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

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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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-, after the injection of insulin, and thus no apparent genetic change. 27 There is a greater concentration of carbohydrates in the membranes around the neural tube, noto- chord, and yolk sac after insulin injection, and a decreased concentration in the tail bud region. 3l Rumplessness seems to be caused by the action of insulin in stopping the yolk sac from transferring glycogen to the embryo, or preventing metabolic processes, and this deficiency causes the undeveloped tail bud. 43 The naturally occurring mutation of rumplessness tCreeper conditionl is due to the genes' interfering with the carbohydrate metabolism and transport in the tail bud of the embryo. The Effects of Hyperbaric Oxygenation on Cerebral Metabolism STEVEN TRIPPEL In tests determining the ability of mice to successfully run through a maze, it has been found that when the run was made in a chamber of high pressure oxygen, the mice ran the maze both faster and with fewer errors than did mice under normal pressure air. It is suggested that this is a result of an increase in cerebral metabolism and a proced- ure for experimentation to test this has been developed. Hyperbaric oxygenation has been in use for clinical pur- poses for a long time and many of its physiological effects are thus, well known and understood. The possibilities of its having any mental effect, however, have not, to my know- ledge, ever been explored. Yet, in the use of HBO fhyper- baric oxygenl in the medical treatment of certain diseased aged people, there has been observed a substantial loss of senility during and shortly following the period of oxygena- tion? Although these observations could possibly be due to a generally improved physical condition resulting from the curing of the disease, it poses an interesting question whether or not they may be due, at least in part, to the HBO itself. In this report, then, my experiments testing this question are analyzed and a method is proposed for evaluating the tentative answer to it. To test whether or not HBO has any effect on mental activity, a number of mice were trained to run through a maze in order to obtain food. They were trained by being fed only in the maze and only after having successfully run through it. Motivation was supplied to them by feeding them only once a day and thus, by keeping them hungry. The trained mice were then divided into two groups: a con- trol group and an HBO groupf In testing, each mouse was placed at the entrance to the maze and allowed to proceed as it pleased. Its progress was timed and the number of errors twrong turnsl were recorded. In the case of the HBO group, this maze run was made inside a hyperbaric chamber at three atmospheres iabsolutel ygressure of pure oxygen. The control was run in the same maze but under normal atmospheric conditions. The results of running each group through the maze twice with approximately and hour and a half interval be- tween runs showed that the HBO group ran the maze in a shorter average time and with an average of fewer errors than did the control group. tSee graphs and tables for spe- cific resu1ts.l This seems to indicate that HBO makes mice smarter, or at least more alert than they are under normal oxygen partial pressure inormal pO,l. This one set of tests, however, is by no means sufficient to determine whether or not HBO actually does have the standard effect of increasing mental alertness. Even if such extensive tests were made Originally there were four groups: a control, a one- atmosphere pure oxygen group, a two-atmosphere pure HBO group, and a three-atmosphere pure HBO group. The four groups together consisted of forty-seven trained mice, and were to determine whether there was an increase in mental activity corresponding to the increase in pO,. Unfortunately, however, a dog kept in the same hospital lab with these mice escaped from its cage and devoured every one of them. The ten mice that could be obtained afterward were not suffi- cient to make four new groups. so as to show beyond reasonable doubt that such effects are the result of increased oxygen pressure, they still would give no indication of just why and how: thus, a more thorough examination of the present results is called for. The following graphs and tables illustrate the results of the above-described experimentation. un hu no ll:-ulsoxoa TIHE 'M Gm-.5 M u ' - ll' Rn.. Aung. ' on-.ll neva,-,- XP I I i l Lac' R... A-a-UV, . ' .QI X kiss in-an-a :Hia Is Yiwu. llvnhu- Kia Qvn ,, A TuMElcoNraoLp:'::-fl-M. use H ' TQ 'Qf','2'f, M .... .. at l J N i i W H U rd' R-nt Hvu-mme. A a c- n ' : ' Hun-. Latin ani lun T E S 'I' co urlox. 2 mer suewcs lsr RVN Frau. 6134... :vt Ru- ao --. 3 7... we Ov'--dl as 50 zz The mice under three atmospheres tabsolutel pure oxygen iunder fifteen times the normal oxygen partial pressure! ran the maze an average of about twenty-two percent faster than the control mice under normal atmospheric conditions. w ERRORSBTESTHf.'T5I'w'mi Ell0l.S 3 A 1 . 1 R-an Average 1'4 lun marqu- Q 1 1. l -.t l k-- r 1. l ? ' I 'Z ' 3 xW 35' Hausa Nuubu- mul Run Q Emmons 5 coNTnoLlLi.'::.l-fa Q. YKRORS 2. L A ln Q Annan I I '-VA R-la huermge, O I a. 1 at rs a x 'z Q' A B C D E Nusa Leiter and Run I Run -'l I-6 44, ar' R... -'ff - 9 al TEST CONTROL. ZDIFFERENCE Ovev-all .93 l-2 31

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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