You also want an ePaper? Increase the reach of your titles
YUMPU automatically turns print PDFs into web optimized ePapers that Google loves.
<strong>53</strong> WINTER 2018<br />
NEWSLETTER<br />
••<br />
••<br />
••<br />
••<br />
••<br />
••<br />
••<br />
••<br />
••<br />
Toxicity and Toxicokinetic Study of Subcutaneously<br />
Administered RPh201 in <strong>Göttingen</strong> <strong>Minipigs</strong> page 3<br />
Testing cognitive abilities over different life stages<br />
in <strong>Göttingen</strong> <strong>Minipigs</strong> page 6<br />
Obesity and diabetes result in pathological bone repair<br />
around dental implants:<br />
Demonstration in a <strong>Göttingen</strong> <strong>Minipigs</strong> models page 9<br />
Using <strong>Göttingen</strong> <strong>Minipigs</strong> as model for peritoneal dialysis page 14<br />
Vascular Access Buttons in <strong>Göttingen</strong> <strong>Minipigs</strong> page 15<br />
Fifty Years with <strong>Göttingen</strong> <strong>Minipigs</strong>: Let’s Celebrate! page 18<br />
<strong>Ellegaard</strong> <strong>Göttingen</strong> <strong>Minipigs</strong> Going Global page 19<br />
<strong>Ellegaard</strong> <strong>Göttingen</strong> <strong>Minipigs</strong> Enters into a License<br />
and Commercialization Agreement for <strong>Göttingen</strong> <strong>Minipigs</strong><br />
Research Models in Partnership with Exemplar Genetics page 19<br />
Creating a stimulating and cooperative environment<br />
for <strong>Göttingen</strong> <strong>Minipigs</strong> page 20<br />
••<br />
••<br />
Invitation to join The 13th Minipig Research Forum<br />
22-24 May 2019 in Vienna, Austria page 21<br />
New scientific publications on <strong>Göttingen</strong> <strong>Minipigs</strong> page 22<br />
See<br />
where you<br />
can meet us<br />
in 2019<br />
Clean pigs<br />
for clear results<br />
page 24
Dear<br />
Reader<br />
The days are getting shorter and soon we will<br />
be saying goodbye to 2018 and hello to a new<br />
year. At <strong>Ellegaard</strong> <strong>Göttingen</strong> <strong>Minipigs</strong>, we will<br />
especially remember 2018 for two important<br />
landmark events: Approval of the Veterinary<br />
Health Certificates for the export of <strong>Göttingen</strong> <strong>Minipigs</strong><br />
to both China and India! Read more about the availability<br />
of <strong>Göttingen</strong> <strong>Minipigs</strong> in these new markets in this <strong>Newsletter</strong>.<br />
We are getting ready to enter 2019 with the expectations of a<br />
special year to come celebrating Fifty Years of <strong>Göttingen</strong> <strong>Minipigs</strong><br />
by, among other things, a roadshow of scientific events around<br />
Europe as well as a few places in the USA and Asia. We reveal<br />
more on the plans for our year of celebration at page 18, and I<br />
recommend that you follow us on LinkedIn to stay updated on<br />
venues and dates for the meetings and other exciting news.<br />
Looking back at the old year, in my humble opinion, some highlights<br />
at <strong>Ellegaard</strong> <strong>Göttingen</strong> <strong>Minipigs</strong> stood out: At the Minipig<br />
Research Forum in Barcelona in May, the <strong>Ellegaard</strong> <strong>Göttingen</strong><br />
<strong>Minipigs</strong> Research Foundation announced a substantial grant<br />
to a very promising and exciting scientific project entitled<br />
“Speedgene targeted knock out of the PCSK-9 gene in <strong>Göttingen</strong><br />
<strong>Minipigs</strong>”; and in September, the GENISYST Project Steering<br />
Group assembled for a successful two-day meeting in Denmark<br />
including a visit to our location in Dalmose (see photo).<br />
On 1 October 2018, we proudly turned 20 years of continued<br />
and full AAALAC accreditation; a very important achievement<br />
which emphasizes our passion and tireless engagement in the<br />
welfare of our animals.<br />
Finally, I am pleased to announce that early November 2018, <strong>Ellegaard</strong><br />
<strong>Göttingen</strong> <strong>Minipigs</strong> signed an agreement with Exemplar<br />
Genetics to develop genetically modified animal models based<br />
on our <strong>Göttingen</strong> <strong>Minipigs</strong>. Read more about this at page 19.<br />
Further, during 2019, <strong>Ellegaard</strong> <strong>Göttingen</strong> <strong>Minipigs</strong> will offer<br />
new services from our research barrier in Denmark; I look<br />
forward to sharing more news with you on this in due course.<br />
GENISYST Steering Group Meeting in Dalmose, Denmark. From left: Roy Forster<br />
(Citoxlab France), Jaya Krishnan (Genome Biologics), Andy Makin (Citoxlab<br />
Denmark), Jonathan Ward (Genome Biologics), Lars Friis Mikkelsen (<strong>Ellegaard</strong><br />
<strong>Göttingen</strong> <strong>Minipigs</strong>), Jens <strong>Ellegaard</strong> (<strong>Ellegaard</strong> <strong>Göttingen</strong> <strong>Minipigs</strong>), Marianne<br />
Kronborg Bracken (Citoxlab Denmark), Peter Vestbjerg (<strong>Ellegaard</strong> <strong>Göttingen</strong><br />
<strong>Minipigs</strong>) and Henrik Duelund Pedersen (<strong>Ellegaard</strong> <strong>Göttingen</strong> <strong>Minipigs</strong>)<br />
A warm thank you from all of us at <strong>Ellegaard</strong> <strong>Göttingen</strong> <strong>Minipigs</strong><br />
to customers and collaboration partners for working with us<br />
during 2018 and also a big thank you for interesting talks, chats,<br />
meetings and conferences around the world throughout 2018:<br />
See you again next year, maybe at the MRF 2019 from 22-24<br />
May in Vienna, Austria?<br />
Happy Holidays and Happy Reading!<br />
Lars Friis Mikkelsen, CEO<br />
<strong>Ellegaard</strong> <strong>Göttingen</strong> <strong>Minipigs</strong> A/S<br />
New Finance Manager<br />
On 1 October 2018, we welcomed Klaus Kvist Rasmussen in<br />
a new position as Finance Manager. Klaus holds a master’s<br />
degree in economics from the University of Southern Denmark<br />
and has previously worked for international consulting firms<br />
such as PwC and Deloitte as well as for Novo Nordisk, where,<br />
among other things, he advised on the optimization of financial<br />
management and reporting. Since 2016, Klaus has worked as<br />
an independent business consultant and added even more to<br />
his extensive experience within his areas of financial<br />
expertise.<br />
At <strong>Ellegaard</strong> <strong>Göttingen</strong> <strong>Minipigs</strong>, in addition<br />
to being responsible for our financial<br />
management, Klaus will no doubt play<br />
an important role in the future develop-<br />
ment and further internationalization<br />
of our company. In his spare time, Klaus<br />
is a passionate hunter and also enjoys<br />
other outdoor activities.<br />
CONTENTS<br />
Toxicity and Toxicokinetic Study of Subcutaneously<br />
Administered RPh201 in <strong>Göttingen</strong> <strong>Minipigs</strong> . . . . . . . . . . 3<br />
Testing cognitive abilities over different life stages<br />
in <strong>Göttingen</strong> <strong>Minipigs</strong> . . . . . . . . . . . . . . . . . . . . . . . 6<br />
Obesity and diabetes result in pathological bone repair<br />
around dental implants: Demonstration in a <strong>Göttingen</strong><br />
<strong>Minipigs</strong> models . . . . . . . . . . . . . . . . . . . . . . . . . 9<br />
Using <strong>Göttingen</strong> <strong>Minipigs</strong> as model for peritoneal dialysis . . 14<br />
Vascular Access Buttons in <strong>Göttingen</strong> <strong>Minipigs</strong> . . . . . . . . 15<br />
Fifty Years with <strong>Göttingen</strong> <strong>Minipigs</strong>: Let’s Celebrate! . . . . . 18<br />
<strong>Ellegaard</strong> <strong>Göttingen</strong> <strong>Minipigs</strong> Going Global . . . . . . . . . . 19<br />
<strong>Ellegaard</strong> <strong>Göttingen</strong> <strong>Minipigs</strong> Enters into a License and<br />
Commercialization Agreement for <strong>Göttingen</strong> <strong>Minipigs</strong><br />
Research Models in Partnership with Exemplar Genetics . . . 19<br />
Creating a stimulating and cooperative environment<br />
for <strong>Göttingen</strong> <strong>Minipigs</strong> . . . . . . . . . . . . . . . . . . . . . 20<br />
Invitation to join The 13th Minipig Research Forum<br />
22-24 May 2019 in Vienna, Austria . . . . . . . . . . . . . . 21<br />
New scientific publications on <strong>Göttingen</strong> <strong>Minipigs</strong> . . . . . . 22<br />
Meeting Calendar 2019 . . . . . . . . . . . . . . . . . . . . . 24<br />
2
Toxicity and Toxicokinetic Study<br />
of Subcutaneously Administered RPh201<br />
in <strong>Göttingen</strong> <strong>Minipigs</strong><br />
By Vanessa Ross 1 , Sydney Mukaratirwa 1 , Abraham Nyska 2 , Yuval Ramot 3 , Zadik Hazan 4 , Andre Lucassen 4 & Konstantin Adamsky 4<br />
1<br />
Envigo Ltd, Alconbury, Cambridgeshire, UK,<br />
2<br />
Timrat and Tel Aviv University, Tel Aviv Israel,<br />
3<br />
Hadadssah Hebrew University Medical Center, Jerusalem, Israel,<br />
4<br />
Regenera Pharma, Nes-Ziona, Israel<br />
Introduction<br />
At Envigo, we ran a long-term (39 weeks) toxicity study in<br />
<strong>Göttingen</strong> <strong>Minipigs</strong> in conjunction with Regenera Pharma. The<br />
material investigated was an extract of mastic gum and such<br />
extracts have traditionally been used as a dietary additive and<br />
as a flavouring agent. These compounds are being developed<br />
for a variety of clinical indications, which include treatment<br />
of Crohn’s disease and the healing of peptic and dental ulcers.<br />
RPh201 is an extract of mastic gum which has been formulated<br />
and stabilised in a proprietary method, and is being developed<br />
for a wide range of neurological indications.<br />
Experimental design<br />
32 male and 32 female <strong>Göttingen</strong> <strong>Minipigs</strong> were administered<br />
subcutaneous injections of RPh201 (0, 3.1, 12.5. 50 mg/kg/occasion)<br />
twice weekly for 39 weeks. The dose volumes were 0.062,<br />
0.25 or 1 mL/kg/occasion respectively and the controls received<br />
the vehicle at a dose volume of 1 mL/kg/occasion. There was a<br />
26-week interim phase and a 6-week recovery period.<br />
The protocol included routine clinical observations, hematology,<br />
clinical chemistry, toxiciokinetics, blood evaluations, electroretinogram<br />
and tonometry, electrocardiography, and macroscopic<br />
and microscopic evaluations of selected organs.<br />
Results<br />
Clinical observations (including ocular ERG and tonometry,<br />
cardiac electrocardiography) and organ weights<br />
There were no test article-related clinical signs or effects on<br />
body weight or food consumption. During ophthalmic examination,<br />
posterior suture line opacity was observed in the lens of<br />
the eye of four animals. However, there was no effect on the<br />
retina at electroretinogram examination and no ocular hypotension<br />
or hypertension was evident. There were no effects on<br />
electrocardiography parameters.<br />
Clinical pathology<br />
Hematology investigations conducted in Week 26 of treatment<br />
revealed slightly higher mean neutrophil counts observed in<br />
both sexes receiving 12.5 or 50 mg/kg/occasion when compared<br />
to controls. There was full recovery during the 6 week off-dose<br />
period. In Week 39, a lower mean erythrocyte count and hemoglobin<br />
concentration was observed in females receiving 50 mg/<br />
kg/occasion in comparison with the controls. There was partial<br />
recovery in both parameters following 6 weeks off-dose.<br />
Toxicokinetics<br />
The rate of systemic exposure of minipigs to RPh201, measured<br />
as the two components masticadienonic acid (MDA) and isomasticadienonic<br />
acid (IMDA), was characterized by nonlinear,<br />
subproportional, (dose dependent) kinetics over the dose range<br />
from 3.1 to 50 mg/kg/day on Day 1 and during Week 13,<br />
Week 26 and Week 39 of the 39 week subcutaneous toxicity<br />
study. However, the extent of systemic exposure of minipigs<br />
to MDA and IMDA was characterized by dose independent<br />
(linear) kinetics over the same dose range and period. Systemic<br />
exposure of minipigs to MDA was generally comparable to that<br />
of IMDA.<br />
Macroscopic observations<br />
Clear oily liquid was found at the injection sites of all groups<br />
at the 26 and 39-week sacrifice points, and was also observed<br />
in controls and animals previously treated with 50 mg/kg/<br />
occasion with a similar incidence in the 6-week recovery group.<br />
Additional findings at the injection sites included skin thickening<br />
(39-week sacrifice point), pale areas (26, 39-week and recovery<br />
sacrifice points) and raised areas (week 26, week 39 and recovery<br />
sacrifice points).<br />
Clear oily liquid, enlargement and pale areas were seen at 26-<br />
week, 39-week and 6-week recovery sacrifice points in various<br />
lymph nodes (including axillary, inguinal, mandibular and mediastinal)<br />
in animals treated with 50 mg/kg/occasion and in the<br />
control group. Enlargement was also seen in one female animal<br />
treated with 12.5 mg/kg/occasion and pale areas were also seen<br />
in a male and female animal in this group.<br />
Microscopic observations<br />
Treatment-related findings were observed at injection sites<br />
of animals from all sacrifice points, and included abscesses at<br />
the 26 week and 39 week sacrifice and recovery phase and<br />
recovering abscesses and an increase in fibrous tissue in the<br />
39 week sacrifice and recovery phases. Abscesses graded as<br />
moderate or marked were confined to animals receiving 50<br />
mg/kg/occasion. Resolving abscesses were seen at the 39-<br />
week sacrifice point in females receiving 50 mg/kg/occasion.<br />
These were characterized by fibrous tissue, mineralization<br />
and with few neutrophils present. At the 6 week recovery<br />
sacrifice, the severity of the abscesses, resolving abscesses<br />
and fibrosis was of a minimal or slight degree, which indicated<br />
partial recovery from the findings seen at 26 and 39<br />
weeks.<br />
<br />
3
Figure 1 legend:<br />
A. Histopathological section of the subcutaneous injection site<br />
in a <strong>Göttingen</strong> Minipig from the high-dose group (50 mg/<br />
kg), main phase of the study. The tissue reaction consists of<br />
chronic inflammation (asterisks). These findings were comparable<br />
to the control group, and were considered as related<br />
to the vehicle. In addition, abscesses (arrow) in the subcutis<br />
(minimal grade) were seen, characterized by a cystic space<br />
containing aggregates of neutrophils with a variable chronic<br />
inflammatory component and a fibrous capsule of varying<br />
thickness. H&E.<br />
B. Histopathological section of the subcutaneous injection<br />
site in minipig from the high-dose group (50 mg/kg), main<br />
phase of the study. The tissue reaction consists of chronic<br />
inflammation (asterisks), and intermixed with empty spaces<br />
reflecting the washed-out cotton-seed oil vehicle. Note the<br />
abscess (arrows) in the subcutis (marked grade). This grade of<br />
lesion is considered treatment related. H&E.<br />
C. Histopathological section of the subcutaneous injection site<br />
in a <strong>Göttingen</strong> Minipig from the high-dose group (50 mg/kg),<br />
recovery phase of the study. Another example of a resolving<br />
abscess. The tissue reaction is characterized by fibrous tissue<br />
(arrow) and with only few neutrophils present (asterisk). H&E.<br />
D. Histopathological section of the subcutaneous injection site<br />
in a <strong>Göttingen</strong> Minipig from the high-dose group (50 mg/kg),<br />
recovery phase of the study. Area of fibrosis (asterisks). The<br />
increase in subcutaneous fibrosis was also seen in the 39-<br />
week and 6-week recovery sacrifice, mainly in the high dose<br />
animals. H&E.<br />
Vehicle-related findings<br />
Cystic spaces and chronic inflammation were seen at injection<br />
sites of all treated groups and controls with the severity<br />
generally correlating with the volume of vehicle administered.<br />
The chronic inflammation was characterized by mononuclear<br />
cells (lymphocytes and macrophages), foreign body giant cells,<br />
fibrosis and occasional neutrophils. Incidence and severity of<br />
the findings did not show signs of recovery after 6 weeks.<br />
Cystic spaces and chronic inflammation were seen in the left<br />
inguinal lymph node of all treated groups and controls at 26<br />
week, 39 week and recovery phase with the severity generally<br />
correlating with the volume of vehicle administered. Abscesses,<br />
graded as minimal, were seen in the left inguinal lymph node<br />
of a few animals killed at 26 weeks in all treated groups but<br />
also in a female control killed after 39 weeks of treatment and<br />
4
in the recovery group. They were also seen in other lymph<br />
nodes in all three phases of this study that were examined<br />
histopathologically due to being macroscopically abnormal.<br />
Considering the overall incidence of abscesses in the left inguinal<br />
lymph node and other lymph nodes in the study, this finding<br />
was considered to be related to the vehicle. The incidence and<br />
severity of the findings at the 6-week sacrifice point were<br />
similar to the 39 week sacrifice point, therefore not showing<br />
signs of recovery. Similar findings were also seen in the right<br />
inguinal lymph node and in the axillary, mediastinal, medial<br />
iliac, aortic and superficial inguinal lymph nodes when a macroscopic<br />
abnormality was seen. Such abnormalities were present<br />
most commonly in controls and animals receiving 50 mg/kg/<br />
occasion.<br />
Conclusions<br />
RPh201 was well tolerated for 39 weeks with no clinical or<br />
dose-related signs observed and with no changes in body<br />
weight, food consumption, electroretinography, intraocular<br />
pressure, electrocardiography or blood chemistry and urinalysis<br />
parameters.<br />
Treatment-related findings were seen at the injection sites and<br />
included abscesses that correlated with a small increase in peripheral<br />
neutrophil count. In animals given 50 mg/kg/occasion<br />
abscesses graded above slight corresponded with macroscopically<br />
thickened or raised areas (which contained either dark<br />
fluid or green purulent material). The subcutaneous fibrosis<br />
seen at the injection sites was considered secondary to chronic<br />
inflammation and was again more pronounced in animals given<br />
50 mg/kg/occasion and to a lesser extent in animals given<br />
12.5 mg/kg/occasion. There was partial recovery from both<br />
the abscess formation and fibrosis. Necrosis was not seen in<br />
any of the injection sites, and bacterial colonies were not seen<br />
in the abscesses. Therefore, these abscesses were concluded<br />
to be sterile, and attributed to irritation from the presence<br />
of the drug in a location with a relatively slow rate of absorption.<br />
Under the conditions of this study, the No Observed Adverse<br />
Effect Level (NOAEL) was considered to be 12.5 mg/kg/occasion,<br />
after 39 weeks of administration followed by a 6-week recovery<br />
period. RPh201 is a promising new and safe drug candidate for<br />
the treatment of several neurological indications.<br />
Figure 1E legend:<br />
E. Histopathological section of the inguinal lymph-node (regional<br />
to the injection site) in a <strong>Göttingen</strong> minipig from the<br />
intermediate-dose group (12.5 mg/kg), main phase of the<br />
study. The tissue reaction consists of chronic inflammation<br />
(asterisks), and intermixed with empty spaces reflecting the<br />
washed-out cotton-seed oil vehicle. These findings were<br />
comparable to the control group, and were considered related<br />
to the vehicle. In addition, abscesses (arrows) (minimal grade)<br />
were seen, characterized by a cystic space containing aggregates<br />
of neutrophils with a variable chronic inflammatory<br />
component and a fibrous capsule of varying thickness. H&E.<br />
5
Testing cognitive abilities over different<br />
life stages in <strong>Göttingen</strong> <strong>Minipigs</strong><br />
By Caroline Clouard & Inonge Reimert<br />
Wageningen University & Research, Department of Animal Sciences, Adaptation Physiology Group,<br />
Wageningen, The Netherlands<br />
Introduction<br />
The pig is a highly intelligent species and is, therefore, a good<br />
animal model to investigate cognitive functions [1,2,3] . In the wild,<br />
pigs have a mixed omnivorous diet, which requires high spatial<br />
cognitive abilities to remember spatial location of food patches<br />
[4] . This has also been shown by the plethora of pig studies<br />
using spatial cognitive tasks such as the holeboard task [5,6,7,8,9,10] .<br />
In these studies, however, pigs have been subjected to the<br />
holeboard task during only one life stage, usually the juvenile or<br />
pubertal life stage, and nothing is known about their cognitive<br />
abilities in adulthood or the stability of their cognitive abilities<br />
over life stages. It would therefore be interesting to determine<br />
(1) whether pigs’ cognitive abilities are stable over time (i.e. from<br />
juvenility to puberty to adulthood) and (2) whether pigs, which<br />
are curious and novelty-seeking animals [11] , can be motivated<br />
to perform the same task – which consists of a large number of<br />
trials repeated over many testing days – multiple times throughout<br />
life. To answer these research questions, we used <strong>Göttingen</strong><br />
<strong>Minipigs</strong> because their small size, even at the adult stage, makes<br />
them very easy to handle and allows for longitudinal studies<br />
in the same individuals, and because they have already been<br />
tested in the holeboard task before [12,13,14] .<br />
Methodology<br />
Animals and housing<br />
In total, 8 female <strong>Göttingen</strong> <strong>Minipigs</strong> (<strong>Ellegaard</strong>, Denmark) were<br />
subjected to the holeboard task during three consecutive life<br />
stages: the juvenile stage (from 7 to 9 weeks of age), the pubertal<br />
stage (from 17 to 20 weeks of age) and the adult stage (from<br />
41 to 45 weeks of age). The animals were distributed over 3<br />
consecutive batches, with a 2-week interval between batches.<br />
In the first week after birth, piglets were kept with their sow<br />
in individual farrowing pens (1.8 × 1.5 m). Pens had shredded<br />
straw as bedding and contained a separate heated nest for the<br />
piglets. From 1 week of age onwards, pigs were separated from<br />
the sows and non-related pigs were housed in pairs in pens<br />
(2.5 × 1 m) with shredded straw as bedding, and enriched with a<br />
squeeze ball, a dog bed and 2 metallic chains per pen. Water was<br />
available ad libitum and feed was given according to <strong>Ellegaard</strong><br />
recommendations.<br />
All pigs were habituated to being handled by experimenters<br />
and to the apple rewards (offered in a bucket in the home pen)<br />
before the start of the holeboard task in the juvenile stage.<br />
Holeboard task<br />
Principle and apparatus<br />
The holeboard arena (3 × 3 m; Figure 1) had black, wooden,<br />
80-cm-high walls and 4 entrances with guillotine doors that<br />
could be operated from the southwest corner of the arena with<br />
a system of ropes and pulleys. The arena was surrounded by<br />
a corridor for the pig to access the 4 entrances, a waiting area<br />
containing a jute bag and some toys in the southeast corner of<br />
the room and an area for the observers on the southwest corner<br />
of the room. In the arena, 16 grey metallic buckets (juvenile stage,<br />
Ø10.5 cm – H10 cm; pubertal and adult stage, Ø12 cm – H12 cm)<br />
were screwed to the floor in a 4 × 4 matrix (Figure 2). During the<br />
task, 4 of the buckets were baited with a piece of apple (juvenile<br />
stage ~ 12 × 12 × 12 mm; pubertal and adult stage ~ 12 × 12 ×<br />
20-40 mm) according to 1 out of 4 different configurations. To<br />
prevent the use of visual cues to find the rewards, the rewards<br />
were hidden under a thin layer of shredded straw. All buckets<br />
also had a perforated false bottom under which fresh pieces of<br />
apple were placed at the start of the day to also prevent the use<br />
of odour cues to locate the baited buckets. Pigs were deprived<br />
from feed overnight during the whole period of holeboard testing.<br />
Procedure<br />
Before the start of the holeboard test, the juvenile piglets were<br />
gradually habituated to the experimenters, the buckets and<br />
rewards, the corridor leading to the test room, the test room<br />
and the task in sessions of 10-15 min per day. At the end of this<br />
8-day habituation period, piglets liked/ate the rewards, showed<br />
no extreme stress responses (high-pitched vocalizations, standing<br />
alert, escape attempts) when alone in the holeboard arena,<br />
and performed the task (i.e. looking into the buckets).<br />
Figure 1. Lay out of the holeboard arena.<br />
6
Figure 2. From left to right, a pig in the holeboard arena in the juvenile, pubertal and adult stage, respectively.<br />
After the habituation was completed, testing started with acquisition<br />
trials.<br />
In the juvenile stage, piglets were individually subjected to 2<br />
massed trials (i.e.performed a few minutes apart) per day on 14<br />
consecutive working days, i.e. 28 acquisition trials. Each testing<br />
day, both piglets from a pen were brought to the test room.<br />
While one piglet was being tested, its pen mate was kept in the<br />
waiting area. The test pig was led to the starting box (at the<br />
south door) or released into the corridor where it could walk<br />
freely to the correct entrance door, which was then opened. Two<br />
different entrances were used on a testing day (i.e. 1 entrance<br />
per trial), to prevent piglets from developing a fixed pattern of<br />
visits that would reduce the working memory (WM) load. A trial<br />
started when the piglet had its 4 legs in the holeboard arena<br />
and ended when the piglet found all 4 rewards or after 180 s.<br />
Every time the piglet visited a baited bucket for the first time,<br />
a clicker sound was produced to facilitate learning. If the piglet<br />
completed the task (i.e. found the 4 rewards in fewer than 180<br />
s), the exit (south) door was opened, the piglet was congratulated<br />
(“good job!”, “well done!”), and received half of a white<br />
grape. If the piglet did not complete the task within the 180 s,<br />
a police siren sound was produced for 1-2 s; the piglet was not<br />
congratulated and did not receive a reward. After the trial, the<br />
piglet was led back into the waiting area, and the other piglet<br />
was tested. After the 2 piglets per pen had been tested twice,<br />
they were brought back to their home pen and allowed to eat.<br />
For the acquisition phase, 4 different configurations of baited<br />
buckets were used in total (the 4 configurations were rotated<br />
versions of the one shown in Figure 1). Each piglet was tested on<br />
a fixed configuration throughout the acquisition phase, with the<br />
configuration of baited buckets differing between the 2 piglets<br />
in each pen and balanced over all piglets. Testing order within<br />
and between pens was changed daily.<br />
In the pubertal stage, the pigs were individually subjected to<br />
2 massed trials per day on 12 consecutive working days, i.e.<br />
24 re-acquisition trials. Pigs were tested according to the same<br />
procedures and with the same configurations as in the juvenile<br />
stage. However, after the re-acquisition phase was completed,<br />
the pigs were also individually subjected to 16 reversal trials,<br />
with 2 massed trials per day on 8 consecutive working days. The<br />
procedures were the same as in the re-acquisition phase, but the<br />
pigs were assigned to another, mirrored, configuration of baited<br />
buckets. In the reversal phase, the pigs’ cognitive flexibility can<br />
be assessed, i.e. how well pigs learn a new configuration.<br />
In the adult stage, the number of trials and the procedures were<br />
completely similar to those used in the pubertal stage. In the<br />
re-acquisition phase, piglets were, however, tested with the<br />
same configurations as those used in the reversal phase of the<br />
pubertal stage, but in the reversal phase, 4 new configurations,<br />
with completely different patterns, were used.<br />
Measurements<br />
During each trial, all visits and revisits to all buckets, latencies<br />
to all bucket visits, trial duration, and the total number of<br />
defecations, urinations, high-pitched vocalizations and escape<br />
attempts were scored using The Observer XT 10 software<br />
(Noldus Information Technology, Wageningen, The Netherlands).<br />
From these parameters, the following variables were calculated<br />
a posteriori according to van der Staay et al. (2012 [5] ): working<br />
memory scores, reference memory scores, trial duration, inter-visit-interval<br />
and total number of visits.<br />
Results and Conclusions<br />
<strong>Göttingen</strong> <strong>Minipigs</strong> seemed to be able to perform the task equally<br />
in each life stage, as shown by the consistent average working<br />
and reference memory scores over the 3 life stages (Figure 3).<br />
Figure 3.<br />
Working and reference<br />
memory scores of the<br />
pigs in the different life<br />
stages and test phases.<br />
<br />
7
Figure 4. Trial duration, inter-visit-interval and total number of visits of the pigs in the different life stages and test phases.<br />
It is worth noting, however, that in the juvenile stage only 1 of<br />
8 piglets managed to complete finding the 4 rewards in all 28<br />
trials, and that 4 of 8 piglets failed more than 50% of the trials.<br />
In the pubertal and adult stages, all 8 pigs completed 100% of<br />
the 24 acquisition and 16 reversal trials (except 1 pig which<br />
failed 1 trial in the pubertal stage). Furthermore, in the juvenile<br />
stage, piglets took more time to finish the trial and had longer<br />
inter-visit-intervals, but did not visit more buckets than in the<br />
other stages (Figure 4). These data suggest that the juvenile<br />
piglets were less focussed and/or less motivated for the task,<br />
i.e. explored the arena more or were more distracted in between<br />
2 bucket visits, compared to the older pigs. It is also possible<br />
that the juvenile piglets were performing worse as they were<br />
less trained, but that is less likely as there were no differences<br />
between the pubertal and the more trained adult pigs. These<br />
findings may also explain why many piglets failed to find the 4<br />
rewards before the end of the 180-s trial in the juvenile phase.<br />
In conclusion, while juvenile <strong>Göttingen</strong> <strong>Minipigs</strong> appear to show<br />
lower attention/motivation for the task, cognitive performance<br />
and motivation for the task remained high and stable in the<br />
pubertal and adult stages, even after many days of testing.<br />
Moreover, in each life stage the pigs did not show any clear<br />
stress responses such as escape attempts and high-pitched<br />
vocalizations anymore after several days of testing.<br />
References<br />
1 Marino, L., Colvin, C.M. 2015. Thinking pigs: A comparative review of cognition, emotion, and personality in Sus domesticus.<br />
International Journal of Comparative Psychology, 28.<br />
2 Gieling, E.T., Nordquist, R.E., van der Staay, F.J. 2011. Assessing learning and memory in pigs. Animal Cognition, 14:151-173.<br />
3 Kornum, B.R., Knudsen, G.M. 2011. Cognitive testing of pigs (Sus scrofa) in translational biobehavioral research. Neuroscience &<br />
Biobehavioral Reviews, 35:437-451.<br />
4 Nawroth, C, Langbein J, Puppe B. 2018. Swine cognition. In: Vonk, J and Shackelford, TK (eds), Encyclopedia of Animal Cognition<br />
and Behavior. Springer International Publishing AG.<br />
5 van der Staay, F.J., Gieling, E.T., Pinzón, N.E., Nordquist, R.E., Ohl, F. 2012. The appetitively motivated “cognitive” holeboard: a family<br />
of complex spatial discrimination tasks for assessing learning and memory. Neuroscience & Biobehavioral Reviews, 36:379-403.<br />
6 Arts, J.W., van der Staay, F.J., Ekkel, E.D. 2009. Working and reference memory of pigs in the spatial holeboard discrimination task.<br />
Behavioural Brain Research, 205:303-306.<br />
7 Antonides, A., Schoonderwoerd, A.C., Nordquist, R.E., van der Staay, F.J. 2015. Very low birth weight piglets show improved<br />
cognitive performance in the spatial cognitive holeboard task. Frontiers in Behavioral Neuroscience, 9:43.<br />
8 Bolhuis, J.E., Oostindjer, M., Hoeks, C.W., de Haas, et al. 2013. Working and reference memory of pigs (Sus scrofa domesticus) in a<br />
holeboard spatial discrimination task: the influence of environmental enrichment. Animal Cognition, 16:845–850.<br />
9 Clouard, C., Kemp, B., Val-Laillet, D., Gerrits, W.J., Bartels, A.C., et al. 2016. Prenatal, but not early postnatal, exposure to a Western<br />
diet improves spatial memory of pigs later in life and is paired with changes in maternal prepartum blood lipid levels. The FASEB<br />
Journal, 30:2466-2475.<br />
10 Clouard, C., Le Bourgot, C., Respondek, F., Bolhuis, J.E., Gerrits, W.J. 2018. A milk formula containing maltodextrin, vs. lactose, as<br />
main carbohydrate source, improves cognitive performance of piglets in a spatial task. Scientific Reports, 8:9433.<br />
11 Wood-Gush, D.G.M., Vestergaard, K. 1991. The seeking of novelty and its relation to play. Animal Behaviour, 42:599-606.<br />
12 Gieling, E., Wehkamp, W., Willigenburg, R., Nordquist, R.E., Ganderup, N.-C., et al. 2013. Performance of conventional pigs and<br />
<strong>Göttingen</strong> miniature pigs in a spatial holeboard task: effects of the putative muscarinic cognition impairer Biperiden. Behavioral<br />
and Brain Functions, 9:4.<br />
13 Haagensen, A.M., Grand, N., Klastrup, S., Skytte, C., Sørensen, D.B. 2013. Spatial discrimination and visual discrimination: two<br />
methods evaluating learning and memory in juvenile <strong>Göttingen</strong> minipigs. Behavioural Pharmacology, 24:172-179.<br />
14 Haagensen, A.M., Klein, A.B., Ettrup, A., Matthews, L.R., Sørensen, D.B. 2013. Cognitive performance of <strong>Göttingen</strong> minipigs is affected<br />
by diet in a spatial hole-board discrimination test. PLoS One, 8:e79429.<br />
8
Obesity and diabetes result in pathological<br />
bone repair around dental implants:<br />
Demonstration in a <strong>Göttingen</strong> <strong>Minipigs</strong> models<br />
Rebecca Sandgren 1 & Benjamin E. Pippenger 2<br />
1<br />
Biomedical Center, Lunds University, Lund, Sweden<br />
2<br />
Institut Straumann, Preclinical & Translational Research, Basel, Switzerland<br />
Introduction<br />
The number of obese and diabetic patients in need of medical<br />
treatment, especially for the oral cavity, is growing steadily.<br />
Obesity (metabolic syndrome) and, by extension, type 2 diabetes<br />
mellitus are known risk factors for oral diseases including<br />
periodontal disease [1] , alveolar bone loss [2] and gum disease [3] ,<br />
with adipose-derived pro-inflammatory cytokines thought to<br />
be the common underlying basis for the degeneration in oral<br />
health [4] . Not only do obesity and diabetes often result in oral<br />
health deterioration, but these same systemic diseases can also<br />
be considered contraindications for dental implantation procedures<br />
[5] , having been shown to result in a higher incidence of<br />
implant failure [6] . While the treatment of obesity and diabetes<br />
aims to avoid the development of oral health complications, the<br />
risk of severe periodontal disease remains 3-4 times higher in<br />
these patients [7] . Therefore, novel dental materials and/or material<br />
surface treatments are needed that can reliably perform in<br />
compromised patients, but their development is hindered by a<br />
lack of clinically relevant obese/diabetic animal models.<br />
<strong>Göttingen</strong> Minipig models have long been used in translational<br />
research, surgical models, and procedural training (particularly<br />
in the dental field). Their specificity for experimental studies has<br />
excluded them from low-fat producing, selective breeding programs<br />
and their normal adult weight remains within a manageable<br />
range ( ~ 40 kg)[8] . Additionally, obese/metabolic syndrome<br />
<strong>Göttingen</strong> Minipig models have already been developed through<br />
a high energy feeding diet for periods up to 3 months [9,10] .<br />
While these <strong>Minipigs</strong> become extremely obese, their adult body<br />
weight rarely exceeds 80-90 kg, demonstrating that even in<br />
an obese condition, this animal model remains manageable for<br />
the experimentator. A type 2-like diabetes can also be induced<br />
in <strong>Göttingen</strong> <strong>Minipigs</strong> by administration of Streptozotocin, a<br />
chemical agent which damages the insulin-producing beta-cells<br />
of the pancreas [11] , demonstrating the potential to model a com-<br />
Figure 1: Physical and critical organ<br />
weight gain upon obesity induction<br />
followed by stabilization following<br />
diet control. A) Animal weight profiles<br />
throughout the duration of the experiment<br />
(47 weeks) demonstrating rapid<br />
weight gain for cafeteria diet animals<br />
(labeled obese and diabetic) in the first<br />
phase followed by weight stabilization<br />
in the remainder of the experiment.<br />
STZ: beginning of Streptozotocin<br />
administration. B) Heart (left and right<br />
ventricles weighed separately) and<br />
lung weights at 47 weeks (sacrifice)<br />
demonstrating higher average weights<br />
for critically affected organs in obese<br />
and diabetic animals. C) Liver, spleen<br />
and kidney wet weights at 47 weeks<br />
(time of sacrifice).<br />
<br />
9
promised patient condition using an obese/diabetic <strong>Göttingen</strong><br />
Minipig. However, while stable metabolic syndrome/diabetes<br />
animal models exist in the dog [12] , no stable pathological changes<br />
have yet been reported for the <strong>Göttingen</strong> Minipig. Finally, it is<br />
unknown what consequence/s such an optimized model would<br />
have on dental implant materials.<br />
In the present pilot study, we aimed to: 1) demonstrate the proof<br />
of principal of the induction of both stable metabolic syndrome<br />
and diabetes in a <strong>Göttingen</strong> Minipig animal model and 2) validate<br />
both of these disease-optimized <strong>Göttingen</strong> Minipig models<br />
for the testing of dental materials. Evidence of stable metabolic<br />
syndrome and type 2 diabetes induction is demonstrated<br />
through blood analysis, tissue histology and weight progression<br />
monitoring. We then implanted bone level dental implants into<br />
the mandibles of these compromised animal models to determine<br />
whether the induction of one or both systemic diseases<br />
affects the osseointegration and short term performance of the<br />
implant material.<br />
Materials and Methods<br />
Establishment of obese & diabetic Minipig models<br />
All experimentation was conducted in the Magneten building<br />
for Surgical Research, Lund University, Lund-Malmö, Sweden<br />
(ethical approval number M-206-11 Malmö-Lunds djurförsöks<br />
etiska nämnd). A total of nine female <strong>Göttingen</strong> <strong>Minipigs</strong><br />
(<strong>Ellegaard</strong>, Dalmose, Denmark) of 18 months of age (33-38 kg<br />
in weight) were used for this study. <strong>Minipigs</strong> were split into 3<br />
groups: 1) control (normal diet) (n = 3), 2) obese (cafeteria diet)<br />
(n = 3) and 3) diabetic (cafeteria diet + Streptozotocin) (n = 3).<br />
The animals were fed twice a day with either a combination<br />
of Minipig Expanded Standard Diet (SDS Special Diets Services,<br />
UK) and a custom made RDS Control Diet (control group) or RDS<br />
Cafeteria Diet (Research Diet Services , NL) (obese and diabetic<br />
groups), as previously described [13] . For the Minipig diets, we<br />
split the timing of the different diets into 3 phases: conversion,<br />
growing and maintenance phases. To induce obesity, <strong>Minipigs</strong><br />
(n = 6; groups 2 and 3) were gradually introduced to the cafete-<br />
Figure 2: Blood and plasma marker profiles demonstrate effective induction of metabolic syndrome and a diabetic phenotype. A) Blood glucose levels following STZ<br />
administration. Glucose levels are particularly elevated in diabetic animals but obese animal levels are comparable to control. B) Blood ketone levels are elevated in<br />
the diabetic group. Obese and control groups remain at basal levels. C-F) Average plasma levels of Tumor necrosis factor-α (TNF-α), C-reactive protein (CRP), Cortisol<br />
and Insulin taken at the time of implantation and termination. Plasma levels of all proteins demonstrated the same overall trend in that the obese group had the<br />
highest levels of all the groups (except for TNF-α-highest in the diabetic group). * = p ≤ 0.05. G) Insulin staining and H) quantification after 47 weeks. Data represent<br />
means ± SD.<br />
10
ia diet over a period of 4 weeks (25% decrease in normal diet<br />
on a weekly basis and restricted feeding to 2 x 500 g/day;<br />
conversion phase), after which time they remained at 100%<br />
cafeteria diet for 8 months and were allowed to feed ad libitum<br />
(growth phase). Once obese and diabetic group animals reached<br />
the desired body weight (roughly doubling in weight), the cafeteria<br />
diet was then halved with control diet to maintain the<br />
animals at the desired weight (maintenance phase). Control animals<br />
were fed standard diet and water throughout the duration<br />
of the experiment. A type 2 diabetic state in the pigs was then<br />
induced by slow injection (over a period of 1 minute; through<br />
the ear vein catheter) of filter-sterilized β-cell cytotoxin Streptozotocin<br />
solution (STZ, Enzo Life Sciences, Raamsdonksveer,<br />
the Netherlands) (20 mg/kg in 0.1 mol/l Na-citrate, pH 4.5) on<br />
two consecutive days after overnight fasting, as previously<br />
described [13] . STZ-injected swine were given free access to food<br />
during the day time and after the second STZ injection, during<br />
day and night. At the end of each of the first 2 days of STZ<br />
treatment, 25 g glucose was fed to offset insulin release from<br />
β-cells, thereby preventing hypoglycemia.<br />
other groups (Figure 1B). Liver and kidney weights increased<br />
from control to diabetic, with obese group weights being intermediate.<br />
However, the liver seemed to be the most effected<br />
of the two organs with a marked increase of weight the more<br />
compromised the animal became. Spleens decreased in weight<br />
with the severity of the compromised condition (Figure 1C).<br />
Blood analysis corroborated macroscopic evidence that obese<br />
animals were indeed suffering from metabolic syndrome<br />
with evidence of inflammation and stress, and that STZ induced<br />
animals were suffering a mild form of type 2 diabetes<br />
(Figure 2A-H).<br />
Compromised animals demonstrate decreased bone healing<br />
around implants<br />
Experimental bone chamber and removal torque implants were<br />
placed into the 3 animal model groups and animals were allowed<br />
to heal for 12 weeks. Maximum torque-out is commonly used<br />
as an indirect measure of the osseointegration around a dental<br />
Surgical procedure<br />
All surgeries were performed as previously described [14]. Briefly,<br />
teeth (P1, P2, P3 and M1) were extracted bilaterally from the<br />
mandibles of 6 <strong>Minipigs</strong>. Custom designed 4.2 x 6 mm implants<br />
(bone chamber and removal torque experimental implants,<br />
Titanium-SLA, Straumann, Switzerland) were placed bilaterally<br />
in transverse orientation and the soft tissue was closed.<br />
Implant and tissue analytic procedures<br />
Removal torque and histology<br />
Immediately after sacrifice, mandibles were collected for either<br />
removal torque measurements or histological analysis. Mandibles<br />
were excised and the left and right halves separated. Right<br />
hemi-mandible implants (side with Torque-out implants) were<br />
then subjected to biomechanical removal torque measurements<br />
according to a previously established method [16] . Oral hard and<br />
soft tissue for histological slide preparation was performed<br />
and histomorphometric measurements were calculated (Bone<br />
area to toal area – BATA and bone to implant contact – BIC), as<br />
previously described [15] .<br />
Results<br />
Induction of stable metabolic syndrome<br />
and mild type 2 diabetes<br />
Animal weights from both the cafeteria diet groups (obese and<br />
diabetic) continued to climb beyond that of the control group,<br />
which plateaued at 45 kilograms at 14 weeks after the beginning<br />
of the study and remaining at this weight for the duration<br />
of the study. By week 27 (STZ administration for diabetic group),<br />
obese and diabetic groups had almost tripled in weight, having<br />
gained an average of 37 kg and 44 kg, respectively (both groups<br />
completely tripled in weight by week 35). Diluting the cafeteria<br />
diet 50% with control diet combined with STZ administration had<br />
the overall effect of weight stabilization on the diabetic group<br />
(Figure 1A). Organ wet weights demonstrated the obese group<br />
having heavier left ventricle and right ventricle wet weights<br />
than both the control and diabetic groups (Figure 1B). Lung<br />
wet weights for the diabetic group were higher than for both<br />
Figure 3: Biomechanical and histomorphometric measurements. A) Maximum<br />
torque-out values for implants at time of sacrifice. Lower torque-out vales<br />
demonstrate that the osseointegration of implants in obese and diabetic<br />
animals is significantly less than in the control group. Furthermore, there are<br />
no significant differences between obese and diabetic groups. B) Histomorphometric<br />
analysis of tissue/implant sections. BATA corresponds to the new bone<br />
area per total area within a defined region of interest (ROI), here defined as the<br />
total area from the defect border to the implant surface. BIC corresponds to<br />
the total bone to implant contact and is expressed as a percentage of the bone<br />
physically attached to the implant surface as compared to the total implant<br />
surface. Histological evidence supports the biomechanical measurements and<br />
demonstrates the significantly less new bone formation around dental implants<br />
is obese and diabetic groups as compared to the control group. * = p ≤ 0.05; **<br />
= p ≤ 0.01; *** = p ≤ 0.001; ns = not significant. <br />
11
implant, with higher values demonstrating a higher degree<br />
of osseointegration. While the control group reached average<br />
levels of approximately 260 Ncm, both metabolic syndrome and<br />
diabetic groups had much lower average values (90 Ncm and 60<br />
Ncm, respectively). Importantly, not only did the compromised<br />
groups measure removal torque values that were significantly<br />
less than the control group, but the metabolic syndrome group<br />
values were not statistically different from those of the diabetic<br />
group (Figure 3A).<br />
Histomorphometric analysis on bone chamber implants confirmed<br />
the trend seen in the biomechanical removal torque<br />
experiment. Two different histomorphometric parameters were<br />
measured: bone area to implant area (BATA) and bone to implant<br />
contact (BIC). The control group, for both BATA and BIC<br />
measurements, had significantly higher values than both of the<br />
compromised groups (BATA- control: 40%, obese: 25%, diabetic:<br />
25%; BIC- control: 45%, obese: 22%, diabetic: 18%). Importantly,<br />
there was no significant difference between the compromised<br />
groups for both BATA and BIC measurements (Figure 3B). These<br />
measurements were also evident by visual examination of the<br />
histological slides, especially in terms of BIC, in that the new<br />
bone growth around the implant is less than compared to both<br />
compromised groups (Figure 4).<br />
Conclusion<br />
This study, using an animal model considered physiologically<br />
similar to that of humans, demonstrates that bone remodeling<br />
is indeed severely affected in obese individuals. Not only was<br />
the bone regeneration around implants placed into bone defects<br />
less pronounced in obese animals as compared to control, but<br />
the biomechanical stability of the newly formed bone seems<br />
to be less mature, as demonstrated by mechanical torque out<br />
measurements. In line with Doucette et al., the inflammatory<br />
factor TNF-α was also found to be only slightly affected by the<br />
induction of an obese phenotype. However, previous studies<br />
have shown that an alternative inflammatory factor (C-reactive<br />
protein (CRP)) is particularly increased in obese humans and<br />
suggests a source of infection or inflammation is more common<br />
among obese subjects than in nonobese subjects [16] . This study<br />
also measured CRP levels and found significantly higher levels<br />
of CRP in obese animals, suggesting Minipig models do indeed<br />
mirror the pathological events associated with metabolic syndrome<br />
found in humans.<br />
We demonstrate that after induction of obesity, the animal<br />
weight can indeed be stabilized without reversing the disease<br />
process itself. Interestingly, this suggests that <strong>Minipigs</strong> manifest<br />
a similar disease process to humans in that the deleterious<br />
clinical effects of obesity in human subjects are reversible upon<br />
concerted weight loss, not weight stabilization [17] .<br />
Biomechanical analysis of implant osseointegration (torqueout)<br />
showed significant differences between the control and<br />
compromised groups. This further demonstrated that implant<br />
osseointegration, and therefore secondary stability, is greatly<br />
affected by the animals’ compromised state. Based on the<br />
present data, it appears that implant osseointegration is equally<br />
compromised in obese and diabetic animals. This corresponds to<br />
recent work in humans demonstrating that bone mineral density<br />
is already reduced in metabolic syndrome adolescents [18] .<br />
Here, we demonstrate it is possible to induce a mild state of<br />
diabetes (with detectable glucose metabolism deficiency) using<br />
a dose (twice 20 mg/kg STZ) previously reported to have no metabolic<br />
effect of <strong>Göttingen</strong> <strong>Minipigs</strong>. However, we first induced<br />
metabolic syndrome in the animals before STZ administration,<br />
contrary to previous studies that administered STZ on healthy<br />
Figure 4: Histological sections demonstrating the differences between the study groups in terms of new bone formation surrounding implants. Top row = Hematoxylin<br />
and eosin stained sections; bottom row = Region of interest defined for BATA calculations. New bone is clearly distinguished from existing bone by color (new bone<br />
= slightly darker pink) and morphology (new bone = higher percentage of trabeculae; less mature).<br />
12
individuals. It appears that healthy individuals are resistant to<br />
low doses of STZ, whereas metabolic syndrome animals are not,<br />
further highlighting that a compromised state is present already<br />
in obese animals. While this remains a pilot study, these data<br />
also suggest that the aberrant bone remodeling previously<br />
reported in type 2 diabetic individuals could have its origins in<br />
obesity rather than diabetes. In line with this, the aberrant bone<br />
remodeling may be related to the pro-inflammatory status present<br />
during metabolic diseases, with severe pro-inflammation<br />
at diabetes and more mild pro-inflammation at pre-diabetes or<br />
metabolic syndrome. In the present study, the minipigs were fed<br />
a cafeteria diet containing substantial amounts of hydrogenated<br />
oils, consisting of trans-fatty acids which are known to induce<br />
chronic inflammation [19] . It may well be that the aberrant bone<br />
remodeling in obese pigs is amplified by using dietary trans fatty<br />
acids, thereby creating a more severe state of pro-inflammation<br />
at obesity. The increase of inflammatory factors also shown to<br />
be increased in human obese patients shows that the systematic<br />
inflammation present in this animal model more closely resembles<br />
that of the human disease state [20] .<br />
References<br />
1 Suresh S, Mahendra J. Multifactorial relationship of obesity and periodontal disease. J Clin Diagn Res 2014;8:ZE01–3.<br />
2 Alabdulkarim M, Bissada N, Al-Zahrani M, Ficara A, Siegel B. Alveolar bone loss in obese subjects. J Int Acad Periodontol 2005;7:34–8.<br />
3 Engebretson S, Chertog R, Nichols A, Hey-Hadavi J, Celenti R, Grbic J. Plasma levels of tumour necrosis factor-alpha in patients with<br />
chronic periodontitis and type 2 diabetes. J Clin Periodontol 2007;34:18–24.<br />
4 Di Benedetto A, Gigante I, Colucci S, Grano M. Periodontal disease: linking the primary inflammation to bone loss. Clin Dev Immunol<br />
2013;2013:503754.<br />
5 National Institutes of Health Consensus Development Conference statement on dental implants June 13-15, 1988. J Dent Educ<br />
1988;52:824–7.<br />
6 Marchand F, Raskin A, Dionnes-Hornes A, Barry T, Dubois N, Valéro R, et al. Dental implants and diabetes: conditions for success.<br />
Diabetes Metab 2012;38:14–9.<br />
7 Mellado-Valero A, Ferrer García JC, Herrera Ballester A, Labaig Rueda C. Effects of diabetes on the osseointegration of dental<br />
implants. Med Oral Patol Oral Cir Bucal 2007;12:E38–43.<br />
8 Bollen P, <strong>Ellegaard</strong> L. The <strong>Göttingen</strong> Minipig in pharmacology and toxicology. Pharmacol Toxicol 1997;80 Suppl 2:3–4.<br />
9 Johansen T, Hansen HS, Richelsen B, Malmlöf R. The obese <strong>Göttingen</strong> Minipig as a model of the metabolic syndrome: dietary effects<br />
on obesity, insulin sensitivity, and growth hormone profile. Comp Med 2001;51:150–5.<br />
10 Larsen MO, Rolin B, Wilken M, Carr RD, Svendsen O. High-fat high-energy feeding impairs fasting glucose and increases fasting<br />
insulin levels in the <strong>Göttingen</strong> Minipig: results from a pilot study. Ann N Y Acad Sci 2002;967:414–23.<br />
11 Larsen MO, Wilken M, Gotfredsen CF, Carr RD, Svendsen O, Rolin B. Mild streptozotocin diabetes in the <strong>Göttingen</strong> Minipig. A novel<br />
model of moderate insulin deficiency and diabetes. Am J Physiol Endocrinol Metab 2002;282:E1342–51.<br />
12 Ionut V, Liu H, Mooradian V, Castro AVB, Kabir M, Stefanovski D, et al. Novel canine models of obese prediabetes and mild type 2<br />
diabetes. Am J Physiol Endocrinol Metab 2010;298:E38–48.<br />
13 Te Pas MFW, Koopmans S-J, Kruijt L, Calus MPL, Smits MA. Plasma proteome profiles associated with diet-induced metabolic<br />
syndrome and the early onset of metabolic syndrome in a pig model.<br />
14 Friedmann A, Friedmann A, Grize L, Obrecht M, Dard M. Convergent methods assessing bone growth in an experimental model at<br />
dental implants in the minipig. Ann Anat 2014.<br />
15 Gottlow J, Dard M, Kjellson F, Obrecht M, Sennerby L. Evaluation of a new titanium-zirconium dental implant: a biomechanical and<br />
histological comparative study in the mini pig. Clin Implant Dent Relat Res 2012;14:<strong>53</strong>8–45.<br />
16 Aronson D, Bartha P, Zinder O, Kerner A, Markiewicz W, Avizohar O, et al. Obesity is the major determinant of elevated C-reactive<br />
protein in subjects with the metabolic syndrome. Int J Obes Relat Metab Disord 2004;28:674–9.<br />
17 Shapses SA, Sukumar D. Bone metabolism in obesity and weight loss. Annu Rev Nutr 2012;32:287–309.<br />
18 Nóbrega da Silva V, Goldberg TBL, Mosca LN, Bisi Rizzo A da C, Teixeira A dos S, Corrente JE. Metabolic syndrome reduces bone<br />
mineral density in overweight adolescents. Bone 2014;66:1–7.<br />
19 Lee L, Alloosh M, Saxena R, Van Alstine W, Watkins BA, Klaunig JE, et al. Nutritional model of steatohepatitis and metabolic<br />
syndrome in the Ossabaw miniature swine. Hepatology 2009;50:56–67.<br />
20 Hotamisligil GS. Inflammation and metabolic disorders. Nature 2006;444:860–7.<br />
13
Using <strong>Göttingen</strong> <strong>Minipigs</strong> as model<br />
for peritoneal dialysis<br />
Anette Blak Gross & Andrew Makin, Citoxlab Denmark, Ejby, Denmark<br />
Peritoneal dialysis is a commonly used treatment in human<br />
patients with severe renal and other diseases. Extended use<br />
of peritoneal dialysis including new peritoneal dialysis formulations<br />
in the pharmaceutical industry requires appropriate<br />
animal models in order to meet the regulatory requirements for<br />
approval of products.<br />
Selection of the correct or most appropriate model depends<br />
on similarities between animals and humans in terms of both<br />
physiology and anatomy. To a degree the relative size of the<br />
animal model can be important. Similar transport properties of<br />
solute and water across the peritoneal membrane in humans<br />
and animals have been important factors in choosing the right<br />
animal model [1] .<br />
Rats, rabbits, and genetically modified mice have been used as<br />
experimental models, but also larger animals such as pigs dogs,<br />
sheep, or even kangaroos have been used [1,2,3] .<br />
The use of each animal model offers advantages and disadvantages<br />
[1] . Rodents are cheap and readily available, but the small<br />
size also complicates catheter insertion and increases the risk<br />
of complications. In addition, the size of the parietal peritoneum<br />
and ratio of peritoneal surface area is different from that of<br />
humans and larger animals. Pigs and other large animal models<br />
also offer the advantage that large volumes of dialysate solution<br />
and human size catheters may be used.<br />
Pigs are an attractive model for peritoneal dialysis due to<br />
similarities in the anatomy and physiology of several of the<br />
abdominal organs and not least the similar size to humans.<br />
In addition, pigs are widely used as a model for many other<br />
procedures involving abdominal tissues and organs (for instance<br />
renal transplantation, intrarenal surgery, artificial bladders and<br />
ureteral stents.<br />
At Citoxlab we have developed an excellent model for peritoneal<br />
dialysis products in the <strong>Göttingen</strong> Minipig.<br />
The minipigs we have used have been around 4 to 5 months old<br />
and with a weight of ca 10 kg at study initiation. Under general<br />
anaesthesia intraperitoneal catheters are placed and tunnelled<br />
subcutaneously to the neck skin. Dosing is performed once<br />
daily for 10-20 minutes using injection pumps while the animals<br />
are walking freely in their pen unaffected by the dosing as the<br />
catheters are sufficiently long to ensure normal behaviour of<br />
the minipigs.<br />
After some time, typically a few hours, the dialysate (infused<br />
formulation included) can be collected by drainage of the<br />
catheters in conscious animals (typically 75%-100% of infused<br />
formulation can be retrieved). Drainage is preferably achieved<br />
by gravity, and suction only used if necessary. Suitable toxicokinetic/clinical<br />
chemistry/hematology parameters can be<br />
measured in the dialysate after retrieval.<br />
Formulations for intraperitoneal dosing should obviously be<br />
sterile, isotonic, non-irritating and heated to body temperature<br />
before use.<br />
Surgery and handling of catheters are only performed by<br />
dedicated, trained personnel using aseptic procedures at all<br />
time. Care should be taken to ensure that the placement of<br />
the catheter does not compromise the organs and tissues in the<br />
abdomen; irritation could lead to inflammation and peritonitis.<br />
To assist in this, the catheter end is curled, and placement is as<br />
far away from the omentum as possible.<br />
Using these methods, we have successfully achieved dosing<br />
daily for up to 10 days.<br />
When performed correctly, intraperitoneal catheters are very<br />
well tolerated and the <strong>Göttingen</strong> Minipig is considered a suitable<br />
model for peritoneal dialysis products.<br />
References<br />
1. Animal models in peritoneal dialysis, Olga Nikitidou,Vasiliki<br />
I. Peppa,Konstantinos Leivaditis, Theodoros Eleftheriadis,Sotirios,<br />
G. Zarogiannis, and Vassilios Liakopoulos, Front Physiol.<br />
2015; 6: 244.<br />
2. Swine as Models in Biomedical Research and Toxicology<br />
Testing, M. M Swindle, A. Makin, A. J. Herron, Veterinary<br />
Pathology, 2012: 49 (2): 344.<br />
3. Pawlaczyk K., Baum E., Schwermer K., Hoppe K., Lindholm B.,<br />
Breborowicz A. (2015). Animal models of peritoneal dialysis:<br />
thirty years of our own experience. Biomed. Res. Int. 2015:<br />
Article ID 261813.<br />
14
Vascular Access Buttons in <strong>Göttingen</strong> <strong>Minipigs</strong><br />
Adrian Zeltner, <strong>Ellegaard</strong> <strong>Göttingen</strong> <strong>Minipigs</strong>, Dalmose, Denmark<br />
Infusion and serial blood sampling are often important technical<br />
aspects of an experimental design. Superficial vessels in the<br />
minipig are few and frequently accessing them is a challenge.<br />
Although minipigs have a convenient size for handling, restraint<br />
and venipuncture can be stressful and affect blood parameters.<br />
Therefore, when experiments require infusion or frequent blood<br />
sampling, catheterization is often the best option, both ethically<br />
and scientifically.<br />
The implantation of Vascular Access ports and Seldinger Catheters<br />
is described and published in various variations. <strong>Ellegaard</strong><br />
already supplies <strong>Minipigs</strong> with pre-implanted systems of that<br />
type. To add another option when choosing the optimal study<br />
design, we tested the Rat Vascular Access Button (VAB) in<br />
<strong>Göttingen</strong> <strong>Minipigs</strong>. The button allows up to three catheters to<br />
be connected and opens the possibility to sample and dose via<br />
separate catheters in one device. In contrast to a VAP this device<br />
allows for pain free access as it is percutaneously implanted.<br />
The device is designed for rats, but we wanted to find out if such<br />
a button can be used in a minipig and whether a decent patency<br />
period can be achieved. Furthermore, it was of interest whether<br />
group housing is an option.<br />
As with all catheters, clotting, biofilm and fibrin build up are<br />
the main factors affecting patency. Therefore, we also tested<br />
different vessels and different types of catheters in the pilot<br />
study to see if there is a significant difference.<br />
Pilot study<br />
Four <strong>Minipigs</strong> at around 15 kg were implanted with a three port<br />
VAB. Each Minipig had three catheters implanted:<br />
1. In the left carotid artery; two <strong>Minipigs</strong> in cranial direction and<br />
in two in caudal direction. A 3fr PU catheter, with a bead at 3<br />
cm was inserted to that length and fixed with a purse string<br />
suture and tissue glue.<br />
2. In the left internal Jugular vein. This vessel runs alongside<br />
the carotid artery. It was ligated and a 3fr PU catheter, with<br />
two lateral perfusion holes, at 3 cm and 2 mm apart was<br />
inserted to a length of 7-8 cm, so the tip was in the vena<br />
cava.<br />
3. In the right internal jugular vein. A 3fr PU catheter, was<br />
inserted in the same manner as in the other vein.<br />
After implantation the <strong>Minipigs</strong> were left to recover for a week<br />
after which regular patency testing started.<br />
The VABs were accessed once a week for two months and any<br />
difficulties were recorded. In week 2 typical catheter related<br />
incidences were observed with the arterial and perfusion holes<br />
catheters. Some were blocked early, others could be revived by<br />
rinsing.<br />
The standard catheters (3) performed best, with only tree rinsing<br />
incidents in two pigs after week 6. All were still patent in week 8<br />
when the study was concluded.<br />
Materials<br />
The Vascular Access Button is small device with one, two or<br />
three ports in the same button. It is available in one size only,<br />
catheters have a size of 3fr but are available in different designs<br />
and lengths. Here, we used catheters with rounded tips, beads<br />
and in some cases added prefusion holes just proximal of the<br />
tip. The ports have a small septum which can be penetrated with<br />
an adapter that fits to any syringe with luer. The smallest inside<br />
diameter of the system is 27 g and is in the adapter.<br />
The ports and the part of the button that is exposed is protected<br />
by an aluminum cap that is fixed with a magnet.<br />
All parts are available from Instech Laboratories, Inc. USA.<br />
Group housing study<br />
In the pilot study we had some issues in the group after recovery.<br />
One of the <strong>Minipigs</strong> was not as social anymore after<br />
recovery and was bothering the others. At the time we decided<br />
to single house them, so the study did not get compromised.<br />
As we did not want to give up altogether in trying to group<br />
house <strong>Minipigs</strong> with VABs we designed a small study where we<br />
could test the group housing situation. We hypothesized that<br />
the pen mates will go after the VAB because it is sticking out<br />
and has a strong color. The idea was to camouflage the bright<br />
red cap of the button and/or add some bitter taste to it, so it is<br />
less attractive.<br />
Rat Vascular Access Button with three ports ready to be implanted.<br />
<br />
15
Four male <strong>Minipigs</strong> of around 20 kg, that were group housed<br />
previously, were implanted with single port Buttons, without the<br />
catheter. Some of the caps were painted in a color closer to pig<br />
skin, others were covered with bitter nail polish. After they were<br />
all fully recovered we grouped them again, observed and filmed<br />
the activity.<br />
There was certainly a lot of activity, typical male pig behavior<br />
and it carried on for several hours before they calmed down a<br />
bit. We did not observe any dedicated attacks on the VAB, nor<br />
any interest in them. Some of the caps did fall off during the<br />
observation period, but after analyzing the videos it was clear<br />
that it was due to random movements or normal interaction<br />
between the animals. We also realized that in some occasions<br />
the cap flew off and we concluded it was because of the two<br />
magnets in the button with different polarization. When the cap<br />
is twisted far enough the cap is repelled by the magnet of the<br />
same polarization.<br />
To avoid this situation, we modified the single port buttons to<br />
avoid the twisting of the cap for the main study and decided to<br />
go ahead and group house the <strong>Minipigs</strong>.<br />
There seemed to be no real difference between the original and<br />
the “camouflaged” caps, therefore we decided to use them in the<br />
original form.<br />
Button implanted behind the ear<br />
Main study<br />
Apart from a larger number we also wanted to test if this system<br />
could be used in a toxicology setting and what the chances are<br />
to have it patent throughout a 13 week period. A further aim<br />
was to evaluate if it is an advantage to have two catheters in<br />
respect to obtaining a longer patency. An obvious advantage<br />
of having two catheters is that one can be used for dosing, the<br />
other for sampling.<br />
We chose 8 kg minipigs and planned to run the study for 3<br />
months. Four groups of four <strong>Minipigs</strong>:<br />
Male, one catheter in left external jugular vein.<br />
Female, one catheter in left external jugular vein.<br />
Male, two catheters in left external jugular vein, tip 2 cm apart.<br />
Female, two catheters in left external jugular vein, tip 2 cm<br />
apart.<br />
Group housing, testing of VAB once a week commencing one<br />
week after surgery. Necropsy with macroscopic pathology at the<br />
end of study or after complete loss of patency.<br />
Surgery<br />
Behind the ear, where the skin is thinnest, a dorsal-ventral skin<br />
incision of around 4 cm is made and a pocket is formed by blunt<br />
dissection so the lower part the button can be placed in it. Then,<br />
after an incision in the jugular grove the external jugular vein<br />
is exposed by blunt dissection, ligated cranially and one or two<br />
catheters inserted to a length where the tip is placed in the<br />
cranial vena cava, just cranially of the right atrium. If there are<br />
two catheters, this position is for the longer one, the other is<br />
2-3 cm more cranial. The catheters are tied to the vessel and<br />
tunneled to the pocket behind the ear. Now they can be cut to<br />
length and connected to the VAB. After testing functionality, the<br />
button is placed in the pocket and the skin fitted into the collar<br />
of the button. The incision is closed tightly and after making<br />
sure the catheters are not kinked the jugular site is closed in<br />
three layers. After final testing and locking the cap is placed on<br />
the button and the Minipig left to recover.<br />
Taking a blood sample<br />
Testing<br />
Sampling, saline and lock solution syringes have been prepared<br />
and fitted with the adapter in an aseptic manner. The <strong>Minipigs</strong><br />
placed in the sling, the cap removed, and the ports disinfected<br />
with alcohol.<br />
The sampling syringe is pushed in the port and the plunger<br />
pulled back. If blood was flowing, around 1 ml was drawn, the<br />
16
catheter flushed immediately with saline, and locked with 0.4 ml<br />
Taurolock under positive pressure.<br />
If it was not possible to draw blood in the first attempt the<br />
Minipig was repositioned slightly and retested. I that did not<br />
help it was flushed with saline and then retested again.<br />
Locking under positive pressure with this device describes the<br />
procedure where the adapter is slowly redrawn from the septum<br />
of the port while continuously pushing the plunger of the<br />
syringe. This ensures that the pressure in the system is always<br />
larger than the blood pressure and no blood enters the lumen<br />
of the catheter. All rinsing and locking should be done like that.<br />
Results<br />
Group Housing<br />
Soon after the groups were established in their pens, the caps<br />
started falling off randomly. The modification of the VAB was<br />
not enough to prevent it. We removed some of the most prone<br />
animals from the group and single housed them as we were<br />
concerned about cleanness and systemic infections if the caps<br />
were off. At the same time, we talked to the manufacturer and<br />
ordered caps with two magnets to have stronger adhesion and<br />
avoid twisting. Even with two magnets we were not completely<br />
successful and added a screw to the cap which did not solve the<br />
problem entirely either. At the end we resigned and left the caps<br />
off permanently. This was not quite as hygienic as we wished,<br />
but it did not seem to be an issue at the end. We had one pair<br />
in each group throughout the study and whether there was a<br />
cap or not the <strong>Minipigs</strong> did not interfere with each other’s VAB.<br />
We have no explanation why in the pilot study the caps stayed<br />
on for the entire two months but fell off in the main study.<br />
Biological Compatibility and Patency<br />
A total of 24 <strong>Göttingen</strong> <strong>Minipigs</strong> were implanted with Buttons.<br />
No Complications were observed in the first two studies. The<br />
incisions in the neck and at the site of the button healed well<br />
and no signs of infection was observed. The Dracon collar of the<br />
button was grown in the subcutaneous tissue to seal the exit site<br />
completely. In the main study some infections around the button<br />
appeared in 6 cases (25%) four weeks post-surgery. In four of<br />
them recovered after treatment with antibiotics but two animals<br />
(8%) were euthanized, as infection did not clear and the button<br />
was rejected by the tissue.<br />
No systemic infections were observed<br />
Blood sampling performed by one person with Minipig in a sling<br />
Accessing the ports was easy and painless and with the Minipig<br />
in a hammock it could be performed by one person only.<br />
In the main Study there were a total of 24 catheters in 16 pigs.<br />
All catheters worked fine initially, but some typical catheter<br />
related issues appeared along the way<br />
Patency main study:<br />
After one month: 23/24 patent 1 never worked properly<br />
After two months: 16/24 patent 1 never worked properly,<br />
second blocked,<br />
2 infected VAB,<br />
4 in 2 animals blocked<br />
After three months: 10/24 patent as above and rest blocked.<br />
Conclusion and discussion<br />
The amount of infection in the main study, relatively late after<br />
surgery, might be because of a small change in procedure. In<br />
some cases, the Dracon cuff was placed in the subcutaneous fat<br />
rather than below the fat layer. That might have slowed down<br />
the ingrowing process and made it more prone to infections.<br />
It can be concluded that Rat Vascular Access ButtonsTM can<br />
successfully be implanted in <strong>Minipigs</strong> and provide long term<br />
vascular access.<br />
Because of the small diameters in the system flow speed is reduced<br />
to a certain degree compared with larger bore catheters.<br />
Typical catheter related issues are to be expected like: temporary<br />
blockage, rinsing before patency is restored and fibrin build<br />
up around catheter. This is no different to other systems. Proper<br />
training and experience with catheter handling will give better<br />
results and it is possible that patency could be improved if the<br />
system was accessed and rinsed in a higher frequency. Testing<br />
this hypothesis was not a the goal of this study.<br />
The buttons can accommodate 1-3 catheters which gives to<br />
opportunity to infuse and sample trough the same unit without<br />
cross-contamination. It might be possible to use the VAB when<br />
cannulating other vessels, like portal vein or bile duct, but this<br />
has not been tested yet.<br />
<strong>Minipigs</strong> with pre-implanted VAB, VAP or Seldinger catheters<br />
are available from <strong>Ellegaard</strong> <strong>Göttingen</strong> <strong>Minipigs</strong> or you can learn<br />
to implant them by joining one of our courses in Denmark.<br />
Minipig in pen after surgery<br />
For further information please contact Adrian Zeltner:<br />
az@minipigs.dk<br />
17
Fifty Years with <strong>Göttingen</strong> <strong>Minipigs</strong>:<br />
Let’s Celebrate!<br />
During the 1960’s, the Georg-August-University in <strong>Göttingen</strong>,<br />
Germany, worked on the creation of a small pig model and in<br />
1969, they managed to establish the first barrier-bred population<br />
of <strong>Göttingen</strong> <strong>Minipigs</strong> at the University’s experimental farm<br />
in Relliehausen, Germany.<br />
In 1992, <strong>Ellegaard</strong> <strong>Göttingen</strong> <strong>Minipigs</strong> entered into a licensing<br />
agreement with the University, giving the exclusive world-wide<br />
right to breed and sell <strong>Göttingen</strong> <strong>Minipigs</strong> to <strong>Ellegaard</strong> <strong>Göttingen</strong><br />
<strong>Minipigs</strong>, and the first colony of barrier-bred, microbiologically<br />
and genetically defined <strong>Göttingen</strong> <strong>Minipigs</strong> was established<br />
through Caesarean section at <strong>Ellegaard</strong> <strong>Göttingen</strong> <strong>Minipigs</strong> in<br />
Denmark. Since then, <strong>Ellegaard</strong> <strong>Göttingen</strong> <strong>Minipigs</strong> has extended<br />
its operations and sublicensed the breeding of <strong>Göttingen</strong> <strong>Minipigs</strong><br />
to Marshall BioResources, the US, and OYC, Japan.<br />
During the coming year we will host several scientific events<br />
as a roadshow around the world to celebrate Fifty Years with<br />
<strong>Göttingen</strong> <strong>Minipigs</strong>.<br />
The roadshow events will take place at several locations in<br />
Europe, as well as a number of locations in the US and Asia,<br />
respectively, together with our global partners. The events<br />
will feature speakers and topics for discussion of interest<br />
to researchers and users of <strong>Göttingen</strong> <strong>Minipigs</strong>, including an<br />
outlook on future needs and the further development of<br />
<strong>Göttingen</strong> <strong>Minipigs</strong>.<br />
Roadshow events being planned<br />
The roadshow events are currently in the planning phase, and<br />
invitations with venue, dates and final program will be published<br />
at our website and LinkedIn during 2019.<br />
We look forward to welcoming you to join us celebrating the<br />
first Fifty Years with <strong>Göttingen</strong> <strong>Minipigs</strong>!<br />
THE BIRTH OF THE FIRST COLONY OF GÖTTINGEN MINIPIGS:<br />
P. Glodek und B. Oldigs, 1981, Das Göttinger Miniaturschwein, p. 12:<br />
1.3. Züchterische Entwicklung nach der Schnittentbindung in Relliehausen<br />
1.3.1. Gewichtsentwicklung<br />
Der Erfolg der Selektion auf geringeres Wachstum und adultes Körpergewicht ist in<br />
starkem Maße vom Gesundheitszustand in den Zuchtpopulationen abhängig, da genetische<br />
Fortschritte nicht mit umweltbedingten Kümmerern erzielt werden können. Die unzureichenden<br />
Stallverhältnisse in der Friedländer Anlage führten mit sinkendem Gewicht der Tiere zu<br />
erhöhten Kümmererfrequenzen, so daß beschlossen wurde, das gesamte Zuchtprogramm<br />
über Hysterektomie und künstliche Aufzucht der schnittentbundenen Ferkel in eine neue<br />
geschlossene SPF-Anlage auf dem Versuchsgut Relliehausen zu überführen, die 1969 mit<br />
Unterstützung der Stiftung Volkswagenwerk errichtet werden konnte.<br />
Follow us on ! www.linkedin.com/company/2864308/<br />
Join <strong>Ellegaard</strong> <strong>Göttingen</strong> <strong>Minipigs</strong> at LinkedIn to stay updated on our scientific events and other exciting initiatives to celebrate<br />
Fifty Years with <strong>Göttingen</strong> <strong>Minipigs</strong><br />
18
<strong>Ellegaard</strong> <strong>Göttingen</strong> <strong>Minipigs</strong> Going Global<br />
<strong>Göttingen</strong> <strong>Minipigs</strong> are now available in all major R&D markets, including China and India!<br />
At <strong>Ellegaard</strong> <strong>Göttingen</strong> <strong>Minipigs</strong>, we are truly pleased to be able<br />
now also to supply local pharmaceutical companies, contract<br />
research organizations and research institutions in both China<br />
and India with <strong>Göttingen</strong> <strong>Minipigs</strong> for their biomedical research<br />
and studies. Thus, we can comply with the great interest and<br />
demand for our high-quality minipig model which comes<br />
well-characterized and with lots of background data as well as<br />
has a very well-defined health and genetic background.<br />
Please contact us via ellegaard@minipigs.dk if you are interested<br />
in learning more about our supply of <strong>Göttingen</strong> <strong>Minipigs</strong> to<br />
China and India. Our expert colleagues are also always ready to<br />
support you scientifically and/or technically.<br />
Head of Business Development, Peter Vestbjerg and CEO, Lars<br />
Friis Mikkelsen attended the Society of Toxicology Pathology-India<br />
(SPS-I) conference in Hyderabad, India, in October 2018 to<br />
present and talk about <strong>Göttingen</strong> <strong>Minipigs</strong> to the Indian research<br />
community.<br />
Lars Friis Mikkelsen, CEO at the MoU signing ceremony at the Royal Danish<br />
Embassy in Beijing, China with Li Genping, Beijing Administration Office of<br />
Laboratory Animal (BAOLA), attended by the Danish Minister for Environment<br />
and Food, Jakob Ellemann-Jensen, and the Ambassador of Denmark to China,<br />
A. Carsten Damsgaard. Photo: Royal Danish Embassy Beijing, September 2018<br />
The participants of STP-I conference 26-28 October 2018, Hyderabad, India. Photo: Glenmark Pharmaceuticals Limited, November 2018<br />
Do you spot Peter and Lars in the picture...?<br />
<strong>Ellegaard</strong> <strong>Göttingen</strong> <strong>Minipigs</strong> Enters into<br />
a License and Commercialization Agreement for <strong>Göttingen</strong> <strong>Minipigs</strong><br />
Research Models in Partnership with Exemplar Genetics<br />
Late November 2018, <strong>Ellegaard</strong> <strong>Göttingen</strong> <strong>Minipigs</strong> A/S announced the signing of a license and commercialization agreement<br />
with Exemplar Genetics to develop and commercialize genetically modified <strong>Göttingen</strong> <strong>Minipigs</strong> models of human disease on<br />
a target-by-target basis.<br />
“Adding <strong>Göttingen</strong> Minipig genetics to our offerings will allow us to better serve our customer needs,” said John R. Swart,<br />
President of Exemplar Genetics. “We are very excited to partner with <strong>Ellegaard</strong> <strong>Göttingen</strong> <strong>Minipigs</strong> in providing models to the<br />
research community that we believe will translate well to the clinic.”<br />
Lars Friis Mikkelsen, CEO, added: “At <strong>Ellegaard</strong> <strong>Göttingen</strong> <strong>Minipigs</strong>, we are looking forward to working with Exemplar in providing<br />
genetically modified animal models based on our high-quality and well-defined <strong>Göttingen</strong> <strong>Minipigs</strong>. We, too, are very<br />
excited to launch this new partnership supporting the development and commercializing of transgenic <strong>Göttingen</strong> <strong>Minipigs</strong>.”<br />
Read the full press release here:<br />
https://minipigs.dk/fileadmin/filer/Info/EGM_-_Exemplar_licence_agreement_-_EGM_press_release_26.11.2018.pdf<br />
19
CREATING A STIMULATING AND COOPERATIVE ENVIRONMENT<br />
FOR GÖTTINGEN MINIPIGS<br />
Kirsten Rosenmay Jacobsen & Lars Friis Mikkelsen, <strong>Ellegaard</strong> <strong>Göttingen</strong> <strong>Minipigs</strong> A/S, Denmark<br />
INTRODUCTION<br />
When developing behavioral management and husbandry procedures for laboratory minipigs, it is important to recognize that minipigs are highly intelligent<br />
animals with a unique perspective about their environment. This intelligence might cause a challenge to the provision of a stimulating and appropriate environment.<br />
The poster gives examples on how to stimulate natural behaviors providing species specific and appropriate environmental enrichment. Daily socialization<br />
and positive human interaction is furthermore an important part of creating a stimulating and cooperative environment for minipigs.<br />
The recommendations described in the poster are based on years of in-house observation and experience from breeding, housing, and socializing several<br />
thousand <strong>Göttingen</strong> <strong>Minipigs</strong> in a barrier breeding environment.<br />
NATURAL BEHAVIOR<br />
ENVIRONMENTAL ENRICHMENT<br />
SOCIAL INTERACTION<br />
<strong>Göttingen</strong> <strong>Minipigs</strong> (GM) are curious and friendly animals. As conventional<br />
swine, GM possess a great explorative and investigative<br />
motivation, and they will spend much of their awake time rooting<br />
and exploring the surroundings. The active behavior is highly related<br />
to feeding and the time afterwards.<br />
Floor feeding facilitates the natural rotting process. Furthermore,<br />
floor feeding allows easy access to the diet and ability to feed at<br />
the same time.<br />
Natural products such as straw prompt and stimulate natural behaviors<br />
such as rooting, manipulation and exploration, and can be<br />
used in a variety of ways to maintain novelty and remaining interest.<br />
The use of straw hacks increase the time engaged in these behaviors.<br />
Furthermore long fibered hay placed in racks increases<br />
nesting behavior the day before farrowing for pregnant sows.<br />
GM are socialized from birth and intensively after weaning. However<br />
GM can be socialized at any time in life, with a combination<br />
of treats and positive experiences with humans.<br />
GM should be housed in social groups due to a strong social disposition.<br />
The hierarchy within the group is typically stable and agonistic<br />
behavior is mainly related to changes in group composition.<br />
The success of mixing new animals can be enhanced by appropriate<br />
enrichment, a neutral area, reducing the olfactory stimulation<br />
by alternative smells, the use of nanny-sows for young females<br />
etc.<br />
Homemade devices such as ice cubes with or without flavor are a<br />
good supplement to engage minipigs in short term active behaviors.<br />
The cubes must be adapted to the size of the animals e.g<br />
small ice cubes for weaning animals, and large for adult animals.<br />
Many types of commercial enrichment devices can be used to<br />
stimulate GM. Items that stimulate multiple behaviors are more<br />
likely to engage the minipigs for a longer time. E.g. toys that dispense<br />
food items. Currently we are testing Porcichew enrichment<br />
device (ERFS, UK) that besides manipulation and biting also stimulates<br />
olfaction, as it comes with various smells. To add a little extra<br />
comfort especially during shedding, we are currently also testing<br />
the Scratch-n-All pads (Scratchnall, USA).<br />
Furthermore, GM can easily be trained to engage in study or husbandry<br />
related procedures. They respond very well to the Positive<br />
Reinforcement training technique, and most food items can be<br />
used as rewards. However, new flavours might need a little habituation<br />
time. In general, GM have a strong preference of sucrose<br />
and they reject bitter things. Various aromas can be used to camouflage<br />
an unwanted taste e.g. a special diet or medicine for voluntary<br />
ingestion.<br />
GM will naturally divide their pen into three zones: a sleeping area,<br />
a eating/active area and a elimination area. Any of these areas<br />
need be modified to match the individual needs e.g. piglets and<br />
newly weaned pigs need additional heat and shelter in the sleeping<br />
area to avoid hypothermia and to give them a feeling of protection<br />
and cover. Sexually mature animals that are very active<br />
during heat benefit from pen dividers to give small hiding areas,<br />
and farrowing sows need a calm environment to reduce farrowing<br />
related complications.<br />
Dedicated play pens can be implemented to minipigs of any age to<br />
stimulate play and active behavior. Furthermore, these pens can<br />
be used during mixing of animals to reduce the amount of<br />
fighting.<br />
Locomotion and exploration can be stimulated simply be letting<br />
the pigs out of the pens. Everything will potentially be manipulated<br />
! A water hose, empty buckets, old rubber boots etc. will be<br />
used for play.<br />
The positive relationship with humans must be maintained by continuous<br />
positive interaction with humans. The minipigs are not as<br />
forgiving as dogs. The relationship starts at the arrival of the minipigs.<br />
During the acclimation period, the minipigs can be further<br />
socialized and adapted to the new environment—both from a microbiological<br />
and behavioral perspective. Habituation to new routines<br />
and potential training of procedures can start a few days<br />
after. Proper socialization, habituation and training results in calm<br />
animals and reduces stress—both for the animals and for the staff<br />
working with then. Both aspects are important in order to ensure<br />
optimal welfare of the animals and to gain valid study results<br />
when using GM in experimental research.<br />
For more information, contact Kirsten Rosenmay Jacobsen at krj@minipigs.dk<br />
20
19<br />
Invitation to join<br />
THE 13TH MINIPIG RESEARCH FORUM<br />
22-24 MAY 2019 IN VIENNA, AUSTRIA<br />
MAIN SCIENTIFIC TOPICS:<br />
• Toxicology<br />
• Animal training & welfare<br />
• Better understanding of the <strong>Göttingen</strong> Minipig<br />
• Transgenic models<br />
• Immune system<br />
WORKSHOPS:<br />
• Species selection in regulatory toxicology<br />
• Designing regulatory toxicology studies<br />
• Identifying disease model gaps<br />
The program also features poster presentations and time for networking with minipig users from all around<br />
the world. We accept posters now with technical (e.g. tips & tricks) and/or scientific (including data)<br />
content. View the poster guidelines at www.minipigresearchforum.org and send your poster as pdf to<br />
contact@minipigresearchforum.org.<br />
ONLINE CONFERENCE REGISTRATION OPENS MID-DECEMBER 2018: FEE € 350<br />
Including: 5 scientific sessions, 1 workshop of choice, get-together evening (Wednesday), social event with<br />
dinner (Thursday), lunches, coffee breaks and conference material.<br />
The full program with speakers will be ready during February/March 2019.<br />
START YOUR PLANNING NOW<br />
Duration: 22 May 2019 at 14:00 hrs. CEST to<br />
24 May 2019 at 13:00 hrs. CEST<br />
Venue: Novotel Wien Hauptbahnhof<br />
(15 min. from Vienna Airport by direct train)<br />
Accommodation is also available at Ibis Wien<br />
Hauptbahnhof (situated next to the venue hotel).<br />
Visit www.minipigresearchforum.org<br />
to get the booking form for rooms<br />
at special conference rates at Novotel/Ibis.<br />
Feedback from participants 2018:<br />
“<br />
My first MRF:<br />
Impressed<br />
how friendly<br />
and inclusive<br />
all members were<br />
”<br />
“<br />
Appreciated<br />
the diversity<br />
of topics and<br />
numerous opportunities<br />
to network<br />
”<br />
The MRF is a non-profit organization with more than 500 members worldwide<br />
working with minipigs in industry, academia and regulatory bodies.<br />
Participation in the annual MRF conference requires membership (free of charge).<br />
Read more and apply for membership at www.minipigresearchforum.org<br />
21
New scientific publications<br />
on <strong>Göttingen</strong> <strong>Minipigs</strong><br />
<strong>Ellegaard</strong> <strong>Göttingen</strong> <strong>Minipigs</strong> gives high priority to collaborative projects that aim to better characterize<br />
and validate <strong>Göttingen</strong> <strong>Minipigs</strong> as a translational animal model and which facilitate and refine the use of<br />
<strong>Göttingen</strong> <strong>Minipigs</strong> in research projects and safety testing. Please contact us if you have an idea for such<br />
a collaborative project. Below is a list of a few recent articles on <strong>Göttingen</strong> <strong>Minipigs</strong>.<br />
• Gauthier BE, Penard L, Bordier NF, Briffaux JJ, Ruty BM. Specificities of the Skin Morphology in<br />
Juvenile <strong>Minipigs</strong>. Toxicol Pathol. 2018 Oct 22. [Epub ahead of print]<br />
https://www.ncbi.nlm.nih.gov/pubmed/30348062<br />
• Carbonero F, Mayta-Apaza AC, Yu JZ, et al. A comparative analysis of gut microbiota disturbances in<br />
the Gottingen minipig and rhesus macaque models of acute radiation syndrome following bioequivalent<br />
radiation exposures. Radiat Environ Biophys. 2018 Nov;57(4):419-426.<br />
https://www.ncbi.nlm.nih.gov/pubmed/30343431<br />
• Christoffersen B, Straarup EM, Lykkegaard K, et al. FGF21 decreases food intake and body weight in<br />
obese <strong>Göttingen</strong> minipigs. Diabetes Obes Metab. 2018 Oct 17. [Epub ahead of print]<br />
https://www.ncbi.nlm.nih.gov/pubmed/30328263<br />
• Inomata K, Tajima K, Yagi H, et al. A Pre-Clinical Large Animal Model of Sustained Liver Injury and<br />
Regeneration Stimulus. Sci Rep. 2018 Oct 9;8(1):14987.<br />
https://www.ncbi.nlm.nih.gov/pubmed/30301901<br />
• Fiebig U, Fischer K, Bähr A, et al. Porcine endogenous retroviruses: Quantification of the copy number<br />
in cell lines, pig breeds, and organs. Xenotransplantation. 2018 Jul;25(4):e12445.<br />
https://www.ncbi.nlm.nih.gov/pubmed/30264881<br />
• Andreasen LJ, Krog S, Ludvigsen TP, et al. Dietary normalization from a fat, fructose and cholesterol-rich<br />
diet to chow limits the amount of myocardial collagen in a <strong>Göttingen</strong> Minipig model of obesity.<br />
Nutr Metab (Lond). 2018 Sep 25;15:64.<br />
https://www.ncbi.nlm.nih.gov/pubmed/30263039<br />
• Ronnander P, Simon L, Spilgies H, Koch A. Modelling the in-vitro dissolution and release of sumatriptan<br />
succinate from polyvinylpyrrolidone-based microneedles. Eur J Pharm Sci. 2018 Dec 1;125:54-63.<br />
https://www.ncbi.nlm.nih.gov/pubmed/30223035<br />
• Lingner M, Seidling R, Lehmann LJ, et al. Osseointegrative effect of rhBMP-2 covalently bound on<br />
a titan-plasma-spray-surface after modification with chromosulfuric acid in a large animal bone<br />
gap-healing model with the <strong>Göttingen</strong> minipig. J Orthop Surg Res. 2018 Aug 30;13(1):219.<br />
https://www.ncbi.nlm.nih.gov/pubmed/30165865<br />
• Mahan B, Moynier F, Jørgensen AL, et al. Examining the homeostatic distribution of metals and Zn<br />
isotopes in <strong>Göttingen</strong> minipigs. Metallomics. 2018 Sep 19;10(9):1264-1281.<br />
https://www.ncbi.nlm.nih.gov/pubmed/30128473<br />
22
• Bech J, Glud AN, Sangill R, et al. The porcine corticospinal decussation: A combined neuronal<br />
tracing and tractography study. Brain Res Bull. 2018 Sep;142:2<strong>53</strong>-262.<br />
https://www.ncbi.nlm.nih.gov/pubmed/30086351<br />
• Measey TJ, Pouliot M, Wierzbicki W, et al. Expanded Characterization of a Hemi-Body Shielded <strong>Göttingen</strong><br />
Minipig Model of Radiation-induced Gastrointestinal Injury Incorporating Oral Dosing Procedures.<br />
Health Phys. 2018 Jan;114(1):32-42.<br />
https://www.ncbi.nlm.nih.gov/pubmed/30085968<br />
• Meier K, Qerama E, Ettrup KS, et al. Segmental innervation of the <strong>Göttingen</strong> minipig hind body. An<br />
electrophysiological study. J Anat. 2018 Oct;233(4):411-420.<br />
https://www.ncbi.nlm.nih.gov/pubmed/30040118<br />
• Lillethorup TP, Glud AN, Landeck N, et al. In vivo quantification of glial activation in minipigs overexpressing<br />
human α-synuclein. Synapse. 2018 Dec;72(12):e22060.<br />
https://www.ncbi.nlm.nih.gov/pubmed/30009467<br />
• Kenchegowda D, Legesse B, Hritzo B, et al. Selective Insulin-like Growth Factor Resistance Associated<br />
with Heart Hemorrhages and Poor Prognosis in a Novel Preclinical Model of the Hematopoietic Acute<br />
Radiation Syndrome. Radiat Res. 2018 Aug;190(2):164-175.<br />
https://www.ncbi.nlm.nih.gov/pubmed/29809108<br />
• Figueiredo C, Carvalho-Oliveira M, Chen-Wacker C, et al. Immunoengineering of the vascular endothelium<br />
to silence MHC expression during normothermic ex vivo lung perfusion. Hum Gene Ther. 2018<br />
Sep 27. [Epub ahead of print]<br />
https://www.ncbi.nlm.nih.gov/pubmed/30261752<br />
23
52 AUTUMN 2018<br />
NEWSLETTER<br />
••<br />
••<br />
••<br />
••<br />
••<br />
••<br />
••<br />
••<br />
••<br />
••<br />
Towards 3Rs: Multiplex protein profiling<br />
from minipig, cyno, dog and rat samples,<br />
to get the most out of preclinical safety<br />
studies page 3<br />
Experience with mixed groups of intact<br />
female and castrated male <strong>Göttingen</strong> <strong>Minipigs</strong><br />
See<br />
where you<br />
can meet us<br />
in 2018<br />
page 28<br />
Minipig<br />
Research<br />
Forum 2018<br />
page 20<br />
for pharmacokinetic studies in drug discovery research<br />
– summary of behavioral observations and collected<br />
back-ground data page 6<br />
Anti-cancer drug development: Comparison of toxicity<br />
in <strong>Göttingen</strong> <strong>Minipigs</strong> and mouse page 10<br />
Body surface area measurement in <strong>Göttingen</strong> <strong>Minipigs</strong><br />
using a computed tomography scanner page 14<br />
The <strong>Göttingen</strong> Minipig for the study of buccal and sublingual<br />
products page 17<br />
Update from Oriental Yeast Co., Ltd., Japan page 24<br />
Update from WOOJUNGBIO, Korea page 25<br />
Update from Marshall BioResources North America page 26<br />
New scientific publications on the <strong>Göttingen</strong> Minipig page 27<br />
Join the CONFIRM Initiative page 28<br />
Clean pigs<br />
for clear results<br />
The next <strong>Newsletter</strong> delivered<br />
directly to your inbox<br />
Our <strong>Newsletter</strong> is published three times a year. We welcome new and former subscribers<br />
that we might have lost contact with during the GDPR process, and<br />
ask you to sign up at https://minipigs.dk/contact-us/sign-up-for-our-newsletter/.<br />
If you have suggestions for topics and/or authors, for a coming issue of the <strong>Newsletter</strong>,<br />
please email the editor: Søs Pihl-Poulsen at spp@minipigs.dk.<br />
Meeting Calendar 2019<br />
Name Date Location<br />
SOT Annual Meeting & ToxExpo 10-14 March Baltimore, MA, USA<br />
Janssen Juvenile Toxicity Symposium 25-26 April Beerse, Belgium<br />
Minipig Research Forum 22-24 May Vienna, Austria<br />
FELASA 10-13 June Prague, Czech Republic<br />
TALAS 24-28 June Bangkok, Thailand<br />
EUROTOX 8-11 September Helsinki, Finland<br />
SPS 22-25 September Barcelona, Spain<br />
AFSTAL 2-4 October La Rochelle, France<br />
ACT 17-20 November Phoenix, AZ, USA<br />
CALAS TBA TBA, China<br />
Follow us on ! www.linkedin.com/company/2864308/<br />
Join <strong>Ellegaard</strong> <strong>Göttingen</strong> <strong>Minipigs</strong> at LinkedIn to stay updated on our scientific events and other exciting initiatives to celebrate<br />
Fifty Years with <strong>Göttingen</strong> <strong>Minipigs</strong><br />
Europe and Asia<br />
<strong>Ellegaard</strong> <strong>Göttingen</strong> <strong>Minipigs</strong> A/S<br />
Sorø Landevej 302,<br />
DK-4261 Dalmose,<br />
Denmark<br />
Tel.: +45 5818 5818<br />
ellegaard@minipigs.dk<br />
North America<br />
Marshall BioResources<br />
North Rose, NY 14516, USA<br />
Tel.: +1 315 587 2295<br />
Fax: +1 315 587 2109<br />
infous@marshallbio.com<br />
Japan & Taiwan<br />
Oriental Yeast Co. Ltd.<br />
3-6-10, Azusawa, Itabashi-ku<br />
Tokyo, 174-8505, Japan<br />
Tel.: +81 3 3968 1192<br />
Fax: +81 3 3968 4863<br />
fbi@oyc.co.jp<br />
Korea<br />
WOOJUNGBIO<br />
B-3F, 145 Gwanggyo-ro,<br />
Yeongtong-gu, Suwon, Korea<br />
Tel.: +82 31 888 9369<br />
Fax: +82 31 888 9368<br />
wj@woojungbsc.co.kr<br />
www.minipigs.dk<br />
JANNERUP GRAFISK