Illinois Junior Academy of Science - Yearbook (Urbana, IL)

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To Dr. Jack van Elk, of Lutheran General Hospital and Northwestern University Medical School, I wish to express my thanks for enabling me to use the John Hartford hyperbaric oxygen facilities at Lutheran General Hospital in Park Ridge, Illinois. My most sincere appreciation is extended to my sponsors at Evanston Township High School, Mr. G. Kolb and Mr. S. Del1'Aria, for their guidance. Without the help of these instructors I could not have done this project. Homotransplantation of Mouse Tissue by Parabiosis CAROL EGEL Niles Township Community H.S. Skokie j. .. In doing my experiment, I continued research in perma- nent homotransplants, using parabiosis to overcome the tendency for rejection of the grafts. More specifically, my branch of study is in the transplanting of tails and skin from mice of one strain to mice of another strain. Normally such transplants are rejected by the recipient, and to estab- lish permanency in the graft, parabiotic union between donor and host mice was evolved before transplantation was attempted. Apparatus used is as follows: white, brown, and -black domestic mice, eight weeks of age: an operating surface composed of pegboard and a system of strings passed around the legs of the mice, through pegboard holes, and secured beneath the pegboardg USP ether for anesthesia: a metal can with a cover and containing ether for anes- thesia: anesthetizing masks consisting of ether-drenched cotton in small paper funnelsg an antiseptic solution of cetylcide in which to soak surgical instrumentsg merthiolate and applicatorsg 4x4 sterile gauze pads used for sponges and sterile drape: plastic adhesive draping material to help re- strain mice during surgery: a cst cutterg hair clippers: and rubber surgical gloves. Surgical equipment consisted of thumb forceps, needle holders, scissors, eye suture of 6-0 plain catgut, eye-surgery needles, and size 5-0 black silk suture.1-2 Postoperatively, cast material was used to keep mice together during parabiotic union while also keeping some strain off of the suture line of anastomosis. Also used at this time was phenobarbital solution, IA grain to 4cc. of water, furacin powder, and penicillin G solution t300,000 units!cc.l. In the experimentation reported here, mice of three different strains were used. To be assured of the absence of any chance genetic kinship among strains used, which might affect rejection of grafts, direct skin switch grafts were made among strains of mice used with complete rejec- tion in all cases? The two mice to be joined are removed one at a time from their cage and each is placed in the Uanesthetizing chamber until it reaches a state of relaxation and responds very little to outside stimulus. It is then removed from the can and secured to the operating surface with a system of strings and adhesive drape. An anesthetizing mask is placed a few inches from the mouse's head to prevent its return to a wakeful state. The second mouse is then anesthetized and fastened to the operating area. The operative site of both mice is then closely shaved with a hair clippers. Excess hair is picked up with tape. The site of the incision is liberally painted with merthiolate, and sterile gauze is draped to expose only the actual area of incision. The first mouse shall be referred to as mouse A: the second shall be referred to as mouse B. The incision is made using a thumb forcepts to hold the skin away from the body while using an eye scissors to actually cut. Because of loose attachment of the skin to the muscle and the paucity of fat tissue, it was not difficult to free the skin from underlying tissue. A flap of skin and subcutaneous tissue approximately 5x5 milli- meters in length was formed by cutting with a surgical scissors through mouse A's skin to the faschia in such a manner that a dorso-lateral flap was formed. This flap was sewn to an existing defect in mouse B prepared by cutting an antero-lateral flap from mouse B. The flap of mouse B was sutured to the defect in mouse A's side caused by the formation of a flap on mouse A, as shown in diagram I. A method of interrupted suture was used to facilitate the healing of the flaps to the corresponding defects and to permit increased blood flow between the mice. Sutures were placed about one millimeter apart and ten sutures were used on an average union. During the operation, anesthesia was kept at a minimum, though the animals were not allowed to awaken completely at any time during the operative procedure. While a cast was being put on the mice, no anesthesia was given, and it was ,at this point that consciousness returned. While under the effect of the anesthesia, twitching of the mice's extremi- ties was noticed. This was probably due to cerebral anoxia or a stage of hyperexcitability also observed in humans under anesthesia. The cast was of plaster-impregnated gauze and encircled the thorax of both mice. Because their bodies are remark- ably flexible, the casts were secured tightly so as to prevent the escape of one or more of the mice from the cast. The mice in the first case escaped the cast and tore the sutures before they could again be encompassed with a restraining device. The death of the dark mouse five days post-opera- tively was probably a result of the escape. At this time a new method of wrapping the cast using a figure-eight!cir- cular wrapping was introduced and found to be an improve- ment. Post-operative care included bedding of clean paper twood chips could enter and infect the woundl, food, and water. A small amount of furacin powder was put directly on the wound. Several drops of penicillin G solution were placed on food and in the water of the mice to combat in- fection. To sedate the mice so they would put less strain on the sutures, several drops of phenobarbital solution were added to the water. The sedative also was used to lessen any fighting between the joined animals since they would have to be in close proximity during the union. However, at no time was any fighting noticed between the parabiotic partners while joined, though the stronger of the two would pull the other around. The mice actually seemed to turn to each other for assurance. After seven days of parabiotic union preparation was made for actual grafting. At this time sufficient vascular anastomosis was assumed to be established? The mice were anesthetized together initially and continued sedation was induced using separate anesthesia masks. Merthiolate was applied to the operative site and area around this site was covered with sterile gauze drape. Using the eye scissors, the base of each flap was cut, severing the remaining con- nection of the flap with its donor. The freshly cut base was sewn to the skin of the host mouse with one or two sutures. This resulted in the formation of a dark skin graft on the light mouse and a graft of light skin on the dark mouse. Furacin powder was put on the wound and penicillin G medi- cation was continued for approximately seven more days. In transplant of a mouse tail, formation of a parabiotic union was identical to that formed in skin grafting. The union was composed of skin flaps and the period that the partners were kept in a union of parabiosis was seven days. After seven days, the mice were separated and skin flap switch transplants were executed as previously described. A slit about 3 milimeters in length was cut perpendicular to the spine and about 1 centimeter from the normal tail of mouse A. With the eye scissors mouse B's tail was severed between vertabrae. A small portion of skin and muscle from the base of the tail was included in the graft. The edges of the tail graft were sutured to the edges of the slit in mouse A's back as shown in diagram II. About twelve sutures are needed. The interrupted suture method is used to aid anastomosis of blood vessel between the graft and host.' A group of 12 controls was used. This group went through the same operative procedures as that of the ex- perimental group with the following exception: rather than forming a parabiotic union, skin flaps when formed were sutured to the mouse from whose skin they had been formed. Pairs of light and dark mice were then bound together using the figure-eightfcircular wrapping. After seven days the cast binding the pairs was cut and free grafts of dark skin were sutured to light mice while free grafts of light skin were

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The mice under three atmospheres Cabsolutel 100 percent oxygen ffifteen times the pO, of the controll ran the maze with an average of about 30 percent fewer errors than did the control mice. These results seem to indicate that mice under three atmospheres partial pressure of oxygen are able to function both faster and more accurately-than those under the normal one-fifth atmosphere oxygen partial pressure. In analyzing these results, however, it must'be recognized that they are not statistically as valuable as they should be if any con- clusions are to be drawn from them. This is a result of the fact that each mouse was run only twice and that there was a shortage of mice due to the loss of the original set. Thus these results must not be regarded as showing that HBO does definitely increase mental activity, but rather as hinting that this might be the case. They are, however, ade- quate to warrant further experimentation that could better test the possible merit of the idea that HBO increases mental activity. In addition, it should be noted that under HBO, there exist certain physiological conditions pertinent to this ex- periment which are independent of the results obtained but which seem to support them. For example, one of the recog- nized effects of subjecting mammals to HBO is an increased oxygen content of the blood. This, in accordance with Hen- ry's law lmoles of gas dissolved I k x gas pressurel, is because the blood plasma dissolves oxygen in quantities pro- portionate to its partial pressure lpO,J. Thus, in this experi- ment, the plasma oxygenation was increased fifteen fold and the overall blood oxygenation by about 31 percent. lThe slight overall increase relative to the large plasma increase is a result of the fact that the blood's hemoglobin, whose amount of oxygen is not much affected by pressure increases, carries the large majority of the blood's oxygenfl. It is interesting to note that the overall difference in blood oxygenation C31 percentl between the test and control groups in these experiments is very close to the overall dif- ference in efficiency of their maze runs f27fZ:J. The significance of this increased pO of the blood is that it means that when an animal such as a mouse for a humanl is put under HBO, its tissues, including the brain tissues, are bathed in the extra oxygen from the blood plasma. This suggests that the ixidative processes consti- tuting the metabolism of such tissues as the brain could increase under HBO conditions. The experimentation done thus far serves as an empirical observation which might support the hypothesis that HBO increases brain metabolism in that it shows how mental activity could be affected by HBO. However, since, as mentioned previously, this experi- ment's results are statistically somewhat cloudy, and because it does not test the hypothesis directly tthis present hypo- thesis was formulated after these results were obtainedl, further experimentation is required. For this reason, I have developed the following procedure and am making arrange- ments for its execution. After a good deal of formulating and collecting of ideas, I decided that the best feasible means of testing whether HBO increased cerebral metabolism is to measure the arterio-venous difference in the quantity of oxygen in the blood of the major artery and vein serving the brain. Briefly, this amounts to performing an operation on the neck of an animal such as a dog and attaching to each its internal carotid artery and internal jugular vein the electrode of a gas analyzer' and the probe of a flowmeterf From the re- sults obtained by these two instruments, the A-V difference in oxygen quantity can be determined. The gas analyzer is an instrument designed to measure the pressure of such gases as oxygen and carbon dioxide in the tissues of animals. It operates by means of an electrode sheathed in a thin gas-permeable membrane which is inserted directly into the tissue fblood in this easel to be analyzed. The presence of gases at the electrode tip initiates a current which is amplified and calibrated on a meter and which gives the partial pressure of the gas at the electrode tip. The electrode can be inserted into the artery Cveinl through the use of a T tube as shown. 2 rm, T .TuT ' mini! ini -- M -.N Hr-fu-y fveinl august Ti' The flowmeter is an instrument designed to measure the rate of flow fvolume per unit time! of a liquid through -L a tube such as an artery or vein. It does this by measuring the voltage produced when flowing blood fwhich is slightly ionizedl cuts the electromagnetic field produced by a probe encompassing the artery fveinl. This voltage is proportion- ate to the rate at which the field is cut and thus also to the rate of blood flows. The probe can be placed on the artery fveinl as shown. 62 The absolute quantity iexpressible in molesl of oxygen pass- ing a given point per unit time in the artery and the vein can be determined from the product of the flowmeter read- ings lin volumeftimel and the gas analyzer readings fin at- mospheres of oxygen at a given temperaturel through a series of relatively simple calculations. The difference in moles of oxygen found between the in- ternal cartoid artery and internal jugular vien will give a good approximation of the amount of oxygen being consumed by the brain i.e. brain metabolism. lIt will be but an approxi- mation because there are other minor arteries and veins feeding and emptying the brain whose services cannot be measured. The i. carotid and i. jugular would have to be the only vessels to the brain and would have to serve only the brain if the results were to be a precise measurement of cerebral metabolism.l The hypothesized results, then, should show the amount of oxygen consumed by the brain under EEO conditions to be greater than that under normal con- 'tions. The experiments testing this will be run with a control group consisting of dogs anesthetized and operated upon fwith instruments attachedl but under normal atmospheric conditions, and with a test group of dogs anesthetized, operated upon, and under HBO. In an effort to reduce the variables introduced by such factors as anesthetic effect, etc., the two groups will consist of as similar dogs as can possibly be obtained. In addition, each group will serve as both a control and a test against itself in that after readings on the control group have been made, it will be tested under HBO to see how the readings change. Similarly, after readings have been made with the test group under HBO, it will be made to serve as control. This not only means that each group will serve as a control both against itself and the other group, but also that any effect of time on the physical condition of the operated dogs will be averaged out by the alternation of oxygenation. It was not until just recently that I gained access to the equipment necessary to carry out the above-described procedure, apd as a result, the tests necessary to confirm the hypothesis that HBO increases cerebral metabolism have not yet been made. However, tests run to determine the possibility of HBO having an effect on mental activity in- dicate a degree of likelihood that this further experimenta- tion will confirm this hypothesis. Footnotes and Acknowledgements 1. From the work of Dr. J. Williams of Sunninghill, England. 2. See page two of this paper. 3. Boerema, I., Meijne, N. G., Brummelkamp, W. K. Bouma, S., Mensch, M. H., Lamarmans, F., Hanf, M. S. and Van Aaleren, W.: Life without blood. J. Thoracic and Cardiovascular Surgery. 1: 133, 1960. 4. My thanks to Dr. Paul Nora of Northwestern University Medical School, Chicago, Illinois, for permitting me to use his gas analyzer and to Mr. Frank Cieslak for demonstrating its operation. 5. My thanks to Dr. F. John Lewis of Northwestern Uni- versity Medical School, Chicago, for making available to me his flowmeter and for showing me how to use it. 6. Cordell, R. A. and Spencer, M. P.: Electromagnetic Blood Flow Measurement in Extracorporeal Cir- cuits. Annals of Surgery Vol. 151 No. 1. pp. 71-74. January 1960.

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sutured to dark mice. The same medication was given to the control group that was given to the experimental group. All grafts in the control group were rejected. In the experimental group results were favorable. Fifty experi- mental mice were used. Forty of these were used for skin grafts. The seven mice that died were not counted as failures since the deaths were before or during formation of a para- biotic union. One mouse died after grafting. However, the death was caused by infection at a site other than that of the graft. Also, the graft had not been rejected at this time nor did it show any signs of rejection. Therefore, this sub- ject was not called a failure though it was not counted as a successful graft. Rejection of a graft is the only factor indicative of failure, just as the acceptance of a graft is the only indication' of success. All thirty-two of the other mice used for skin grafting survived. However, each mouse which survived operative procedures died by the seventeenth post- operative day, on an average. These mice met apparently violent deaths as attested to by these observations: nasal hemorrhage, flexion of forepaws, hind paws, and neck, pre- ceded by clonic movement of the body. Ten of the experi- mental mice received tail transplants and skin transplants. All of these survived operative procedures. However, on the seventeenth day post-operative, on an average, these mice, too, met apparently violent deaths, as did the mouse used to test the reason for tail atrophy seen in these mice before their death. Rejection of a graft is caused by death of its cells. Dead cells are sloughed off by the host animal. Cells of a donor animal die when grafted to the host because of either mechanical or chemical interference with cell metabolism. Mechanical difficulties which may arise are poor blood sup- ply or infection. Either of these is capable of killing cells. These factors are overcome by using sterile technique and developing an adequate circulation of blood between the graft and its host before transplantation. Though no rejection by mechanical means took place during my experimentation, the transplanted tails were atrophied. Since no infection was present, the atrophy was thought to be due to insufficient blood supply. To check this hypothesis a pair of mice were united parabiotically using skin flaps. After one week of para- biosis, the tail of one mouse was partially amputated., The cut surface was sutured to an incision on the back of the host mouse. In this manner, the tail received nourishment from the donor mouse while circulatory connection with the host mouse was being established. After seven days, when sufficient vascular anastomosis was assumed to be estab- lished, the tail was completely amputated from its donor? It was then sutured to an extension of the incision that was previously cut on the host's back. The tail graft did not atrophy in this case, and it is assumed to be established that insufficient blood supply was the cause of the inanation atrophy in the other tails. CThis last test mouse was not in- cluded in results.l Cells foreign to the host may also be killed by anti- bodies which are formed in the blood serum of the host when the host is subjected to foreign substances, notably proteins or carbohydrates. The principle behind using para- biosis to offset rejection must now be explained. When parabiotic union of host and donor is established, there is a free exchange of blood and substances in the blood between parabiotic union of host and donor is established, there is a antibodies, the alien tissue when introduced in gradually increasing amounts, conditions the mice to tissue from their parabiotic partners. In this way tolerance is built and the immunological mechanism of the mice no longer recognizes the donor cells as foreignf' According to the data I have gathered, the inhibition of the immunological mechanism in parabiotic partners for each other's cells is not permanent. The length of time of inhibition of antibody production for a seven day parabiotic union of host and donor is twenty- four days, on an average, for both skin and tail transplants. These time intervals include the period of parabiotic union since cells foreign to an animal were being introduced into the animals at this time. The use of parabiosis to overcome rejection of tissue transplants was first developed in 1936 on inbred, litter-mate white mice' Success with homotransplants using parabio- sis among inbred strains of mice was reported by Eichwald and Silmser in 1955.6 Unsuccessful attempts at transplanting skin homografts in non litter-mate rats were carried out by Gifford and Crossfield in 1957.' Only in recent years have successful homotransplants using parabiosis been made between unrelated animals. I have found no experimental study which parallels mine. However, related studies show a similar incidence of success in both formation of parabiotic unions though in actual acceptance of skin grafts data is not similar to my own. Results other than my own, relating to tail grafting could not be found. In conclusion, parabiotic union of mice preceding homo- transplants of mouse tissue favorably influences the accept- ance of grafts both at the sight of union' and also at other sites. Tolerance of parabionts to each other's tissue results in temporary inhibition of antibody synthesis, and, thereforeg conditional acceptance of homotransplants. Systematic error is inherent in all animal experimenta- tion. The behavior of any living thing is always unpredict- able to a certain degree. Other error might occur because of the dependence on visual observation in some aspects of experimentation, such as the appearance of a scab preceding rejection. Bibliography 1. Shwind, J. V., personal correspondence, The Sansum Research Foundation, February 2, 1965. 2. Martinez, C., F. Shapiro, and R. A. Good, Essential Duration of Parabiosis and Development of Toler- ance to Skin Homografts in Mice, Proceedings Soc. Exptl. Biol. Med., vol. 104, 1960, pp. 256-269. 3. Shwind, J. V., Homotransplantation of Extremities by Parabiosis, Annals of New York Academy of Science, vol. 99, article 3, October 24, 1962, pp. 933-942. 4. Gifford, H. and J. H. Crossfield, The Effect of Con- tinued Parabiosis on Switch Homotransplants in Rats, Stanford Medical Bulletin, vol. 15, Feb- ruary 1957, pp. 28-32. 5. Eichwald, E. and C. R. Silmser, Transp. Bulletin, vol. 2, 1955, p. 148. 6. Shwind, J. V., Successful Transplantation of a Leg in Albino Rats with Reestablishment of Muscular Control, Science, vol. 84, October 16, 1936, p. 355. 7. Billingham, R. D., P. L. Krohn, and P. B. Medawn, Effect of Cortisone of the Survival of Skin Homo- grafts in Rabbits, British Medical Journal, vol. 1, 1951, pp. 1157-1163. 8. Calne, R. Y., The Rejection of Renal Homografts In- hibition in Dogs by 6-Mercaptopurinef' Lancet, vol. 1. 1960. pp. 417-418. 9. Dammin, G. J., N. D. Couch, and J. E. Murray, Pro- longed Survival of Skin Homografts in Uremic Patients, Annals of the New York Academy of Science, vol. 64, 1957. p. 967. 10. Haurowitz, Feliz, The Template Theory of Antibody Formation, pp. 22-31. 11. Main, J. M. and R. T. Prehn, Successful Skin Homo- grafts after the Administration of High Dosage X-irradiation and Homologous Bone Marrow, Journal Nat'l. Cancer Inst., vol 15, 1955, p. 1023. 12. Marianne, T. C. Martinez, J. M. Smith, and R. A. Good, Induction of Immunological Tolerance to Male Skin Isografts Subsequent to Neonatal Period, Proceedings Soc. Exptl. Biol. Med., vol. 101, 1959, pp. 596-599. 13. Raffel, Sidney, Immunity, Appleton-Century-Croft, Inc., New York, copyright 1953. 14. Woodruff, M. F. A. and B. Lennox, Reciprocal Skin Grafts in a Pair of Twins Showing Blood Chimer- ism, Lancet, vol. 2, 1959, pp. 476-478. ,I ,Q X N ' s ,f lu-1-mnunnxmaun m-xnmumnummnuhx