1. For the purpose to obtain parabiotic rats having well maintained humoral circulation, the author observed parabionts having coerio-anastomosis and vascular anastomosis.
2. In the parabiotic rats having coerio-auastomosis when one of the parabionts was prevented from taking food and water by mouth sealing, the animals died within 5 to 6 days just as the control animals subjected to complete starvation, indicating that in coerio-anastomosis no appreciable humoral exchange was established between the two parabionts. 3. In vascular parabiosis having cross anastomosis of the aortas with polyethylene tubules, the animals died about 24 hours after the operation
because of blocking thrombosis formed in the polyethylene tubules. 4. In the vascular parabiosis having cross anastomosis of the aortas by the homologous thoracic aorta animals did not survive through the operation but those having parallel anastomosis of the aortas survived after the operation for 3 weeks at largest and they seem to serve as useful tool for the parabiosis experiment.</p

Kawai, Toru

Okayama University Scientific Achievement Repository

Acta Medica OkayamaVolume 23, Issue 4 1969 Article 1AUGUST 1969Stem cell in peripheral blood: a study byparabiosis of irradiated and non-irradiatedanimals. I. New approach to parabiosis as amethod for hematologic studyToru Kawai∗∗Okayama University,Copyright c©1999 OKAYAMA UNIVERSITY MEDICAL SCHOOL. All rights reserved.Stem cell in peripheral blood: a study byparabiosis of irradiated and non-irradiatedanimals. I. New approach to parabiosis as amethod for hematologic study∗Toru KawaiAbstract1. For the purpose to obtain parabiotic rats having well maintained humoral circulation, the au-thor observed parabionts having coerio-anastomosis and vascular anastomosis. 2. In the parabioticrats having coerio-auastomosis when one of the parabionts was prevented from taking food andwater by mouth sealing, the animals died within 5 to 6 days just as the control animals subjected tocomplete starvation, indicating that in coerio-anastomosis no appreciable humoral exchange wasestablished between the two parabionts. 3. In vascular parabiosis having cross anastomosis ofthe aortas with polyethylene tubules, the animals died about 24 hours after the operation becauseof blocking thrombosis formed in the polyethylene tubules. 4. In the vascular parabiosis havingcross anastomosis of the aortas by the homologous thoracic aorta animals did not survive throughthe operation but those having parallel anastomosis of the aortas survived after the operation for 3weeks at largest and they seem to serve as useful tool for the parabiosis experiment.∗PMID: 4243558 [PubMed - indexed for MEDLINE] Copyright c©OKAYAMA UNIVERSITYMEDICAL SCHOOLActa Med. Okayama 23, 257-263 (1969)STEM CELL IN PERIPHERAL BLOOD: A STUDY BYPARABIOSIS OF IRRADIATED AND NON.IRRADIATED ANIMALSI. NEW APPROACH TO PARABIOSIS AS A METHODFOR HEMATOLOGIC STUDYToru KAWAIDepartment of Pathology, Okayama University Medical School, Okayama, Japan(Director: Prof, S. Seno)Received for publication, April 30, 1969Parabiosis, the surgical union of two living animals, has been origi.nally developed as a valuable tool for the research 'of the transmission ofhumoral factors aud is widely used in a variety of research fields, e. g.hematology, immunology, endocrinology, etc. In the hematologic field,this technique has been applied for the research of the transmission ofcellular elements, e. g. the transmission of "Stem Cells" from non-irradia.ted to lethally irradiated animals (1).At preesent, we have three methods of parabiosis; skin anastomosiswith the union of skin only (BENT, 1863) (2,3) skin and muscle anastomosismeaning the union of both muscle and skin, and coerio·anastomosis withthe union of skin, muscle and abdominal cavities, SAUERBRUCH andHEYDE, 1908 (4) Among these the coerio·anastomosis is considered to bethe best in respect to the humoral communication between two animalsand is now most widely used. However, it is quite uncertain to what extentthe two animals may be kept under the well communicated humoralexchange condition by these methods.For example, the body fluid exchange test as estimated by observingthe distribution of various substances such as organic dyes, bacteria,nucleated red cells, red cells labelled with Fe~9, pS2, Cr~\ etc, introducedinto one of the animals often gave poor results (7, 8, 15, 16) a markeddifference was found in the cellular constituents of the peripheral bloodbetween non.irradiated and lethally irradiated parabionts jointed by coerioanastomosis; i. e. red cell and white cell counts of the normal partnerwere usually 2 times greater than those the irradiated one, though theaverage rate of cross exchange of blood was estimated as 0.64 per cent ofthe blood volume per minute (9). All these data indicate some incomple.teness of humoral and cellular communication between two parabionts2571Kawai: Stem cell in peripheral blood: a study by parabiosis ofProduced by The Berkeley Electronic Press, 1969258 T. KAWAIeven in the coerio-anastomosis. So, the author aimed to observe how longthe mouth-sealed rat would survive through the supply of nourishmentfrom the normal parabiont in coerio.anastomosis.I) Observations on the Mouth-Sealed Rat with Unsealed Partner in Coerio-Anasto-moszs:Fifteen adult male rats of Wister strain of about 200 g body weight,about 3 months old, were used. Ten of these animals were used for coerio-anastomosis, making 5 pairs of parabionts. The other five animals servedas controls having their mouths sealed. As the technique for the parabiosis,the method devised by BUNSTER add MEYER 1933 (10) was used. Eachanimal had his littermate as parabiont.Ten days after the union, the mouth of one animal was sealed com·pletely by suturing both lips to keep the animal from taking food andwater. In the five starving controls their mouth was sutured. The intactpartners were kept on standard diet (Oriental Kobo) and water, but all themouth-sealed parabionts died from starvation within 5-6 days after themouth suture, and one day later the normal par tner died. All the controlanimals also died 5 to 6 days after the mouth suture as in the case of themouth sealed parabiont. The results indicated that there developed noappreciable humoral communication between the two animals joined bythe coerioanastomosis.Then, the author tried to join two animals by vascular parabiosiswith arterial anastomosis so as to obtain the parabiotic animals having acomplete exchange circulatory system.II) The Rats Joined with Vascular Parabiosis by Using Polyethylene Tubules:Forty adult male rats of Wister of about 3 months old, 200-300 gbody weight, were used. With these animals 20 pairs of litter mate para-biotic animals having aortic anastomosis were obtained (Fig. 1).11 111 IVFig. I Schematic drawing of the procedure of the vascular parabiosisby polyethylene tubes2Acta Medica Okayama, Vol. 23 [1969], Iss. 4, Art. 1http://escholarship.lib.okayama-u.ac.jp/amo/vol23/iss4/1VascuIar Parabiosis 259To connect the aorta, polyethylene tubules of 1.5 mm in diameterwere used. Prior to the operation, the animals were anesthesized withsodium Nembutal (60 mg/ml Abbott) by injecting a diluted solution (l :10 vIv) intramusculary, 0.5 cc per 100 g body weight. The supposedpartners received lateral abdominal insisions, about 2.5 cm in length, leftor right, respectively, and then cross sections in the middle of the firstincisions reaching to the left and right lumbar muscle were made to form"T" sections. (Fig. I-I). Two animals were joined by suturing the skin ofthe dorsal sides of the wounds and then the lumbar muscle (Fig. I-II).The abdominal cavities were opened and the abdominal aorta wasexposed dissociating the retroperitoneum from the retroperitoneal tissues.Then the abdominal aorta was clamped with bulldog clamps at two pointsjust above the bifurcation of the iliac artery and just under the renalartery and transected in the middle (Fig. I-III). Then the heparinizedtubules of 2 cm in length were inserted first into the distal end of aortal inboth animals and ligated with 3-0 silk suture and the tubules were filledwith blood by slightly loosening the clamps. Then each tubule conitainingblood was connected to the proximal end of the aorta of the partners. Thetwo upper bulldog clamps were removed at the same time. The conjugatedvessels were covered carefully with the peritoneum and the muscles andskin were sutured.All the animals died within 24 hours and autopsy revealed the thrombo.tic obstruction of the polyethylene tubules.III) The Rats Joined with Vascular Parabiosis by Transplanting HomologousAorta:In this experiment 100 male 'Wister rats of about 3 months old weigh-ing 250-300 g were used. Parabiotic partners were of the same litter andchosen from those of nearly the same body weight, less than 10 g in differ-ence. The animals were divided into two groups; one group consisting of40 animals was parabiosed by cross anastomosis of the aorta by the routinemethod (Fig. 2A). and another group of 60 animals was parabiosed in theopposite direction by pararell anastomosis of the aorta (Fig. 2 B).The thoracic aorta from young rats of 30-40 days old served as graft-ing materials. Rats were sacrificed by decapitation, opening the thoracicand abdominal cavities, the lung, heart, liver and diaphragm wereremoved in order to expose the aorta. Then the abdominal aorta was cutoff at the middle point. A stainless needle core inserted from the cut endinto thoracic and arc aorta was slightly raised and each intercostal arterieswas ligated and cut off from the aorta in the order from the lower to theupper ones. Then the aorta was taken out and preserved in cold saline3Kawai: Stem cell in peripheral blood: a study by parabiosis ofProduced by The Berkeley Electronic Press, 1969260 T. KAWAI---Needle CannulaaFig. 3 Mode of anastomosis of thegrafting aorta with the distal end of thehost aorta by using needle coreFig. 2 Diagrammatic drawing of two ways of vascular parabiosisA: Cross anastomosis of aortaB: Parallel anastomosis of aortaand used within 30 minutes. Before use, the aorta was cut off at bothends, just under the subclavical branch and just above the coeliacal branch,and the thoraice aorta was used as the graft.A) The Rats Joined with Cross Anastomosis of Aortas: Twenty parabioticcouples were formed by this method. Prior to the operation the animalswere anesthesized by the method asdescribed in the foregoing anastomo-sis and they underwent the operationin nearly the same way as those in thevascular anastomosis with polyethy-lene tubules with some modification.After dissociating the retroperito-neum, the left kidney was removedafter ligating the left renal artery.Then the aortas were clamped at twopoints just as in the rats that had theaortic anastomosis with polyethylenetubules and transected at the middlepoint of the abdominal aortas. Theabdominal aortas were filled withheparin solution (100 units) and theneedle core having fresh aorta to begrafted was inserted into the distalend of aorta of the host so as to have4Acta Medica Okayama, Vol. 23 [1969], Iss. 4, Art. 1http://escholarship.lib.okayama-u.ac.jp/amo/vol23/iss4/1Vascular Parabiosis 261the ends of two aorta come in close contact, then loosening the clamps (Fig.3). The junction of two aortas was sutured at two points by 8-0 nontrau.matic monofilament nylon suture under the binocular stereomicroscopeand the cemented with Alon Alpha (SANKYO Co), avoiding the adventitialtissue to turn into the inner part of the aorta.Finally, the needle cores were removed and the grafted aortas clamped.In the similar way into the proximal end of the aorta was inserted theneedle core through the cut end of the left renal artery. The end of theneedle core was further inserted into the end of the aorta just grafted tothe partner animal (Fig. 4) and the suture of the aorta was made just asR.n.Dex.Fig. 4 Diagramatic picture of the anastomosis of the aorta of theparabionts by grafting a homologous thoracic aorta. The anastomosis iscompleted by removing needle core and ligating the left renal arterydescribed. Eventually the needle core was removed and the renal arterywas ligated. In the same way another end of aorta was anastomosed withthe proximal end of the partners aorta by means of another fresh aorta-graft. Covering the operated part of the aorta with the retroperitoneum,the muscle and skin were sutured. In this series of experiments all theanimals died about one hour after the operation, probably because of theshort length of grafted aorta, resulting in the serious disorientation of theaortas and attached vessels and organs. No thrombotic change was observedexcepting the case of technical errors which resulted in the turning of theadventitial tissue into the inside of the anastomosed aorta.B) The Rats Jointed with Parallel Anastomosis of Aortas: For this seriesof experiment about 70 animals were used forming more than 30 parabioticcouples.5Kawai: Stem cell in peripheral blood: a study by parabiosis ofProduced by The Berkeley Electronic Press, 1969262 T. KAWAIThe animals were ·anesthesized and operated as in the cases of thoseof group A, however, the animals were placed in the opposite direction.In this situation, the proximal end of the aorta of one animal came closerto the distal end of the aorta of the partner, making it easier to anastomosethem, in spite of the limited length of the thoracic aortas to be grafted.After completion of the vascular conjugation, the two grafted aortas wereput parallel in close contact, covered with the peritoneum, and the muscleand skin were sutured leaving only a narrow space for the grafted aortasso as to keep the intestine into the individual abdominal cavities, which isnecessary to avoid accidental ileus seen occationally in coerio-parabiosis.In this group, the animals survived throgh the operation for morethan 3 weeks. And they died by developing symptoms of the so-calledparabiotic intoxication (5, 13, 14, 15, 16) but not directly by the ill effect ofvascular anastomosis itself.C) Grafting of aorta: In a few animals the abdominal aortas wereremoved and grafted with homologous thoracic aorta by same method asthose in groups A and B. The animals survived through the operationquite well. Observations over 30 days after operation proved successfultransplantation without any rejecting reaction.Through these observations, it seems that the parabiotic rats havingparallel anastomosis of aorta serve as useful tools for the parabiosis ex-periment.SUMMARY1. For the purpose to obtain parabiotic rats having well maintainedhumoral circulation, the author observed parabionts having coerio-anasto-mosis and vascular anastomosis.2. In the parabiotic rats having coerio-auastomosis when one of theparabionts was prevented from taking food and water by mouth sealing,the animals died within 5 to 6 days just as the control animals subjected tocomplete starvation, indicating that in coerio-anastomosis no appreciablehumoral exchange was established between the two parabionts.3. In vascular parabiosis having cross anastomosis of the aortas withpolyethylene tubules, the animals died about 24 hours after the operationbecause of blocking thrombosis formed in the polyethylene tubules.4. In the vascular parabiosis having cross anastomosis of the aortasby the homologous thoracic aorta animals did not survive through theoperation but those having parallel anastomosis of the aortas survived after6Acta Medica Okayama, Vol. 23 [1969], Iss. 4, Art. 1http://escholarship.lib.okayama-u.ac.jp/amo/vol23/iss4/1Vascular Parabiosis 263the operation for 3 weeks at largest and they seem to serve as useful toolfor the parabiosis experiment.ACKNOWLEDGEMENTThe author is much indebted to Professor SATIMARU SENO for his invaluable advicesthroughout this work and painstaking proof reading of the paper. Many thanks are also due toMiss MASAKO HIRATA for her technical assistance and the members of the Pathology Depart-ment for their valuable suggestions.REFERENCES1. TYLER, R. W. C. and N. B. EVERETT: A radioautographic study of hemopoietie-repopulation using irradiated parabiotic rats; Relation to the stem cell problem. Blood,28, 873, 19662. BERT, P.: Greffe animal. Compt. rend. Soc. de Biol. 15, 20, 18633. BERT, P.: Experiences et considerations sur la greffe animal. ]. Anat., Paris. 169,18644. SAUERBRUCH, F., and HEYDE, M.: Ueber parabiose kunstlich vereinigter WambluterMunch. med. Wschr. 55, 153, 19085. RANZI, E. and H. EHRLICH: Ueber die Wirkung von Toxinen und die Bildung vonAntikorpern bei parabiotischen Tieren, Z. Immunitasforsch. 3, 38, 19096. CRISTEA, G. M. and W. DENK: Beitrag zur Parabiose. Med. Klinik. 6, 146, 19107. DRAGSTEDT, L. R. and E. F. COOPER: Parabiosis in the study of defficiency diseases.Amer. ]. Physiol. 67, 48, 19238. DIEPENHORST, M. J. and O. M. de VAAL: Hematological change in parabiotic rats.Acta Physiol. pharm. neerl. 1, 342, 19509. VAN DYKE, D. C., R. L. HUFF and H. M. EVANS: The efficiency of the vascular unionin parabiosis. Stamf. med. Bull. 6, 271, 194810. BUNSTER, E., and R. K. MEYER: An improved method method of parabiosis. Anat.Rec. 57, 339, 193311. M. DALE CHASE, SEYMOUR I. SCHWART:<::, and CHARLES R.: A technique of smallartery anastomosis. Surg. Gyneco. Obst. 116, 381, 196312. JACOBSON, JULIUS H., SUAREZ, E.: Microsurgery in anastomosIs of small vessels. SurgicalForm. Clinical Congress 1960. Vol. XI, p.243, Chicago, American College of Sugeons,196013. EICHWALD, E. J., E. C. LUSTGRAAF and M. STRAINER: Genetic factors in parabiosis.]. Nat. Cancer Inst. 23, 1193, 195914. EICHWALD, E.J., E. C. LUSTGRAAF and R. B. FUSON: The anemia of parabiotic intoxica-tion. Ann. N. Y. Acad. Sci. 87, 119, 196015. RAYMOND, A. McBRIDE, and MORTEN SIMONSEN: Cellular and humoral phenomenaduring the inductive phase of parabiosis tolerance. Transplantation. 3, 140, 196516. G. A. GRANGER, R. S. WEISER, and BEULAH HOLMES.: The pathogenesis of acuteallogeneic disease in mice studied by parabiosis. Transplantation. 3, 524, 19657Kawai: Stem cell in peripheral blood: a study by parabiosis ofProduced by The Berkeley Electronic Press, 1969

Stem cell in peripheral blood: a study by parabiosis of irradiated and non-irradiated animals. I. New approach to parabiosis as a method for hematologic study

Stem cell in peripheral blood: a study by parabiosis of irradiated and non-irradiated animals. I. New approach to parabiosis as a method for hematologic study

Abstract

Similar works

Full text

Available Versions

Okayama University Scientific Achievement Repository

Okayama University Digital Information Repository