This paper describes various aortic parabiotic procedures and discusses various problems concerning these procedures. The most satisfactory results, nearly 100 % of survival rate, can be achieved using longer sections of aortae of 2 to 5.month old rats. In these rats blood circulation between the parabionts has completely been established.</p

Fang, C. H.

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Improvement of the method of rats parabiosis with aortic anastomoses

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Acta Medica OkayamaVolume 25, Issue 6 1971 Article 2OCTOBER 1971Improvement of the method of rats parabiosiswith aortic anastomosesC. H. Fang∗∗Okayama University,Copyright c©1999 OKAYAMA UNIVERSITY MEDICAL SCHOOL. All rights reserved.Improvement of the method of rats parabiosiswith aortic anastomoses∗C. H. FangAbstractThis paper describes various aortic parabiotic procedures and discusses various problems con-cerning these procedures. The most satisfactory results, nearly 100 % of survival rate, can beachieved using longer sections of aortae of 2 to 5.month old rats. In these rats blood circulationbetween the parabionts has completely been established.∗PMID: 4264053 [PubMed - indexed for MEDLINE] Copyright c©OKAYAMA UNIVERSITYMEDICAL SCHOOLActa Med. Okayama 25, 597-603 (971)IMPROVEMENT OF THE METHOD OF RATS PARABIOSISWITH AORTIC ANASTOMOSESC. H. FANGDepartment of Pathology, Okayama University Medical School, Okayama, Japan-(Director: Prof. S. Seno)Received for publication, Oct. 11, 1971Parabiosis, the surgical union of the animals or grafting of one animalonto another has proved to be often an indispensable tool in researchfield of hematology, immunology and so on. As pointed out by KAWAI(1), parabiosis by skin and muscle anastomosis and celio-anastomosis ofskin (2, 3, 4) does not give satisfactory results. By these methods, noone can expect the free exchange of the blood components or body fluidbetween two animals although capillary anastomosis may occur after thethird postoperative day (5). And the average rates of the blood variedbetween 0.64 and 1.8 per cent of the blood volume per minute (6), andthe time needed for complete mixing of cross.exchange of the labelledred cell ranges from 1 1/2 (7) to 24 (8) hours. All these data of the cellularconstituents of the peripheral blood in irradiated parabionts by celio-anastomosis showed a great difference; i. e. red cell and white cell countsof the non.inadiated partner were usually 2 times greater than the irradi-ated one. But sealing the mouth of one rat of the parabionts 10 daysafter celio-anastomosis resulted in the death of the mouth sealed one by5 to 6 days (l). Then, through these observations KAWAI tried to givecomplete blood exchange between parabionts by aortic anastomoses.Consequently, he has established the method of arterial parabiosis with"parallel anastomoses" by transplanted homologous thoracic aortae, inwhich abdominal aorta of one rat is transversely anastomosed with that ofthe other by using the aortae obtained from different rats of the samestrain (Fig. 1).When operation was accomplished, naturally peripheral blood showedthe equal distribution of red cells and white cells in both animals, indioeating the complete blood exchange between the parabionts. This methodis a valuable tool in various fields, e. g., hematology, immunology, etc. Butthe successful rate of the operation was very low because of the technicaldifficulties.The author therefore tried to raise the successful rate, and finally,5971Fang: Improvement of the method of rats parabiosis with aorticProduced by The Berkeley Electronic Press, 1971598 C. H. FANGsucceeded in obtaining nearly 100 % of survival rate by improving thetechnique, Various technical problems of the operation concerning theoriginal and the improved techniques will be described in this paper.MATERIA'_ S AND METHODS1. Animals:Seventy-six pairs of male Wistar rats of 4-5-month-old weighing 380 to 420 gwere used. Parabiotic partners were of the same litter chosen from those ofnearly the same body weight, less than 20 g in difference. In this experiment, allparabiosis was made between healthy rat and x.ray 0,000 r) whole body irradia.ted rat on the third day after irradiation.2. Procedures of parabiosis:(A) Preparation for aQrtae transpLwtation :Some pairs were parabiosed in the same direction by cross-anastomoses ofaortae. This way of parabiosis was unadaptable because of technical difficultiesand early death of the parabionts. Therefore, the parabiosis in the oppositedirection by parallel anastomoses of aortae with transplantation of the homolo.gous aortae was adapted for experimental pairs.The aorta for grafting was cut at the adequate portion as shown in Fig. 3.The canule was inserted into the aorta, the branches of the aorta were ligatedwith gut (No.1) at the root, and were cut at the distal portion together withsurrounding tissues. By this procedure, a pair of transplanting aortae was takenout. After taking out the aortae, the aortae were washed by injecting salinesolution containing a small quantity of sodium heparin and agglutinated bloodmasses in aortae were removed. On this condition the removed aortae are readyto be transplanted. Up to this step it took about 20-30 minutes for one pair.The aortae were preserved in cold physical saline solution (Fig. 4). Simultane-ously one pair of the rats for parabiosis was carried to a good anesthesized con·dition for operation. For anesthesia non.irradiated rat received the injectionof Nembutal (50 mg/ml, Abott) 0.4-0.6 ml and atropin sulfate (0.5 mg/ml) 0.1ml into gluteal muscle, the irradiated partner was injected less dose 0.3-0.5 mlof Nembutal. The anesthesized rats were disinfected by bathing with dilutedosban solution.Two.cm incision in length was made along left postauricular line about onecm below costal margin. On the middle point of the line and at right angleanother incision reaching to vertebral column was made (Fig. 2a). When thesubcutaneous tissues and muscles were cut with scissors and abdominal aorta wasexposed by tearing of fat tissues with fingers, the abdominal aorta and rt. renalartery were carefully separated from renal vein and surrounding connective tissuesfor the following operation of anastomosis. And the rt. renal artery was ligatedat the distal portion with gut (No.2), the ends of which were left about 5cm andtied together at the furthermost ends. Pulling up sEghtly the ends of the gut, rt.kidney was denuded from the surrounding tissues and capsule with fingers, thert. ureter and rt. renal vein were ligated together with gut (No.5), and the rt.2Acta Medica Okayama, Vol. 25 [1971], Iss. 6, Art. 2http://escholarship.lib.okayama-u.ac.jp/amo/vol25/iss6/2Rat Parabiosis with Aortic Anastomoses 599kidney was finally removed. The abdoimnal aorta of the partner rat was alsoexposed, and the rt. kidney was removed by the same procedures just described.Two animals were placed side by side in opposite direction, and the abdomi-nal opening weres conjugated on both sides sewing up together, skin to skin, andmuscle to muscle, at the three points with gut (No.7).(B). Transplantation of aortae (Fig. 2b and 2c) :(1) Dissected distal end of aorta of the ho.;ts in each parabiont :By using two small clips, the exposed abdominal aorta was clipped afterseparating carefully with tweezers from its surrounding tissues at two pointsjust below the point of the bifurcation of abdominal aorta and ileolumbalartery and at the lower part of the abdominal aorta. Then the aorta was cut atthe middle point as shown in Fig. 2b. The portion of the bifurcation of It. andrt. common iliac artery was depressed with a forefinger to prevent bleeding, theclip of the distal end was taken away and the aorta prepared for transplantationwith a steel canule inserted into the distal abdominal aorta. End-to-end anasto-mosis was performed with 8.0-nontraumatic filament nylon suture. The anasto-mosed portion was cemented with Alone Alpha (Sankyo Co.) in order to avoidthrombus formation due to infolding of the adventitial tissues into the inside ofthe anastomosed aortae (Fig. 2-c). Then the canule was removed, and the graftedaorta was perfused with saline containing a small quantity of sodium heparin.Immediately after these procedures the proximal end of transplanted aorta wasclipped.(2) Proximal end of dissected aorta of the hosts (Fig. 2b, 2c, 2d and 2e) :The abdominal aorta just above rt. renal artery was separated gently withsmaller tweezers from abdominal vein and surrounding connective tissues. Theportion was held with clip (c) to prevent bleeding. Then the clip (a) on theproximal end of dissected aorta was taken off (Fig. 2b). A steel canule insertedinto the dissected proximal end of the host abdominal aorta and was pushedout through the wall of rt. renal artery nearby ligated portion. The clip at theend of the previously grafted aorta was moved to the distal side (clip (d)) andthat canule was inserted into the grafted aorta of the partner (Fig. 2d). Bothends of the aortae were drawn close to meet. When the diameter of an aortawas larger than another, the diameter of the smaller one was extended by mak-ing a minor cut to adjust to the larger one. This will be described again in thesection of discussion.Anastomosis was made by 2-5 sutures followed by cementing with AloneAlpha. Immediately the canule was drawn out from rt. renal artery and about0.5 ml of saline containing 0.1 ml of sodium heparin was injected into the renalartery and the clip (d) on the transplanted aorta was removed (Fig. 2e). Whenthe anastomosis was not well made either leakage of injected saline or obstruc-tion of flowing of the injected saline due to thrombus occulusion would beobserved. Therefore, completely successful anastomosis is always required.After completion of the anastomosis, rt. renal artery was ligated with No.2 silkat its origin.Immediately after completing all processes of aortic anastomoses the clips (d)3Fang: Improvement of the method of rats parabiosis with aorticProduced by The Berkeley Electronic Press, 1971600 C. H. FANG~ 2-b2-eMrb\ 1h....i U4Acta Medica Okayama, Vol. 25 [1971], Iss. 6, Art. 2http://escholarship.lib.okayama-u.ac.jp/amo/vol25/iss6/2Rat Parabiosis with Aortic Anastomoses 601Fig. 1. Diagrammatic drawing of the parabiosis of rats with aortic anastomoses. a :transplanted aorta, b: abdominal aorta, c: heart, d: resected rt. kidney.Fig. 2. Diagrammatic drawing of the process of operationa): Site of incision for parabiosis. "T" indicates the site where the incision is made onlateral abdomen to open the peritoneal cavity.b): Abdominal aorta is separated from the surrounding tissues and the abdominal aortais clippd bl 2 clipes at (a) and (b), and dissected on the middle line. Then the lower cutend is connected with the aorta for transplantation. Then the aorta is clipped at (c) andthe clip (a) is removed. Next, rt. renal artery is ligated and the rt. kidney is cut off. Theupper cut-end of the abdominal aorta is conjugated with the free end of the transplantedaorta of the partner. Finally the clip (c) is removed.c): For the conjugation of aorta a steel canule, about 5 cm in length and 0.8 cm indiameter, is inserted into aorta, ligated with nylon fiber and cemented with Alone Alpha.a: transplanted aorta, b: abdominal aorta, c: steel canule.d): Conjugation of the upper cut-end of aorta with the aorta for transplantation.From the upper cut-end of aorta the needle is inserted into rt. renal artery and the needleend is pushed out through renal artery. Another end of the needle is inserted into the freeend of the transplanted aorta.e): End-to-end anastomosis was done, then the needle is pulled out from the renal artery,and the conjugated area is washed with saline by using curved injection-syringe, and theroot of the renal artery is closed by ligation. a: transplanted aorta, b: abdominal aortaof the partner, c: heart, d: It. kidney.£): Diagrammatic drawing of the parabiosis of rats with aortic anastomoses. a: trans-planted aorta, b: aorta, c: heart, d: resceted rt. kidney.Fig. 3. Relation between the rat age and the useful part of the aorta for transplanta-tion. (a)-(f); see te., t.Fig. 4. Picture of two aortae for transplantation. Taken from healthy adult rats justbefore transplantation and preserved in cold saline.Fig. 5. Picture of the parabiosed rats 3 days after operation.5Fang: Improvement of the method of rats parabiosis with aorticProduced by The Berkeley Electronic Press, 1971602 C. H. EANGon both transplanted aortae were removed first, followed by the removal of theclip. (c) on the aortae of both hosts (Fig. 2-e and Fig. 1). At the end of successfulprocedures the pulses of transplanted aortae were clearly observable. Fig. 5shows the parabionts with aortic anastomoses on third-postoperative day.DISCUSSIONA parabiosis made by aortic anastomoses developed by KAWAI is avery useful tool in the studies of hematology, immunology and othervarious fields of experimental pathology. Nevertheless, the originalKAWAI'S method proves to be very difficult to duplicate by the author.He used thoracic aortae of I to 2.month old rats for transplantation sincethe diameter of the thoracic aortae was almost the same as the diameterof the host abdominal aortae.The author recognized the difficulties in using thoracic aortae mainlydue to its short length and often resulting in splitting apart on thewalls of anastomosing aortae during the procedures. In addition to theseproblems, the diameter and the thickness of the walls of thoracic aortaeare not always similar to those of the host abdominal aortae. The author,therefore, attempted to improve the original method in respect to obtain-ing enough length of transplanting aortae and adjusting these diametersto the host abdominal aortae, as previously mentioned. The adjustmentof two aortae having different diameter was carried by making minorincision in adequate length on the smaller one. By this procedure, theaortae of various aged rats were anastomosed satisfactorily.The most useful parts of the aortae for transplantation were deter.mined depending on various ages of rats used and given as follows (Fig. 3):One to 2-month old rats: The part between the upper end of thoracicaorta (a) and just above the aortae at the origin of rt. renal artery (c).In this case both resected ends of aorta have the same inner diameter ofthree to four-month old abdominal aorta, as already described by KAWAI.Two to 3.month old rats: The part between the upper portion of theorigin of rt. renal artery (d) and upper end of the thoracic aorta (a).Three to 4-month old rats: The part between the upper portion of theorigin of the lumbal artery (e) and the middle portion of thoracic aorta(b).Four to 5-month old rats: The part between the lower portion ofabdomial aorta (f) and the lower portion of thoracia aorta (c).One to 2.month old rats supplied too short aortae, while other olderrats supplied aortae long enough to be well transplanted without formingthrombus or splitting apart the anastomosed aortae. This is the key to be6Acta Medica Okayama, Vol. 25 [1971], Iss. 6, Art. 2http://escholarship.lib.okayama-u.ac.jp/amo/vol25/iss6/2Rat Parabiosis with Aortic Anastomoses 603accomplished in this operation.KAW AI reported that the cross-anastomoses with polyethylene tubeswere not suitable tool because of vascular obstruction in the anastomosedportions within 24 hours. The author tried it again, and obtained thesame results. Further, the author tried the cross-anastomoses in the samedirection of two animals. It became clear that it's not a suitable methodbecauce of various technical difficulties. In addition to this, in this in.stance, no symmetrical blood exchanges could be obtained and it meansearly death of parabiotic animals.SUMMARYThis paper describes various aortic parabiotic procedures and discus.ses various problems concerning these procedures. The most satisfactoryresults, nearly 100 % of survival rate, can be achieved using longersections of aortae of 2 to 5.month old rats. In these rats blood circulationbetween the parabionts has completely been established.ACKNOWLEDGEMENTThe author is indebted much to Professor SATIMARU SZNO for valuable advices through-out this wor£ and painsta\ing proof-reading of the paper. Many thanks are also due to mywife for her encouragement and assistance and r; r. KAWAI for the demonstration of "aorticanastomoses" by his method.REFERENCES1. KAWAI, T.: Stem cell in peripheral blood: A study by parabiosis of irradiated andnon·irradiated animals; I. New approach to parabiosis as a method for hematologicstudy. Acta Med. Okayama 23, 257, 19692. BERT, P.: Greffe animale, Compt. rend. Soc. de Bioi. 15, 20, 18633. BERT, P.: Experience et consideration sur la greffe animale. j. Anat., Paris. 1, 69, 18644. SAUERBRUCH, F. and HEYDE, M.: Ueber parabiose kunstlich vereinigter WarmblUter.Munch. med. Wschr. 55, 153, 19085. EICHWALD, E.J., LUSTGRAF, E. C. and STEINER, M.: Genetic factors in parabiosis. j.Nat. Cancer Inst. 23, 1193, 19596. VAN DYKE, D. C., HUFF, R. L. and EVANS, H. M.: The efficiency of the vascular unionin parabiosis. Stanj. med. Bull. 6, 271, 19487. FLEMING, D. C., DALDWELL, L. and JACOBS, R.: Determination of rate of cross circula-tion in parabiotic rats with P32-1abeled erythrocytes. Amer. j. Physiol. 196, 753, 19598. DOMENICO, A. D. and ANDREOTTI, H. T.: Mechanism of splenic protection against ))Bartonella muris )) in the parabiotic rat. Amer. j. Physiol. 197, 795, 19597Fang: Improvement of the method of rats parabiosis with aorticProduced by The Berkeley Electronic Press, 1971

Improvement of the method of rats parabiosis with aortic anastomoses

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