />
control group and test groups of the same size (10<br />
animals). The recordings were taken directly after<br />
drug admission and also an hour and two hours<br />
afterwards.<br />
All the measured and calculated variables were<br />
then used to determine if the pharmacological<br />
modification of movement shows statistically<br />
significant results and wheatear these results can be<br />
applied to previously known data. To determine this,<br />
Welch's t test was used, with statistical significance<br />
at 0.05.<br />
RESULTS<br />
Serotonin in pure form had a statistically<br />
significant influence on variables describing<br />
motility. Directly after injection 82.05% increase in<br />
the animals’ movement speed and 35.13 % in the<br />
value of epithelium fold coefficient was observed.<br />
These changes remained statistically significant up<br />
to an hour after admission. In comparison, the same<br />
serotonin in a creatinine complex did not cause a<br />
statistically significant change in movement speed,<br />
but affected the pedal wave generation, increasing<br />
their length by 38.34% and their frequency by<br />
19.63%.<br />
Epinephrine also did not show influence on the<br />
animals’ movement speed, causing changes only in<br />
pedal wave generation. Wave length increased by<br />
19.66% and the interval between waves increased by<br />
19.28%, which in turn resulted in lesser epithelium<br />
contraction levels. On the other hand,<br />
norepinephrine caused a different set of changes,<br />
resulting in a major increase of 66.39% in<br />
movement speed, followed by a 24.65% increase in<br />
wave length.<br />
Table I. Movement variables after substance injection<br />
compared to control<br />
Tabela I. Paramerty ruchu po wstrzyknięciu substancji<br />
w porównaniu z kontrolą<br />
* the increase in speed during the next hour was<br />
statistically significant. (w następnej godzinie zmiana<br />
była statystycznie istotna)<br />
** results at p=0.05 (wyniki dla poziomu istotności<br />
p=0,05)<br />
Values described as % difference according to control<br />
directly after neurotransmitter/amborxol admission.<br />
Numbers represent mean values and standard deviation.<br />
Statistically significant values are placed on gray<br />
background. (Wartości przedstawiono jako % różnicę w<br />
stosunku do kontroli bezpośrednio po podaniu<br />
neuroprzekaźników/ambroksolu. Wartości reprezentują<br />
średnie plus odchylenia standardowe. Wyniki istotne<br />
statystycznie przedstawiono na szarym tle.)<br />
Fig. 2. The effects of neurotransmitters and ambroxol on<br />
A. achatina pedal wave length directly after<br />
admission. The values shown are mean including<br />
standard deviation. Statistically significant results<br />
at p=0.05 are marked with *<br />
Ryc. 2. Efekt oddziaływania neuroprzekaźników i ambroksolu<br />
na długość fali skurczu u A. achatina<br />
bezpośrednio po podaniu. Pokazane wartości<br />
to średnie plus odchylenie standardowe.<br />
Istotne statystycznie wartości oznaczono *
30<br />
Wojciech Pawłowicz<br />
The dopamine was used in this test as a reference<br />
to the previous studies. The only statistically<br />
significant changes observed were a 15.29%<br />
reduction in pedal wave interval length and a<br />
29,15% increase in the folded area/total foot area<br />
ratio. Other motility variables remained below<br />
statistically significant levels.<br />
subtypes as this enables to fully evaluate the model.<br />
The proposed method is precise and repeatable<br />
enough to be considered for practical use. Also, the<br />
easiness of the process and animal breeding speed<br />
give chances for constituting a cheap and numerous<br />
research groups.<br />
Finally, the role of mucus and its physical<br />
prosperities in the locomotors activity is not to be<br />
excluded. It has been proven before that mucus<br />
plays a key role in snail movement, and here it was<br />
shown that its modification drastically changes the<br />
movement pattern. A question of interest is if, and to<br />
what extent, such changes can be applied to model<br />
epithelium i.e. to modify the adhesive locomotion.<br />
CONCLUSIONS<br />
Fig. 3. Sole of the snail foot with visible pedal waves<br />
(darker areas). 1 – spontaneous movement, 2 –<br />
directly after abroxol injection. The wave length<br />
and intervals between waves have increased<br />
Ryc. 3. Stopa ślimaka z widocznymi falami skurczu<br />
[ciemne rejony]. 1 – ruch spontaniczny, 2 –<br />
bezpośrednio po podaniu ambroksolu. Długość<br />
fali i odległości pomiędzy nimi uległy<br />
zwiększeniu<br />
After the injection of ambroxol, a variety of<br />
changes in the movement pattern could be observed.<br />
The speed of the animals increased by 43,51%, with<br />
both pedal wave length and frequency increasing.<br />
DISCUSSION<br />
The results show a possibility for the use of A.<br />
achatina snail as a study model to test if the<br />
substances influence epithelial motility. The<br />
adhesive locomotion process responded to the<br />
pharmacological modification and thus, leads to<br />
believe that snails use an analogous set of receptors<br />
and neurotransmitters as mammals. The specific set<br />
of changes in movement speed and other variables<br />
differs according to the substance used, but follows<br />
a unique and characteristic pattern.<br />
Being able to use the proposed model efficiently<br />
depends on the specific types of receptors and ion<br />
channels in the snail’s body. The key to the further<br />
studies is to pinpoint their location and specific<br />
1. Serotonin, serotonin-creatinin complex,<br />
norepineprhine and ambroxol injected into snail’s<br />
body cause an overall increase in movement speed<br />
while epinephrine and dopamine have no effect or<br />
decrease movement speed.<br />
2. The increase in movement speed depends on<br />
the increase of pedal wave length, total distance<br />
covered per pedal wave, the pedal wave frequency<br />
and the epithelium fold percentage. Decrease in<br />
movement speed does not cause such changes or is<br />
followed by a decrease of those parameters.<br />
3. It is proposed that in the process of adhesive<br />
locomotion of land snails an important role is played<br />
by epithelial motility regulated by the nervous<br />
system and caused by muscle contraction.<br />
4. The above mentioned snail foot activities<br />
are a valuable biological model for testing if a<br />
substance influences the epithelial motility.<br />
REFERENCES<br />
1. Barker G. M.:. The biology of terrestrial mollusks,<br />
CABI Publishing, New Zeland 2001<br />
2. Lissmann H.W.: The mechanism of locomotion in<br />
gastropod mollusks I. Kinematics, J. Exp. Biol., 1945,<br />
21, 58-69<br />
3. Lissmann H.W.: The mechanism of locomotion in<br />
gastropod mollusks II. Kinetics, J. Exp. Biol., 1945,<br />
21, 37-50,<br />
4. Tyrakowski T., Kaczorowski P., Pawłowicz W.,<br />
Ziółkowski M.:, Discrete movements of foot<br />
epithelium during adhesive locomotion of land snail,<br />
Folia Biol. (Krakow) 2012, 60, 99-106
Model study of epithelial motility 31<br />
5. Lai J.H., del Alamo J.C, Rodriguez-Rodriguez J.: The<br />
mechanics of the adhesive locomotion of terrestrial<br />
gastropods. J. Exp. Biol., 2010, 213, 3920-3933<br />
6. Denny M. W.: A Quantitive Model for the Adhesive<br />
Locomotion of The Terrestial Slug, Ariolimax<br />
Columbians, J. Exp. Biol., 1981, 91, 195-217<br />
7. Denny M. W.: Mechanical Prosperities of Pedal<br />
Mucus and Their Consequences for Gastropod<br />
Structure and Performance, Amer. Zool., 1984, 24,<br />
23-36<br />
8. Denny M. W.: The Physical Prosperities of the Pedal<br />
Mucus of the Terrestrial Slug Ariolimax columbianus,<br />
J. Exp. Biol., 1980, 88, 375-393<br />
9. Lauga E., Hosoi A.E.: Tuning gastropod locomotion:<br />
Modeling the influence of mucus rhelology on the cost<br />
of crawling, Phys. Fluids, 2006, 18:113102<br />
10. Pavlova G.A.: Effects of serotonin, dopamine and<br />
ergometrine on locomotion in the pulmonate mollusk<br />
Helix lucorum, J. Exp. Biol., 2001 204, 1625-1633<br />
Address for correspondence:<br />
e-mail: wojciech-pawlowicz@wp.pl,<br />
tomtyr@cm.umk.pl<br />
Phone:<br />
Author (+48) 664-194-557<br />
Department (+48) 52-585-35-98<br />
Received: 25.07.2012<br />
Accepted for publication: 18.09.2012
Medical and Biological Sciences, 2013, 27/2, 33-39<br />
ORIGINAL ARTICLE / PRACA ORYGINALNA<br />
Zuzanna Piekorz 1 , Irena Bułatowicz 1 , Agnieszka Radzimińska 1 , Andrzej Lewandowski 2 , Szymon Piekorz 2 ,<br />
Grzegorz Grabarczyk 2 , Monika Ciesielska 3<br />
THE INFLUENCE OF HATHA YOGA EXERCISE ON ARTERIAL PRESSURE AND PULSE<br />
WPŁYW TRENINGU HATHA JOGI NA WARTOŚCI CIŚNIENIA TĘTNICZEGO<br />
I CZĘSTOŚĆ TĘTNA<br />
1 Z Katedry i Zakładu Kinezyterapii i Masażu Leczniczego UMK w Toruniu Collegium Medicum<br />
im. L. Rydygiera w Bydgoszczy<br />
p. o. kierownika: dr Irena Bułatowicz<br />
2 Z Katedry i Zakładu Podstaw Kultury Fizycznej UMK w Toruniu Collegium Medicum<br />
im. L. Rydygiera w Bydgoszczy<br />
p. o. kierownika: dr Andrzej Lewandowski<br />
3 Z Katedry i Kliniki Anestezjologii i Intensywnej Terapii UMK w Toruniu Collegium Medicum<br />
im. L. Rydygiera w Bydgoszczy<br />
kierownik: Dr hab. Krzysztof Kusza, prof. UMK<br />
S u m m a r y<br />
I n t r o d u c t i o n . Hypertension constitutes a great<br />
problem in modern medicine. Due to it being widespread in<br />
the society, it seems justified to introduce complex treatment<br />
and prevent from this disease. Hypotensive qualities of yoga<br />
exercise described in literature constitute an interest as an<br />
alternative form of cardiological rehabilitation.<br />
M a t e r i a l a n d m e t h o d s. In order to evaluate its<br />
influence on the values of blood pressure and pulse, a group<br />
of people attending hatha yoga classes was studied. It was<br />
assumed that circulatory system parameters would decrease<br />
when influenced by training, and the size of those changes<br />
would depend on age, health condition and selected<br />
environmental factors. Before, during and after the classes<br />
arterial blood pressure and pulse were measured. The study<br />
was supplemented by the author's questionnaire consisting of<br />
15 questions with given response categories.<br />
R e s u l t s . Great age difference, majority of female<br />
subjects and those with higher education and white collar<br />
jobs were observed during the study.<br />
The results proved good perception of subjects' own<br />
health condition and their little knowledge concerning<br />
realistic parameters of their circulatory system.<br />
Decrease of arterial blood pressure and pulse due to hatha<br />
yoga exercise were detected during the study, as well as a<br />
greater decreasing tendency in subjects with high arterial<br />
blood pressure.<br />
C o n c l u s i o n s formulated as a consequence of the<br />
study concern the influence of hatha yoga exercise on<br />
circulatory system parameters and its possible use in order to<br />
obtain better hypotensive effect.<br />
S t r e s z c z e n i e<br />
W s t ę p . Nadciśnienie tętnicze stanowi duży problem<br />
dla współczesnej medycyny. Z uwagi na znaczne<br />
rozpowszechnienie w społeczeństwie zasadne wydaje się<br />
wdrożenie kompleksowego leczenia i przeciwdziałania tej<br />
chorobie. Wykazane w piśmiennictwie właściwości<br />
hipotensyjne treningu jogi spowodowały zainteresowanie<br />
możliwością wykorzystania go jako alternatywnej formy<br />
rehabilitacji kardiologicznej.<br />
M a t e r i a ł i m e t o d y . W celu oceny jego wpływu na<br />
wartości ciśnienia tętniczego i częstość tętna badaniu<br />
poddano grupę osób uczęszczających na zajęcia hatha jogi.<br />
Założono, że parametry układu krążenia ulegną obniżeniu<br />
pod wpływem treningu, a wielkość tych zmian będzie<br />
uzależniona wiekiem, stanem zdrowia i wybranymi<br />
czynnikami środowiskowymi. Dokonano pomiaru ciągłego<br />
ciśnienia tętniczego i częstości tętna przed rozpoczęciem
34<br />
Zuzanna Piekorz et al.<br />
zajęć, w trakcie trwania oraz po ich zakończeniu. Badania<br />
uzupełniono autorską ankietą, składającą się z 15 pytań<br />
z podanymi kategoriami odpowiedzi.<br />
W y n i k i . Zaobserwowano duże różnice wieku osób<br />
podejmujących trening hatha jogi oraz przewagę płci<br />
żeńskiej, wyższego wykształcenia i umysłowego charakteru<br />
pracy.<br />
Stwierdzono stosunkowo dobre odczucie stanu zdrowia<br />
badanych osób i małą świadomość o rzeczywistych<br />
parametrach własnego układu krążenia.<br />
Wykazano obniżenie wartości ciśnienia tętniczego<br />
i częstości tętna pod wpływem treningu hatha jogi, oraz<br />
większą tendencję spadkową badanych parametrów u osób<br />
z wysokimi wartościami ciśnienia tętniczego.<br />
W n i o s k i . Sformułowano wnioski dotyczące wpływu<br />
treningu hatha jogi na parametry układu krążenia oraz<br />
możliwości jego stosowania w celu uzyskania efektu<br />
hipotensyjnego.<br />
Key words: yoga, circulatory system, hypertension, changes, environmental determinants<br />
Słowa kluczowe: joga, układ krążenia, nadciśnienie, zmiany, uwarunkowania środowiskowe<br />
INTRODUCTION<br />
Yoga is one of the oldest sciences practiced by<br />
man, while centuries old tradition and numerous<br />
supporters confirm that it is very up to date and<br />
universal. Its condition, which is continuous self<br />
improvement, takes place in eight stages and leads to<br />
achieving absolute cognition – samadhi [1]. However,<br />
one of the side effects of yoga is its healing influence<br />
on the organism. As a result, practicing it is a sort of<br />
antidote for life nuisances in the 21 st century. It is very<br />
likely that nowadays yoga can offer even more to us –<br />
the Europeans [2].<br />
Taking into account high frequency of hypertension<br />
occurrence and the complexity of its etiology, it was<br />
decided that yoga's influence on circulatory system<br />
parameters should be evaluated. The only other trial<br />
found in literature was performed by L. Kulmatycki,<br />
who studied arterial pressure before and after<br />
relaxation training of yoga in subjects with its high<br />
values [3]. Present studies on yoga are conducted in<br />
very few academic facilities in Poland, and one of the<br />
most vibrant is Academy of Physical Education in<br />
Katowice, where the Academic Yoga Society was<br />
established. The work of members of the society and<br />
especially its president, professor J. Szopa, has resulted<br />
in publications concerning yoga and its influence on<br />
the organism [4, 5, 6, 7, 8, 9, 10]. However, a<br />
significant majority of available literature is of popular<br />
scientific character [1, 11, 12, 13, 14, 15, 16].<br />
The issue of non-pharmacological methods of<br />
treating hypertension is widely described in the subject<br />
literature [17, 18, 19]. Trials of evaluating the influence<br />
of various forms of cardiological rehabilitation [20, 21,<br />
22, 23], as well as relaxation exercise [24] and massage<br />
on the circulatory system parameters [25, 26] were<br />
found in literature. The analysis of collected literature<br />
allows the conclusion that yoga training is connected<br />
with the influence of both physical exercise and<br />
relaxation on the organism. A certain bipolarity of this<br />
exercise system explains the interest in its hypotensive<br />
properties. Unfortunately, yoga as a tool of preventing<br />
and treating hypertension, in significant majority of<br />
literature appears as two separate issues, and only few<br />
authors notice the possibility of taking advantage of<br />
healing properties of yoga in terms of arterial pressure<br />
[1, 2, 25, 27]. Having considered the aforementioned<br />
ideas, trying to synthesize the knowledge derived from<br />
literature and evaluating the influence of yoga training<br />
on circulatory system parameters seems justified.<br />
MATERIAL AND METHODS<br />
The study was conducted in Bydgoszcz from June<br />
2010 to end of February 2011, and included the<br />
subjects participating in hatha yoga classes conducted<br />
by a physical therapist and a yoga teacher. The group<br />
consisted of 30 people at the age between 21 and 60,<br />
28 of whom (93.33%) were women. The classes lasted<br />
90 minutes and included taking poses known as asanas,<br />
in which a crucial element is calm, steady breathing.<br />
Each session ended with a seven – minute relaxation.<br />
Utilizing holter blood pressure monitor, arterial<br />
pressure and pulse values were taken before, during<br />
and after the yoga session. The readings began five<br />
minutes after subjects took a comfortable sitting<br />
position. The time of reading before and after the class<br />
was 30 minutes. Obtained readings were supplemented<br />
by a survey, utilizing author's questionnaire including<br />
15 multiple choice questions. First five questions<br />
concerned social identification of the subjects, the<br />
following ones related to their hatha yoga or other<br />
physical activity training, while the last three questions<br />
allowed a subjective evaluation of the respondents'<br />
health condition in the context of specific diseases.<br />
Collected data was compiled by means of basic<br />
statistical methods, such as Student t test. Statistical<br />
significance of differences was established on the level
The influence of hatha youga exercise on arterial pressure and pulse 35<br />
of p
36<br />
Zuzanna Piekorz et al.<br />
Due to a significant number of subjects whose<br />
arterial pressure was high, exceeding normal, when<br />
evaluated in first reading, further study material was<br />
differentiated according to its values. As a result, a<br />
group of 13 subjects with normal values of blood<br />
pressure was selected – from this point on referred to<br />
as subjects with normal arterial pressure, and a group<br />
of 17 subjects with arterial pressure (systolic pressure<br />
≥ 140, diastolic pressure ≥ 90) – from this point on<br />
referred to as subjects with high arterial pressure.<br />
Comparative characteristics of the studied parameters<br />
are presented in table III.<br />
Table III. Comparative characteristics of the changes in<br />
arterial pressure and pulse of subjects with<br />
normal and high values of arterial pressure<br />
Tab. III. Charakterystyka porównawcza zmian ciśnienia<br />
tętniczego i częstości tętna osób z prawidłowymi<br />
oraz wysokimi wartościami ciśnienia tętniczego<br />
Category M 13 M 17 σ 13 σ 17 D 13 D 17 t 13 t 17<br />
Pulse I<br />
73.54 79.82 8.37 16.09<br />
(beats/min)<br />
Pulse II<br />
66.15 69.00<br />
8.85<br />
(beats/min)<br />
8.09<br />
7.39 10.82 3.11** 2.88*<br />
Systolic<br />
pressure I 121.23 146.12 8.47 14.47<br />
(mmHg)<br />
Systolic<br />
6.38 8.47 1.83 2.42*<br />
pressure<br />
II<br />
114.85 137.65 11.48 17.91<br />
(mmHg)<br />
Diastolic<br />
pressure I 83.23 103.53 4.44 12.45<br />
(mmHg)<br />
Diastolic<br />
6.61 10.88 2.84* 4.80**<br />
pressure<br />
II<br />
(mmHg)<br />
76.62 92.65 6.83 15.86<br />
* Statistically significant difference p< 0.05<br />
** Statistically significant difference p < 0.01<br />
For 13 persons the value of t test is: for alfa 0.05=2.18, for alfa<br />
0.01=3.06<br />
* Statistically significant difference p< 0.05<br />
** Statistically significant difference p < 0.01<br />
For 17 persons the value of t test is: for alfa 0.05=2.12, for alfa<br />
0.01=2.92<br />
As it may be concluded from the table, studied<br />
parameters of subjects with normal arterial pressure<br />
decreased after hatha yoga session. Statistically<br />
significant differences on the level of 0.01 were<br />
discovered only in the evaluation of the changes on<br />
pulse. In the group of subjects with high arterial<br />
pressure, means of all parameters decreased. All<br />
differences of means were statistically significant, and<br />
the lowest, as far as significance level 0.05 is<br />
concerned, was that of systolic pressure.<br />
DISCUSSION<br />
When this study was initiated, it was assumed that<br />
hatha yoga training would result in decrease of the<br />
values of arterial pressure and pulse. Readings<br />
obtained after data analysis confirmed this assumption.<br />
Decrease of both pressure readings as a result of yoga<br />
training was stated also by Kulmatycki, who studied its<br />
influence arterial pressure in subjects with<br />
hypertension. However, results of own study provided<br />
a different picture of downward tendency than the<br />
presented in similar studies published in available<br />
literature. Particularly essential difference was that of<br />
significantly greater decrease in diastolic pressure<br />
before and after the session, when compared to<br />
decrease of this parameter noted in Kulmatycki's study<br />
[3]. A positive influence of yoga exercise on<br />
circulatory system presented in both studies may<br />
become a foundation for using its elements in struggle<br />
against hypertension. It should be remembered that the<br />
obstacle against reliable evaluation of hypotensive<br />
influence of yoga is the fact that such small group of<br />
subjects participated in the study.<br />
A possible explanation of yoga's hypotensive effect<br />
is its toning influence on the nervous system, which is<br />
frequently and extensively discussed in literature [1, 2,<br />
9, 29]. Yoga, along with Schultz's Autogenic Training<br />
and Jacobson's Method are the relaxation methods<br />
most frequently described in literature [29, 30, 31, 32],<br />
hence it seems relevant to compare results of own<br />
study with the influence of relations on the circulatory<br />
system parameters. This issue became of interest to the<br />
Wrocław's Academy of Physical Education. In their<br />
studies they observed that autogenic training developed<br />
by Szyszko-Bohusz resulted in decrease of both arterial<br />
blood pressure and pulse. The authors pointed out<br />
those methods of relaxation ought to be more<br />
frequently adapted as a form of prophylactic and<br />
treatment of hypertension [24]. Taking into account<br />
aforementioned study results, it can be assumed that in<br />
case of presently studied subjects, too, approaching a<br />
relaxed condition in a significant manner influenced<br />
the circulatory system parameters, and the methods<br />
utilized to achieve such condition are characterized by<br />
a similar effectiveness.<br />
In order to evaluate hypotensive effect of yoga, its<br />
effectiveness and results of studies concerning the<br />
influence of therapeutic massage on the circulatory<br />
system parameters were compared. A downward<br />
tendency discovered in the analysis of data proved<br />
analogous to the results obtained in the studies on the<br />
influence of therapeutic massage on the selected<br />
parameters of the circulatory system, in which the<br />
greatest decrease of the studied parameters applied to<br />
pulse, then diastolic and systolic pressure [25]. When
The influence of hatha youga exercise on arterial pressure and pulse 37<br />
analyzing obtained data, a significantly higher<br />
hypotensive influence of hatha yoga than massage was<br />
observed [25, 26].<br />
An important aspect of the conducted study was<br />
propagating yoga exercise as a prophylactic and<br />
therapeutic method, primarily concerning high blood<br />
pressure. In Poland, basic methods of treating this<br />
disease is pharmacotherapy and a change of lifestyle as<br />
far as diet and undertaking physical activity is<br />
concerned [17, 18, 19, 33, 34, 35]. Publications are<br />
available in comprehensive medical literature<br />
concerning studies on changes occurring in blood<br />
pressure when affected by cardilogical rehabilitation<br />
including work-out and utilizing cycle ergonometers.<br />
In those studies it was proven that controlled physical<br />
effort caused or contributed to decrease of arterial<br />
blood pressure [20, 21, 22]. Hypotensive effect,<br />
obtained during the study seems to confirm this<br />
relation. Yoga exercise used as a form of cardiological<br />
rehabilitation might exhibit a similar effectiveness.<br />
Acquired results and available publications prove that<br />
it is relevant to conduct other, frequently more<br />
attractive for patients, forms of physical activity<br />
directed at lowering arterial pressure [23]. In<br />
consequence, it seems fully justified to use elements of<br />
yoga as an alternative method of cardiological<br />
rehabilitation combining hypotensive influence of<br />
physical activity with relaxation.<br />
In the course of the study, it became apparent that<br />
the objective evaluation of the level of blood pressure<br />
significantly deviates from the subjects' awareness. The<br />
group of subjects with diagnosed hypertension<br />
constituted only 20% of subjects in total, however, the<br />
results obtained before hatha yoga training proved that<br />
in more than 56% of subjects the readings of arterial<br />
pressure exceed norms. This issue resulted in dividing<br />
subject into two groups. The percentage of subjects in<br />
whom readings reached values classified as<br />
hypertension exceeds the frequency of this disease<br />
occurring in Polish society, in which 50% of men and<br />
40% of women suffer from hypertension [34]. Lack of<br />
awareness concerning circulatory system parameters<br />
and such wide spreading of its high values in the group<br />
of subjects may imply that this condition occurs more<br />
frequently than suggested by the statistics. It needs to<br />
be pointed that the division was executed solely for the<br />
requirements of the study, and actual picture of<br />
hypertensive subjects can be evaluated only with<br />
comprehensive medical examination. As indicated by<br />
the analysis, decrease of readings of both systolic and<br />
diastolic pressure, as well as pulse, is more explicit in<br />
the group of subjects with high blood pressure. This<br />
result is reflected in other publications, in which<br />
decrease of arterial pressure in subjects with its high<br />
readings was observed [20].<br />
The thesis concerning the influence of age, time<br />
span and frequency of practicing hatha yoga on<br />
changes in the readings of arterial pressure and pulse<br />
occurring after the session was not fully verified. Most<br />
correlations are characterized by nearly insignificant<br />
relation. Significant relation occurred solely between<br />
the results of the studied parameters and age. Relying<br />
on this, one could indirectly conclude that decrease of<br />
pressure exhibited after hatha yoga exercises were<br />
more often marked in young subjects. It may be<br />
assumed that the circulatory system of the young<br />
operates more efficiently, and its parameters undergo<br />
changes resulting from effort or relaxation in a more<br />
explicit manner. Material gathered from the survey<br />
allowed characterization of the group of hatha yoga<br />
participants. A significant majority of subjects were<br />
women, which points to the conclusion that men much<br />
more seldom select yoga as recreational physical<br />
activity. A similar tendency can be observed in other<br />
publications [3,6]. A tendency not related to sex was<br />
age of the subjects. Studied group was very varied age<br />
– wise, which seems to confirm the thesis that hatha<br />
yoga training is suitable for participants at any age [6].<br />
Social characteristics state that majority of members of<br />
hatha yoga group in Bydgoszcz had higher education<br />
and performed white collar jobs. The characteristics of<br />
this group may be related to their higher level of self<br />
awareness and desire of growth, as well as greater<br />
knowledge about pro health influence of yoga, when<br />
compared to those not as well-educated. Respondents,<br />
when choosing hatha yoga classes, expected mainly<br />
improving their physical, then mental comfort.<br />
Subjects' expectations seemed to have been fulfilled,<br />
since a great majority of the respondents pointed to a<br />
positive influence on both physical and mental<br />
comfort. A similarly positive effect of yoga was<br />
described in other publications [3,6].<br />
Obtained results, due to their relative value,<br />
connected with low headcount of the group, do not<br />
allow drawing unequivocal conclusions concerning<br />
tackled problems. However, they confirm hypotensive<br />
influence of hatha yoga and they justify executing<br />
exercise using this method as a non pharmacological<br />
method of treating hypertension. A positive influence<br />
of yoga training was reinforced by the subjective
38<br />
Zuzanna Piekorz et al.<br />
evaluation via survey. A successful verification of the<br />
majority of posed hypotheses ought to be treated as a<br />
confirmation of relevance of further studies in this<br />
area. Procured results may be utilized in preliminary<br />
evaluation of the problem and they can form a<br />
teajectory for further studies with expected equally<br />
successful result, which should be widely spread and<br />
included in treatment and prophylactic. Those<br />
presently presented allowed formulation of the<br />
following conclusions.<br />
CONCLUSIONS<br />
1. Confirmed in the study decrease in readings of<br />
arterial pressure and pulse attests to a positive effect of<br />
hatha yoga training on the circulatory system, and<br />
aforementioned changes in the parameters of subjects<br />
with high readings of arterial pressure justify executing<br />
yoga exercise in order to gain hypotensive effect.<br />
2. A positive influence of hatha yoga training on<br />
the organism is confirmed by subjective perception of<br />
the subjects in the scope of physical and mental<br />
comfort, which can result in improving the quality of<br />
life and reducing stress, and furthermore, it can<br />
intensify hypotensive effect and constitute a<br />
prophylactic for cardio-vascular diseases.<br />
3. Objectively read arterial pressure, contrasting<br />
with the knowledge and perception of its value in<br />
subjects, justifies the need for its regular reading and<br />
improving society's awareness concerning prophylactic<br />
of hypertension.<br />
4. The lack of influence of time span of<br />
practicing yoga and the frequency of training on the<br />
changes in values of arterial pressure and pulse<br />
occurring after session, attests to the possibility of<br />
selective and irregular utilizing elements of this<br />
method during sessions, which aim at gaining<br />
hypotensive effects.<br />
5. A domination of female participants, higher<br />
education and white collar professions in subjects may<br />
prove the upper classes' higher awareness of the health<br />
advantages of hatha yoga training and justify the<br />
necessity of its propagation among the remaining part<br />
of the society.<br />
BIBLIOGRAPHY<br />
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Wpływ rehabilitacji kardiologicznej na zachowanie się<br />
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Korzeniowska J., Janczak J., Adamus J.: Efekt<br />
hipotensyjny sześciomiesięcznej rehabilitacji<br />
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Popielewicz-Kautz A., Markuszewski L., Chorębała A.,<br />
Korzeniowska J., Janczak J., Adamus J.: Wpływ<br />
ambulatoryjnego kontrolowanego treningu<br />
kardiologicznego na ciśnienie tętnicze u <strong>chorych</strong> na<br />
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Polski Merkuriusz Lekarski, 2007, 22/127, 9-14<br />
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Błaszczyk J., Błaszczyk-Suszyńska J.: Trening na stołach<br />
rehabilitacyjno-rekondycyjnych i jego wpływ na wybrane<br />
parametry krążeniowo-oddechowe. Acta Bio-Optica et<br />
Informatica Medica, 2009, 2/15, 116-120<br />
24. Dziubek V., Dąbrowska G., Drozd-Dworak I.,<br />
Wysokiński W.: Wpływ relaksacji na wybrane parametry<br />
hemodynamiczne u osób z nadciśnieniem tętniczym.<br />
Fizjoterapia, 2000, 8/2, 46-50<br />
25. Andrzejewski W., Kassolik K., Grabowski W.,<br />
Maligranda M., Trzęsicka E.: Wpływ masażu<br />
medycznego na wybrane parametry układu krążenia.<br />
Family Medicine & Primary Care Review, 2006, 8/1, 48-<br />
51<br />
26. Walaszek R., Kasperczyk T., Nowak Ł.: Wpływ masażu<br />
klasycznego na zmiany wartości ciśnienia tętniczego<br />
krwi i częstości tętna u osób zdrowych w wieku 21-26<br />
lat. Fizjoterapia, 2008, 17/1, 11-19<br />
27. Niedaszkowski B.: Rola jogi w leczeniu i prewencji<br />
pierwotnego nadciśnienia tętniczego. Portal<br />
www.yogamedica.pl,15.03.2011<br />
28. Stanisz A. Przystępny kurs statystyki z zastosowaniem<br />
Statistica PL na przykładach z medycyny. Tom 1.<br />
Statystyki podstawowe. Kraków: StartSoft Polska; 2006.<br />
29. Kulmatycki L., Szczuka E.: Rola relaksacji w relacji<br />
fizjoterapeuta – pacjent. Fizjoterapia 2007, 15/1, 75-84<br />
30. Kulmatycki L.: Stany relaksu – wymiary i poziomy.<br />
Postępy Rehabilitacji, 2007, 21/3, 37-43<br />
31. Sadowska J.: Starożytne, orientalne metody<br />
profilaktyczno-lecznicze i współczesne ich postrzeganie.<br />
Archiwum historii i filozofii medycyny, 2007, 70, 46-49<br />
32. Sharma S.: Terapia metodą jogi i trening autogenny.<br />
Studium porównawcze. Sztuka Leczenia, 1995, 1/3, 97-<br />
102<br />
33. Chintanadilok J., Lowenthal D.T.: Rola ćwiczeń<br />
fizycznych w leczeniu nadciśnienia tętniczego.<br />
Medycyna po Dyplomie, 2002, 11/6, 91-106<br />
34. Jegier A.: Wysiłek fizyczny u osób z nadciśnieniem<br />
tętniczym. Medycyna Sportowa, 2003, 19/3, 99-106<br />
35. Shahnazaryan K., Gaciong Z.: Wysiłek fizyczny i<br />
nadciśnienie tętnicze. Terapia, 2010, 4/2, 26-29<br />
Address for correspondence:<br />
mgr Zuzanna Piekorz<br />
Katedra i Zakład Kinezyterapii i Masażu Leczniczego<br />
UMK w Toruniu<br />
Collegium Medicum im. L. Rydygiera w Bydgoszczy<br />
ul. M. Curie Skłodowskiej 9<br />
85-094 Bydgoszcz<br />
Received: 7.02.2012<br />
Accepted for publication: 12.04.2013
Medical and Biological Sciences, 2013, 27/2, 41-47<br />
ORIGINAL ARTICLE / PRACA ORYGINALNA<br />
Adrian Reśliński 1 , Stanisław Dąbrowiecki 2 , Katarzyna Głowacka 3 , Jakub Szmytkowski 1<br />
THE INFLUENCE OF OCTENIDINE DIHYDROCHLORIDE ON BACTERIAL BIOFILM<br />
ON THE SURFACE OF A POLYPROPYLENE MESH<br />
WPŁYW ROZTWORU DICHLOROWODORKU OKTENIDYNY NA BIOFILM<br />
WYTWORZONY NA POWIERZCHNI SIATKI POLIPROPYLENOWEJ<br />
1 Department of General and Endocrine Surgery, Ludwik Rydygier College of Medicine in Bydgoszcz,<br />
Nicolaus Copernicus University in Torun, Poland<br />
Acting Head: Jacek Szopiński PhD<br />
2 Department of Nutrition and Dietetics, Ludwik Rydygier College of Medicine in Bydgoszcz,<br />
Nicolaus Copernicus University in Torun, Poland<br />
Acting Head: Justyna Przybyszewska PhD<br />
3 Department of Plant Physiology, Genetics and Biotechnology,<br />
Warmia – Mazury University in Olsztyn, Poland<br />
Head: Ryszard Górecki prof.<br />
S u m m a r y<br />
Deep surgical site infections (DSSI’s) involving the<br />
implanted biomaterial remain an important issue in hernia<br />
surgery. The etiological factors include Staphylococcus<br />
aureus and Escherichia coli. The ability of these<br />
microorganisms to create a biofilm on the surface of the<br />
implant is considered to be one of the main reasons why<br />
successful treatment of DSSI’s is not an easy task. It is<br />
widely agreed that an important element of a successful<br />
treatment plan for infections involving biofilm formation is<br />
the use of agents capable of penetrating the biofilm matrix,<br />
and displaying a satisfactory efficacy against the<br />
microorganisms present within. Antiseptic agents meet the<br />
above criteria.<br />
The goal of this study was to evaluate the influence of a<br />
solution of octenidine dihydrochloride (Octenisept) on the<br />
biofilm present on a surface of a monofilament<br />
polypropylene mesh implant.<br />
The study involved 140 bacterial strains from the<br />
collection of the Department of Microbiology Nicolaus<br />
Copernicus University Collegium Medicum in Bydgoszcz,<br />
Poland. The strains included 70 (50%) S. aureus isolates and<br />
70 (50%) E. coli isolates. The influence of an antiseptic<br />
agent on the created biofilm was evaluated with the use of a<br />
qualitative and quantitative method, as well as by scanning<br />
electron microscopy (SEM).<br />
In the qualitative assessment, the observed effect of<br />
octenidine was a diminished intensity of biomaterial staining<br />
after incubation with TTC in comparison to samples from the<br />
control group. In the quantitative study, the live cell counts of<br />
S. aureus and E. coli isolated from the biofilm present on the<br />
implant surface, after exposure to the antiseptic agent were<br />
found to be diminished. SEM studies have shown that<br />
exposure to octenidine hydrochloride decreases the number<br />
of bacteria adhering to the biomaterial surface.<br />
The Octenisept octenidine dihydrochloride solution<br />
displays bactericidal activity against S. aureus and E. coli<br />
bacteria present in the biofilm created on the surface of<br />
monofilament polypropylene mesh.
42<br />
Adrian Reśliński et al.<br />
S t r e s z c z e n i e<br />
Głębokie zakażenie miejsca operowanego (GZMO)<br />
obejmujące wszczepiony biomateriał stanowi istotny<br />
problem w chirurgii przepuklin. Czynniki etiologiczne tego<br />
powikłania, wśród których wymienia się m.in.:<br />
Staphylococcus aureus i Escherichia coli tworzą biofilm na<br />
powierzchni implantów uznawany za jedną z głównych<br />
przyczyn trudności w leczeniu GZMO. Uważa się, że<br />
istotnym elementem leczenia zakażeń przebiegających<br />
z powstaniem biofilmu jest stosowanie substancji aktywnych<br />
wobec drobnoustrojów w biofilmie oraz charakteryzujących<br />
się zdolnością do przenikania przez jego macierz. Związkami<br />
chemicznymi posiadającymi wymienione właściwości są<br />
antyseptyki.<br />
Celem pracy była ocena wpływu roztworu dichlorowodorku<br />
oktenidyny w postaci preparatu antyseptycznego<br />
Octenisept na biofilm wytworzony na powierzchni<br />
monofilamentowej siatki polipropylenowej.<br />
Badaniem objęto 140 szczepów bakterii z kolekcji<br />
Katedry i Zakładu Mikrobiologii Collegium Medicum im.<br />
L. Rydygiera w Bydgoszczy Uniwersytetu Mikołaja<br />
Kopernika w Toruniu, wśród których było 70 (50%) izolatów<br />
S. aureus oraz 70 (50%) E. coli. Ocenę wpływu antyseptyku<br />
na wytworzony biofilm wykonano metodą jakościową,<br />
ilościową oraz z użyciem skaningowego mikroskopu<br />
elektronowego (SEM).<br />
W badaniu metodą jakościową efektem działania<br />
roztworu dichlorowodorku oktenidyny na wytworzony<br />
biofilm było zmniejszenie intensywności zabarwienia<br />
powierzchni biomateriału po inkubacji z TTC w stosunku do<br />
grupy kontrolnej. W badaniu ilościowym stwierdzono<br />
zmniejszenie liczby żywych komórek S. aureus i E. coli<br />
izolowanych z biofilmu na powierzchni implantu po<br />
ekspozycji na działanie antyseptyku. Natomiast badanie<br />
z użyciem SEM wykazało, że roztwór dichlorowodorku<br />
oktenidyny powoduje zmniejszenie liczby bakterii<br />
przylegających do powierzchni biomateriału.<br />
Roztwór dichlorowodorku oktenidyny w postaci<br />
preparatu Octenisept wykazuje działanie bakteriobójcze<br />
wobec S. aureus i E. coli w biofilmie wytworzonym na<br />
powierzchni monofilamentowej siatki polipropylenowej.<br />
Key words: octenidine hydrochloride, polypropylene mesh, biofilm, TTC, SEM<br />
Słowa kluczowe: dichlorowodorek oktenidyny, siatka polipropylenowa, biofilm, TTC, SEM<br />
INTRODUCTION<br />
Deep surgical site infections (DSSI’s) involving the<br />
implanted biomaterial remain an important issue in<br />
hernia surgery [1, 2]. One of the main factors<br />
responsible for the difficulties in the treatment of this<br />
complication is the creation of a bacterial biofilm on<br />
the implant surface [3]. The typical etiological factors<br />
of DSSI’s with biofilm – forming capabilities include<br />
Staphylococcus aureus and Escherichia coli [4].<br />
It is widely agreed that an important element of a<br />
successful treatment plan for infections involving<br />
biofilm formation is the use of agents capable of<br />
penetrating the biofilm matrix [5], and displaying a<br />
satisfactory efficacy against the microorganisms<br />
present within [6]. Antiseptic agents used in local<br />
treatment of chronic problem wounds and biomaterial<br />
infections meet the above – listed criteria [1, 7].<br />
In theory, the ideal antiseptic agent should display a<br />
broad spectrum of antimicrobial activity immediately<br />
after application. Moreover, it cannot be toxic,<br />
teratogenic or carcinogenic, and should not negatively<br />
influence wound healing [7,8]. Such an ideal antiseptic<br />
has not yet been reported despite ongoing research [7].<br />
One of the chemical compounds meeting most of the<br />
above criteria is [N,N'-(1,10-decanediyldi-1[4H]-<br />
pyridinyl-4-ylidene)-bis-(1-octanamine)<br />
dihydrochloride, or octenidine dihydrochloride [8, 9].<br />
It is characterized by a particular affinity to the lipid<br />
components of cell membranes, such as cardiolipin;<br />
this property allows it to display good antimicrobial<br />
efficacy with a minimal cytotoxic effect on the living<br />
tissue [9]. In its commercially available form (the<br />
Octenisept antiseptic solution containing 0.1%<br />
octenidine dihydrochloride, 2.0% phenoxyethyl<br />
alcohol and additives) it has been demonstrated to<br />
exert a bactericidal effect on planktonic forms of S.<br />
aureus and E. coli after exposure of as little as one<br />
minute [10, 11]. A review of the available literature has<br />
not yielded any publications on the influence of a oneminute<br />
exposure to an octenidine dihydrochloride<br />
solution on the biofilm created by S. aureus and E. coli<br />
on the surface of biomaterial implants used in hernia<br />
surgery.<br />
The aim of this study was to evaluate the influence<br />
of a solution of octenidine dihydrochloride on the<br />
biofilm present on a surface of a monofilament<br />
polypropylene mesh implant.<br />
MATERIALS AND METHODS<br />
Bacterial strains. The study involved 140 bacterial<br />
strains from the collection of the Departament of<br />
Microbiology of Nicolaus Copernicus University
The influence of octenidine dihydrochloride on bacterial biofilm on the surface of a polypropylene mesh 43<br />
Collegium Medicum in Bydgoszcz, Poland. The strains<br />
included 70 (50%) S. aureus isolates and 70 (50%) E.<br />
coli isolates. The microorganisms had been isolated in<br />
the period 2008–2009 from wound smears and pus<br />
samples collected from various patients hospitalized at<br />
the Departments of: General and Endocrine Surgery,<br />
General and Vascular Surgery and General Surgery<br />
and Transplantology of the Dr A. Jurasz University<br />
Hospital No.1 in Bydgoszcz, Poland. Initial<br />
identification of the S. aureus strains was based on<br />
colony morphology on Columbia Agar with 5% sheep<br />
blood (Becton Dickinson, Sparks, USA) and the<br />
presence of clumping factor and / or free coagulase.<br />
Species requiring verification were identified using the<br />
ID32 Staph (bioMérieux S.A. RCS Lyon, France) test<br />
for staphylococci. In the identification of E. coli<br />
strains, colony morphology on the MacConkey Agar<br />
(Becton Dickinson) was considered along with ID32 E<br />
test results (bioMérieux). The ID32 Staph and ID32 E<br />
tests were performed according to the manufacturer’s<br />
instructions and the results were read after a 24 – hour<br />
incubation in the oxygen – containing atmosphere at<br />
37ºC in the ATB Expression system (bioMérieux),<br />
with the use of the V 2.8.8 database. The isolates<br />
included in the study material had different<br />
chromosomal DNA patterns within one species, which<br />
was verified in the initial stage of the study by pulsed –<br />
field gel electrophoresis (PFGE). The isolates were<br />
stored in brain heart infusion, (BHI; bioMérieux) with<br />
an addition of 15% glycerol (Polskie Odczynniki<br />
Chemiczne, POCH) at a temperature of -70ºC.<br />
Evaluation of the influence of octenidine<br />
hydrochloride solution on biofilm. The testing was<br />
performed using a qualitative [12,13] and quantitative<br />
method [14]; scanning electron microscopy was also<br />
performed [15]. The investigated implant was<br />
composed of a monofilament polypropylene mesh, as<br />
this type of biomaterial is the most widely used in<br />
hernia surgery [4].<br />
In the quantitative method, utilizing the property of<br />
metabolically active microorganisms to reduce<br />
colorless 2,3,5-triphenyltetrazolium chloride (TTC;<br />
POCH) to red formazan [12,13], sterile fragments of<br />
the implant, measuring 2 x 1 cm, were incubated in 4<br />
ml of tryptic soy broth (TSB, Becton Dickinson,<br />
Sparks, USA) containing a bacterial inoculum<br />
equivalent to the 0.5 McFarland turbidity standard.<br />
Implant incubation at 37ºC continued for 48 hours in<br />
the oxygen – containing atmosphere, with a change of<br />
the growth medium to sterile substrate after 24 hours.<br />
The biomaterial samples were then rinsed with 0.9%<br />
phosphate buffered saline (PBS) at a pH of 7.2 and<br />
placed in 4 mL of the antiseptic agent – a<br />
commercially available octenidine dihydrochloride<br />
solution (Octenisept, containing 0.1% octenidine<br />
dihydrochloride, 2.0% phenoxyethyl alcohol and<br />
additives; Schülke & Mayr). After one minute, the<br />
implant samples were transferred to a neutralizing<br />
solution, containing: 3% Tween 80 (Sigma), 3%<br />
saponin ((Fluka, Steinheim, Germany), 0.1% histidine<br />
(Sigma) and 0.1% lecithin – 0.1 % (Sigma) [16]; this<br />
solution does not influence bacterial cells [10]. After<br />
rinsing with PBS (pH 7.2) and placing in 4 mL of<br />
sterile TSB with the addition of 20 L of a 1% TTC<br />
solution, the samples were further incubated for 24<br />
hours at 37ºC in the oxygen – containing atmosphere.<br />
The degree of TTC reduction to red formazan,<br />
indicating the presence of live bacteria in the biofilm,<br />
was graded on a following scale: 0 – no TTC<br />
reduction; 1 – slight pink dotting of the mesh surface, 2<br />
– solid pink coloring of the entire surface, 3 – red<br />
coloring of the mesh surface, clouding and red coloring<br />
of the substrate (very strong TTC reduction). For each<br />
isolate, the test was repeated three times. The control<br />
group consisted of implant fragments incubated with<br />
the bacterial inoculum without subsequent exposure to<br />
the antiseptic solution.<br />
In the quantitative assessment, the monofilament<br />
mesh fragments were coated with bacterial biofilm<br />
using the same procedure as described above. Samples<br />
were rinsed with 0.9% phosphate buffered saline<br />
(PBS) at a pH of 7.2 and placed in 4 mL of the<br />
antiseptic agent. After one minute, the implant samples<br />
were transferred to the neutralizing solution [16]. Next,<br />
they were rinsed with PBS (pH 7.2) and shaken (1100<br />
rpm) in 1 ml of 0.5% saponin for 2 minutes [17].<br />
Serial 10-fold dilutions of the suspension thus obtained<br />
were performed, with subsequent inoculation of three<br />
Petrie dishes containing trypticase soy agar for every<br />
dilution; 100 l of the solution were used per each<br />
Petrie dish. After 24 hours of incubation of the implant<br />
fragments at 37ºC, the result (average of three<br />
measurements) was recorded as the number of colonyforming<br />
units (CFU’s) per one milliliter of suspension<br />
(CFU/ml). Again, the control group consisted of<br />
implant fragments incubated with the bacterial<br />
inoculum without subsequent exposure to the antiseptic<br />
solution.
44<br />
Adrian Reśliński et al.<br />
The influence of octenidine dihydrochloride on the<br />
biofilm was also evaluated by scanning electron<br />
microscopy (SEM). Random fragments of the<br />
biomaterial, coated with a biofilm in a procedure<br />
identical to that described above, were rinsed with PBS<br />
(pH 7.2) and placed in 4 mL of the antiseptic agent.<br />
After one minute, the implant samples were transferred<br />
to the neutralizing solution [16], rinsed with PBS (pH<br />
7.2) and fixed in a 2.5% glutaraldehyde solution<br />
(POCH, Gliwice, Poland) in a 0.1 M phosphate buffer<br />
at a pH of 7.4 for 48 hours at 4 0 C. After fixation, the<br />
material was rinsed for 2x 20 min in phosphate buffer<br />
at room temperature. The samples were then<br />
dehydrated in a graded series of ethanol<br />
concentrations: 30, 50, 70, 80, 96%, 10 minutes in each<br />
solution, and twice for 30 minutes in 99.8% ethanol.<br />
After dehydration, the implant fragments were placed<br />
in a solution of hexamethyldisilazane (HMDS;<br />
Polysciences) for 45 minutes, and dried at room<br />
temperature. The dried material was placed on copper<br />
tables and sputter – coated with gold in an atmosphere<br />
of argon in an ionic coater (Fine Coater, JCF-1200,<br />
JEOL, Tokyo, Japan). The sputter-coated material was<br />
placed in a SEM column (JSM-5310LV, JEOL, Tokyo,<br />
Japan) and analyzed at a voltage of 15 -20 kV [15].<br />
The results were analyzed and registered using the<br />
NSS Version 3.0 software package (Thermo Fisher<br />
Scientific). Like in two previous cases, the control<br />
group consisted of implant fragments incubated with<br />
the bacterial inoculum without subsequent exposure to<br />
the antiseptic solution.<br />
Statistical analysis. The statistical analysis of the<br />
results was performed using the Statistica 10.0<br />
software package (StatSoft Poland). The codependence<br />
of qualitative variables was tested using the<br />
nonparametric χ 2 test. The statistical analysis of non –<br />
normally distributed quantitative variables was<br />
performed using the nonparametric Wilcoxon test for<br />
codependent samples. The adopted significance level<br />
was p≤0.05.<br />
RESULTS<br />
octenidine dihydrochloride for one-minute, no TTC<br />
reduction was observed in 2.9% of the strains; none of<br />
the strains exhibited very strong TTC reduction. A<br />
statistically significant (p
The influence of octenidine dihydrochloride on bacterial biofilm on the surface of a polypropylene mesh 45<br />
Fig. 1. The influence of the antiseptic solution on the biofilm<br />
created by S. aureus strains (n=70) and E. coli<br />
strains (n=70) – a quantitative analysis<br />
Ryc. 1. Wpływ roztowru antyseptyku na biofilm wytworzony<br />
przez szczepy S. aureus i E. coli (n=70) – ocena<br />
metodą ilościową<br />
Fig. 4. Biofilm of E. coli (control group) on the surface of<br />
a monofilament polypropylene mesh; SEM –<br />
magnification 3500x<br />
Ryc. 4. Biofilm E. coli (kontrola) na powierzchni<br />
monofilamentowej siatki polipropylenowej; SEM<br />
– powiększenie 3500x<br />
Fig. 2. Biofilm of S. aureus (control group) on the surface of<br />
a monofilament polypropylene mesh; SEM –<br />
magnification 3500x<br />
Ryc. 2. Biofilm S. aureus (kontrola) na powierzchni<br />
monofilamentowej siatki polipropylenowej; SEM –<br />
powiększenie 3500x<br />
Fig. 5. The effect of octenidine dihydrochloride on the biofilm<br />
of E. coli; SEM – magnification 3500x<br />
Ryc. 5. Efekt działania dichlorowodorku oktenidyny na<br />
biofilm E. coli; SEM – powiększenie 3500x<br />
DISCUSSION<br />
Fig. 3. The effect of octenidine dihydrochloride on the biofilm<br />
of S. aureus; SEM – magnification 3500x<br />
Ryc. 3. Efekt działania dichlorowodorku oktenidyny na<br />
biofilm S. aureus; SEM – powiększenie 3500x<br />
The influence of a solution of octenidine<br />
dihydrochloride (Octenisept) on the biofilm present on<br />
a surface of a monofilament polypropylene mesh<br />
implant was evaluated in this study. Based on the<br />
results of a quantitative and qualitative evaluation and<br />
scanning electron microscopy we have ascertained that<br />
it exerts a bactericidal effect on S. aureus and E. coli<br />
present within a biofilm.<br />
According to Chen and Wen [5], proper treatment<br />
of infections associated with biofilm formation must<br />
include agents which are efficacious against<br />
microorganisms protected by the biofilm. Our own<br />
results, as well as the observations of other authors
46<br />
Adrian Reśliński et al.<br />
[16,18], indicate that octenidine dihydrochloride<br />
displays such properties. The antiseptic agent used in<br />
our study showed bactericidal properties against S.<br />
aureus and E. coli after an exposure of as little as one<br />
minute, which is indicative of its ability to penetrate<br />
into the biofilm. Other researchers have confirmed the<br />
good penetration of octenidine dihydrochloride<br />
solutions through the biofilm matrix [16,18].<br />
In our qualitative study, exposure to octenidine<br />
dihydrochloride resulted in a decreased intensity of the<br />
color reaction on the surface of the biofilm – coated<br />
implant after incubation with TTC, compared to the<br />
control group. This effect corresponded with<br />
a decrease in TTC reduction by the bacteria within the<br />
biofilm; other authors also report this finding [16].<br />
In our quantitative study, decreased live bacterial<br />
cell counts were found in isolates from biofilm –<br />
coated polypropylene mesh fragments after exposure to<br />
a solution of octenidine dihydrochloride. Similar<br />
outcomes have been reported by Bartoszewicz et al.<br />
[16], who studied the effect of octenidine<br />
hydrochloride on bacterial biofilm covering catheters<br />
and suturing material.<br />
In their SEM studies, Bartoszewicz et al. [16]<br />
showed octenidine dihydrochloride to decrease the<br />
number of bacteria adhering to the biomaterial surface.<br />
Our findings are similar.<br />
The results presented here need in vivo<br />
verification, since in vitro conditions cannot accurately<br />
replicate a clinical situation. However, one may<br />
assume that octenidine dihydrochloride solutions may<br />
be efficacious in local therapy of DSSI’s involving an<br />
implanted biomaterial; it has been documented that<br />
neither the presence of blood nor plasma proteins<br />
decreases the efficacy of this agent [19]. Moreover, no<br />
resistance to octenidine dihydrochloride has been so far<br />
reported, even among MRSA strains [9]. The well –<br />
documented synergy of this compound with antibiotics<br />
may also positively influence treatment outcomes [9].<br />
CONCLUSIONS<br />
A solution of octenidine dihydrochloride,<br />
commercially available as Octenisept, displays<br />
bactericidal properties against S. aureus and E. coli<br />
within a biofilm present on the surface of a<br />
monofilament polypropylene hernia mesh.<br />
The study funded by research grant NCU 06/2009.<br />
ACKNOWLEDGEMENTS<br />
The authors wish to thank Professor Eugenia<br />
Gospodarek, Head of the Department of Microbiology<br />
of Nicolaus Copernicus University Collegium<br />
Medicum in Bydgoszcz for making this study possible.<br />
REFERENCES<br />
1. Jezupors A, Mihelsons M. The analysis of infection<br />
after polypropylene mesh repair of abdominal wall<br />
hernia. World J Sur 2006; 30: 2270-2278<br />
2. Stremitzer S, Bachleitner-Hofmann T, Gradl B et al.<br />
Mesh graft infection following abdominal hernia<br />
repair: risk factor evaluation and strategies of mesh<br />
graft preservation. A retrospective analysis of 476<br />
operations. World J Surg 2010; 34: 1702-1709<br />
3. Sanchez VM, Abi-Haidar YE, Itani KM. Mesh<br />
infection in ventral incisional hernia repair: incidence,<br />
contributing factors, and treatment. Surg Infect<br />
(Larchmt) 2011; 12: 205-210<br />
4. Engelsman AF, van der Mei HC, Ploeg RJ et al. The<br />
phenomenon of infection with abdominal wall<br />
reconstruction. Biomaterials 2007; 28: 2314-2327<br />
5. Chen L, Wen YM. The role of bacterial biofilm in<br />
persistent infections and control strategies. Int J Oral<br />
Sci 2011; 3: 66-73<br />
6. Donlan RM. Biofilm elimination on intravascular<br />
catheters: important considerations for the infectious<br />
disease practitioner. Clin Infect Dis 2011; 52: 1038-<br />
1045<br />
7. White RJ, Cutting K, Kingsley A. Topical<br />
antimicrobials in the control of wound bioburden.<br />
Ostomy Wound Manage 2006; 52: 26-58<br />
8. Krzemiński M, Bartoszewicz M, Czarniak E et al. The<br />
use of octenidine dihydrochloride in the treatment of<br />
musculoskeletal infections. Adv Clin Exp Med 2010;<br />
19: 631–636<br />
9. Hübner NO, Siebert J, Kramer A: Octenidine<br />
dihydrochloride, a modern antiseptic for skin, mucous<br />
membranes and wounds. Skin Pharmacol Physiol 2010;<br />
23: 244-258<br />
10. Müller G, Kramer A. Biocompatibility index of<br />
antiseptic agents by parallel assessment of<br />
antimicrobial activity and cellular cytotoxicity.<br />
J Antimicrob Chemother 2008; 61: 1281-1287<br />
11. Koburger T, Hübner NO, Braun M et al. Standardized<br />
comparison of antiseptic efficacy of triclosan, PVPiodine,<br />
octenidine dihydrochloride, polyhexanide and<br />
chlorhexidine digluconate. J Antimicrob Chemother<br />
2010; 65: 1712-1719<br />
12. Gallimore B., Gagnon RF, Subang R et al. Natural<br />
history of chronic Staphylococcus epidermidis foreign<br />
body infection in a mouse model. J Infect Dis 1991;<br />
164: 1220-1223
The influence of octenidine dihydrochloride on bacterial biofilm on the surface of a polypropylene mesh 47<br />
13. Różalska B, Sadowska B, Więckowska M et al.<br />
Wykrywanie biofilmu bakteryjnego na biomateriałach<br />
medycznych. Med Dośw Mikrobiol 1998; 50: 115-122<br />
14. Saygun O, Agalar C, Aydinuraz K et al.: Gold and<br />
gold-palladium coated polypropylene grafts in a S.<br />
epidermidis wound infection model. J Surg Res 2006;<br />
131: 73-79<br />
15. Araujo JC, Téran FC, Oliveira RA et al. Comparison of<br />
hexamethyldisilazane and critical point drying<br />
treatments for SEM analysis of anaerobic biofilms and<br />
granular sludge. J Electron Microsc (Tokyo) 2003; 52:<br />
429-433<br />
16. Bartoszewicz M, Rygiel A. Biofilm jako podstawowy<br />
mechanizm zakażenia miejsca operowanego - metody<br />
prewencji w leczeniu miejscowym. Chir Pol 2006; 8:<br />
171–178<br />
17. Kwiecińska-Piróg J, Bogiel T, Gospodarek E.<br />
Porównanie dwiema metodami tworzenia biofilmu<br />
przez pałeczki Proteus mirabilis na powierzchni<br />
różnych biomateriałów. Med. Dośw Mikrobiol 2011;<br />
63:131-138<br />
18. Bartoszewicz M, Rygiel A et al. Penetration of a<br />
selected antibiotic and antiseptic into a biofilm formed<br />
on orthopedic steel implants. Ortop Traumatol Rehabil<br />
2007; 9: 310-318<br />
19. Pitten FA, Werner HP, Kramer A. A standardized test<br />
to assess the impact of different organic challenges on<br />
the antimicrobial activity of antiseptics. J Hosp Infect<br />
2003; 55: 108-115<br />
Address for correspondence:<br />
Adrian Reśliński PhD<br />
Department of General and Endocrine Surgery<br />
Nicolaus Copernicus University in Torun<br />
Ludwik Rydygier College of Medicine in Bydgoszcz<br />
M. Skłodowskiej-Curie 9 Str.<br />
85-094 Bydgoszcz, Poland<br />
tel. 00 48 52 585-47-30<br />
fax 00 48 52 585-40-16<br />
email: bigar@wp.pl<br />
Received: 26.03.2013<br />
Accepted for publication: 28.05.2013
Medical and Biological Sciences, 2013, 27/2, 49-55<br />
ORIGINAL ARTICLE / PRACA ORYGINALNA<br />
Ewa Joanna Szymelfejnik, Anna Chiba<br />
THE INTERDEPENDENCE OF NUTRITIONAL STATUS AND BLOOD PRESSURE<br />
IN FEMALE STUDENTS<br />
WSPÓŁZALEŻNOŚĆ MIĘDZY STANEM ODŻYWIENIA A CIŚNIENIEM TĘTNICZYM<br />
U STUDENTEK<br />
Department of Nutrition and Dietetics of the Collegium Medicum in Bydgoszcz,<br />
Nicolaus Copernicus University of Toruń<br />
Head: prof. dr hab. Roman Cichon<br />
S u m m a r y<br />
I n t r o d u c t i o n : The value of blood pressure is<br />
affected by a number of factors, nutritional status being of<br />
utmost importance.<br />
T h e a i m o f t h e s t u d y was an assessment of the<br />
interdependence between the nutritional status and systolic<br />
blood pressure (SBP) as well as diastolic blood pressure<br />
(DBP) in female students.<br />
M a t e r i a l a n d m e t h o d : The research included 66<br />
women aged 20.5±0.71, studying in Bydgoszcz. The systolic<br />
and diastolic blood pressure was taken. The nutritional status<br />
of the students was estimated with the use of anthropometric<br />
parameters. To assess the status, nutritional indexes such as<br />
the Body Mass Index (BMI) and the percentage of fat in the<br />
body (%FM) were applied.<br />
R e s u l t s : The mean systolic and diastolic pressure of<br />
the female students was optimal. Hypertension was identified<br />
only in one person (1.5% of the female students) and high<br />
normal blood pressure - in the eight all those (12% of the<br />
female students). The normal BMI was found in 42.4% of the<br />
female students. However, low body mass was found in<br />
every 5th person and undernutrition in every 3rd person. The<br />
percentage of body fat was high (31.1±4.6%), and obesity<br />
was identified in about 60% of the students (%FM>30%). A<br />
significant correlation was observed between systolic<br />
pressure and body mass (r=0.4 p
50<br />
Ewa Joanna Szymelfejnik, Anna Chiba<br />
(BMI) wykazano u 42,4% studentek (BMI 20,0-24,9 kg/m 2 ).<br />
Jednak u co 5 osoby wykazano niską masę ciała, a u co<br />
3 niedożywienie. Procentowa zawartości tłuszczu w ciele<br />
studentek była wysoka (31,1±4,6%), a nadmierne<br />
otłuszczenie odnotowano aż u ok. 60% studentek (%FM).<br />
Odnotowano istotną korelację pomiędzy ciśnieniem<br />
skurczowym oraz masą ciała (r=0,4 p
The interdependence of nutritional status and blood pressure in female students 51<br />
RESULTS<br />
The mean systolic pressure (SBP) of the female<br />
students in Bydgoszcz was at 117.2 ± 9.8 mm Hg, and<br />
the diastolic pressure (DBP) was 75.4 ± 7.7 mm Hg.<br />
The mean heart rate was 74.9 ±11.1 beats/minute<br />
(Tab. I).<br />
Table I. Average value of systolic blood pressure (SBP),<br />
diastolic blood pressure (DBP) and pulse in female<br />
students<br />
Tabela I. Średnia wartość ciśnienia tętniczego skurczowego<br />
(SBP), rozkurczowego (DBP) i tętna studentek<br />
Parametr/ parameter x± SD Me Min Max<br />
SBP [mm Hg] 117.2±9.8 117.5 92.0 137.0<br />
DBP [mm Hg] 75.4±7.7 75.0 57.0 90.0<br />
Tętno [uderzeń/min]<br />
/ Heart rate<br />
74.9±11.1 72.0 57.0 120.0<br />
[beats/minute]<br />
x – średnia, SD - odchylenie standardowe, Me – mediana, Min –<br />
minimum, Max – maximum<br />
x – mean, SD - standard deviation, Me – median, Min – minimum,<br />
Max – maximum<br />
The optimum arterial SBP and DBP pressure was<br />
found respectively in 59.1% and 69.7% of students<br />
(Tab.II). The normal value of the systolic blood<br />
pressure occurred in nearly one-third of all the students<br />
(31.8%), and the normal value of diastolic pressure – in<br />
16.7% of the population. High normal SBP and DBP<br />
pressure was identified in 9.1% and 12.1% of the<br />
students respectively. Among all the students, the SBP<br />
value does not show hypertension. Hypertension was<br />
identified only in one person. Only in 1.5% of the<br />
students an increase in the value of DBP above the<br />
limit was observed and hypertension was found<br />
(Tab. II).<br />
Table II. Classification of blood pressure in female students<br />
Tabela II. Klasyfikacja ciśnienia tętniczego wśród studentek<br />
Kategoria / Category N=66 %N<br />
Optymalne / Optimal 39 59.1<br />
SBP<br />
Normalne / Normal 21 31.8<br />
[mm<br />
Wysokie prawidłowe / High normal 6 9.1<br />
Hg]<br />
Nadciśnienie / Hypertension 0 0<br />
DBP<br />
[mm<br />
Hg]<br />
Optymalne / Optimal 46 69.7<br />
Normalne / Normal 11 16.7<br />
Wysokie prawidłowe / High normal 8 12.1<br />
Nadciśnienie / Hypertension 1 1.5<br />
SBP – ciśnienie tętnicze skurczowe, DBP – ciśnienie tętnicze<br />
rozkurczowe, N – liczebność, %N - odsetek populacji,<br />
SBP - systolic blood pressure, DBP - diastolic blood pressure, N –<br />
number, %N – percentage of population<br />
Table III. The average value of the anthropometric<br />
parameter measurements and nutritional status<br />
in female students<br />
Tabela III. Średnie wartości parametrów antropometrycznych<br />
i wskaźników stanu odżywienia wśród<br />
studentek<br />
Parametr/ parameter x± SD Min Max<br />
Wysokość/Weight<br />
[cm]<br />
166.5<br />
± 5.1 152.0 181.0<br />
Masa ciała/Body mass<br />
[kg] 56.6 ± 10.1 42.4 98.0<br />
A [cm] 24.4 ± 3.0 20.0 30.5<br />
W [cm] 73.9 ± 6.3 64.0 96.0<br />
H [cm] 90.8 ± 5.0 82.5 107.5<br />
TSF [mm] 20.7 ± 7.7 9.7 37.6<br />
BSF [mm] 15.6 ± 7.7 4.0 35.1<br />
SCSF [mm] 14.8 ± 5.5 8.2 31.5<br />
SISF [mm] 20.7 ± 7.5 6.4 36.1<br />
% FM [%] 31.1 ± 4.6 19.6 40.1<br />
WHR 0.8 ± 0.1 0.7 1.0<br />
BMI [BMI kg/m 2 ] 20.3 ± 2.7 17.0 29.9<br />
AMC [cm] 17.9 ± 2.6 10.7 25.6<br />
x - średnia; SD - odchylenie standardowe; Min - minimum; Max -<br />
maximum; A – obwód ramienia, W – obwód talii, H – obwód<br />
bioder; grubość fałdu skórno-tłuszczowego nad: TSF – tricepsem,<br />
BSF - bicepsem; SCSF - dolnym kątem łopatki; SISF - grzebieniem<br />
kości biodrowej; % FM - procentowa zawartość tłuszczu w ciele;<br />
WHR - wskaźnik talia -biodro; BMI - wskaźnik masy ciała; AMC -<br />
obwód mięśni ramienia<br />
x – mean, SD - standard deviation, Me – median, Min – minimum,<br />
Max – maximum; A - Arm circumference, W - Waist circumference,<br />
H – Hip circumference,TSF- triceps skinfold thickness, BSF- biceps<br />
skinfold thickness; SCSF- subscapular skinfold thickness; SISFsuprailiac<br />
skinfold thickness; % FM - the percentage of fat in the<br />
body; WHR- Waist to Hip Ratio; BMI- Body Mass Index; AMCarm<br />
muscle circumference<br />
The characteristics of the anthropometric<br />
parameters and indicators of nutritional status were<br />
shown in the Tab. III. Statistical analysis showed no<br />
statistically significant differences between blood<br />
pressure among students with waist circumferences<br />
52<br />
Ewa Joanna Szymelfejnik, Anna Chiba<br />
Table IV. Blood pressure and pulse of the sample depending<br />
on nutritional status<br />
Tabela IV. Ciśnienie tętnicze i tętna studentek w zależności<br />
od stanu odżywienia<br />
Kategoria/Category N %N<br />
SBP<br />
[mm Hg]<br />
DBP<br />
[mm<br />
Hg]<br />
Tętno<br />
[uderz./min.] /<br />
Heart rate<br />
(beats/minute)<br />
x± SD x± SD x± SD<br />
Obwód talii (cm) / Waist circumference (cm)<br />
The interdependence of nutritional status and blood pressure in female students 53<br />
The analysis of interdependence between blood<br />
pressure and body fat showed a high positive<br />
correlation between systolic pressure and body fat<br />
(r=0.5 p
54<br />
Ewa Joanna Szymelfejnik, Anna Chiba<br />
among the students of School of Medicine (9-10%).<br />
The results of research among Polish adults<br />
LIPIDOGRAM [8], WOBASZ [13] and the NATPOL-<br />
PLUS [15,16] indicated a significant prevalence of<br />
hypertension (29-42%) and a significant percentage of<br />
people at risk of its development (11-30%).<br />
The mean nutritional status of female students from<br />
Bydgoszcz according to the BMI was adequate<br />
(BMI=20.3±2.7 kg/m2). However, the analysis of<br />
distribution in nutritional status classes showed low<br />
body mass in every 5th person and underweight in<br />
every 3rd person. Despite the underweight and low<br />
body weight, the concern was body composition of<br />
young women, as the average percentage of fat tissue<br />
in the body was very high indeed (31.1±4.6%). Obesity<br />
was identified in about 60% of the students. High<br />
content of androidal fat in the body of students with a<br />
low or normal BMI was observed in research<br />
[17,18,19]. For all those with high androidal fat in the<br />
body the authors suggest the presence of metabolic<br />
hazards is similar to the one in obese people.<br />
The assessment of interdependence between blood<br />
pressure and nutritional status showed a significant<br />
relationship between the systolic pressure and body<br />
mass, the % FM and the BMI. The strongest<br />
correlation was found between the content of fat in the<br />
body and the systolic pressure (r=0.5 p
The interdependence of nutritional status and blood pressure in female students 55<br />
13. Tykarski A., Posadzy Małaczyńska A., Wyrzykowski B.,<br />
et al.: Rozpowszechnienie nadciśnienia tętniczego oraz<br />
skuteczność jego leczenia u dorosłych mieszkańców<br />
naszego kraju. Wyniki programu WOBASZ. Kardiol Pol,<br />
2005, 63, 6 (supl.4), 614-619<br />
14. Zdrojewski T.: Nadciśnienie tętnicze w Polsce. Terapia,<br />
2002,10,7/8, 4-7<br />
15. Zdrojewski T.: Rozpowszechnienie głównych czynników<br />
ryzyka chorób układu sercowo-naczyniowego w Polsce.<br />
Wyniki badania NATPOL PLUS. Kardiol Pol, 2004, 61,<br />
IV-5-IV-19<br />
16. Zdrojewski T., Bandosz P., Szpakowski P., et al.:<br />
Rozpowszechnienie głównych czynników ryzyka chorób<br />
układu sercowo-naczyniowego w Polsce. Wyniki<br />
Badania NATPOL PLUS. Kardiol Pol, 2004, 61,IV-5.<br />
17. Szczepańska J., Wądołowska l., Słowińska M.A., et al.,<br />
Badanie wpływu częstości spożycia wybranych źródeł<br />
błonnika na skład ciała studentek. Probl Hig Epidemiol<br />
2011, 92(1): 103-109.<br />
18. Conus F, Alisson DB, Rabasa-Lhoret R., et al.. Metabolic<br />
and behavioral characteristics of metabolically obsese but<br />
normalweight women. JCEM 2004, 89(10): 5013-5020.<br />
19. Conus F, Rabasa-Lhoret R, Peronnet F. Characteristics of<br />
metabolically obese normal weight (MONW) subjects.<br />
Appl Physiol Nutr Metab 2007, 32: 4-12.<br />
Address for correspondence:<br />
dr inż. Ewa Joanna Szymelfejnik<br />
Katedra i Zakład Żywienia i Dietetyki<br />
UMK w Toruniu<br />
Collegium Medicum im. L. Rydygiera w Bydgoszczy<br />
ul. Dębowa 3, 85-626 Bydgoszcz<br />
Tel. (052) 585 54 01 w.45<br />
e-mail: szymelfejnik@wp.pl<br />
Received: 9.05.2012<br />
Accepted for publication: 25.09.2012
Medical and Biological Sciences, 2013, 27/2<br />
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przekraczać 15 stron maszynopisu przy<br />
pracach poglądowych oraz 12 stron przy pracach<br />
oryginalnych i kazuistycznych. Przekroczenie<br />
objętości skutkuje opłatą 100 zł od dodatkowej<br />
strony.<br />
4. Praca powinna być napisana jednostronnie<br />
w programie Word (na jednej stronie może być<br />
do 32 wierszy, tj. 1800 znaków, margines z lewej<br />
strony – 4 cm), czcionką 12 pkt., interlinia<br />
– 1,5.<br />
5. W nagłówku należy podać:<br />
a) imiona i nazwiska autorów oraz tytuły naukowe,<br />
b) tytuł pracy (również w j. pol.),<br />
c) nazwę kliniki (zakładu) lub innej instytucji,<br />
z której praca pochodzi, w j. ang.,<br />
d) tytuł naukowy, imię i nazwisko kierownika<br />
kliniki (zakładu), innej instytucji,<br />
e) adres do korespondencji, który powinien<br />
zawierać również e-mail, tel i faks.<br />
6. Każda praca powinna zawierać streszczenie<br />
w języku polskim i angielskim oraz słowa kluczowe<br />
w j. polskim i angielskim, a także piśmiennictwo.<br />
7. Prace oryginalne powinny mieć następujący<br />
układ: streszczenie w języku polskim i angielskim,<br />
słowa kluczowe w j. polskim i angielskim,<br />
wstęp, materiał i metody, wyniki, dyskusja,<br />
wnioski, piśmiennictwo.<br />
8. Tabele i ryciny należy ograniczyć do niezbędnego<br />
minimum. Tabele numerujemy cyframi<br />
rzymskimi. Tytuł tabeli w jęz. polskim i angielskim<br />
umieszczamy nad tabelą. Opisy wewnątrz<br />
tabeli zamieszczamy w języku polskim i angielskim.<br />
9. Ryciny (fotografie, rysunki, wykresy itp.) numerujemy<br />
cyframi arabskimi. Tytuł ryciny<br />
w jęz. polskim i angielskim umieszczamy pod<br />
ryciną. Opisy wewnątrz rycin zamieszczamy<br />
w języku polskim i angielskim.<br />
10. Odnośniki do piśmiennictwa zaznaczamy<br />
w tekście cyframi arabskimi i umieszczamy<br />
w nawiasie kwadratowym.<br />
11. Streszczenie powinno mieć charakter strukturalny,<br />
tzn. zachować podział na części, jak tekst<br />
główny. Objętość streszczenia zarówno w języku<br />
polskim jak i angielskim – ok. 250 wyrazów.<br />
12. Autor dostarcza pracę na płycie CD lub DVD<br />
oraz 3 egzemplarze, w tym 1 kompletny, zgodny<br />
z płytą, zawierający nazwiska autorów i nazwę<br />
instytucji, z której praca pochodzi (patrz<br />
pkt. 5 i 9) oraz 2 egz. przeznaczone dla recenzentów<br />
bez nazwisk autorów, nazwy instytucji<br />
i innych danych umożliwiających identyfikację.<br />
13. Na dyskietce w odrębnych plikach powinny być<br />
umieszczone:<br />
a) tekst pracy,<br />
b) tabele,<br />
c) ryciny (fotografie w formacie BMP, TIF,<br />
JPG lub PCX; ryciny w formacie WMF,<br />
EPS lub CGM),<br />
d) podpisy pod ryciny i tabele w formacie<br />
MS Word lub RTF.<br />
14. Fotografie powinny mieć postać kontrastowych<br />
zdjęć czarno-białych na błyszczącym (ewentualnie<br />
matowym) papierze. Na odwrocie należy<br />
podać imię i nazwisko autora, tytuł pracy, numer<br />
oraz oznaczyć górę i dół.<br />
15. Należy zaznaczyć w tekście miejsca, w których<br />
mają być zamieszczone ryciny. Wielkość ryciny:<br />
podstawa nie powinna przekraczać 120 mm<br />
(z opisami).<br />
16. Piśmiennictwo – tylko prace cytowane w tekście<br />
(maksymalnie 30 pozycji) – powinno być<br />
ponumerowane i ułożone wg kolejności cytowania,<br />
każdy tytuł od nowego wiersza. Pozycja<br />
piśmiennictwa dotycząca czasopisma musi zawierać<br />
kolejno: nazwisko, inicjał imienia autora<br />
(ów) – maksymalnie trzech – tytuł pracy, tytuł<br />
czasopisma wg skrótów stosowanych w „Index<br />
Medicus”, rok, numer tomu i stron. Przy cytowaniu<br />
pozycji książkowej (monografii, podręczników)<br />
należy podać nazwisko i inicjały<br />
imion autorów, tytuł dzieła, wydawcę, miejsce<br />
i rok wydania.<br />
17. Z pracą należy przesłać oświadczenie, iż nie<br />
była ona dotąd publikowana, a także że nie została<br />
złożona do innego wydawnictwa oraz<br />
zgodę kierownika zakładu na publikację.
Medical and Biological Sciences, 2013, 27/2<br />
18. Do każdej pracy należy dołączyć oświadczenie<br />
podpisane przez wszystkich współautorów, że<br />
aktywnie uczestniczyli w jej realizacji i przygotowaniu<br />
do druku oraz akceptują bez zastrzeżeń<br />
tekst pracy w formie przesłanej do redakcji.<br />
19. Prace niespełniające wymogów regulaminu<br />
będą zwracane autorom.<br />
20. Redakcja zastrzega sobie prawo poprawiania<br />
usterek stylistycznych oraz dokonywania skrótów.<br />
21. Za prace zamieszczone w Medical... autorzy nie<br />
otrzymują honorarium.<br />
22. Redakcja nie przekazuje autorom bezpłatnych<br />
egzemplarzy Medical...<br />
23. Prace publikowane w Medical... są oceniane<br />
przez dwóch recenzentów.<br />
24. Medical and Biological Sciences są punktowane<br />
zgodnie z listą czasopism Ministerstwa Nauki<br />
i Szkodnictwa Wyższego.<br />
Redakcja:<br />
Medical and Biological Sciences<br />
ul. Powstańców Wielkopolskich 44/22<br />
85-090 Bydgoszcz<br />
Dyżury sekretarza Redakcji: wtorek 11.00-13.00<br />
tel.: 52 585 33 26<br />
Opracowanie redakcyjne i realizacja wydawnicza:<br />
Redakcja w Bydgoszczy<br />
ul. Powstańców Wielkopolskich 44/22, 85-090 Bydgoszcz<br />
tel./faks: 52 585 33 25, e-mail: wydawnictwa@cm.umk.pl<br />
COLLEGIUM MEDICUM im. LUDWIKA RYDYGIERA<br />
BYDGOSZCZ 2013<br />
Nakład: 100 egz.<br />
Druk i oprawa: Drukarnia cyfrowa UMK, ul. Gagarina 5, 87-100 Toruń, tel.: 56 611 22 15