Moccasin venom as a test for petechial susceptibility in the cheek pouch and mesoappendix of the hamster

Thesis (Ph.D.)--Boston UniversityA method has been devised for determination of the hemorrhagic tendency in vivo. Cottonmouth moccasin snake venom (Agkistrodon piscivorus piscivorus) is used as a quantitative test to measure the petechial susceptibility of blood vessels in the cheek pouch and mesoappendix of the golden hamster (Mesocricetus auratus). The test has been applied under Nembutal anesthesia to more than 330 hamsters treated in groups of 10 or more with beta and gamma irradiations, the hemostatic agent Adrenosem, rutin, cortisone, calcium; free Ringer's solution, hyaluronidase, antihyaluronidase, and nor-epinephrine. As applied to the cheek pouch, the venom test consisted of the intrapouch injection of .02 cc. of a standard test solution (0.01 percent moccasin venom in mammalian Ringer's solution) by means of a Krogh-Keyes syringe pipette. Exact petechial counts were made at 30-minute intervals for periods of 2 hours or less. The response to be expected in normal hamsters was established in a group of 50 animals. An average of 80 petechiae (standard deviation 36) formed within 1 hour following application of venom. Within 2 hours 150 or more petechial hemorrhages developed. This figure was therefore considered as the end-point value and used as a basis for comparison with experimental groups. As applied to the mesentery, the venom test consisted of irrigating the preparation with pre-heated venom for 25 seconds. Observations were made at 15-minute intervals for a period of 1 hour. The end-point count in the mesoappendix of the normal hamster was established as 50 petechiae at 1 hour. The mechanism involved in petechial formation was studied by Kodachrome motion picture recording of the actual process at 900 magnifications. Erythrocytic extravasation occurred without apparent rupture of the vessel wall and ceased abruptly without platelet plug or fibrin clot formation. Petechiae occurred principally at venous junctions. The effect of snake venom was local and transitory, resolution of the hemorrhages usually being complete within 24 to 48 hours. Various vascular effects in addition to the hemorrhagic phenomenon were observed. Within 1 to 3 minutes after application of the venom, the erythrocytes appeared packed and swollen. This pre-petechiation phase was designated as the period of "beaded flow". An increase occurred in the numbers of leukocytes adhering to the venular endothelium. Also arteriolar vasoconstriction and vasomotion were often encountered. An increased bleeding tendency in x-irradiated hamsters was shown by the moccasin venom test. At 5 days after total body x-irradiation with 1200r, an average of 117 petechiae (standard deviation 30) were counted in the cheek pouch within 1 hour. Increased petechial susceptibility was also demonstrated in the blood vessels of the mesoappendix after similar treatment with x-irradiation as shown by an average of 92 petechiae (standard deviation 20) at 15 minutes following application of venom. No change in petechial susceptibility was detected on the 2nd post-irradiation day. However, a marked increase in susceptibility was observed in hamsters in a near-terminal condition at 7 to 9 days following 1200r total body x-irradiation. Petechial counts averaged 146 (standard deviation 5) at 1 hour. X-irradiation of the total cheek pouch with 500r produced an immediate increase in petechial susceptibility, as indicated by an average of 145 hemorrhages at 1 hour (standard deviation 7). Dosages of 50r delivered to the total pouch produced no significant immediate effects, but on the 2nd day after irradiation petechial counts at 90 minutes averaged 129 with a standard deviation of 31. Intrapouch implantations of Sr90 (141 microcurie bead; 24 to 48 hour exposure) and Co60 (92 microcurie needle; 4 weeks exposure) resulted in an increased petechial response to topically applied moccasin venom. End-point counts were obtained within 45 minutes and 90 minutes respectively, following application of the venom. Adrenosem (1.5 mg. intramuscularly) administered shortly before topical application of the snake venom test solution increased the resistance of cheek pouch and mesenteric blood vessels to the action of venom. Petechial counts in the cheek pouch averaged 72 (standard deviation 47) at 2 hours. Ten petechiae (standard deviation 3) were counted at 1 hour in the mesentery. Pre-irradiation treatment with Adrenosem effectively reduced the petechial susceptibility of the blood vessels of the cheek pouch to snake venom applied on the 5th day after x-irradiation (lOOOr and 1200r). An average of 48 hemorrhages (standard deviation 36) was recorded 2 hours after application of moccasin venom. The protective value of pre-irradiation treatment with Adrenosem was not as great in the mesentery. Petechial counts averaged 89 (standard deviation 8) 1 hour after application of the venom test solution. Treatment with 25 mg. of rutin before intrapouch administration of moccasin venom proved ineffectual in decreasing the petechial susceptibility of the blood vessels. At 2 hours the mean petechial value was 146 with a standard deviation of 8. However, rutin gave protection against hemorrhaging when administered prior to total body x-irradiation with 1200r as demonstrated in hamsters tested with moccasin venom solution on the 5th post-irradiation day. The average count at 1 hour was 83 petechiae (standard deviation 17). This response was essentially the same as that determined for the non-irradiated control group, although significantly lower than the mean value obtained at 1 hour in unprotected irradiated animals. The effect of prolonged treatment with cortisone upon the petechial susceptibility of blood vessels in the cheek pouch was studied. Ten daily injections of cortisone acetate (5 mg. subcutaneously) were administered before intrapouch application of the snake venom test. The petechial response did not differ materially from that of the control group. Average petechial counts of 147 (standard deviation 9) were recorded at 2 hours. No demonstrable modification occurred in the resistance of cheek pouch blood vessels in hamsters treated with calcium-free mammalian Ringer's solution. Intrapouch and intravenous injections of this solution alone were almost wholly ineffective in eliciting a petechial response as indicated by average 2-hour counts of 1.7 (standard deviation 2.4) and 5 (standard deviation 2) respectively. Intrapouch injection of calcium-free Ringer's solution and moccasin venom combined, resulted in the formation of 140 petechiae (standard deviation 12) in 2 hours. A similar response was also observed in animals which were treated with intravenously administered calcium-free Ringer's solution and intrapouch injections of moccasin venom. The counts at 2 hours were comparable with those in the control group, averaging 146 petechiae (standard deviation 6). Topically-applied hyaluronidase (75 TR units) was ineffective in producing petechiae in the cheek pouch, but intra-pouch injection of a combination of only 7.1 TR units of hyaluronidase and .02 cc. of 0.01 percent moccasin venom solution produced a marked increase in petechial formation. End-point counts of 150 or more petechiae were recorded at 1 hour in all 10 hamsters comprising this group. In the mesoappendix 75 TR units of hyaluronidase were only slightly effective. An average petechial count of 7 (standard deviation 3) was obtained at 1 hour. Three experimental antihyaluronidase solutions (SN 40-B; SC-4892; SC-5345, G. D. Searle, Inc.) produced no significant change in the petechial susceptibility of cheek pouch blood vessels as compared with the control group. Simultaneous intrapouch injections of nor-epinephrine (.03 cc. of a .001 percent solution) and the standard amount of the venom test solution produced an average of 14S petechial hemorrhages (standard deviation 4) in 2 hours. Intravenous injection of a vasoconstrictor dose of norepinephrine produced a very significant increase in petechial formation. The mean petechial count at 1 hour was 108 (standard deviation 32). The effects of intramuscular and intravascular administrations of venom were also studied. Intramuscular (flank) injections of .2 cc. of a concentrated solution of moccasin venom (1 percent) did not produce petechiae in the cheek pouch although arteriolar constriction and beaded flow were prevalent. Similar results were obtained with intravascular administration of moccasin venom accomplished by cannulating the external maxillary artery and perfusing the cheek pouch with approximately 2 cc. of a 1 percent venom solution over a period of 2 hours