GYNECOLOGIC ONCOLOGY 3, 233-243 (1975)
Radioprotection of the Digestive Tract by Intravenous Infusion of Vasopressin 1,2 aGuY J. F. JUILLARD, M.D., bHARTMUTH. PETER, M.D., CTHOMASH. WEISENBURGER,M.D., dALAN S. TESLER, M.D., eEDWARD A. LANGDON, M.D., qVIOgRIS BARENFUS, D.V.M., OLEo D. LAGASSE, M.D., hWATSON E. WATRING,M.D., AND ~MCCLURE L. SMITH, M.D. °Associate Professor of Radiological Sciences, Division of Radiation Therapy, UCLA ;~'4 blnstructor, Division of Radiation Therapy, UCLA; 3 CAssistant Professor of Radiological Sciences, Division of Radiation Therapy, UCLA; 3 aResident, Division of Radiation Therapy, UCLA; 3 eProfessor of Radiological Sciences; Chief Division of Radiation Therapy, UCLA; 3 eChief Animal Care Facility, UCLA; 3 gAssociate Professor of Obstetrics and Gynecology, Director, Division of Gynecologic Oncology, UCLA;s hAssistant Professor of Obstetrics and Gynecology, Division of Gynecologic Oncology, UCLA; ~ ~Assistant Professor of Obstetrics and Gynecology, Division of Gynecologic Oncology, UCLA. 3 The effect of venous infusions of vasopressin during fractionated abdominal radiation exposures was evaluated in four pairs of dogs. In each pair, the control dog was given venous infusion of saline during irradiation. The results were analyzed from clinical observation, autopsy findings, and pathological examination. It appears that venous infusion of vasopressin has a definite and reproducible effect of radioprotection on the gastrointestinal tract, the dose modifying factor (DMF) being 1.5. Radiation therapy of the gynecologic malignancies would be one major application since the radiosensitivity of the intestinal tract is often a limiting factor in delivering high doses to the tumor, and further investigations are being done to study the effects of vasopressin on the radiosensitivity of malignant tumors.
Because of the specificity of the pharmacologic activity of vasopressin on the splanchnic circulation and the interest of radiation therapists to increase the tolerance of the bowel to radiation while maintaining usual fractionation; the U C L A Division of Radiation Therapy has instituted animal experimentation in attempt to define the possible clinical benefits of the use of vasopressin in conjunction with radiation therapy. The "oxygen effect" on tissues which are subjected to radiation was noticed thirty-two years ago [19] and clearly analyzed in 1961 [10]. Attempts to take advantage of oxygen effect in radiation therapy have been developed in two main directions: (1) By attempting to equalize the oxygenation of the normal tissues 1 This work was supported by United States Public Health Service Grants #5 T01 CA-05117 CAN and #RR-00865. From the Division of Radiation Therapy (Department of Radiological Sciences) and the Division of Gynecologic Oncology (Department of Obstetrics & Gynecology), U C L A School of Medicine. 3 University of California, Los Angeles, Center for the Health Sciences, Los Angeles, California 90024. 4 Send reprint requests to: Guy Juillard, M.D., Division of Radiation Therapy, U C L A Center for the Health Sciences, Los Angeles, California 90024. 233 Copyright© 1975by AcademicPress, Inc. All rightsof reproductionin any formreserved.
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and tumors [5,31], or (2) utilizing a more selective oxygen effect by modifying the oxygenation of either the tumor [36] or the normal tissues [17, 14, 30] in order to increase the "therapeutic ratio." These methods are sometimes hazardous and often technically difficult. As a consequence, the routinely accepted fractionation is modified, compromising the quality of radiation. The advantage of fractionation is that it allows reoxygenation of tumor cells and increases the tolerance of normal tissues. It would be advantageous to use an approach which selectively modified the oxygenation of tissues without requiring alteration of optimal fractionation. Imaginative techniques have been developed in gastroenterology to control bleeding by creating regional vasoconstriction through cooling [35], administration of norepinephrine [20] or vasopressin [21, 22, 27, 28]. Vasopressin for our studies seems to be the most satisfactory vasoconstrictor because of its specificity for the splanchnic area [26]. METHOD Four pairs of dogs, one experimental and one control animal in each pair, were submitted to abdominal irradiation. The experimental animal received intravenous vasopressin during radiation, and the control received intravenous saline. The radiation used was the 1.25 MeV gamma beam of a cobalt-60 source (Thetatron 780). The dogs were irradiated in the supine position. An anterior beam was used in all cases. The source skin distance was 80 cm, and the dose was calculated at 5 cm depth. Simulation films, IVP's, and port films were taken in all cases. Each paired dog received the same amount of radiation using the same technique and the same fractionation. The radiation technique used was as follows: Pair I = 22 x 30 cm field encompassing the whole abdomen. 1500 rads over 2 days (24 hour interval), 2 fractions of 750 rads. Pair II = 22 x 30 cm encompassing the whole abdomen. 1500 rads over 5 days, 3 fractions of 500 rads. Pair III = 22 x 30 cm encompassing the whole abdomen. 3500 rads over 16 days, 7 fractions of 500 rads. Pair IV = 11 × 11 cm field. 7500 fads over 45 days, 15 fractions of 500 rads. Venous infusion (saline and vasopressin in saline) was initiated 2 minutes prior to radiation exposure. Blanching of the buccal mucosa was noted on 90 seconds after starting vasopressin infusion, and radiation was started as soon as this change was documented. The infusion was stopped immediately at the completion of the irradiation (Fig. 1). The duration of infusion varied between 5 and 7 minutes at the rate of .76 units per minute. The total amount of vasopressin at each fraction varied between 3.8 and 5.3 units for a 25 kg animal. The radiation exposure per fraction was 2.95 to 4.40 minutes according to the field size and the dose. The tolerance to radiation was evaluated by clinical symptoms, medications required to control symptoms, and by autopsy findings and the histologic examination of the irradiated tissue.
RADIOPROTECTION OF THE DIGESTIVE TRACT I
I~. ~.\ ~ , ~ II I Radiation
I I ! I I 0 , i I 2 3 4 5 J l Infusion II I'\X\ ~\ \ \ ~,k \\\ \ \.\ N.\\~, I
235
~,\1
~
Time (Minutes)
FIo. 1. Diagram shows the timing of radiation and infusion.
CLINICAL RESULTS 1. Whole Abdomen Irradiation
One pair of dogs received 1500 rads over 2 days (23 hours) in 2 fractions of 750 fads. On day 3 the control animal became anorexic, and on day 5 bloody diarrhea was noted. He expired on day 6. The dog who had received vasopressin during irradiation was behaving normally on day 3 but was less alert on day 5 and 6. He was sacrificed on day 6 when the control died. Although autopsies showed radiation effects in both dogs, there were obvious differences in the extent of the lesions (Fig. 2). Dramatic changes were noted on the entire intestine of the control dog, which appeared congested, dark, contracted, and approximated one-half of its normal diameter. The intestine of the
FIG. 2. Acute changes of the intestinal mucosa five days after irradiation to the whole abdomen (750 rads x 2); the radiated control (#545) animal exhibits much more severe hemorrhagic intestinal changes than the dog receiving vasopressin (#547).
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dog receiving vasopressin was somewhat congestive, especially at the level of the duodenum and jejunum. Samples were taken on each dog for histopathological examination. The second pair of dogs received 1500 rads over 5 days in 3 fractions. The irradiated control dog had watery diarrhea on day 5 and 6, became prostrated and died on day 7. The "protected" dog had diarrhea on day 5 without alteration of his general condition. He was sacrificed on day 7 when the control died. (No gross modification was found; samples were taken. The delay of several hours between death and autopsy of the control dog precluded definitive interpretation of the severe damage which was found.) The third pair of dogs was submitted to the same fractionation. The control had diarrhea after 1500 rads, which became bloody after 2000 fads on day 9. Intravenous hydration was administered on day 9 (1000 cc of lactated Ringer's solution, amino acids, antibiotics). This treatment controlled the diarrhea for two days. On day 12 (2500 rads) the symptoms reappeared, and a second venous infusion was given again with a beneficial effect; but the dog remained apathetic, anorexic, and irritable. A third intravenous infusion for hydration was given on day 13 (at 3000 rads) with beneficial effects. During the same period of time, the dog receiving vasopressin was doing extremely well. On day 11 (at 2500 rads) he had diarrhea, which subsided spontaneously without medication although irradiation was continued. After 3500 rads on day 16, the "protected" dog expired from an overdose of anesthesia. Autopsies were performed. The intestine of the control showed radiation changes but less than expected from observation of his clinical course. It is speculated that this might be due to the supportive therapy with intravenous fluids. The delay in post mortem examination of the treated dog did not permit a valid pathological examination for comparison.
2. Irradiation of Part of the Abdomen: (Fourth pair of dogs, 7500 rads over 45 days, 15 fractions). The control dog developed diarrhea after 2500 rads on day 16. Apathy, anorexia, and profuse diarrhea started on day 17. The symptoms became more severe after day 19 (3500 rads). On day 31 (5500 rads) the weight loss was 17%, and a first intravenous infusion of 100 cc of lactated Ringer's solution and amino acids was given, which had a dramatic effect on controlling diarrhea for seven days although radiation was continued. A second intravenous rehydration was given after 5500 rads on day 30 with the same success for one week. Bloody diarrhea was noted after 7000 rads on day 39. The dog was rehydrated for the third time. After the end of radiation (7500 rads), diarrhea or loose stools persisted for two months with occasional remissions of several days during the second month following irradiation. The total weight loss was 21%. The weight was stable during the last month. The dog receiving vasopressin tolerated radiation extremely well. Diarrhea was noted only once, two days after the end of radiation (7500 rads). No medication was given. During the experiment his weight loss was 12%. Both dogs were sacrificed on day 109, 64 days after the completion of irradia-
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tion. Macroscopic examination of the digestive tract of the control dog revealed severe changes. The entire intestinal tract showed diffuse and patchy hyperemia progressing to focal hemorrhages. The upper jejunum revealed two circumscribed ulcers. The ileocecal region was severely hemorrhagic, and the ileocecal junction was marked by a circumferential ulcer which had abscessed and perforated through the mesenteric attachment. The serosal surface was granulomatous in appearance. Contrasting with these findings, the macroscopic examination of the gastrointestinal tract in the dog who had received vasopressin was substantially less marked. The stomach was filled with recent feeding, the entire intestinal tract evidenced patchy physiologic hyperemia. The terminal ileum and upper colon revealed petechial hemorrhages (Fig. 3). MICROSCOPIC RESULTS
Microscopic examination was available in six dogs. In four paired dogs (two who received whole abdomen irradiation and two who had partial irradiation and who both survived until euthanasia on day 109) an accurate comparison is possible because there were no post mortem alterations.
1. Whole Abdomen Irradiation: (1500 rads/2 days). (Pair I) In the control dog all samples of the intestine revealed a destruction of surface and crypt epithelium. The residual epithelial cells were dead or dying, evidencing
FIG. 3. Chronic changes two months after irradiation (7500 rads, 65 days, 15 fractions) to part of the abdomen. Circumferential ulcer in the ileocecal region of the radiated control dog (left).
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F I 6 . 4 . Microscopic findings five days after irradiation of control dog (750 rads x 2); devastation of surface and crypt epithelium.
FIG. 5. Microscopic findings five days after irradiation and concurrent intravenous infusion of vasopressin. Many areas reveal distinct epithelial resistance.
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nuclear fragmentation and condensation. Few mitotic figures were observed. Hyperemia and hemorrhage were present, and there was some round cell infiltration of the lamina propria. In the protected dog, although there was loss of epithelium in some areas, there were many areas revealing distinct epithelial resistance. Intact epithelium was noted both on the surface of villi and in crypts. Samples of colon revealed almost total retention of epithelium. Many mitotic figures were observed in the crypts (Figs. 4 and 5).
2. Partial Abdominal Irradiation: (7500 fads~45 days~15 fractions). (Pair IV) Microscopic examination of the intestine of the control dog showed scattered areas of collapsed, denuded villi-some areas revealing frank necrosis. Crypts were sparse and atrophied. The lamina propria was heavily infiltrated with macrophages and mononuclear and eosinophil leukocytes. The jejunal and cecal ulcers, which were noted grossly, were a mass of necrotic mucosa supported by proliferated inflammatory lamina propria. The inflammatory cells included mostly neutrophils and macrophages. The cecal ulcer revealed necrosis penetrating through the muscular tunics. In contrast, the findings in the "protected" dog were minimal. Microscopic examination revealed foci of atrophied or absent crypts. The lamina propria was moderately infiltrated with macrophages and mononuclear and eosinophilic leu-
FIG. 6. Control dog-Photomicrograph 100x. Severe mucosal necrosis of small intestine accompanied by heavy inflammatory cellular infiltration.
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FIG. 7. Experimental dog-Photomicrograph 100x. Retention of small intestine's crypt epithelium. Infiltration of lamina propria by inflammatory cells.
kocytes. Based on the radiation enteritis, it appeared conclusively that the control dog was the more severely affected (Figs. 6 and 7). DISCUSSION The efficacy of intravenous infusion of vasopressin in increasing the tolerance of the gastrointestinal tract to irradiation has, to us, been demonstrated impressively in dogs. The difference in the clinical symptoms becomes striking after 3000 rads in 11 days when a portion of the abdomen is radiated (Pair IV) and after 1500 rads in 5 days when the field encompasses the entire abdomen (Pairs I, II, and III). This does not mean that one should expect no histologic alteration of the intestinal mucosa when vasopressin is administered. There would still be s o m e alteration in the normal histology even under total anoxia. An attempt to evaluate the increase of tolerance with vasopressin may be proposed by comparing the doses necessary to produce the first symptoms in "protected" and control dogs. Fractionation obviously influences the evaluation of the effects of vasopressin and for a better comparison of the results, an attempt to express dosage in rets seems to have value. Although this is not above criticisms when applied to the digestive tract in dogs, the results seem to deserve some attention. Mild symptoms were noticed in "protected" dogs at 3000 rads, 12 days, 6 fractions (1485 rets) when the entire abdomen was irradiated and at 7500 rads, 45 days,
241
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15 fractions (2572 rets) when ionizing radiation was given to part of the abdomen. Severe symptoms were observed respectively in control dogs at 1500 rads, 5 days, 3 fractions (966 rets) and 3500 fads, 18 days, 7 fractions (1596 rets). When these doses in rets are compared, it appears that vasopressin increases the tolerance by a similar factor when irradiating part of the abdomen (1.6) and the whole abdomen (1.5) (Fig. 8). Besides reporting the tolerance increase of the digestive tract to radiation, it should be stressed that the same amount of vasopressin was given during each radiation exposure, even when reported 15 times over 45 days to the same animal with the same effect. There was no need to increase the dose, and blanching of the buccal mucosa was constantly observed 90 seconds to 2 minutes after the infusion was started. On the other hand, the well-known remarkable effect of intravenous hydration in correcting the intestinal symptoms was observed in the control dogs. In resume, the increase of the tolerance of the gastrointestinal tract to radiation, the reproducibility of identical effects of vasopressin up to 15 times to the same animal, and the efficacy of venous hydration in controlling intestinal symptoms are three positive aspects of this study. But there is no doubt that further observations are necessary. The doses of vasopressin which we used induced a 25% elevation of the blood pressure, bradycardia, and sometimes arrhythmia. Ongoing experiments seem to show that .3 units of vasopressin per minute in a 30 kg animal give the same radioprotection as .76 units per minute with less side effects (no significant modification of the cardiac rhythm but still a 20% rise of the blood pressure). Furthermore, a major concern is to avoid the radioprotection of a tumor if vasopressin is administered during radiation therapy. Current investigations seem to indicate that this major problem might be overcome in certain gynecologic malignancies. CONCLUSION Venous infusion of vasopressin increases the tolerance of the digestive tract to irradiation in dogs. The dose modifying factor (DMF) is estimated to be 1.5. The
Treated Area
Venous Infusion During Irradiation
Dose of Radiation When I s t Symptom Noted I .. 3000 rads, 12 days, 6 fractions
Rets
Ratio (DMF)
i
Vasopressin Whole Abdomen
1485 -
Saline
]500 rads, 5 days, 3 fractions
966
Vasopressin
7500 rads, 45 days, 15 fractions
2572
Sa]ine
3500 rads, 18 days, 7 fractions
1596
Part of the Abdomen
1.53
1.61
FIG. 8. Tentative evaluation of the Dose Modifying Factor (DMF) from intravenous vasopressin.
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radioprotective effect of vasopressin is reproducible in the same animal without modifying the amount of the drug. The efficacy of infusion of polyionic electrolyte solutions and amino acids was remarkable in controlling gastrointestinal symptoms from radiation in unprotected dogs. ACKNOWLEDGMENTS The authors are grateful for the technical assistance of Tom Patin, John Robert, Sue Wall, Donna Intrator, and Julia Smith,
REFERENCES 1. Amory, I. I., and Brick, I. B. Irradiation damage of the intestine following 1000 KV roentgentherapy-evaluation of tolerance dose, Radiology 56, 49-57 (1951). 2. Appel, A. J., Lintermans, J. P., Mullins, G. L., and Guntheroth, W. G. The effects of vasoactive drugs on mesenteric blood flow and volume, Surg., Gyn. & Ob. 123, 755-764 (1966). 3. Carter, D. B., Adair, H. M., and Grove, C. A. Effects of vasomotor drugs and mediators of the inflammatory reaction upon the oxygen tension of tumors and tumor blood flow, Brit. J. Cancer 20, 504-516 (1966). 4. Churchill-Davidson, I. Oxygen effect on radiosensitivity, Am. J. Roent. 82, 688-692 (1959). 5. Conard, R. A. Experimental therapy of the gastrointestinal syndrome produced by lethal doses of ionizing radiation, J..dppl. Physiol. 9, 227-233 (1956). 6. Dewey, D. L. Effect of oxygen and nitric oxide on the radiosensitivity of human cells in tissue culture, Nature (London) 186, 780-782 (1960). 7. DuSault, L. A. Application of the oxygen factor to clinical radiotherapy, Radiology 100, 675-678 (1971). 8. Elkind, M. M., Swain, R. W., Alescio, T., Sutton, H., and Moses, W. B, Oxygen, nitrogen, recovery, and radiation therapy, in Cellular Radiation Biology Williams and Wilkins, Baltimore, Md., pp. 442-446 (1965). 9. Ellis, F. Dose, time, and fractionation-a clinical hypotheses, Clinical Radiology 20, 1-7 (1969). 10. Gray, L. H. Radiobiologic basis of oxygen as a modifying factor in radiation therapy, Am. J. Roent. 85, 803-815 (1961). 11. Hewitt, H. B., and Wilson, C. W. Survival curves for tumor cells irradiated in vivo, Am. N.Y. Acad. Sci. 95, 318-327 (1961). 12. Howard-Flanders, P., and Alper, T. The sensitivity of microorganisms to irradiation under controlled gas conditions, Rad. Res. 7, 518-540 (1957). 13. Howard-Flanders, P. Physical and chemical mechanisms in the injury of cells by ionizing radiation, Adv. Biol. Med. Phys. 6, 553-603 (1958). 14. Johnson, R. E., Doppman, J. L., Harbert, J. C., et al. Prevention of radiation nephritis with renal artery infusion of vasoconstrictors: Experimental and preliminary clinical studies, Radiology 91, 103-108 (1968). 15. Kaplan, H. S. Clinical potentialities of recent advances in cellular radiobiology, in Cellular Radiation Biology, Williams and Wilkins, Baltimore, Md., pp. 584-598 (1965). 16. Kehne, J. H., Hughes, F. A., and Gompertz, M. L. The use of surgical pituitrin in the control of esophageal varix bleeding, Surgery 39, 916-925 (1956). 17. Klingerman, M. M., Bond, V. P., Held, B. T., Mather, G., and Nielsen, N. Anoxic protection of small intestine of dogs by intra-arterial catheterization, Radiology 78, 284-285 (1962). 18. Kohn, P. M., Charms, B., and Brofman, B. L. Effect of Epinephrine and posterior pituitary extract on the wedged-hepatic-vein pressure in normal patients and in those with liver disease, N e w Eng. J. Med. 261, 323-327 (1959). 19. Lacassagne, A. Chute de la sensibilte aux rayons x chez la souris nouveaune en etat d' dsphyxie, Compt. Rend. Acad. Sc. 215, 231-232 (1942). 20. Le Veen, H., et al. Control of gastrointestinal bleeding, Am. J. Surg. 123, 154-159 (1972). 21. Nusbaum, M., Baum, S., Sakiyaka, P., and Blakemore, W. Pharmacologic control of portal hypertension, Surgery 62, 299-310 (1967).
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22. Nylander, G. Vascular response to vasopressin as reflected in angiography, Acta Radiologica, Suppl. 266, 79 (1967). 23. Orrego, H., Mena, I., Sepulveda, G., Maggiolo, C., Munoz, A., and Duran, F. Effects of pituitrin and vasopressin on hepatic circulation, Am. J. Dig. Dis. 9, 109-120,(1964). 24. Regaud, C. L., Nogier, T. H., and Lacassagne, A. Sur les effets redoutables des irradiations etendues de 1' abdomen et sur les lesions du tube disestif determinee par les rayons de rontgen, Arch. D' Electr. Med. 343, 321-334 (1912). 25. Roswit, B., Malsky, S. J., and Reid, C. B. Severe radiation injuries of the stomach, small intestine, colon, and rectum, Am. J. Roent. 114, 460-475 (1972). 26. Schwabe, A., Professor of Medicine, Chief of Gastroenterology Division, U C L A School of Medicine (Personal Communication). 27. Shaldon, S., and Sherlock, S. The use of vasopressin ("Pitressin") in the control of bleeding from esophageal varices, Lancet 222-225 (1960). 28. Shaldon, S., Dolle, W., Guevara, L., Iber, F. L., and Sherlock, S. Effect of Pitressin on the splanchnic circulation in man, Circulation 24, 797-807 (1961). 29. Shapiro, H., and Britt, L. G. The action of vasopressin on the gastrointestinal tract, Digest. Dis. 17, 667-669 (1972). 30. Steckel, R. J., Ross, G., and Grollman, J. H. A potent drug combination for producing construction of the superior mesenteric artery and its branches, Radiology 91, 579-581 (1968). 31. Suit, H. D., and Lindberg, R. D. Radiation therapy administered under conditions of tourniquetinduced local tissue hypoxia, Am. J. Roent. 102, 27 (1968). 32. Thombrison, R. H., and Gray, L. H. Histological structure of some human lung cancers and possible implications for radiotherapy, Brit. J. Cancer 9, 539-549 (1955). 33. Tsakiris, A., Haemmerli, P., and Buhlmann, A, Reduction of portal venous pressure in cirrhotic patients with bleeding from esophageal varices by administration of a vasopressin derivative, phenylalanine-lysine-vasopressin, Am. J. Med. 36, 825-839 (1964). 34. Van den Brenck, H. A. S. The tourniquet technique in radiotherapy of limb tumors, Nuntius Radiologicus 34, 207 (1968). 35. Wangensteen, O. H., Salmon, P. A., Griffen, W. O., et al. Studies of local gastric cooling as related to peptic ulcer, Ann. Surg. 150, 346-360 (1959). 36. Whittle, R. J. M. Regional oxygenation by infusion of hydrogen peroxide, in Modern Trends in Radiotherapy (T. J. Deeley and C. A. P. Wood, Eds.) Butterworth, London, pp. 99-106 (1967). 37. Wright, E. A., and Bewley, D. K. Whole body radioprotection of mice to 8 MEV electrons produced by breathing nitrogen for brief periods, Rad. Res. 13, 649-656 (1960).
Radioprotection of the Digestive Tract by Intravenous Infusion of Vasopressin 1,2 aGuY J. F. JUILLARD, M.D., bHARTMUTH. PETER, M.D., CTHOMASH. WEISENBURGER,M.D., dALAN S. TESLER, M.D., eEDWARD A. LANGDON, M.D., qVIOgRIS BARENFUS, D.V.M., OLEo D. LAGASSE, M.D., hWATSON E. WATRING,M.D., AND ~MCCLURE L. SMITH, M.D. °Associate Professor of Radiological Sciences, Division of Radiation Therapy, UCLA ;~'4 blnstructor, Division of Radiation Therapy, UCLA; 3 CAssistant Professor of Radiological Sciences, Division of Radiation Therapy, UCLA; 3 aResident, Division of Radiation Therapy, UCLA; 3 eProfessor of Radiological Sciences; Chief Division of Radiation Therapy, UCLA; 3 eChief Animal Care Facility, UCLA; 3 gAssociate Professor of Obstetrics and Gynecology, Director, Division of Gynecologic Oncology, UCLA;s hAssistant Professor of Obstetrics and Gynecology, Division of Gynecologic Oncology, UCLA; ~ ~Assistant Professor of Obstetrics and Gynecology, Division of Gynecologic Oncology, UCLA. 3 The effect of venous infusions of vasopressin during fractionated abdominal radiation exposures was evaluated in four pairs of dogs. In each pair, the control dog was given venous infusion of saline during irradiation. The results were analyzed from clinical observation, autopsy findings, and pathological examination. It appears that venous infusion of vasopressin has a definite and reproducible effect of radioprotection on the gastrointestinal tract, the dose modifying factor (DMF) being 1.5. Radiation therapy of the gynecologic malignancies would be one major application since the radiosensitivity of the intestinal tract is often a limiting factor in delivering high doses to the tumor, and further investigations are being done to study the effects of vasopressin on the radiosensitivity of malignant tumors.
Because of the specificity of the pharmacologic activity of vasopressin on the splanchnic circulation and the interest of radiation therapists to increase the tolerance of the bowel to radiation while maintaining usual fractionation; the U C L A Division of Radiation Therapy has instituted animal experimentation in attempt to define the possible clinical benefits of the use of vasopressin in conjunction with radiation therapy. The "oxygen effect" on tissues which are subjected to radiation was noticed thirty-two years ago [19] and clearly analyzed in 1961 [10]. Attempts to take advantage of oxygen effect in radiation therapy have been developed in two main directions: (1) By attempting to equalize the oxygenation of the normal tissues 1 This work was supported by United States Public Health Service Grants #5 T01 CA-05117 CAN and #RR-00865. From the Division of Radiation Therapy (Department of Radiological Sciences) and the Division of Gynecologic Oncology (Department of Obstetrics & Gynecology), U C L A School of Medicine. 3 University of California, Los Angeles, Center for the Health Sciences, Los Angeles, California 90024. 4 Send reprint requests to: Guy Juillard, M.D., Division of Radiation Therapy, U C L A Center for the Health Sciences, Los Angeles, California 90024. 233 Copyright© 1975by AcademicPress, Inc. All rightsof reproductionin any formreserved.
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ET AL.
and tumors [5,31], or (2) utilizing a more selective oxygen effect by modifying the oxygenation of either the tumor [36] or the normal tissues [17, 14, 30] in order to increase the "therapeutic ratio." These methods are sometimes hazardous and often technically difficult. As a consequence, the routinely accepted fractionation is modified, compromising the quality of radiation. The advantage of fractionation is that it allows reoxygenation of tumor cells and increases the tolerance of normal tissues. It would be advantageous to use an approach which selectively modified the oxygenation of tissues without requiring alteration of optimal fractionation. Imaginative techniques have been developed in gastroenterology to control bleeding by creating regional vasoconstriction through cooling [35], administration of norepinephrine [20] or vasopressin [21, 22, 27, 28]. Vasopressin for our studies seems to be the most satisfactory vasoconstrictor because of its specificity for the splanchnic area [26]. METHOD Four pairs of dogs, one experimental and one control animal in each pair, were submitted to abdominal irradiation. The experimental animal received intravenous vasopressin during radiation, and the control received intravenous saline. The radiation used was the 1.25 MeV gamma beam of a cobalt-60 source (Thetatron 780). The dogs were irradiated in the supine position. An anterior beam was used in all cases. The source skin distance was 80 cm, and the dose was calculated at 5 cm depth. Simulation films, IVP's, and port films were taken in all cases. Each paired dog received the same amount of radiation using the same technique and the same fractionation. The radiation technique used was as follows: Pair I = 22 x 30 cm field encompassing the whole abdomen. 1500 rads over 2 days (24 hour interval), 2 fractions of 750 rads. Pair II = 22 x 30 cm encompassing the whole abdomen. 1500 rads over 5 days, 3 fractions of 500 rads. Pair III = 22 x 30 cm encompassing the whole abdomen. 3500 rads over 16 days, 7 fractions of 500 rads. Pair IV = 11 × 11 cm field. 7500 fads over 45 days, 15 fractions of 500 rads. Venous infusion (saline and vasopressin in saline) was initiated 2 minutes prior to radiation exposure. Blanching of the buccal mucosa was noted on 90 seconds after starting vasopressin infusion, and radiation was started as soon as this change was documented. The infusion was stopped immediately at the completion of the irradiation (Fig. 1). The duration of infusion varied between 5 and 7 minutes at the rate of .76 units per minute. The total amount of vasopressin at each fraction varied between 3.8 and 5.3 units for a 25 kg animal. The radiation exposure per fraction was 2.95 to 4.40 minutes according to the field size and the dose. The tolerance to radiation was evaluated by clinical symptoms, medications required to control symptoms, and by autopsy findings and the histologic examination of the irradiated tissue.
RADIOPROTECTION OF THE DIGESTIVE TRACT I
I~. ~.\ ~ , ~ II I Radiation
I I ! I I 0 , i I 2 3 4 5 J l Infusion II I'\X\ ~\ \ \ ~,k \\\ \ \.\ N.\\~, I
235
~,\1
~
Time (Minutes)
FIo. 1. Diagram shows the timing of radiation and infusion.
CLINICAL RESULTS 1. Whole Abdomen Irradiation
One pair of dogs received 1500 rads over 2 days (23 hours) in 2 fractions of 750 fads. On day 3 the control animal became anorexic, and on day 5 bloody diarrhea was noted. He expired on day 6. The dog who had received vasopressin during irradiation was behaving normally on day 3 but was less alert on day 5 and 6. He was sacrificed on day 6 when the control died. Although autopsies showed radiation effects in both dogs, there were obvious differences in the extent of the lesions (Fig. 2). Dramatic changes were noted on the entire intestine of the control dog, which appeared congested, dark, contracted, and approximated one-half of its normal diameter. The intestine of the
FIG. 2. Acute changes of the intestinal mucosa five days after irradiation to the whole abdomen (750 rads x 2); the radiated control (#545) animal exhibits much more severe hemorrhagic intestinal changes than the dog receiving vasopressin (#547).
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dog receiving vasopressin was somewhat congestive, especially at the level of the duodenum and jejunum. Samples were taken on each dog for histopathological examination. The second pair of dogs received 1500 rads over 5 days in 3 fractions. The irradiated control dog had watery diarrhea on day 5 and 6, became prostrated and died on day 7. The "protected" dog had diarrhea on day 5 without alteration of his general condition. He was sacrificed on day 7 when the control died. (No gross modification was found; samples were taken. The delay of several hours between death and autopsy of the control dog precluded definitive interpretation of the severe damage which was found.) The third pair of dogs was submitted to the same fractionation. The control had diarrhea after 1500 rads, which became bloody after 2000 fads on day 9. Intravenous hydration was administered on day 9 (1000 cc of lactated Ringer's solution, amino acids, antibiotics). This treatment controlled the diarrhea for two days. On day 12 (2500 rads) the symptoms reappeared, and a second venous infusion was given again with a beneficial effect; but the dog remained apathetic, anorexic, and irritable. A third intravenous infusion for hydration was given on day 13 (at 3000 rads) with beneficial effects. During the same period of time, the dog receiving vasopressin was doing extremely well. On day 11 (at 2500 rads) he had diarrhea, which subsided spontaneously without medication although irradiation was continued. After 3500 rads on day 16, the "protected" dog expired from an overdose of anesthesia. Autopsies were performed. The intestine of the control showed radiation changes but less than expected from observation of his clinical course. It is speculated that this might be due to the supportive therapy with intravenous fluids. The delay in post mortem examination of the treated dog did not permit a valid pathological examination for comparison.
2. Irradiation of Part of the Abdomen: (Fourth pair of dogs, 7500 rads over 45 days, 15 fractions). The control dog developed diarrhea after 2500 rads on day 16. Apathy, anorexia, and profuse diarrhea started on day 17. The symptoms became more severe after day 19 (3500 rads). On day 31 (5500 rads) the weight loss was 17%, and a first intravenous infusion of 100 cc of lactated Ringer's solution and amino acids was given, which had a dramatic effect on controlling diarrhea for seven days although radiation was continued. A second intravenous rehydration was given after 5500 rads on day 30 with the same success for one week. Bloody diarrhea was noted after 7000 rads on day 39. The dog was rehydrated for the third time. After the end of radiation (7500 rads), diarrhea or loose stools persisted for two months with occasional remissions of several days during the second month following irradiation. The total weight loss was 21%. The weight was stable during the last month. The dog receiving vasopressin tolerated radiation extremely well. Diarrhea was noted only once, two days after the end of radiation (7500 rads). No medication was given. During the experiment his weight loss was 12%. Both dogs were sacrificed on day 109, 64 days after the completion of irradia-
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tion. Macroscopic examination of the digestive tract of the control dog revealed severe changes. The entire intestinal tract showed diffuse and patchy hyperemia progressing to focal hemorrhages. The upper jejunum revealed two circumscribed ulcers. The ileocecal region was severely hemorrhagic, and the ileocecal junction was marked by a circumferential ulcer which had abscessed and perforated through the mesenteric attachment. The serosal surface was granulomatous in appearance. Contrasting with these findings, the macroscopic examination of the gastrointestinal tract in the dog who had received vasopressin was substantially less marked. The stomach was filled with recent feeding, the entire intestinal tract evidenced patchy physiologic hyperemia. The terminal ileum and upper colon revealed petechial hemorrhages (Fig. 3). MICROSCOPIC RESULTS
Microscopic examination was available in six dogs. In four paired dogs (two who received whole abdomen irradiation and two who had partial irradiation and who both survived until euthanasia on day 109) an accurate comparison is possible because there were no post mortem alterations.
1. Whole Abdomen Irradiation: (1500 rads/2 days). (Pair I) In the control dog all samples of the intestine revealed a destruction of surface and crypt epithelium. The residual epithelial cells were dead or dying, evidencing
FIG. 3. Chronic changes two months after irradiation (7500 rads, 65 days, 15 fractions) to part of the abdomen. Circumferential ulcer in the ileocecal region of the radiated control dog (left).
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F I 6 . 4 . Microscopic findings five days after irradiation of control dog (750 rads x 2); devastation of surface and crypt epithelium.
FIG. 5. Microscopic findings five days after irradiation and concurrent intravenous infusion of vasopressin. Many areas reveal distinct epithelial resistance.
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nuclear fragmentation and condensation. Few mitotic figures were observed. Hyperemia and hemorrhage were present, and there was some round cell infiltration of the lamina propria. In the protected dog, although there was loss of epithelium in some areas, there were many areas revealing distinct epithelial resistance. Intact epithelium was noted both on the surface of villi and in crypts. Samples of colon revealed almost total retention of epithelium. Many mitotic figures were observed in the crypts (Figs. 4 and 5).
2. Partial Abdominal Irradiation: (7500 fads~45 days~15 fractions). (Pair IV) Microscopic examination of the intestine of the control dog showed scattered areas of collapsed, denuded villi-some areas revealing frank necrosis. Crypts were sparse and atrophied. The lamina propria was heavily infiltrated with macrophages and mononuclear and eosinophil leukocytes. The jejunal and cecal ulcers, which were noted grossly, were a mass of necrotic mucosa supported by proliferated inflammatory lamina propria. The inflammatory cells included mostly neutrophils and macrophages. The cecal ulcer revealed necrosis penetrating through the muscular tunics. In contrast, the findings in the "protected" dog were minimal. Microscopic examination revealed foci of atrophied or absent crypts. The lamina propria was moderately infiltrated with macrophages and mononuclear and eosinophilic leu-
FIG. 6. Control dog-Photomicrograph 100x. Severe mucosal necrosis of small intestine accompanied by heavy inflammatory cellular infiltration.
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FIG. 7. Experimental dog-Photomicrograph 100x. Retention of small intestine's crypt epithelium. Infiltration of lamina propria by inflammatory cells.
kocytes. Based on the radiation enteritis, it appeared conclusively that the control dog was the more severely affected (Figs. 6 and 7). DISCUSSION The efficacy of intravenous infusion of vasopressin in increasing the tolerance of the gastrointestinal tract to irradiation has, to us, been demonstrated impressively in dogs. The difference in the clinical symptoms becomes striking after 3000 rads in 11 days when a portion of the abdomen is radiated (Pair IV) and after 1500 rads in 5 days when the field encompasses the entire abdomen (Pairs I, II, and III). This does not mean that one should expect no histologic alteration of the intestinal mucosa when vasopressin is administered. There would still be s o m e alteration in the normal histology even under total anoxia. An attempt to evaluate the increase of tolerance with vasopressin may be proposed by comparing the doses necessary to produce the first symptoms in "protected" and control dogs. Fractionation obviously influences the evaluation of the effects of vasopressin and for a better comparison of the results, an attempt to express dosage in rets seems to have value. Although this is not above criticisms when applied to the digestive tract in dogs, the results seem to deserve some attention. Mild symptoms were noticed in "protected" dogs at 3000 rads, 12 days, 6 fractions (1485 rets) when the entire abdomen was irradiated and at 7500 rads, 45 days,
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15 fractions (2572 rets) when ionizing radiation was given to part of the abdomen. Severe symptoms were observed respectively in control dogs at 1500 rads, 5 days, 3 fractions (966 rets) and 3500 fads, 18 days, 7 fractions (1596 rets). When these doses in rets are compared, it appears that vasopressin increases the tolerance by a similar factor when irradiating part of the abdomen (1.6) and the whole abdomen (1.5) (Fig. 8). Besides reporting the tolerance increase of the digestive tract to radiation, it should be stressed that the same amount of vasopressin was given during each radiation exposure, even when reported 15 times over 45 days to the same animal with the same effect. There was no need to increase the dose, and blanching of the buccal mucosa was constantly observed 90 seconds to 2 minutes after the infusion was started. On the other hand, the well-known remarkable effect of intravenous hydration in correcting the intestinal symptoms was observed in the control dogs. In resume, the increase of the tolerance of the gastrointestinal tract to radiation, the reproducibility of identical effects of vasopressin up to 15 times to the same animal, and the efficacy of venous hydration in controlling intestinal symptoms are three positive aspects of this study. But there is no doubt that further observations are necessary. The doses of vasopressin which we used induced a 25% elevation of the blood pressure, bradycardia, and sometimes arrhythmia. Ongoing experiments seem to show that .3 units of vasopressin per minute in a 30 kg animal give the same radioprotection as .76 units per minute with less side effects (no significant modification of the cardiac rhythm but still a 20% rise of the blood pressure). Furthermore, a major concern is to avoid the radioprotection of a tumor if vasopressin is administered during radiation therapy. Current investigations seem to indicate that this major problem might be overcome in certain gynecologic malignancies. CONCLUSION Venous infusion of vasopressin increases the tolerance of the digestive tract to irradiation in dogs. The dose modifying factor (DMF) is estimated to be 1.5. The
Treated Area
Venous Infusion During Irradiation
Dose of Radiation When I s t Symptom Noted I .. 3000 rads, 12 days, 6 fractions
Rets
Ratio (DMF)
i
Vasopressin Whole Abdomen
1485 -
Saline
]500 rads, 5 days, 3 fractions
966
Vasopressin
7500 rads, 45 days, 15 fractions
2572
Sa]ine
3500 rads, 18 days, 7 fractions
1596
Part of the Abdomen
1.53
1.61
FIG. 8. Tentative evaluation of the Dose Modifying Factor (DMF) from intravenous vasopressin.
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radioprotective effect of vasopressin is reproducible in the same animal without modifying the amount of the drug. The efficacy of infusion of polyionic electrolyte solutions and amino acids was remarkable in controlling gastrointestinal symptoms from radiation in unprotected dogs. ACKNOWLEDGMENTS The authors are grateful for the technical assistance of Tom Patin, John Robert, Sue Wall, Donna Intrator, and Julia Smith,
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