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Effect of a rattlesnake venom (Crotalus viridis helleri) on bone marrow
Toxkon, VoL 19, pp . 181-183 . m Pergamon Pleas Ltd 1981 . Printed m Great Britain.
0041-0101/81/0101-0181 $02 .00/0
EFFECT OF A RATTLESNAKE VENOM
VIRIDIS HELLERI)
(CROTALUS
ON BONE MARROW
A.
WINGERT,' TAMMANUR R. PATTABHIRAMAN,' DARLENE POWERS' and FINDLAY E . RUSSELL' ' Laboratory of Neurological Research and 2 Department of Pediatrics, Los Angeles County/University of Southern California Medical Center, Los Angeles, CA 90033, U.S .A. WILLIS
(Accepted for publication 16 September 1980) W . A . WINGERT, T . R . PATTABHIRAMAN, D . POWERS and F. E . RUSSELL . Effect of a rattlesnake venom (Crotales piridis helleri) on bone marrow . Toxicon 19, 181-183,1981 .-Crotales oiridis helleri venom produces a fall in peripheral platelet count in rats but does not appear to damage the precursors in the bone marrow . The major response is a myeloid hyperplasia .
by Crotalus oiridis helleri causes a decrease in circulating platelets in both humans and experimental animals (LA GRANGE and RUSSELL, 1970) . Following injection of the venom into rabbits, there is a three-phase platelet response : an initial rapid drop followed by a rise at 36 hr, a subsequent overshoot, and a return toward normal levels at 144-282 hr (LA GRANGE and RUSSELL, 1970). Since this effect appears to be both time- and dose-related, it may be suggested as an index to determine the level ofpoisoning following bites by this snake, and thus be of value in determining antivenin administration . The loss ofplatelets may be due to (1) direct intravascular destruction or to consumption during the intravascular coagulation initiated by the direct action of the venom on some clotting component, such as thrombin or fibrinogen ; (2) toxin-induced damage to platelet precursors in the bone marrow ; or (3) some combination of these factors. The direct effect of megakaryocyte kinetics following envenomation has not been clearly defined, although isotope-tagged pit viper venom is known to be distributed to the marrow (GENNARo and RAMSEY, 1959 ; WINGERT et al., 1980) . The present study was designed to observe the progressive changes that occur in bone marrow cells, particularly the megakaryocytes, following the injection of various doses of a crotalid venom. Ten white male rats, weighing 300-387 g each, were injected subcutaneously with Crotalus viridis helleri venom reconstituted following 6 months storage at 5°C . The venom doses varied from 2 to 8 mg/kg. (In a previous study, the subcutaneous LDSO at 24 hr was 4.0 mg/kg body wt.) Bone marrow from the femur was obtained by killing the rats 2-24 hr after injection, sectioning the bone, and transferring the marrow directly to a slide. The marrow was fixed and stained with Wright's stain, using standard techniques. Platelet counts were obtained prior to envenomation and complete blood and platelet counts were obtained before sacrifice. One rat injected with 4 mg/kg of venom died at 15 hr. All other rats survived until sacrificed. The control platelet counts were normal, ranging from 100,000 to 520,000 per ENVENOMATION
182
Short Communications TABLE 1 . EFFECT OF CrOWIY3
Virldls helleri
Dose Time to Pre-envenomation (mg/kg) sacrifice (hr) platelet count
VENOM ON PLATELET COUNT AND HONE MARROW
Post-envenomation final platelet count
Pathological marrow findings
2
16
210,000
90,000
No abnormalities noted
2
24
430,000
420,000
2
24
230,000
14,000
Increased number of plasma cells
4
2
480,000
50,000
Megakaryocytic erythrophagocytosis. Eosinophils : 10'/
4
4
500,000
438,000
4
15'
145,000
-
Vacuolization of red cell precursors. Eosinophils : 10'/. Occasional megakaryocytic erytbrophagocytosis
4
16
510,000
80,000
Normal except increased number of mast cells
4
24
230,000
14,000
No abnormalities noted
8
12
100,000
5000
8
24
520,000
70,000
Megakaryopoiesis active and normal. Eosinophils : 5'/. Microvacuohintion of myeloblasts, promyelocytes and erythroblasts . Marked mastocytosis
No abnormalities
Marked vacuolization of red cell precursors, moderate vacuolization of myelocytes. Megakaryocytis normal Eosinophils : 20'/ . erythrophagocytosis
Megakaryocytic
" Died at 15 hr.
mm3. Following envenomation, eight of the ten rats had a significant fall in platelets, while white blood counts were not affected. The bone marrow of all rats showed a normal maturation progression of myelocytic cells from myeloblasts to mature polymorphonuclear leukocytes. However, the myeloid/erythroid ratio was markedly shifted from the normal 2 :1 ratio (SCHALM, 1965) to 8-10 :1 . Erythropoiesis showed normal maturation. Megakaryocytes were normal in number and appeared morphologically unchanged . There was little nuclear division, no vacuolization, and no hyalinization ofthecell borders. The structure ofthe chromatin appeared normal . No platelet fragmentation was observed. In two rats, the marrow showed erythrophagocytosis by afewmegakaryocytes. An increased number ofmast cells were noted in the marrow oftbvo rats and increased eosinophils in three other rats (Table 1). Reduction of platelets following severe rattlesnake envenomation may be due to several mechanisms, possibly mediated through the extrinsic coagulation pathway by release of tissue factor from injured cells or by activation ofthe intrinsic pathway through exposure of the basement membrane, due to damaged vascular epithelial cells. There may be a direct action of the venom on thrombin (DENSON et al., 1972) or direct damage to the megakaryocytes of the marrow. The fall in platelet count usually occurs too soon after envenomation to appear to implicatedirect megakaryocytedestruction as the primary cause.
Short Communications
183
Observation ofseverely envenomated humans indicates thatthenadir occurs at 72 hr and the platelet count may not return to normal for over 144 hr (LA GRANGE and RUSSELL, 1970). Transient damage to marrow megakaryocytes may be a factor in this delay. In the present study, no decrease in the number of megakaryocytes was noted in rat marrow, regardless of the quantity of venom injected. No morphological evidence of megakaryocyte injury-vacuolization, fragmentation or hyalinization of the membrane-was observed 24 hr after envenomation. It appears that the decrease in platelets was probably associated with peripheral consumption. Other hematopoietic elements of the marrow appeared damaged, as indicated by vacuolization ofred and white cell precursors. This did not occur in all marrows and could not be correlated with either dose or duration of exposure to the toxin. A consistent finding was a marked physiological myelocytic-erythrocytic ratio shift from normal to 10 :1, which possibly reflected a stress reaction . Further evidence ofa stress reaction might be apparent by the megakaryocytic erythrophagocytosis . The mastocytosis and eosinophilia observed in several of the marrows require further investigation ; they could be the response to an ammunologic insult . Acknowledgements--This work was supported by a grant from the National Institute of General Medical Sciences (5R01 GM24141-02 TOX~ and a grant (5S07RR0-5466-11) from the Professional Staff Association of the Los Angeles County University of Southern California Medical Center . We thank JoHN Q. PATroN for his technical assistance . REFERENCES DENsoN, K. W. E, RUSSELL, F. E, ALMAOL4, P. and BisHor, R. C. (1972) Characterization of the coagulant activity of some snake venoms . Toxicon 10, 557. GENNARo, J, Jr. and RAMsEY, H. W. (1959) Distribution in the mouse of lethal and sublethal doses of cottonmouth moccasin venom labeled with I" 1. Nature . Lond. 184, 1244. LA GRANGE, R. G. and RUSSELL, F. E. (1970) Blood platelet studies in man and rabbits following Crotalus envenomation. Proc. West. Plurrmac. Soc. 13, 99. SCHALM, O. W. (1965) Veterinary Hematology, 2nd Edn, p. 312. Philadelphia : Lea & Febiger. WmaERT,W. A, PATTABHrRAMAN, T.R., CLELAND, R. A., MEYER, P, PATPABHmAMAK R and RUSSELL, F. E. (1980) Distribution and pathology of copperhead (Agkistrodon contortrix) venom. Toxicon 18, 591.
0041-0101/81/0101-0181 $02 .00/0
EFFECT OF A RATTLESNAKE VENOM
VIRIDIS HELLERI)
(CROTALUS
ON BONE MARROW
A.
WINGERT,' TAMMANUR R. PATTABHIRAMAN,' DARLENE POWERS' and FINDLAY E . RUSSELL' ' Laboratory of Neurological Research and 2 Department of Pediatrics, Los Angeles County/University of Southern California Medical Center, Los Angeles, CA 90033, U.S .A. WILLIS
(Accepted for publication 16 September 1980) W . A . WINGERT, T . R . PATTABHIRAMAN, D . POWERS and F. E . RUSSELL . Effect of a rattlesnake venom (Crotales piridis helleri) on bone marrow . Toxicon 19, 181-183,1981 .-Crotales oiridis helleri venom produces a fall in peripheral platelet count in rats but does not appear to damage the precursors in the bone marrow . The major response is a myeloid hyperplasia .
by Crotalus oiridis helleri causes a decrease in circulating platelets in both humans and experimental animals (LA GRANGE and RUSSELL, 1970) . Following injection of the venom into rabbits, there is a three-phase platelet response : an initial rapid drop followed by a rise at 36 hr, a subsequent overshoot, and a return toward normal levels at 144-282 hr (LA GRANGE and RUSSELL, 1970). Since this effect appears to be both time- and dose-related, it may be suggested as an index to determine the level ofpoisoning following bites by this snake, and thus be of value in determining antivenin administration . The loss ofplatelets may be due to (1) direct intravascular destruction or to consumption during the intravascular coagulation initiated by the direct action of the venom on some clotting component, such as thrombin or fibrinogen ; (2) toxin-induced damage to platelet precursors in the bone marrow ; or (3) some combination of these factors. The direct effect of megakaryocyte kinetics following envenomation has not been clearly defined, although isotope-tagged pit viper venom is known to be distributed to the marrow (GENNARo and RAMSEY, 1959 ; WINGERT et al., 1980) . The present study was designed to observe the progressive changes that occur in bone marrow cells, particularly the megakaryocytes, following the injection of various doses of a crotalid venom. Ten white male rats, weighing 300-387 g each, were injected subcutaneously with Crotalus viridis helleri venom reconstituted following 6 months storage at 5°C . The venom doses varied from 2 to 8 mg/kg. (In a previous study, the subcutaneous LDSO at 24 hr was 4.0 mg/kg body wt.) Bone marrow from the femur was obtained by killing the rats 2-24 hr after injection, sectioning the bone, and transferring the marrow directly to a slide. The marrow was fixed and stained with Wright's stain, using standard techniques. Platelet counts were obtained prior to envenomation and complete blood and platelet counts were obtained before sacrifice. One rat injected with 4 mg/kg of venom died at 15 hr. All other rats survived until sacrificed. The control platelet counts were normal, ranging from 100,000 to 520,000 per ENVENOMATION
182
Short Communications TABLE 1 . EFFECT OF CrOWIY3
Virldls helleri
Dose Time to Pre-envenomation (mg/kg) sacrifice (hr) platelet count
VENOM ON PLATELET COUNT AND HONE MARROW
Post-envenomation final platelet count
Pathological marrow findings
2
16
210,000
90,000
No abnormalities noted
2
24
430,000
420,000
2
24
230,000
14,000
Increased number of plasma cells
4
2
480,000
50,000
Megakaryocytic erythrophagocytosis. Eosinophils : 10'/
4
4
500,000
438,000
4
15'
145,000
-
Vacuolization of red cell precursors. Eosinophils : 10'/. Occasional megakaryocytic erytbrophagocytosis
4
16
510,000
80,000
Normal except increased number of mast cells
4
24
230,000
14,000
No abnormalities noted
8
12
100,000
5000
8
24
520,000
70,000
Megakaryopoiesis active and normal. Eosinophils : 5'/. Microvacuohintion of myeloblasts, promyelocytes and erythroblasts . Marked mastocytosis
No abnormalities
Marked vacuolization of red cell precursors, moderate vacuolization of myelocytes. Megakaryocytis normal Eosinophils : 20'/ . erythrophagocytosis
Megakaryocytic
" Died at 15 hr.
mm3. Following envenomation, eight of the ten rats had a significant fall in platelets, while white blood counts were not affected. The bone marrow of all rats showed a normal maturation progression of myelocytic cells from myeloblasts to mature polymorphonuclear leukocytes. However, the myeloid/erythroid ratio was markedly shifted from the normal 2 :1 ratio (SCHALM, 1965) to 8-10 :1 . Erythropoiesis showed normal maturation. Megakaryocytes were normal in number and appeared morphologically unchanged . There was little nuclear division, no vacuolization, and no hyalinization ofthecell borders. The structure ofthe chromatin appeared normal . No platelet fragmentation was observed. In two rats, the marrow showed erythrophagocytosis by afewmegakaryocytes. An increased number ofmast cells were noted in the marrow oftbvo rats and increased eosinophils in three other rats (Table 1). Reduction of platelets following severe rattlesnake envenomation may be due to several mechanisms, possibly mediated through the extrinsic coagulation pathway by release of tissue factor from injured cells or by activation ofthe intrinsic pathway through exposure of the basement membrane, due to damaged vascular epithelial cells. There may be a direct action of the venom on thrombin (DENSON et al., 1972) or direct damage to the megakaryocytes of the marrow. The fall in platelet count usually occurs too soon after envenomation to appear to implicatedirect megakaryocytedestruction as the primary cause.
Short Communications
183
Observation ofseverely envenomated humans indicates thatthenadir occurs at 72 hr and the platelet count may not return to normal for over 144 hr (LA GRANGE and RUSSELL, 1970). Transient damage to marrow megakaryocytes may be a factor in this delay. In the present study, no decrease in the number of megakaryocytes was noted in rat marrow, regardless of the quantity of venom injected. No morphological evidence of megakaryocyte injury-vacuolization, fragmentation or hyalinization of the membrane-was observed 24 hr after envenomation. It appears that the decrease in platelets was probably associated with peripheral consumption. Other hematopoietic elements of the marrow appeared damaged, as indicated by vacuolization ofred and white cell precursors. This did not occur in all marrows and could not be correlated with either dose or duration of exposure to the toxin. A consistent finding was a marked physiological myelocytic-erythrocytic ratio shift from normal to 10 :1, which possibly reflected a stress reaction . Further evidence ofa stress reaction might be apparent by the megakaryocytic erythrophagocytosis . The mastocytosis and eosinophilia observed in several of the marrows require further investigation ; they could be the response to an ammunologic insult . Acknowledgements--This work was supported by a grant from the National Institute of General Medical Sciences (5R01 GM24141-02 TOX~ and a grant (5S07RR0-5466-11) from the Professional Staff Association of the Los Angeles County University of Southern California Medical Center . We thank JoHN Q. PATroN for his technical assistance . REFERENCES DENsoN, K. W. E, RUSSELL, F. E, ALMAOL4, P. and BisHor, R. C. (1972) Characterization of the coagulant activity of some snake venoms . Toxicon 10, 557. GENNARo, J, Jr. and RAMsEY, H. W. (1959) Distribution in the mouse of lethal and sublethal doses of cottonmouth moccasin venom labeled with I" 1. Nature . Lond. 184, 1244. LA GRANGE, R. G. and RUSSELL, F. E. (1970) Blood platelet studies in man and rabbits following Crotalus envenomation. Proc. West. Plurrmac. Soc. 13, 99. SCHALM, O. W. (1965) Veterinary Hematology, 2nd Edn, p. 312. Philadelphia : Lea & Febiger. WmaERT,W. A, PATTABHrRAMAN, T.R., CLELAND, R. A., MEYER, P, PATPABHmAMAK R and RUSSELL, F. E. (1980) Distribution and pathology of copperhead (Agkistrodon contortrix) venom. Toxicon 18, 591.