Give Me Some Oxygen! - Oklahoma Biological Survey

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Give Me Some Oxygen! - Oklahoma Biological Survey

Give Me Some Oxygen!

The effects of hyperbaric oxygen therapy on the

tissue regeneration of Lumbriculus variegatus.

A Biomedical Paper

Presented to

The Oklahoma Junior Academy of Science

February 21, 2006

Alex Burroughs

Grove Public Schools

Grove, Oklahoma


Abstract

Hyperbaric oxygen therapy (H.B.O.T) has long been established as the

primary treatment of medical disorders such as carbon monoxide poisoning and

gas gangrene. H.B.O.T. is now increasingly being used in problem wounds,

chronic bone infections and radiation injury.(1) H.B.O.T is a means of providing

additional oxygen to the tissues of the body. These increased oxygen levels further

the body’s ability to kill germs and decrease healing time.(2)

Does a simulated hyperbaric chamber have beneficial effects on the

regeneration of Lumbriculus variegatus tissue

To test this, a simulated hyperbaric chamber was made from an autoclave.

Using oxygen to pressurize the chamber, cut segments of lumbriculus were set in

the chamber and pressurized to 0.35kg/cm 2 or 0.7kg/cm 2 (depending on the group),

for time intervals of 15,30,60,or 120 minutes each day for twelve days, counting

each section of worm on the 6 th and 12 th day.

The hypothesis that the higher pressure would have greater regeneration

rates was supported. The hypothesis that the greater amount of time spent in the

chamber would increase regeneration rates, was not supported.

The purpose of this project was to explore the efficiency of H.B.O.T. on the

regeneration of tissue. The project supported the fact that the simulated H.B.O.T.

does increase the rate of tissue regeneration in Lumbriculus variegatus.

Introduction

Hyperbaric oxygen therapy (H.B.O.T) has long been established as the

primary treatment of medical disorders such as carbon monoxide poisoning,

decompression sickness, and gas gangrene. H.B.O.T. is now increasingly being

1


used in problem wounds, chronic bone infections and radiation injury.(1) H.B.O.T

is a means of providing additional oxygen to the tissues of the body. These

increased oxygen levels further the body’s ability to kill germs and decrease

healing time.(2)

Research sited in the Journal of Trauma, injury, Infection and Critical Care

has shown that hyperbaric oxygen treatment increases the rate of healing following

a crush injury. The crush injury patients also required less follow up operations

when H.B.O.T. was used. (3)

Increased pressure allows oxygen to get into tissues better. Oxygen protects

tissues from free radical damage.(4) Hyperbaric oxygen therapy is a conservative

treatment method in which oxygen levels can be returned to comprised tissues.

H.B.O.T. uses 100% medical grade oxygen at increased atmospheric pressure to

nourish all tissues and allow the body to heal itself naturally.(5)

Medical studies of diabetic patients with foot wounds showed drastic

reduction in leg amputations in patients treated with hyperbaric oxygen

treatment.(6, 7)

A study by Jeffrey Stone, DO, MPH from the Institute of Exercise and

Environmental Medicine (IEEM), which in included 1,633 patients, suggests that

limb salvage rate was 72% in treated patients and only 53% in untreated

patients.(8)

An interview of diabetic patient, Thelma Bell speaks of her experience with

H.B.O.T. She had gangrene of her lower leg and had already lost two toes. She

had decreased nerve and circulatory damage and was facing amputation. After

receiving treatment, she had pink healthy healed tissue and avoided amputation.(9)

Lumbriculus variegatus, also known as the California Black Worm, is an

aquatic annelid. Cultures of this worm require little maintenance, which makes

them ideal for classroom projects. The lumbriculus were used in this project

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ecause they can vividly illustrate a wide variety of biological phenomenon such

as: Patterned regenerated of lost body parts, blood vessel pulsations, swimming

reflex, and giant nerve fiber action potentials.(10)

A worm’s capacities for asexual reproduction is matched by its ability to self

amputate in response to injury or other types of noxious stimulation. One thing

that causes lumbriculus to atomize is body compression or a sudden compression

stimulus, which stimulates a predatory attack and causes a rapid and clean

dissection of the body in less than 1/5 th of Lumbriculus variegatus.(10)

When Lumbriculus variegatus regenerate segments, all tissues types are

regenerated. These would include epithelial, muscular, vascular, nervous,

connective glandular, excretory and reproductive.(11) Medical research suggests

that hyperbaric oxygen therapy could be beneficial to patients with neurological,

muscular, circulatory, bone and epithelial damage. (1)

For testing hyperbaric pressure on regeneration, the experiment will include;

cutting and placing Lumbriculus variegatus into a small hyperbaric oxygen

chamber devised from an autoclave to test their regeneration rate with different

amounts of pressure and time spent in the chamber. The results should prove

whether H.B.O.T. is effective in tissue regeneration.

Purpose

The purpose of this project is to find out if a simulated hyperbaric chamber

has beneficial effects on the regeneration rate of Lumbriculus variegatus tissue.

3


Hypothesis

It is hypothesized that the increased amounts of oxygen pressure will

increase the rate of regeneration in Lumbriculus variegatus as compared to a

control of Lumbriculus variegatus that will have no increased oxygen pressure.

It is further hypothesized that the Lumbriculus variegatus, which spend

greater amounts of time under oxygen pressure, will also have greater rates of

regeneration.

Materials

Oxygen Tank

Regulator

Air chuck

Air Hose

Lumbriculus variegatus

Carolina Biological Supply #FR-14-1720

Spring Water

Micro well plates

Autoclave adjusted to make simulated

hyperbaric chamber

Scalpel

Aerator

Isopropyl Alcohol

Paper towel

Lumbriculus food

Glass slide

Well slide

Beaker

Pipette

Clock

Regeneration chart

Pencil

Microscope

Laptop

Plastic organism tank

Grease pencil

Distilled water

Procedure

A. Build Hyperbaric Chamber

1. Unscrew the pop off valve from the lid of the auto

clave replacing it with shrader valve.

2. Clean with non-oil based solvent ensuring that

there’s no oil, or oil residues.

4


B. Preparing and Dissecting Worms (12)

1. Clean plastic organism tank, allowing to dry.

2. Place paper towel in bottom of tank, fill tank with spring water.

3. Sterilize micro well plates.

4. Label the lids of the well plates.

5. Using a pipette, measure 5ml of spring water, transferring the water into six

of the wells of the well plates.

6. Collect worms using pipette.

7. Place one worm on a glass slide, and cut it into three

or four sections using a scalpel, sterilizing the scalpel

after each worm by dipping in alcohol then dipping in

distilled water and allowing to dry.

8. Using a clean pipette, place one worm section onto a

well slide.

9. Place the well slide under a microscope and using a 30x, count each segment

of the worm and record data.

10.Using a clean pipette place the worm section in to each of the six wells in

each well plate.

C. Prepare the Hyperbaric Chamber

1. Attach an air chuck and hose to a regulator.

2. Attach regulator to the oxygen tank.

D. Give Lumbriculus Hyperbaric Treatments

1. Place well plate into chamber, seal and lock chamber.

2. Purge chamber for thirty seconds.

3. Pressurize chamber to ten or five pounds (depending on

the group).

4. Release pressure after time lapse (depending on the

group).

E. Record Data

1. Count each worms segments on days 6 and 12.

2. Record

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Data

Group 1

0.7kg/cm 2 (10 lbs) of pressure daily

Started treatment on same day worms were cut

Group 1 Average

segment

increase

A 22.17

B 5.17

C 5.17

D 4.17

E 0.17

It is believed that the worms in this trial did not have ample time for their wound to seal

which was detrimental to them when put under pressure.

6


Group 2

0.7kg/cm 2 (10 lbs) of pressure treatment daily

Started treatment the day after worms were cut

Group 2 Average

segment

increase

A 23.50

B 34.17

C 31.50

D 34.67

E 34.00

All groups in this trial show significant increase regeneration rates over the control group.

The difference is believed to be the extra day to seal their wounds before treatment began.

Lumbriculus with many new segments

7


Group 3

0.35kg/cm 2 (5 lbs) of pressure daily

Started treatment the day after worms were cut

Group 3 Average

segment

increase

A 21.83

B 22.30

C 20.30

D 24.67

Test groups in this trial did not show significant increases in regeneration rates. The difference

is probably the decreased pressure used as opposed to the second test group trial.

Another Lumbriculus with many fresh new segments.

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Analysis

It is theorized that group one did not do well because they were not given a

day to seal over where they were cut. This would be comparable to putting a

person with a fresh bleeding cut into an oxygen chamber. The pressure would

cause excess bleeding. Blood clotting must occur to some degree before being

placed in the chamber. This is why the worms were given an extra day to clot or

seal before treatment for group 2.

All time intervals for group 2 regenerated at a greater rate than the control

group. No significant difference can be seen between the different time intervals.

Group 3 was given half the pressure treatment at the same time intervals.

This group did not do significantly better than the control except the group that

was in the chamber the longest, 1 hour.

The hypothesis that the higher pressure would have greater regeneration

rates was supported.

The hypothesis that the greater amount of time spent in the chamber would

increase regeneration rates, was not supported.

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Conclusion/Applications

The purpose of this project was to explore the efficiency of H.B.O.T. on the

regeneration of tissue. This project supported the fact that the simulated H.B.O.T.

does increase the rate of tissue regeneration in Lumbriculus variegatus.

Skin heals via regeneration so it can be assumed that H.B.O. T. does work

for wound healing, at least wounds involving epithelial tissue.(13) When

Lumbriculus variegatus regenerate segments, all types of tissue are being

regenerated.(11) Some research mentioned H.B.O.T. as being effective for

diabetic wounds. Diabetic wounds would include epithelial, muscular, circulatory,

and nerve tissue. Most research found for this study was favorable for treatment of

diabetic wounds and crush injuries, but the research on bone infections,

neurological radiation damage and treatment for cerebral palsy is still inconclusive.

The questions are: Do doctors use this treatment as a regular prescription for

problem wounds Could the other conditions mentioned benefit from H.B.O.T.

Maybe future research will help answer these questions.

Acknowledgements

Randy Burroughs, who helped design the hyperbaric chamber

and for delivering oxygen.

Shelly Flickenjer, who supplied treatment information and advice for this project.

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Bibliography

1. Whelan, H.T., MD. Professor of Neurology, Pediatrics, Hyperbaric Medicine, Medical College of

Wisconsin. The healing Powers of Hyperbaric Oxygen Therapy. Website: Healthlink,

Medical College of Wisconsin.

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