OMENTAL TRANSPLANTATION and CELL CULTURE.
by: ROSENDO CRIOLLOS, M.D.
A thesis submitted to the faculty of Graduate Studies and Research in partial fulfillment of the requirements of the Master of Science Degree.
Department of Experimental Surgery, McGill University, MONTREAL. APRIL 1964. (i)
P R E F A C E.
The tremendous progress in medicine, especially in cardiovascular surgery during the pBst few decades bas promoted development of measures for the control and cure of various anomalies and diseases by surgical means. While the controversy over the different procedures of revascularization for an ischaemic heart still continues, the rate of surgery in the management of the occlusive coronary artery disease is widely accepted; as James Bryce so ably said, WMedicine is the only profession that labours incessantly to destroy the reason for its own existence". If this be true allow me to thank Dr. Arthur Vineberg for letting me be one of the labourera in his investigations on free omental grafts as a method to revascularize the ischaemic heart. For the 1~ years that I have expended in the field of research under his supervision I wish to extend my appreciation of his thought provoking discussions on the problems encountered throughout this investigation that lead me to develop a method of scientific thinking. These studies afforded me the opportunity to explore more fully the vascularization activities of the omental tissue in re-establishing itself while set free in ectopie environments which resulted in the finding of a (ii)
three day minimum of such free omental grafts to become revascularized. The work certainly has roused my interest and enthusiasm in the importance of experimental medicine, enabling me to complete the training in general surgery with better perspective and understanding. It has been a pleasure and enjoyment to do this work in the fascinating fields of cardiac revascularization, tissue transplantation and tissue culture. Here I must express my gratitude to Dr. D. R. Webster, Chairman of the Department of Experimental Surgery, for permitting this work to be carried out in the laboratories, his interest in cardiac revascularization and kindly spirit. To the Director of Research, Dr. S. c. Skoryna, for his gracious being and willingness to help has always been a source of inspiration. The candidate wishes to thank Dr. A. M. Masson, Associate Professer of the Department of Bacteriology, McGill University for her enthusiastic suggestions and guidance on the tissue culture techniques. Special acknowledgement is due to Dr. W. J. Pirozynski, Associate Professer of Pathology, for his interpretations and criticism of the slides as well as the unflogging zeal with which he supported the pursuit of this work. (iii)
I am indebted to Dr. S. M. Ban~ill, Pro~essor of Anatomy, for the opportunity of relearning my anatomy while a demonstrator to the first year medical students at the University.
I am obliged to Mr. Nagy and his staf~ for their assistance in the operating rooms and care of the dogs. To Mr. Farrell and Mr. s. Byers ~or the eare o~ the small animals. To Mr. C. Hodge and Mr. P. Morin for their photographie work and Mr. J. Szarmany ~or radiologieal aid. I am grateful to Miss N. Johnston, librarian at the Medical Sehool, for her valuable help in the review of the literature and supply o~ copies from libraries abroad and to Miss Nicole Pineau in the preparation of the microscopie sections. I wish to thank Miss B. Murrell for her devoted time and effort in the preparation of this manuscript. Miss c. Fraser deserves a special thanks for her assistance am the typing of my seminar. Finally the candidate wishes to extend his sincere gratitude ~or the interest, ideas and support rendered to me by all members and colleagues of the Department, especially to Dr. D. W. Edwards whose eriticism and talent was weleome and Dr. T. M. Paul whose adviee and ideas were most helpful. (iv)
I owe much to Dr. Alan A. Kane of Brooklyn, New York, it would be incomplete without expressing my indebtedness for the opportunities and encouragement given to me during the course of my surgical residency training under his supervision.
R. CRIOLLOS, M.D. -: TABLE OF CONTENTS .-
Page No.
PREFACE ...... (i)-(iv) CHAPTER I: INTRODUCTION and PURPOSE •••••••••••••••••••• 1.
CHAPTER II: REVIEW OF LITERATURE • • • • • • • • • • • • • • • • • • • • • • • • 6. CHAPTER III: EXPERIMENTAL STUDIES and RESULTS ••••••••••• 16. EXPERIMENT I: Free Omental Transplants in body cavities and subcutaneous tissues •••••••••••••••••••••••• 17. Series 1: Intraperitoneal free omental grafts • • • • • • • • • • • • • • • • • • • • • • 18. Series 2: Intrapleural free omental grafts. 19. Control studies of transplants with other tissues basides omentum ••••••••••••••••••••• 20. Series 3: Intraperitoneal free grafts of omentum, muscle and fat ••••• 22. Series 4: Intrapleural free grafts of omentum, muscle and fat ••••• 24. Series 2: Subcutaneous free omental grafts 26. Series 6: Intrapericardial free omental grafts •••••••••••••••••••••• 28. EXPERIMENT II: Free Omental Transplant in Diffusion Chambers ••••••••••••• 33. Part 1 : In the Transparent Chamber in the dorsal skin of the mouse. 34. Part 2 : In the Anterior Chamber of the Rabbit's Eye •••••••••••••••• 47. EXH:RIMENT III: Omental Cell Culture •••••••••• 57. 1. In Natural Media. 2o In Artificial Media. 3. Cell Suspension Method. Results •••••••••••••••••••••••• 64 (Table of Contents- contd.) Page No.
EXFERIMENT IV: Phagocytic Properties of Free Omental Grafts ànd Mast Cella ••••••••••••••••••••• 70. EXFERIMENT V: Cardiac Revascularization with Free Qnental Grafts and Chemical Epicardiectomy •••••••• 73.
Methods • • • • • • • • • • • • • • • • • • • • • • • • • • • 75. Control Series ••••••••••••••••••••
Resulta • • • • • • • • • • • • • • • • • • • • • • • • • • • 82.
CHAPTER IV: C CHAPTER V: SU~~RY •••••••••••••••••••••••·••••••••••••·••••• 88. CHAPTER VI: CONCLUSIONS ••••••••••·••••••••••••••••••••••••••• 96. CHAPTER ~ BIBLIOGRAPHY ••••••••••••••••••••••••••••••••••••• 98. -: TABLE OF FIGURES :- Fig. Page No. No. 1. A Free intraperitoneal omental graft, 25 days old • • • • 19 2. Same omental graft with its attachments •••••••••••••• 19 3. A free intrapleural omental graft, 25 days old ••••••• 21 4. A free intrapleural omental graft with its attachments . • ...... • . • ...... • • . . • 21 5. Photomicrograph of Rat No. 24 with 25 days of intrapleural free omental graft ••••••••••••••••• 23 6. Photomicrograph of section of intrapleural free omental graft •••••••••••••••••.••••••.•••..••.•• 23 7. Photomicrograph of section of intrapleural free omental gra ft ...... 2 5 8. Rat with intraperitoneal 3 grafts, omentum, muscle and fat ...... • . . . • ...... • ...... • • • • 25 9. Rat with intrapleural 3 grafts, omentum, muscle and fat ...... • ...... 27 10. Subcutaneous free omental graft, 4 weeks old • •••••••• 27 11. Subcutaneous free omental graft, 6 weeks old • • • • • • • • • 29 12. 5 Free omental grafts inside pericardium of dog •••••• 29 13. Same grafts after injection with Schlesinger Mass • • • • 31 14. Section of sorne grafts showing the mass and blood cells in a large omental vessel ••••••••••••••••• 31 15. Intrapericardial free omental grafts attached to internal mammary vessels and heart •••••••••••••• 32 16. Cham ber parts • ...... • ...... • . . • • . • 3 5 17. Diagram of a cross-section of chamber and graft •••••• 35 18. Mouse with assembled chamber, front view ••••••••••••• 37 19. Mouse with assembled chamber, back view •••••••••••••• 37 (Table of Figures - contd.) Fig. Page No. No. 20. Diagram of 18 chambers in mice ...... 40 21. Chamber with 3 omental transplants, 5 days old •••••• 43 22. Same chamber, same grafts, 10 days old •••••••••••••• 43 23. Photomicrograph of a section of omental graft in the chamber . . • • ...... • . . . . . • . 45 24. Diagram of technique of intraocular transplantation.. 45 25. Intraocular omental graft, 3 days old ••••••••••••••• 52 26. Intraocular omental graft after author's injection •• 52 27. Photomicrograph of section of injected intraocular graft • ...... • . 53 28. Close-up of same graft to see dye in omental vessels. 53 29. Photomicrograph of blood supply of iris after injection ...... 55 JO. Photomicrograph of blood supply of iris and graft.... 55 31. Contents of Medium No. 150. ••••••••••••••••••••••••• 65 32. Leighton Tube with omenta1 exp1ants • • • • • • • • • • • • • • • • • 66 JJ. Fibro-b1ast 1ike ce11s growing from explants, 2nd day of incubation •••••••••••••••••••••••••• 66 34. Same exp1ants after 10 days of incubation • • • • • • • • • • • 68 35. Tubes with exp1ants at 3, 6 and 9 days of incubation •...... •...... •...... •. 68 36. Cat's omentum after intraperitonea1 injection of carbon suspension •••••••••••••••••••••••••••••• 71 37. Photomicrograph of intraperitonea1 free omenta1 graft treated with intraperitonea1 injection of carbon suspension ••••••••••••••••••••••••• ·•• 71 38. Mast cel1s in omenta1 tissue •••••••••••••••••••••••• 74 39. Diagram of free omenta1 graft operation ••••••••••••• 74 (Table of Figures- contd.} Fig. Page No. No. 40. Ameroid constrictor on branches of left coronary artery • • ...... • 77 41. Ameroid constrictor on right coronary artery •••••••• 77 42. Chemical epicardiectomy ...... 78 43. Chemical epicardiectomy • •••••••••••••••••••••••••••• 78 44. Heart after being epicardiectomized ••••••••••••••••• 79 45. Root of aorta after cleaning ••••••••••••.••••••••••• 79 46. Free omental graft around the heart ••••••••••••••••• 81 47. Photomicrograph of section of omental graft after injection- 8 weeks with 3 ameroids •••••••••••• 81 48. X-ray of dog's heart after injection with Schlesinger ma ss ••••••••••••••••••••••••••••••••••••••••••• 84 -1- CHAPTER I. INTRODUCTION AND PURPOSE •No research is ever quite complete. It is the glory of a good bit of work that it opens the way for something still better, and thus rapidly leads - to its own eclipse. The object of research is the advancement, not of the investigator, but of knowledge.• quoted by: SIR ALEXANDER FLEMING, M.D. (1881 - 1955}. Revascularization of the ischemie myocardium is one of the major concerna in the field of experimental surgery. The increasing disability and death from obliterative disease of the coronary arteries, which all too frequently attacks men in the prime of life whose other organs are still intact and healthy, has stimulated widespread interest in the field of medicine to produce sorne means of increasing the blood flow to the ischemie myocardium. The causative factors in the genesis of coronary occlusive disease is still far from clear. The death usually occurs due to one or a combination of the following sequelae: 1. Cardiogenic irreversible shock. 2. Congestive heart failure. 3. Ventricular fibrillation. -2- 4. Cardiac aneurysm and rupture. 5. Perforation through infarcted inter ventricular septum. 6. Disruption of papillary muscles usually in mitral insufficiency. Those cases who escape fatal issue probably do so because they have developed intercoronary anastomotic channels. But in the vast majority of patients this new network of vessels is either inadequate or not sufficiently developed to be of value in relieving an area of ischaemic myocardium. Medical treatment has been mostly directed at relief of symtoms. As yet there has been no medication which has Qeen effective in permanently correcting an area of myocardial ischaemia. Thus, patients with coronary artery disease are prone to suffer from repeated attacks of myocardial infarction with progressive diminution of their activities. Failure of medical measures to relieve myocardial ischaemia has resulted in the extensive investigations to do so by surgical means. Established procedures for correcting occlusive arterial diseases elsewhere in the body, such as embolectomy and thromboendarterectomy have been of little value in the coronary arteries. This is because in the majority of instances there is diffuse involvement of the coronary arteries by disease and the probability of recurrence is -3- great. Therefore the tendency of investigators is to search for a rational treatment of coronary artery disease through the establishment of a new blood supply from extracoronary sources. Following this line of thinking Beek in the United States, O'Shaughnessy in England and Vineberg in Canada are among the pioneers who have tried to revascularize the heart from extracoronary sources. Beek in 1929 attempted for the first time to supply more arterial blood to the heart by producing pericardial adhesions between the pericardium and the myocardium. O'Shaughnessy applied a pedicled omental graft to the heart muscle which he brought through the diaphragm creating a cardio-omentopexy. Vineberg's internal mammary artery implant operation has been demonstrated to be of permanent value, especially when the disease is limited to the left coronary arteries. Reports of the Ivalon Sponge operation are encouraging in cases with diffuse involvement of the coronary arterial system and in patients with ventricular failure. Application of the sponge to the right and left posterior ventricular surfaces however are technically difficult. This was the reason for the development of the free omental graft operation which is more easily applied and establishes extra cardiac arterial communications with -4- the circulation of both ventricles. The purpose of this investigation is to study the basic nature and intrinsic properties of omental tissue with particular reference to its ability to grow a new vascular network. Knowledge of these properties may well permit its use in the surgical correction of vascular insufficiency in various parts of the body. With the above objective a series of experimenta both in vivo and in vitro have been designed to study the eventual fate of autologous free omental grafts. The grafts were studied after being placed in various body cavities without producing any inflammation or traumatisation of the surfaces in these locations. Furthermore the established survival rate was compared with that of other tissues which were simultaneously placed in the areas mentioned. These autotransplants of living omental tissue were also studied in subcutaneous transparent diffusion chambers in the mouse and in the anterior chamber of the eye of the rabbit in order to observe under direct vision its inherent vascularizing property. In vitro investigations of omental explants using tissue culture methods were also carried out to determine the nature of omental growth and to find out something about its nutritional requirements. The phagocitic properties of -5- the mast cells of the free omental tissue were tested in an attempt to elucidate the functional viability of the graft. Finally studies were done to revascularize the heart under experimental coronary insufficiency with the use of free omental grafts. -6- CHAPTER II. REVIEW OF THE LITERATURE. HISTORY: The omentum has been compared, because of its deposits of fat along the blood vessels to a Victorian lace curtain, and it was used to ornament the carcasses of sheep hanging in butchers' shops. Reference has been made in the German literature to the vast network of vessels and perforated interstices as "Netz". The word "omentum" may be a derivation of the Latin "omen", since the ancients were said to have derived prophetie inspiration from inspecting it. The origin of the great omentum is said to be due to an excessive redundancy to the left and downward of the dorsal mesogastrium. Its anterior and posterior lamellae at first are each composed of two leaves of peritoneum that easily fused so that in adult life the pouch of the lesser peritoneal cavity usually extends no lower than the greater curvature of the stomach and the posterior layer is fixed to the transverse rn esocolon. There are considerable variations in size, attachments and distribution. The giant salamander is the first vertebrate to posses it. It is well developed in ------~------~ -- -~ -- ~~ -7- carnivorous animals whereas birds have only a rudimentary organ, except birds of prey which have an excellent omental development on account of their liability to develop peritonitis as a result of their combative existence, or secondary to rupture of the intestines from swallowing bones. It is completely absent in fish. In man there are considerable variations in length and fat content, depending on age, sex or race. It can be absent, have congenital clefts, fenestrations, accessary omenta or be divided into two or more portions. McWorter (1928) referred to a case of intestinal obstruction produced by a bifid omentum. Draper (1931} felt such omental aberrations may interfere with proper duodenal function. The functions of the omentum have always been a mystery and have excited interest since earliest times. Hippocrates (460 B.C.) taught that the omentum in sorne way was concerned with the regulation of the amount of free fluid in the peritoneal cavity, either by secretion into it or absorption from it. Aristotle (322 B.C.) regarded it as a fatty apron designed to protect the abdominal viscera from cold. Galen (130 A.D.) was the first to describe the omentum but it was not until 1512 when Vesalius noted its attachments and regarded it as a ligament to support the transverse colon. Fabricius (1537) believed it was an apron spread out in front of the stomach in order to facilitate its movements. Malpighi (1628) felt that besides being the source of fluid in ascites, _g_ the omentum was an important store house for fat. Verhagen (1648) claimed that it was for protection of the intrabdominal organs from friction. Hansen (1S41) believed that it served to keep the loaded stomach downward in order to facilitate the respiratory movements. Morison (1906) called the omentum the "abdominal policeman" stating that "the greater omentum travels about in the abdomen with considerable rapidity and is attached by sorne sort of information to neighbourhoods in which mischief is brewing. It may effect a radical cure of a hernia by blocking the hernial orifice. It surrounds and adheres to a recently reduced and strangulated loop of intestine and may keep it alive and prevent a leak. It is generally found in the neighbourhood of a diseased appendix, by wrapping it up if gangrenous, or blocking up the pus from an appendiceal abscess, it may prevent general peritonitis. In a similar manner it may prevent the perforation of an ulcerating malignant growth of a gastric ulcer or the death of a damaged portion of bowel or the perforation of a suppurating gall bladder. Its effective mobility is shown by the fact that whether the lesion be in the diaphragmatic roof of the abdomen or the floor of the pelvis, there the omentum can and does find its way." However, studies of Florey and Carlenton (1926) indicated that the movements of the omentum were entirely -9- extrinsic depending on the posture, gastrointestinal peristalsis and diaphragmatic excursions, denying any intrinsic ability to move. Wilkie (1911) found that the cat's resistance to the intraperitoneal injection of staphylococci was remarkably diminished in those in which the omentum had been removed. Crile {1914) said that the omentum was of immense utility in abdominal shock through the spleno-pleura system and it renders assistance during gastric and intestinal digestion, by retaining large quantities of blood within the circulation which slowly feeds fresh blood into its structure as required. Crouse (1915) advanced his opinion that the nerve supply of the omentum is from the solar plexus, the right and left semilunar ganglia from which direct distribution is made to it and believed the function of the omentum was somewhat like that of the spleen since embryologically both have analogue relationships. C. H. Mayo (1916} advocated an operation for the prevention and healing of intestinal fistulas with the use of omental grafts and pointed out that the omentum contained a high percentage of cella of the defensive repair type and that in response to intra-peritoneal injections it became heavily infiltrated with phagocytes and thought that such a response would be beneficia! to patients undergoing certain -10- abdominal operations. More recently Mandl (1944) successfully treated a cerebrospinal fistula in the skull by a tab of omentum, such a fistula was closed within 48 hours. The way in which the omentum will phagocyte foreign bodies made Albrecht christen this organ as the "abdominal leucocyte". Bergen and Rankin {1928) carried out pre-operative intraperitoneal vaccination using cultures of B. coli and streptococcus, in patients undergoing bowel resection for carcinoma. These studies marked the beginning of the work on the immunalogical potentialities of the omentum. In 1955 Roberts showed that the omentum is an important site of antibody formation when the route of administration of the antigen was intraperitoneal. More recently Walker {1960) suggested the plasma cells as the site of antibody formation. Also considerable numbers of mast cells have been discovered in the omentum which are easily demonstrated by their intense metachromatic staining with toludine blue. This metachromasia was considered to be due to heparin in the cells, however, it now appears that the granules also contain histamine and 5-Hydroxytryptamine (Serotonin). Though the functions of these cells are not well known they have been noted to become degranulated in sorne anaphylactic reactions (Florey 1925, 1954 and Fancett 1955}. Schutz (1930) found that migration of the omentum is controlled and caused by migration of the omental arteries and besides the abundant blood supply of the omentum it posseses a delicate meshwork of connective tissue bearing minute blood vessels, practically empty but "potential" vessels when needed. -11- The use of free omental grafts in abdominal surgery dates back to the time of Nicholas Senn in 1887 using them to reinforce his intestinal anastomosis. Bennett (1896) used it for the first time as a plug ta seal a perforated gastric ulcer and then Clogg (1905) successfully closed a perforated duodenal ulcer with an omental graft. Springer (1910) demonstrated that the entire omentum may be severed from its attachment and yet not become gangrenons. The hemostatic effect of the free omental graft has been known for sorne time. Lowe (1906) reparted controlling haemorrhage of the liver with free omental grafts. Hesse (1912) declared by experimenting upon thirty cats, that he was able to check haemorrhages from the liver, spleen, gall bladder bed and urinary bladder by transplanting omentum and that within four days vascular formations could be demonstrated in the transplanted omentum. Grouse stated that these experiments proved the ability of the detached omentum to assume increased circulation. Previously De Renzi and Boeri (1903} had published the results on which the splenic vessels were ligated in cats and found in autopsy living spleen tissue vascularized by the omentum. The se studies were confirmed by Wilkie who suggested the omentum to be wrapped around a strangulated portion of the intestine on which the patients condition was unfavourable for resection and anastomosis. Also Morison (1906), Cullen (1907) reparted clinical cases of large uterine fibroids or ovarian tumeurs on -12- which their entire blood supply derived from enlarged and engorged omental vessels instead of the uterine supply. Neuhof and Wiener (1910) revealed that the omentum and particularly the free_ edge, bas the power of being insinuated in a needle hole puncture and finding attachment there. They described a peritoneal suture to avoid peritoneal denudation exposed to the omental surface. Corbett (1916} in his studies on peritoneal adhesions found that the persistance of omental adhesions to suture lines were due to definitely organized new blood vessels furnished by the omentum to devitalized areas. Larkin (1940) advocated removal of the omentum as a safeguard against recurrence of adhesions painting out that the medical literature does not reveal that human subjects having little or no omentum have an increased susceptibility to peritonitis. On the subject of detached omental grafts, Davis (1917) experimentally transplanted free grafts and reported that such transplants irrespective of their size, showed remarkable vitality and persisted with little change whether transplanted free, subcutaneously or in the peritoneal cavity. Finton and Peet (1919) and then Mann (1921) experimentally used omentum to control haemorrhages from the liver, spleen, pancreas and kidney with success. They also -13- found it very effective in replacing lost peritoneum, to patch an opening in the G-1 tract, to strengthen suture lines in the intestines, stomach, or in the ureter, to occlude a portion of the G-I tract, to cover the stump of the cystic duct or fallopian tube, to reinforce the peritoneum in threatened perforations and to prevent adhesions. They advised that the raw edges of the omental graft itself should be turned in to prevent adhesions. The last worker favoured pedicle rather than free grafts. Talma and Morison at the beginning of this century both sutured the omentum (omentopexy} to a~arified surface of the peritoneal wall in order to establish an anastomosis between the portal system and vena cava for the cure of ascites in Banti's disease. Lanz (1911) transplanted the testicle and spermatic cord into the peritoneal cavity and surrounded these structures with omentum as a means of increasing faster, more collateral circulation for the relief of portal hypertension. Pemberton (1945) recommended the placing of the omentum in the splenic bed after removal of the spleen for splenic anemia. He also inserts the omentum to different depths in the abdominal wall ascribing the establishment of new collateral channels for the decrease of portal hypertension. McKechnie in Canada (1948} plastered the omentum to a denuded portal vein and gallbladder bed creating a Porto-Hepato-Omentopexy in cases of portal hypertension secondary to cavernous transformation -14- where portal or splenic vein shunts are of no value. From Puerto Rico there is a report by Passa1acqua (1955) revealing four cases of bleeding esophagea1 varices treated by resection and esophagogastrostomy plus transposition of the omentum into the mediastinal space, as a modification of the posterior mediastinal packing operation of Garlock {1947) to deve1op larger collateral channels between the portal and systemic venous system and then Logacheva {1957) attached the omentum to the kidney producing a nephro-omentopexy for the relief of hypertension. Based on the arterialization properties the omentum has, O'Shaughnessy (1936) attempted to provide the heart with an alternative blood supp1y by tacking the omentum to the myocardium with its vascular pedicle in the abdomen. Experimental and clinical work gave him grounds for supposing that a collateral circulation could be set up in this way. His early death and experiences of other investi- gations developing serious secondary sequelae such as diaphragmatic hernias, intestinal obstruction etc. induced them to abandon this procedure as standard technique. It was in 1958 when Knock revived the O'Shaughnessy cardio-omentopexy in a modified form. She developed multiple omenta1 loops, finger-like, from a pedicle brought through the diaphragm and implanted them in the myocardium, attempting to revascularize its deeper layers. -15- She reported that just as the omentum can gain a new blood supply from the chest wall, pericardial sac and lung, it can also gain a new blood supply directly from the root of the aorta. Vineberg (1962) designed an operation to promote the growth of new coronary arteries, using a detached omental graft in the surgical relief of myocardial ischaemia and uses it as a supplementary means to revascularize both ventricles with the implantation of the internal mammary artery into the left ventricular wall, in cases of generalized coronary disease. -16- CHAPTER III EXPERIMENTAL STUDIES and RESULTS. -17- EXPERIMENT I. FREE OMENTAL TRANSPLANT IN BODY CAVITIES AND SUBCUTANEOUS TISSUES: The following studies were done to determine whether detached omental grafts would be capable of surviving in the absence of an inflammed surface. Materials and Methods: The first five series of experimenta were carried out in male rats of Royal Victoria Hospital strain, approximately twelve weeks old, with an average weight of 240 grams. For series 6 with intrapericardial free omental grafts, adult mongrel dogs of both sexes were used, weighing between JO and 40 pounds. The rats were anesthetized with intra peritoneal injections of Sodium Pentobarbital 6 mgs. per 100 grams weight and the dogs by intravenous injection in the doseage of 25 mgs. per Kg. of body weight. The procedures were performed under aseptic conditions. Small sections of autogenous omental tissue were detached from the greater omentum through a small laparotomy incision and then set loose within the peritoneal pleural and pericardial cavities as well as in the subcutaneous tissues. -18- SERIES 1: INTRAPERITONEAL FREE OMENTAL GRAFTS. (10 Animals}. In this group of rats the grafts, taken from the free edge of the omentum, were dropped into the peritoneal cavity, as far away as possible from the laparotomy wound. Twenty five days later the rats were sacrificed and the grafts studied. RESULTS: Two of the animals died from peritonitis. The other eight rats when studied showed the omental grafts grossly vascularized and were found with tongue-like protrusions (Figures 1 and 2} attaching them firmly to two or more different organs or to the abdominal wall. Histological1y central necrosis occurred in three grafts. This was not present in the other five transplants. In 62.5% of the animals the grafts survived. SERIES 2: INTRAPLEURAL FREE OMENTAL GRAFTS. (10 Animals). The omental grafts were placed in the pleural cavity of the rats through a lower small thoracotomy incision. -19- FIGURE I: A free intraperitoneal omental graft, found grossly vascularized in a rat sacrificed on the 25th day. FIGURE II: The same graft removed from the rat showing firm attachments to mesentery, small bowel and abdominal wall. -20- The animals were sacrificed on the average of twenty five days after transplantation. RESULTS: In all cases the grafts appeared healthy and were found intimately attached either to the lung or chest wall, pericardium or diaphragm. (Figures 3 and 4). Numerous good size vessels coursing apparently from the graft to the tissues of ' the host were grossly noted in seven animals. Under microscopie examination there were signs of necrosis in three of the grafts. One had marked foreign body reaction due to contamination with hair and the rest showed minimal inflammatory reaction with considerable vascularization and good fixation to other tissues. The cells of the pleura were no longer identifiable in most of the specimens. The cells of the detached omental graft appeared intermingled with thos of the sub-pleural structures; this occurred in four grafts. (Figures 5, 6 and 7). In this study 70% of the grafts were viable. CONTROL STUDIES OF TRANSPLANTS WITH OTHER TISSUES BESIDES OMENTUM. -21- FIGURE 3: An intrapleural free omental graft found healthy and intimately attached to pericardium, diaphragm and chest wall in a rat sacrificed on the 25th day. FIGURE 4: An intrapleural free omental graft in situ, attached to chest wall and grossly vascularized. -22- SERIES 3: INTRAPERITONEAL FREE GRAFTS OF OMENTUM, MUSCLE AND FAT. (9 Animals). To rule out an inflammatory reaction (necrosis) set up by the free omental graft as the reason for its adherence to the liver, small and large bowel, spleen, mesentery etc; a group of free autogenous transplants of omentum, muscle and fat were carried out in the following manner. A piece of omentum, a piece of rectus abdominus muscle, and from the groin of the rat a piece of fat tagged with a silk suture, were simultaneously dropped into the peritoneal cavity of nine rats. The grafts were placed there: a) The muscle of the left ileac fossae; b) The fat in the right upper quadrant; c) The omentum in the right ileac fossae. The animals were sacrificed twenty five days lat er. RESULTS: It was observed that: a) The free muscle transplants were seen either free or encased by peritoneal folds in the same location of transfer. They were all both grossly and.microscopically necrotic. -23- FIGURE 5: Photornicrograph of rat No. 24 with 25 days of intrapleural free omental graft. It shows the intirnate attachment of the graft to the lung. FIGURE 6: From the same animal, a photomicrograph of a section of the graft showing the association of healthy omental graft with intercostal muscle. ------24- b) The free fat transplants were in all cases reduced in size and found with poor attachment to other tissues in the right upper quadrant or middle abdomen. Histologically all had necrosis. c) Four of the nine detached omental transplants were microscopically viable and attached to different organs (Figure a); they were histologically vascularized. The survival of the omentum in this series was 44.4%. SERIES 4: INTRAPLEURAL FREE GRAFTS OF OMENTUM, MUSCLE AND FAT. (13 Animale). A piece of omentum, a piece of pectoralis muscle and a piece of fat from the groin were all inserted through a small thoracotomy incision into the left lower pleural cavity of thirteen rats. They were studied on the average of twenty five days after surgery. It was observed that: a) All free muscle transplants were necrotic and had little or no attachment to other tissues, except when the muscle graft happened to come in contact with the free omental graft. -25- FIGURE 7: Photomicrograph of section of omental graft placed freely for 26 days in the pleural cavity of Rat No. 20. It shows viable ornental tissue intimately associated with the heart tissues. FIGURE 8: Animal with intraperitoneal free grafts of omentum, muscle and fat sacrificed 25 days later. Left ruler shows muscle partially necrotic, middle ruler shows fat poorly attached and reduced in size and right ruler with grossly vascularized omental graft. -26- b) The free fat transplants were noted to be necrotic, and were completely absorbed in six cases. c) Of the thirteen intrapleural omental trans plants studied here, eight specimens were found alive, fully vascularized and adherent to one or two structures such as the lung and chest wall, pericardium, thymus or even to the great vessels. Histologically they had a take with minimal inflammatory reaction. {Figure 9 ) • The survival of the omental graft was 61.5%. SERIES 5: SUBCUTANEOUS FREE OMENTAL GRAFTS. (12 Animals). The operation was carried out on rats, in which the detachèd omental grafts were buried in the subcutaneous tissues of the back of the animal. A group of five rats were sacrificed at two, four and six weeks after the procedure. RESULTS: It was noted that the grafts did well and appeared viable in the majority of cases, though their bulk seemed to be slightly reduced in five cases studied at six weeks. Here again the omental -27- FIGURE 9: Rat with 3 intrapleural grafts, sacrificed 25 days after surgery. It shows the omentum attached to the chest wall partially necrotic and muscle and fat completely necrotic. FIGURE 10: Subcutaneous free omental graft, animal sacrificed after 4 weeks. Well preserved omentum. tissue was microscopically well preserved and intimately associated with the subfascial tissues (Figure 10 and 11). Four of the grafts showed necrosis. The vascularization was reafirmed by injecting in vivo the abdominal aorta with India ink solution. The dye was observed in the blood vessels of ten detached omental grafts. (Figure 20). The survival rate of the omental grafts buried in the subcutaneous tissue was 73.2%. SERIES 6: INTRAPERICARDIAL FREE OMENTAL GRAFTS. (9 Dogs). For this study five pieces of free omentum, one inch square and weighing about two grams each, were introduced through a small thoracotomy incision into the pericardial cavity of each animal. One dog died from lobar pneumonia at ten days after the transplantation and the rest of the animals were sacrificed four and six weeks after. RESULTS: The intraperitoneal, intrapleural and subcutaneous transplants have demonstrated that the free omental grafts have the ability of -29- FIGURE 11: Subcutaneous free omenta1 graft p1aced for ~ weeks in the rat. ~ ----- 1 • FIGURE 12: 5 Sections of omentum 1" square, 2 g. each 1eft in the pericardium of dog free. Animal died of pneumonia 10 days after. NOTE pieces of omentum attached to themse1ves and to pericardium and coronary vesse1s. -30- penetrating in some way the different serosae and fasciae in order to obtain their blood supply.• It was presumed that the application of free omental grafts into the pericardial cavity will result in the penetration of blood vessels through the epicardium and fibrous pericardium. However, the results did not show intimate attachment of the grafts to the smooth epicardium or pericardium as was expected but a striking feature was that the five pieces of omentum had attached themselves to one another to form one piece which in turn had itself become attached to the pericardium, and coronary vessels (Figure 12). Such grafts cannot always be separated easily from the coronary vessels and would ooze blood on attempted separation. Histologically there was necrosis in two of the specimens. This was not true of the six intrapericardial omental grafts which were studied at four and six weeks after trans- plantation; although the bulk seemed to have been slightly reduced. The result of injection in the coronary arteries has revealed Schlesinger mass in the free omental grafts in two of the animals (Figuresl3 . and 14). -31- FIGURE 13: Pericardium and fused pieces of omentum from same dog after injection. Dye present in ~"~mental tissue. FIGUl\.:..., .,4·: The same grafts showing Schlesinger Mass in a large omental vessel. -32- In this group of experiments it was frequent1y observed that the omenta1 grafts showed a tendency to become attached to the base of the great vesse1s and origins of coronary vesse1s. In one case the upper end of the pericardectomy was found open and the omenta1 grafts were hanging do~m from the interna1 mammary vesse1s to the epicardium and gross1y vascu1arized. (Figure 15). FIGURE 15: Intrapericardia1 free omental grafts attached to interna1 mammary vesse1s and heart. -33- EXPERIMENT II. FREE OMENTAL TRANSPLANT IN DIFFUSION CH~ffiERS. Part 1: In the Transparent Chamber in the Dorsal Skin of the Mouse. Part 2: In the Anterior Chamber of the Rabbit's Eye. This work so far has described experiment5 in which sections of autologous detached omental tissue can survive when transplanted loosely into the peritoneal, pleural, pericardial cavities and in the subcutaneous tissues of rats and dogs by rapidly acquiring an adequate blood supply. The free omental graft tissue viability was noted and survival compared to other tissues recorded. The desire to obtain a direct observation of growth, behaviour and in particular the vascularization of the free omental grafts in vivo, fragments of autologous omental tissue were transplanted into a transparent chamber in the dorsal skin of the mouse and in another series of experimenta in the anterior chamber of the eye of the rabbit. -34- PART 1: SERIES 7: FREE OMENTAL TRANSPLANT IN THE DORSAL SKIN CHAMBER OF THE MOUSE. Sandison (1924) designed a chamber in the rabbit's ear for visualizing living tissue. Clark (1930) further modified the technique and created the bay-chamber, round table chamber and preformed-tissue chamber. Algire and Leggallis (1949)'designed a chamber in the dorsal skin of the mouse for the study of tumors. Joslin (1952) revamped this deviee by fashioning a slint. More recently Conway, et al (1957} extended the chamber for the study of skin grafts. The transparent windows that were used in this work were made according to those described by Sabet and co-workers (1961) for the study of embryonic whole bane and Ellis' method (1962} in the study of periosteal transplants. Materials and Method: There are four main parts needed to assemble the chamber (Figure 16): a) a telfon sheet about 100 x 50 mm. and 0.1 mm. thick, b} two lucite rings, front and back -35- FIGURE 16: Chamber parts and pre-assembled chamber: a) Teflon; b) front ring; c) back ring; d) cover slip; · e) pre-assembled chamber. FIGURE 17: Diagram of a cross-section through chamber and graft. a) Teflon; b) skin· c) subcutis; d) glue; e) cover slipt fj suture; g) front ring; h) back ring; iJ omentum. - F F A 8 C 8 A R . C. ~ op,) --~==~------36- measuring 19 mm. outside and 12 mm. inside diameters and 1 mm. thick, with five superimposable holes, c) A circular glass cover slip 15 mm. The parts were sterilized chemically by soaking in 1 : 1000 solution of Zephiran Chloride. They were rinsed in sterile saline before use. The mice were anesthetized with intra peritoneal injection of sodium pentobarbital (3 mg./100 gram body weight). Then the back of the animals were first depilated, then rinsed and painted with Zephiran solution. The midline over the dorso-lumbar region was pulled up, making a fold, permitting flexibility without restricting the respiratory movements and legs of the animal. The fold of skin was maintained in its retracted position by means of three bulldog clamps, and then carefully removed while the teflon sheet was glued to the skin fold with rubber cement. Two lucite rings were superimposed and sutured together with silk sutures which were placed through the five openings along the rim -37- FIGURE 18: Mouse with assembled chamber. Front View. FIGURE 19: Same animal. Back View. Back ring sealed with permanent cement. Well tolerated by the mouse. -3$- of the rings. Before tieing the sutures, lucite cement was placed between the rim of the rings and the teflon sheet following which the sutures were tied. This glue will prevent the rings from • being too loose and will prevent air bubbles from developing in the chamber thus interfering with visualization of the bed and the graft. Gare was taken to not tie the sutures too tightly in order to avoid strangulation of the blood vessels between the rings and hence make the preparation useless. By means of a circular incision made in the inner circumference of the· fro~ ring, the teflon sheet, the skin, subcutaneous tissues and fascia all were excised exposing the fascia and subcutanèous tissues of the opposite side. The bed was rinsed with Hanks' balanced salt solution and then the cover glass slip was placed over and sealed with rubber cement (Fi~re 17 and lg). Two days following this procedure, the cover slip was removed and fragments of omentum were rernoved through a small laparotorny incision -39- and then chopped to aval shape of about 1 mm. in size and were then placed on a major vessel in the bed. Two minutes later the chamber was filled again with Hanks balanced salt solution admix with antibiotics. The caver slip was replaced and sealed with permanent cement (Figure 19). Aseptic technique was practiced throughout. Over fifty preparations were carried out in the course of this work with the chambers. They were observed under a regular microscope at different intervals with magnification up to 100 diameters (Figure 20). Higher magnifications with the dissecting microscope or by removing the epidermis from the opposite side to permit better transillumination were not used. During each microscopie examination it was necessary to anesthetize the mice. EXAMINATION OF CHAMBERS: During these experimenta it was observed that :- 1) Within a few minutes the omental grafts became adherent to the bed (perhaps by -40- FIGURE 20: Diagrams of là Chambers. Left representa a major vessel selected for site of transplantation. Right same chamber with the omental grafts and new vascularization scen with naked eye between . 2 and 4 days. M_9 M_11 M_12 M_13 M.;..16 -41- sorne intrinsic exudate from the omentum) {made of white blood cells and fibrin according to Crouse). 2) No intrinsic movemen~s on the grafts were noted, but in sorne cases where 2 or 3 grafts were auto-transplanted in the same chamber and placed not too far apart, they were found to have attached themselves to one another and survived (Figure 21}. 3) When the size of the transplants exceeded 3 mm. in length they underwent central necrosis and sloughed off within 2 days {perhaps this tissue needs a high concentration of oxygen for better survival, or as has been stated (Woodruff) that thin grafts are more resistant to infection). 4) From the transplants that took in all sorne shrinkage was noted (perhaps due to regression of traumatized cells and deficient effective nourishment from the bed). 5) The lost tissue of the grafts that had shrunk -42- was not subsequently replaced by regeneration from the surviving remnant (this may suggest that the omentum, once traumatized, will not regenerate). 6) The grafts that became established and survived, occurred within 4 days after transplantation (this was confirmed by the changes in the vascular pattern of the bed following grafting, and vessels were seen macro and microscopically filled with blood in the graft). It is assumed that these vessels were already in the graft and they may become anastomosed with those in the bed, perhaps this representa a new set of vessels growing in or out of the graft. All we presume is that this circulation was effective, as proven by the injection of India ink solution into a vein of the tail or in the inferior vena cava in vivo. The dye within minutes coloured the vascularized bed and in a couple of animals appeared to have reached the graft (Figure 22). Likewise -43- FIGURE 21: A 5 day old chamber with three omental transplants. It showes the vascularized bed, only one large omental transplant with new set of vessels in different direction. FlGURE 22: Same chamber 10 days old. The mouse was injected in vivo through the inferior vena cava. The dye has coloured the vascularized bed and graft. -44- this assumption was supported by the histological studies of the grafts which revealed well preserved omental cells ~nd JUSt sorne cell infiltration. (Figure 2J). 7) The grafts that took were observed to be healthy up to 10 or 12 days. From then on the degree of vïsibility was lessened and did not permit the events within the graft to be detected any longer due to alteration in the fluid content of the chamber, or due to the coverage of the graft by regenerative host tissues. Sorne times the graft survived for a time and then destroyed itself leaving no trace or a residual scar tissue or necrotic patch. This chamber did not seem to offer the best media for nourishment, temperature, oxygenation and longer survival for direct observation of the omental transplants. 8) The omental cells were readily killed by drying or prolonged exposure to light and warmth and bacterial contaminants which thriv~d inside the chamber. -45- FIGURE 23: Photomicrograph of a section of a transplant 5 days in the chamber. NOTE well established omental graft. FIGURE 24: Technique of intra-ocular transplantation. (Modified by Woodruff). -46- Roughly 50% of the grafts were lost due to mechanical difficulties or infection. 9) Certain other hazards still exist with the use of these chambers. The assembly and introduction of these chambers is a time consuming procedure requiring a high degree of mechanical and surgical skill. Considerable practice was necessary before such dexterity could be achieved. -47- PART 2: SERIES 8: FREE OMENTAL TRANSPLANT IN THE ANTERIOR CHAMBER OF THE RABBIT'S EYE. The anterior chamber of the eye has proven to be an ideal site for tissue transplantation. The technique of transfer is simple, a high percentage of takes is obtained, the transfer tissue can be followed by direct visual observation or even subjected to microscopie examinations and supports the growth of heterologous grafts whereas, in other body sites such tissues fail to survive. (Green 1941}. The iris is apparently unresponsive to the presence of foreign bodies. The transplant derives nourishment from the acqueous humor while seeking for its own blood supply. The inflammatory reaction which follows the introduction of foreign tissue into other regions of the body does not occur. (Green 1942). This method has been extensively used and perfected by Green in the study of homologous and heterologous transplants of -48- embryonic tissues and human tumors and by Medawar in his study of autografts and homografts of skin. Materials: Forty five adult rabbits of both sexes with black or pink eyes, weighing 2 to 2.5 Kg. were used throughout this investigation. The animals were fasted for 16 hours and were maintained under ordinary conditions of cage life after the transplantation. Method: Through the marginal vein of the ear, the animals were anesthetized with 25 mg. of Sodium Pentobarbital per Kg. This was supplemented with ether anesthesia when needed. Through an aseptic small laparotomy incision a segment o~ omentum was excised and the incision closed in 3 layers. The omentum was placed in Hank's salt solution and small fragments of about 4 mm. in diameter were prepared. The eye globe was steadied by a hook in a fold of conjunctiva and then the anterior chamber of the eye was opened by penetrating the cornea inwards and close to -49- the !imbus (sclero-cornea junction) with a single small stab made with the tip of a fine cataract knife, avoiding injuring the iris by making outward pressure. A small amount of fluid was allowed to escape. With a fine curved forceps the omental fragment was tucked through the slit and then with the finger outside the cornea manouvered into the opposite side, to prevent it escaping through the wound or slipping behind the iris into the posterior chamber. The corneal incision closes after withdrawal of the inserting forceps and further care was unnecessary except in few instances when the slit was too long, in this case sutures of fine plain catgut were needed to prevent herniation of the iris and infection. Two drops of an ophthalmic antibiotic were applied to the eye at the end. Most of the animals had both eyes transplanted using the same technique (Figure 24). The animals were sacrificed at different intervals, from one day to fifty six days. To prove that there was a patent blood supply in the grafts, an injection technique -50- was developed. The injection fluid consisted of 20 cc. of India ink (particle size 24 milli microns), 5 ozs. of warm 10% gelatin and 20 cc. of 10% formalin. 30 cc. of thesolution was then injected into each carotid artery via a canula. The animal was frozen for 24 hours. This procedure prevents the India ink from being washed off during histological preparation of the tissue. RESULTS: Seventy five intraocular omental trans plants were macro and microscopically studied. It was observed :- 1) The omental eye transplants could not be moved by applying gentle pressure through the cornea after twenty four hours (possible sign of viability). 2) The pale white-yellowish colouration of the grafts became light pink and shiny with a smooth surface on the 3rd day (possible sign of vascularization). 3) When two transplants were placed fairly close together in the same chamber, the -51- fragments became attached to each other and vascularized. 4) A peculiar observation was that sometimes grafts with previous observed vessels placed with reverse orientation to the radial vessels of the iris, shift their position to have a parallel position with those radial of the iris. 5) No increase of size in the graft was observed. On the contrary slight shrinkage was noticed during the period of vascularization, none became completely necrotic nor disintegrated, even in the presence of sorne infection. 6) The grafts were supplied with large vessels which could be seen with the naked eye to transverse the surface of the graft. (Figure 25 ) • 7) Takes were usually evident on the )rd day and subsequent progress in vascularization was rapid. 8) The maximum degree of vascular proliferation in most grafts was reached from the 6th day to the lOth day of transplantation and -52- FIGURE 25: Typical picture of intraocular omental graft, 3 days after transplantation. Large vessels could be traced with the naked eye. FIGURE 26: Dye solution injected through the carotid artery was recovered in the vessels of the omental graft. / ' -53- FIGURE 27: Photomicrograph of section of intraocular graft in an injected rabbit. NOTE the intimate attachment of the graft to the iris which contains vessels, full of dye ·as well as in those of the omentum. Rx>r attachment to the cornea which has no blood vessels • • FIGURE 28: Close up of same slide, showing major blood vessels in the omental graft with the dye and healthy omental cells. -....----:--- ..,....------54- generally they remained unchanged afterwards. Sometimes no blood vessels were seen even though the graft had a healthy appearance and colour. 9) On the 6th day blood vessels with active blood flow within the graft could be seen with a magnifying lens. 10) Vessels within the graft were observed at the earliest on the 3rd day where previously were none. These proved to be patent by the injection technique. India ink was found in the grafts from the 3rd day. Maximum dye was observed microscopically in many vessels after 6 days. {Figures 46 and 27). 11) In sorne preparations the tortuous and spiky vessels within the graft could be traced clearly and they were found to lead directly to the vessels of the iris, sclera and even to the external ocular muscles leading to the main ciliary arterial trunks which supply the iris. 12} The grafts were strongly attached to the iris and could not be separated from this structure without tearing. -55- FIGURE 29: Photomicrograph of control iris of rabbit No. 401 treated with the author's injection. It shows its normal blood sunply. FtGURE 30: Same rabbit, opposite eye with omental graft. The graft is attached to the iris. The injection clearly visualized on right side of dotted line NOTE the outgrow of new vessels in the omental graft • • -56- 13) Histologically the omental cells were mostly well preserved with minimal inflammatory reaction, no sign of regression or degeneration (vacuolization, cysts, granulation or carnification) was detected·. The attachment to the outer face of the iris or in the angle between the iris, sclera and cornea was intimate (vascularized surfaces), but the attachment to the corneal endothelium was most of the time weak. (Figure 28). 14) Intraocular transfer of autografts of omentum resulted in almost lOO% takes. Necrosis in sorne were believed to be due to faulty technique and infection. 15) Microscopical preparations of iris and attached omental grafts treated with new injection technique demonstrated well the new network of vessels reaching out from the omental grafts. -57- EXPERIMENT III. OMENTAL TISSUE CULTURE IN VITRO. 1. In Natural Media. 2. In Artificial Media. 3. Cell Suspension Method. Because the previous studies in this work did not fulfil all the pre-requisites concerning the growth, regeneration and nutrition of free omental grafts, a long series of studies were carried out under Dr. A. M. Masson's supervision at the department of Virology at McGill University in an attempt to culture cells of omental tissue in vitro and obtain more information about their intrinsic behaviour, making use of natural media such as plasma clot, chick embryo extract and host animal serum; also in defined media like balanced saline solution, Medium No. 199 and Medium No. 150 and finally by the cell suspension technique. Tissue Culture Historical: The invention of the tissue culture technique was introduced by Harrison (1907) based -58- on his observations of growing nerve fibres from a frog embryo in clotted lymph. He was further aided in this work by adhering to the concepts of internal cellular environment (milieu interieur) of Claude Bernard (1878). Since the isolation and growth of the medullary plate of a chick embryo in warm saline by Roux (1$85), many living tissues have been cultured in vitro, such as: connective tissue cells, epithelial cells, cartilage cells, bone cells, nerve cells, muscle cells, reticular cells from bone marrow, lymph nodes, spleen tissue, leucocytes from the blood and tumor cells. The majority of which may be derived either from the embryo or the adult tissues. Among the various pioneers who contributed a great deal to the development and improvement of the culture media were Lewis, Maximow, Champy, Levi, Strangeways and later Eagle, Morgan, Morton, Parker, Sanford and others. Carrel (1914) published his study of the growth of connective tissue cultured in vitro. The new cells were identifiable up to 34 years and kept showing -59- their original morphology, such cells were called fibroblasts. Now it is well understood that fibroblast like cells as a group appear similar. However, although their external appearance may be uniform regardless of the site of origin, the internal characteristics such as nutritional requirements, functional properties and reaction to dyes may be quite different, but are entirely dependant upon their sites of origin, whether they were derived from embryonic or adult tissues. Role of Tissue Culture in Medicine: The method of tissue culture has contributed to science :- a) In Experimental Embryology: To elucidate problems concerning the development of organs by dDferentiation of cells like the ovary, teeth, thyroid and parathyroid, the eye, kidney, salivary glands, hair etc. For instance, heart muscle cells have been observed to acquire striations and even intercalated dises and to start contracting in vitro (Burrows 1912). -60- b) In Virology: By isolation ~nd recognition of new viruses (Enders' work 1945) at the Boston Childrens Iviedical Centre opened a new era in virology by obtaining a propagation of polio virus relatively clean in cultures of extraneural tissue from human embryos. The adenovirus group, recovered from adenoid tissue culture by Hilleman and Wenner (1954) causing different respiratory diseases and the enterovirus group recovered from the intestinal tract in persons with aseptic meningitis syndrome. c) In Immunology: Bx the recognition of the site of antibody production and synthesis in the bone marrow, liver, lymph nodes, thymus and omentum (Suter 1952, Thorbecke 1953, Walker 1960 and Walf 1958), using radioactive tracer techniques, it was found that aminoacids may be the precursors of antibody formation. d) In Bacteriology: By recognizing new nutritional requirements essential for cell growth in cultures like aminoacids by Eagle (1955) -61- glucose, vitamins, minerals, co-enzymes etc. by Morgan (1955) and supporting proliferation substances like serum and chick embryo extract by Sanford (1952). e) In Cancerology: By testing chemotherapeutic agents against cancer. Much of this work was done by Biesele (1957) who investigated the carcinolytic activity of about 300 compounds in vitro for sarcoma cells. Araong them were purines, purinenucleosides, benzimidazoles, antifolics and aminoacids. f} In Biochemistry: By the protein synthesis of cells with aminoacids or proteins. For instance Gerarde (1952) found that the heart tissue had a low rate of aminoacid turnover and that 25 per cent of embryo extract was needed to stimulate protein synthesis and growth. g) In Preventive Medicine: By the development of the poliomylitis vaccine by Salk and collegues (1952) who were able to produce a high yield of poliovirus by growing them in monkey kidney tissue cultures, without nerve tissue antigen and non-virus antigens of any kind. After treating the viruses -62- with formalin and reinoculating monkeys to test for residual infectivity they produced antibodies against each of the three poliovirus types and vaccines. Sabin {1959) developed a live, orally given polio virus vaccine, by 1960 he published the effects of rapid mass immunization on people (Toluca, J).1exico} with prevalent enteroviruses which have been found to interfere with the establish ment of attenuated polio vaccine. The results showed the overcome of the inter fering effect of other viruses present in the alimentary tract with his type of vaccine and method. h) In ExRerimental Histologv: By testing tissue viability of different grafts in vitro. TISSUE CULTURE: The omentum can be described as a trabeculated connective tissue framework of fat cells in a special mesh of abundant blood vessels and lymphatics. Two types of cells are recognized that can be grown in vitro from connective tissues a) Fibroblast-like cells which form a -63- loose meshwork in which the cells may not be intimately associated with one another and, b) Epithelial-like cells that grow in a continuously developing sheet in which the cells tend to arrange themselves in a mosaic (Parker). With this in mind, seeking to learn more about the mechanism of nutrition and cell growth of the omental grafts, it was decided to make many series of explants of omental tissue from the rat and rabbit and test them in different culture media according to the latest techniques and developments of tissue culture. METHOD: The omental tissue was chopped into fine fragments and transferred to Petri-dishes containing Hank's balanced salt solution, to wash the blood clots and debris from them. Then two of the now cleansed explants were placed on the cover slip of the culture tubes. After a short period of time the explants became fixed to the cover slip which were previously sprayed with plasma clot. Subsequently a drop of chick embryo extract was -64- added on top of each explant. Finally 2 cc. of a culture medium consisting of 10 per cent normal animal serum and 90 per cent of a mixture of M-150. Penicillin and Streptomycin were added to give a final concentration of 100 units and 200 mgs. per 100 cc. respectively. The tubes were incubated at 37°C. in a stationery position. The tube cultures were re-fed by replacing the medium with fresh nutrient solution once or twice a week. Each series contained twenty tubes. Strict asepsis was maintained throughout. RESULTS: During the course of these experimenta it was found that:- 1) The combination of natural media such as chick embryo extract, plasma clot and 10 per cent of animal serum and the aid of a synthetic medium like Morgan's Media No. 150 (Figure 31), better than M-199, offered the best suitable medium to grow omental tissue in vitro at incubation of 37°C. in Leighton tubes with cover slip (Figure 32). -65- MORGAN, CM1PBELL AND MORTON'S MEDIUM. No. 150 (1955) •. Milligrams Milligrams per 1000 ml. per 1000 ml. L-Arginine 140 Pyridoxine 25 L-Histidine 40 Pyridoxal 25 L-Lysine 140 Niacin 25 L-Tyrosine 200 Niacinamide 25 DL-Tryptophan 40 Pantothenate 10 DL-Phenylalanine 100 Biot in 10 L-Cystine 100 Folie Acid 10 DL-Methionine 60 Choline 500 DL-Serine 100 Inositol 50 DL-Threonine 120 p-Aminobenzoic Acid 50 DL-Leucine 240 Vitamin A 10 DL-Isoleucine $0 Calciferol (Vit. D) 10 DL-Valine 100 Menadione (Vit. K) 10 DL-Glutamic Acid 300 a-Tocopherol Phosphate 10 DL-Aspartic Acid 120 (Vit. E) DL-Alanine 100 Ascorbic Acid 50 L-Proline $0 Glutathione 50 L-Hydroxyproline 20 Cholesterol 10 Glycine 100 Glucose 1000 Cysteine 100 Sodium Acetate 166 Adenine Sulphate 100 L-Glutamine 100 Guanine 10 Adenosine Triphosphate 27 Xanthine 11.~. Adenylic Acid 10 Hypoxanthine 10 Ferric Nitrate 36 Thymine 10 Ribose 100 Uracil 10 Deoxyribose 100 Thiamin 10 Phenal Red 40 Riboflavin 10 Sodium Bicarbonate 1400 * This medium also contains a balanced salt solution and antibiotics. (FIGURE 31). -66- FIGURE 32: Leighton tubes with omental explants and media. __ _j FIGURE 33: Fibroblast-like cells outgrowing from omental explant. 2nd Day of incubation • • -6?- 2) In general the outgrow of cella from omental tissue was scanty, but more new cella were obtained from rabbit's omentum that rat's. 3) On the second day of incubation fibroblast-like cella could beJseen outgrowing from the e~ges of the explants and cell dividing in the majority of the tubes. Such growth reached a peack after ten days of culturing and then gradually became degenerated, pigmented, separated and disintegrated at the end of fourteen days (Figures 33 and 34). 4) The rapid changing of colour in the tubes that were not contaminated, may suggest the active metabolic rate with high concentration of waste products that this tissue may have. (Figure 35). 5) Attempts to sub-culture the new cells failed due to the low number and quality of cells. 6) Attempts were made to deliver more oxygen to the explants by placing the tubes in • rotating drums for better oxygen distribution, this made it possible to obtain better growth in fewer days but also the meshwork -68- FIGURE 34: Fibroblast-like cells outgrol'ting from omental explant. lOth day of incubation. FIGURE 35: Tubes with omental explants at 3, 6 and 9 days of incubation. NOTE different colour of media. A A -69- of fibroblasts became more rapidly disintegrated with the movements by the end of seven days. 7) Attempts to trypsinize omental tissue and to obtain dispersed omental cells (cell suspension) resulted in poor tissue digestion. 8) Addipose has been always a problem in cell culture. Direct delivery of a current of oxygen to the culture tubes may change this and c~use better proliferation of omental cells. EXPERIMENT IV. PHAGOCYTIC PROPERTY CF FREE OMENTAL TRANSPLANT AND MAST CELLS. (3 Cats). C. H. Mayo (1917) described the omentum as a fatty structure with numerous vessels capable of great distention and storage of blood, many glands and large lymph channels traversing its fat, indicating that it is an organ of extensive absorbent capacity. The following experimenta were carried out . to demonstrate the phagocytic absorptive power of the omentum which it retains even when detached :- Twenty cc. of a carbon suspension were given to a cat intraperitonealy. Twelve hours later the animal was sacrificed and only a few remnants of carbon were found in some pre-pyloric lymph nodes. Another cat received the same amount of carbon but was sacrificed six hours after the injection. The omentum had become jet black -71- FIGURE 36: A cat's omentum 6 hours after intraperitoneal injection of carbon suspension. FIGURE 37: Photomicrograph of section of intraperitoneal free omental graft in a cat treated with carbon suspension. -72- {Figure 36), while the rest of the peritoneal 'surface and mesentery appeared of normal colour. Gentle pressure did not remove the carbon particles. . The omentum was removed from another cat and fragmented into ten pieces. All were dropped freely into the peritoneal cavity. A daily injection of carbon suspension was given to the cat until the sixth day when it died. The grafts were found agglomerated into three pieces, attached firmly to loops of small bowel, spleen and mesentery. Microscopie examination showed the same black masses outlining, blood vessels in the free omental graft. (Figure 37). A section of omental tissue was treated with Toluidine blue to demonstrate the many mast cells which the omentum possesses. (Figure 38). -73- EXPERIMENT V. OMENTAL MYOCARDIAL REVASCULARIZATION: The possibility that free omental grafts could be used for the surgical relief of ventricular myocardial ischaemia due to multiple coronary disease has been considered. Vineberg has devised an operation to promote the growth of new coronary arteries by using a detached omental graft (Figure 39). The free omental graft apparently acts as a vascular network between vessels in the ischaemic heart and systemic vessels. Numerous new vessels rapidly grow from the aorta into the omental graft and then to the cardiac muscle, promoting intercoronary anastomosis. The following basic steps for the omental graft operation for myocardial revascularization were outlined by Vineberg. 1. Removal of the epicardium from both ventricles. 2. Removal of the pericardium and pleura 1 1 1 1 +- -.....J -.....J r r CD CD (J) (J) 1:'=:1 1:'=:1 (J) (J) P> P> (J) (J) s:: s:: CT> CT> (J) (J) ~ ~ ct ct 1-'• 1-'• s s •• •• ~ ~ f f ct ct ...... 1-'· 1-'· CT> CT> • • c:: c:: 0 0 ...... 0 0 ::tl ::tl ct ct "Xj "Xj H H 0 0 ...... w w 100. 100. 1 1 1 1 1 1 1 1 i i ' ' 1 1 : : 1 1 ' ' l I . . " " ~ ~ 'i \ \ . . .. /-" /-" 1 1 ('" ('" J J /" /" ~ ~ \ \ . . ~ ~ - v· v· ~ . . / / .. .. ...... , ,. ,. ., ., l l . . , . . j j • • j "!" "!" c c ( ( • • ' ' • • 1!1'!> 1!1'!> r r . . ~ ~ 1 1 1 1 1 1 j j . . 1 1 1 1 CD CD 1:'=:1 1:'=:1 l-I) l-I) 0 0 ~ ~ Il> Il> "Xj "Xj 11 11 ct ct 1-'• 1-'• c:: c:: 1-t) 1-t) CT> CT> 11 11 ::tl ::tl .. .. H H 0 0 ...... 0 0 0 0 ...... • • 'd 'd >S >S o o C:::OQ C:::OQ t:ICT> t:ICT> CT> CT> ~Il> ~Il> CT> CT> a>S a>S 1-'•11 1-'•11 ~g ~g S::ll> S::ll> o'ct o'ct 1-1)1-t) 1-1)1-t) li li li li 110 110 (J) (J) • • ctll> ctll> S::OQ S::OQ • • ::ret ::ret 0 0 Oll> Oll> 1111 1111 '-t:l '-t:l groft groft in in on on ils ils graft: graft: meet meet ce ce or or s s s s s s t t l l ) ) l t new new a s ostinal ostinal e arter':J arter':J i c i omentum omentum s el f f f of of y y o es omentol omentol vessels vessels myocordial myocordial vesse v med vesse vess ome.ntal ome.ntal enter enter constr arter iol iol coronary coronary ed ed blood blood growth growth rd rd (cornposed (cornposed left left ca ca nd nd vessels vessels ameroid ameroid a a detoch from: from: peri peri myocardial myocardial coronory coronory a a 3) 3) (1) (1) (2.) (2.) blood blood ( omentum omentum 1=2romote 1=2romote to to using using h h · wit ~v\ ~v\ and and groft groft de~igned de~igned vessels vessels orferies orferies actual actual moss moss on on omentol omentol blood blood from from of of cells cells inger inger s QP-eroti coronor'::J coronor'::J blood blood schle showing showing section section drawing drawing -75- from the base of the aorta and pulmonary arteries and removal of the serous layer of the fibrous pericardium. 3. Removal of the omental graft through a small incision in the diaphragm. 4. Opening the free omental graft to a thickness of one layer. 5. Wrapping the entire heart with the detached omentum. 6. Anchoring the graft with a few sutures to the denuded base of the aorta. ?. Closing the pericardium. CARDIAC REVASCULARIZATION WITH FREE OMENTAL GRAFTS AND CHEMICAL EPICARDIECTOMY. 9 Dogs. This study was carried out in adult mongrel dogs. Each weighed between 40 and 50 pounds. The animals were anesthetized with Pente barbital Sodium 6% given intravenously. The dosage was 30.mg. per Kg. of body weight. METHOD: One side of the chest was shaved. An endotracheal tube with occlusion cuff was inserted and then connected to a bird assister, to provide -76- a method of pulmonary ventilation as well as a means of re-expanding the lung at the end of the operation. The left side of the chest was entered through the fifth interspace from the sternum anteriorly to the line of the transverse processes of the vertebrae posteriorly. A longitudinal pericardiectomy anterior and parallel to the left phrenic nerve was done. The arterial coronary dissection was then carried on, with care being taken to assure that all these major trunks were dissected as close to their origins as possible. At the same time about 1 cm. of each three major coronary trunks was freed from the surrounding tissues. An ameroid constrictor was placed around each of the three coronary arteries {Figures 40 and 41). The ventricles were epicardiectomized by surgically scraping off the epicardium after the heart had been exposed to moist sponges soaked in 5% Sodium Salicylate according to Knock's technique (Figures 42, 43 and 44). The same was done with the serous layer of the fibrous pericardium and reflexion over the base of the aorta (Figure 4$). A section of the -77- FIGURE 40: Ameroid constrictor around branches of left coronary artery. FIGURE 41; Ameroid constrictor around the origin of right coronary artery. -7$- FIGURE 42: Sponges soaked in 5% sodium salicylate and placed over the epicardium. FIGURE 43: Appearance of loose epicardium after exposure to chemical agents. -79- FIGURE 44: Appearance of left ventricle after surgical scraping of epicardium. - FIGURE 45: Appearance of root of the aorta after its cleaning. -80- free edge of the omentum was then excised through a small incision in the diaphragm and then the rent closed. The omental graft was unfoltled to a thickness of one layer; wrapped around the entire heart and fixed to the base of the aorta with a few sutures (Figure 46). The pericardium was closed. The thoracotomy wound was also closed after re-expanding the lung; the pleural space was drained with a rubber catheter through a separate stab wound. · This catheter was attached to suction during closure and then removed. Antibiotics were given intramuscularly at the end of the operation for additional protection. They consisted ·of 2 cc. of Fortomycin (400,000 units of penicillin and 1 gm. of streptomycin) and 600,000 units of Bicillin in order to avoid daily injections •. CONTROL SERIES : AMEROIDS ALONE. 3 Dogs. The anterior descending, circumflex and· rigbt coronary arteries were encased close to their origins with an ameroid constrictor in each one in these dogs, in order to produce experimentally gradual and general coronary obstruction. No revascularization -81- FIGURE 46: Free omental graft wrapped around the heart and sutured at the base of the aorta. FIGURE 47: Photornicrograph of section of ornental graft. NOTE the vessels filled with Schlesinger Mass and blood cells. ~----~--~~------82- procedure was carried out in these animals. RESULTS: In the control group of dogs, all died within three weeks which is the maximum survival time recorded in this laboratory after the application of three ameroids. One died ten days after surgery and the others exactly twenty one days post-operatively with no accountable cause other than coronary insufficiency. The injection technique showed that there was not a great deal of intercoronary anastomosis in any of them. On the other hand, of the nine dogs who had all three ameroid constrictors plus the free omental graft, five died of lobar pneumonia or pleural effusion between four and eight weeks after the operation. The mean survival days in these five dogs was forty days. The other four dogs were sacrificed at approximately six months after the procedure. All the hearts were subsequently studied by the injection technique. They showed that there was a filling of the vessels with Schlesinger Mass in the pericardium and omental graft. {Figures 47 and 48). -83- It also revealed numerous intercoronary anastomosis large enough to fill the left coronary artery in part or completely when the right coronary was injected. Attempted separation of the omentum from the pericardium and myocardium was done with difficulty and there was some degree of avascular tight adhesions, which the author believes were due to the chemical irritant (Sodium Salicylate) used over the areas of epicardium that were not completely removed or produced by the remnants of dead epicardium. Perhaps both factors influence the production of avascular adhesions. After ten minutes exposure of the heart to 5% Sodium Salicylate in hypertonie (25%) glucose solution, extra-systoles were frequently observed and in one case ventricular fibrillation, followed by death, occurred. These evidences suggest that the use of Sodium Salicylate in hypertonie glucose for the removal of the epicardium are not without danger even though this chemical had greatly helped the radical extirpation of the epicardium. -84- FIGURE 48: Dog #739. Heart injected after 8 weeks with 3 ameroids and free omental graft. NOTE numerous intercoronary anastomosis, and in lm"'er left vessels of the graft with mass and in those of the pericardium, lower right. (See also Fig. 47). CHAPTER IV. C 0 M M E N T S These experimenta show that detached autologus grafts of omental tissue when. placed loosely in the pleural, peritoneal and pericardial cavities and in the subcutaneous tissues, became attached and were viable in a large percentage of cases. This has occurred because the grafts obtain their own blood supply by rapid formation of anastomosis and growth of extensive new vascular network which draws blood and nourishment from the neighbouring structures. The survival rate of detached omental grafts in different sites of application were .- 1. Intraperitoneal 62.5%. 2. Intrapleural 7o%. 3. Intrapericardial 77~. 4. Subcutaneously 73.3% When the viability of detached omental grafts was compared with other tissues placed under identical experimental conditions, tissues such as muscle and fat, did not reveal any evidence of survival, either macroscopically or microscopically. Davis (1917) reported higher survival in his -86- experimental work with free intraperitoneal omental grafts. But here the transplants were anchored with sutures to previously denuded areas of overlying peritoneum. Freeman (1916) obtained similar results and stated that an advantage of the free omental graft over the undetached omentum was that it avoids entangling bands and injurious traction on the colon and stomach and that if the entire omentum is attached to a given spot, a secondary emergency may develope. Recently, after completing this work, the experiences of Thompson and Pollock {1945), with the use of free omental grafts in the thorax came to our attention. They reported "takes" as high as 95% with free intrapleural omental grafts. Of all the previous workers their free omental grafts were applied over traumatised bared areas of the lung, chest wall, diaphragm, stump of bronchus, esophagus, aorta and vena cava. Their grafts were anchored either with silk or plasma glue. This traumatization of the host surface and the resulting inflammatory reaction may have acted as a stimulant for the free omental transplant and bence higher survival rate in their experimenta. The series reported in this work deals with completely detached omental grafts, which were left free to -87- survive by attaching themselves and in sorne way developed vascular channels capable of penetrating the walls of the pleura, peritoneum, pericardium and superficial fasciae. Intraperitoneal injections of carbon suspension were absorbed by the attached omentum within six hours, colouring it black. Such black masses, described by Florey as not being extracellular, could not be removed by light rubbing because as he says they were bound up by histocytes. This same phagocytic property was retained in our experiments with detached omental grafts. The use of a detached omental graft placed loosely in the pericardial cavity does not seem to have been recorded in the literature. The survival rate of the detached omental grafts in the pericardial cavity was 77% which is striking. This was due to the ability of the free omental grafts to become attached to the coronary vessels, from which they contained a blood supply within ten days. The characteristic of a free omental graft to form arteriolar or larger sized blood vessels between itself and the surrounding tissues may find a definite clinical application. -88- CHAPTER V. SUMMARY 1. Pertinent history of omental function has been reviewed. 2. Autogenous sections of omental tissue have been experimentally transplanted freely in the absence of an inflammed surface, into pleural, peritoneal and pericardial cavities and also subcutaneously in rats and dogs. 3. The omental grafts were not repelled either by the fasciae or the different serosal surfaces. 4. The grafts were examined both macroscopically and microscopically at different intervals, varying from one week to six weeks. The grafts showed remarkable integrity and revascularization in the following percentages of survival : Intrapleural 70% Intraperitoneal 62.5% Intrapericardial 77% Subcutaneously 73.3% These results were proved histologically as well as by the injection technique. -89- 5. In contrast the survival rate of free omental grafts accompanied by free fat and free muscle grafts were : Intrapleural 61.5% Intraperitoneal 44.4% with the free fat and free muscle grafts having 0% survival in both cases. 6. This remarkable viability of the free omental graft was accomplished by their ability to attach quickly to surrounding tissues. 7. The omental tissue implantation was effected by penetration in sorne way into different serosae and fasciae tissues thereby effectively securing an adequate blood supply. 8. On the subject of their rapid attachment and vascular ization, Dr. Arthur Vineberg has suggested that this is analogous to the implantation of the ovum in the endometrium, wherein the chorionic villi invade with proliferative extensions the spiral vessels of the endometrium. This could imply that there exists sorne primitive trophism between the free omental graft and the surrounding tissues. -90- 9. The elastic layer of epicardium prevents firm and quick attachment of the free omental grafts to the underlying myocardium. In spite of this, it was shown that one inch square sections of omental tissue placed free into the pericardial cavity of dogs, became united to form one piece and attached to the roots of great vessels and coronary vessels from which they obtained arterial blood supply. Such grafts would ooze blood on attempted separation from the coronary vessels. 10. A suspension of carbon was given to cats intraperitoneally to reafirm the powerful absorptive function the omentum has and retains when it has been detached and set free in the peritoneal cavity. 11. The numerous mast cells that the omentum possesses containing granules with special metachromatic staining properties were demonstrated in cat's omentum. 12. Omental tissue was transplanted in diffusion chambers, using the transparent chamber in the dorsal skin of the mouse and in the anterior chamber of the eye of the rabbit, with the abject of studying in vivo, -91- under direct vision the mechanism of growth, behaviour and particularly revascularization of the free omental graft. 13. On revascularization of the grafts, the anterior chamber of the eye proved to be the best. Here, by direct microscopie inspection, histological and injection technique, such free omental grafts were well established and revascularized within three days. 14. Throughout these experimenta a new injection method to study arterialization of grafts in the anterior chamber of the eye was developed. 15. Such injection consisted of 10% gelatin in warm water solution to carry India ink particle of 24 millimicrons size and lü% formalin to fix the tissues. 16. The dye solution was injected in the carotid artery of the rabbit via a canula and the animal was immediately frozen for twenty four hours, preventing the India ink from being washed off during histo logical preparation of the grafts. -92- 17. Unstained histological slides made of the graft and the iris showed clèarly massive invasion of a new network of vessels reaching out from the omental grafts to the radial vessels of the iris, sclera, and even to the external occular muscles. lS. The ability of the free omental grafts to grow vessels was demonstrated under direct vision in the diffusion chambers and in particular in the anterior chamber of the eye. 19. Long studies of omental cell growth and nutritional requirements by tissue culture method were attempted. 20. Explants of living omental tissue from rats and rabbits were tested in vitro making use of different natural media, artificial media and by the cell suspension method. 21. Fibroblast-like cells could be seen outgrowing from the edges of the omental explants and cells dividing from the second day of incubation, reaching a peak in ten days. 22. The most favourable method for the omental explants -93- to live and multiply was in Leighton tubes with a caver slip, containing a combination of natural media such as chick embryo extract, plasma clot, 10% animal serum and the supplemented aid of synthetic Morgan's Medium No. 150 at incubation of 37°C. 23. In general the new growth of cells from the omental tissue was scanty, preventing to sub-culture them or obtaining disspersed omental cells by tryptimizing the omentum. These studies suggested this trabecu- lated type of connective tissue with special mesh vessels and lymphatics may have a high metabolic rate. 24. Simultaneous general coronary artery insufficiency was induced experimentally in eleven dogs, by placing an ameroid constrictor around the origins of the right coronary and two major branches of the left coronary arterial trunks. 25. In the control groups with ameroids alone, all animals died within three weeks due to coronary insufficiency and myocardial infarction in one. -94- 26. The graduai occlusion of the three main coronary trunks did not stimulate the development of intercoronary anastomosis in time sufficient to protect the myocardium. 27. The eight dogs treated with free omental graft operation plus radical epicardiectomy survived the total three ameroid coronary constriction. This was accomplished by : 1) The grafts becoming firmly attached to the pericardium, root of the aorta and myocardium in a few days. 2) The grafts forming vascular connections with the aorta, coronary vessels, pericardium and myocardium. 3) The grafts facilitating the opening of the hemocoronary and intercoronary anastomosis. All these fully protected the heart from the heart from the coronary occlusion of the three ameroids. 28. The application of chemicals, sodium salicylate in particular, to the surface of the epicardium, facilitates the surgical removal greatly by loosening it. But the chemicals will also produce -95- tight adhesions where the epicardium was not removed. Such adhesions may not be desirable. 29. The use of sodium salicylate with hypertonie glucose for epicardiectomy may subject theheart to tachycardia, ectopie ventricular beats, or ventricular fibrillation. 30. The main principles of the free omental grafting operation and the application of three ameroid constrictors have been described and illustrated with pictures. -96- CHAPTER VI. C 0 N C L U S I 0 N S The majority of the free omental transplants showed excellent viability in each of the four sites of application described. The rapid and extensive vascularization of the surrounding tissues suggest that the detached omental transplants may find application in the surgical treatment of coronary artery disease, portal hypertension, renal ischaemia and peripheral vascular disease. Also it may be useful in plastic and reconstructive surgery of soft tissue. Experimental myocardial ischaemia in a group of dogs produced by three ameroid constrictors were relieved by the free omental graft operation. It suggests that it is a good method of bringing an extracardiac blood supply to both ventricles within ten days, thus offering a high degree of protection against myocardial infarction. These results are substantially similar to the findings in a large series of experimenta with free omental grafts and two and three ameroid constrictors described by Pifarre and Kato respectively. -97- How does free omental grafting compare in effectiveness with more conventional forms of cardiac revascularization? As yet this question cannot be answered in full, but it would seem that this method of myocardial revascularization may be of great value. CHAPTER VII. B I B L I 0 G R A P H Y 1. ALBRECHT, B., Cited by Jones and McClure. Peritoneum. Lewis's Prac. Surg., 1: 48, 1929. 2. ALGIRE, G. H. and LEGALLAIS, F. Y., Recent developments in the transparent chamber inserted in the rabbit's ear. J. Can. Ins., 10: 225, 1949. 3. BECK, C. S., The development of a new blood supply to the heart by operation. Ann. Surg., 102: 801, 1935. 4. BECK, C. s., The coronary operation. Am. Heart J. 22:539, 1941. 5. BECK, C. S., Coronary Heart Disease: Three dominant causes of death. Am. J. 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The use of free omental grafts in abdominal surgery. Ann. Surg., §1: 87, 1916. 41. GARLOCK, J.H. and SOM, M. L., New approach to treatment of esophageal varices. J.A.M.A., !Ji: 628, 1947. 42. GERARDE, H. W., JONES, M., WINNICK, T., Protein and amino acid turnover in tissue culture. J. Biol. Chem., 196: 51, 1952. 43. GRAY, H., Anatomy. 32nd Ed. The greater omentum, P. 1398. Longmans, Green and Co. 1958. 44. GREEN, H., Heterologous transplantation of mammalian tumors. J. Exp. Med., 11: 461, 1941. -101- 45. GREEN, H., Heteralogous transplantation of a human fibrosarcoma in the anterior chamber of the eye of the guinea pig. Can. Res., 2: 649, 1942. 46. GREEN, H., The participation of the anterior chamber of the eye in resistance phenomeno related to tumor growth. Can. Res., ~: 669, 1942. GRINNELL, R. s., Omentopexy in portal cirrhosis of the liver with ascites. Ann. Surg., 101: 891. 1935. HARRISON, R. E., Observation on the living developing nerve fiber. An. Rec., 1: 116, 1908. 49. HILLEMAN, M. R., WERNER, J. H. Recovery of a new agent from patients with acute respiratory illness. Froc. Soc. Exp. Biol., ~: 183, 1954. 50. JOSLIN, D.,A tissue chamber and splint for the mouse. Science, 115: 601, 1952. 51. KIRSCHNER, P. A., GARLOCK, J., The raionale of routine omentectomy in subtotal gastrectomy. Surg. ~: 884, 1954. 52. KNOCK, F. E., Cardio-omentopexy and implantation of multiple loops for revascularization of the heart. Surg. Forum, 2: 230, 1958. 53. KNOCK, F. E. and BEATTIE, E. J., Chemical and surgical removal of epicardium and viscera pleura. J. Th. Surg., ~: 178, 1961. 54. KNOCK, F. E. and BEATTJE, E. J., Histological aspects of radical cardio-omentopexy: Vascularization of omentum from the aortic root. J. Th. Surg., ~: 387, 1961. 55. LANZ, A., Cited by MACKBY. The surgical treatment of portal hypertension. P. 117, 1960. 56. LARKIN, C. L. and BURNHAM, V. E. Omentumectomy as a safeguard against the recurrence of omental adhesions. New. Eng. Med. 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