J. Comp. Path. 1990Vol. 103
H i p p o c a m p a l Neuronal Necrosis in Control Fischer 344 Rats T. A. B a r b o l t a n d R. M. E v e r e t t
Department of Toxicology, Sterling Research Group, Rensselaer, New York 12144, U.S.A. Summary Brain tissue sections from control male and female Fischer 344 (F344) rats from ten National Toxicology Program (NTP) Bioassays were histomorphologically reviewed for non-neoplastic lesions of the hippocampus. Unilateral segmental hippocampal neuronal necrosis, which has not been reported in normal rats, was observed in 9 of 433 (2"1 per cent) males and 1 of 454 (0"2 per cent) females. Significant coexisting lesions were left-sided atrial and/or valvular thrombosis, metastatic mesothelioma, and large lymphocyte leukaemia. These data suggest that this naturally occurring lesion of predominantly aged male rats may result from an impairment of cerebral perfusion secondary to vascular obstruction by thrombotic emboli or leukaemic cells and haemolytic anaemia concomitant with large lymphocyte leukaemia, which commonly occurs in F344 rats. Introduction Selective neuronal necrosis of the hippocampus of rats has been described following treatment with benzaldehyde (Kluwe, Montgomery, Giles and Prejean, 1983), trimethyltin (Robertson, Gray and de la Iglesia, 1987) and soman (McLeod, Singer and Harrington, 1984). It has been suggested that the mechanism for this toxicity is related to generalized cerebral ischaemia resulting from increased metabolic activity (neuroexcitation) which exceeds the delivery of oxygen and nutrients to the brain (consumptive anoxia). Similar hippocampal lesions have been produced experimentally in rats after combined anoxic anoxia and unilateral carotid ligation (Levine, 1960), further d~monstrating the sensitivity of the hippocampus to ischaemia. ,The purpose of this study was to determine whether hippocampal neuronal necrosis, which to our knowledge has not been reported in untreated rats, occurs naturally as a background lesion in control F344 rats. Carcinogenicity bioassays were chosen as a source of tissue for study because they provided access to ~ large pool of rats which were treated according to a standardized protocol.
Materials and Methods Ten NTP Bioassays using F344 rats were chosen for review at the NTP Archive (Research Triangle Park, NC) and included untreated control rats from five dietary feeding studies and vehicle (corn oil) control rats fi'om five gavage studies. Three 0021-9975/90/070335+ 07 $03.00/0
© 1990AcademicPressLimited
336
T. A. Barbolt and R. M . E v e r e t t
coronal sections of brain, including forebrain, midbrain and cerebellum, which had been fixed by immersion in 10 per cent neutral buffered formalin, embedded in paraffin wax and stained with haematoxylin and eosin (HE), were retrieved from archival storage of approximately 50 male and 50 female rats from each study. The histomorphological review was focused on non-neoplastic lesions of the hippocampus. The Individual Animal Data Records and the Table II Package (computerized data listings and incidence tables) and slides of other tissues from each animal were made available during the histomorphological review so that concomitant lesions for other tissues could be reviewed. Results
In most cases, the anterior and dorsal portions of the hippocampus were included in the standard coronal sections. Normally, the pyramidal neurons of the hippocampus and granular cells of the dentate gyrus form discrete continuous bands (Fig. 1). The hippocampal lesions observed during this review were characterized by unilateral segmental necrosis of pyramidal neurons, sometimes including an adjacent segment of granular cells of the dentate gyrus (Fig. 2). Affected neurons were shrunken and had nuclear and cytoplasmic eosinophilia, with or without a mild reactive gliosis (Figs 3 and 4). Generally, neuronal loss resulted from lysis of eosinophilic neurons without evidence of an inflammatory response. The lesions occurred at different sites in the dorsal hippocampus without a predilection for any specific region of the
Fig. 1, Normal dorsal hippocampus with continuous band of pyramidal neurons (large arrows) and granular cells of the dentate gyrus (small arrows). HE x 23.
Neuronal Necrosis in Control Rats
337
Fig, 2. Segmental necrosis of pyramidal neurons (between large arrows) and granular cells of the dentate gyrus (between small arrows). HE x 22. Fig. 3. Greater magn fication of Fig. 2. Segmental necrosis of pyramidal neurons with reactive gliosis. HE × 70. c o r n u a m m o n u s . O n e lesion was m o r e extensive t h a n the others a n d was c h a r a c t e r i z e d b y a large a r e a o f m a l a c i a with distortion of the c o r n u a m m o n u s (Fig. 5). T h e overall i n c i d e n c e o f these h i p p o c a m p a l lesions was 2"1 per cent (9 o f 433) for m a l e a n d 0"2 per c e n t (1 o f 454) for female F344 rats. F o r males, the r a n g e o f incidences a m o n g studies was 0 o f 49 to 2 o f 39, a n d there was no
338
Fig. 4.
T . A . B a r b o l t a n d R. M . E v e r e t t
Segmental necrosis of pyramidal neurons (between arrows) without reactive gliosis. HE x 92,
Fig. 5. Hippoeampal malacia with loss of the pyramidal band and granular cells of the dentate gyrus. HE x 68.
apparent difference in the incidences between untreated control rats and those given corn oil by garage. The time-on-test for the ten rats with hippocampal lesions ranged from 74 to 108 weeks (Table 1). Three rats were found dead, four were killed in extremis and three were killed at study termination. Significant coexisting lesions were observed in other tissues for nine of ten
Neuronal
N e c r o s i s in C o n t r o l R a t s
339
Table 1 D i s p o s i t i o n and Significant Coexisting Lesions for F344 Rats with H i p p o c a m p a l N e u r o n a l
Necrosis Case No.
Disposition
Weeks on test
1
Killed in extremis
97
2
Found dead
74
3
Killed in extremis
98
4 5 6 7 8 9 10
Killed Killed Killed Killed Killed Found Found
in extremis at termination in extremis at termination at termination dead dead
95 105 80 108 108 101 96
Coexisting lesions Cerebral thrombosis Mononuelear cell leukaemia Left atrial thrombosis Mononuelear cell leukaemia Left atrial thrombosis Mononuclear cell leukaemia Mononuclear cell leukaemia Mononuclear cell leukaemia Mononuclear cell leukaemia None Metastatic mesothelloma Mononuelear cell leukaemia A-V valve thrombosis Mononuclear cell leukaemia
rats with hippocampal lesions (Table 1). Mononuclear cell leukaemia (recorded by the original N T P pathologist) was observed for eight of ten rats, left atrial thrombosis was observed for two of ten rats, and thrombosis at the atrio-ventricular valve and" thrombosis of a superficial cerebral artery were each observed for one of ten rats. Metastatic mesothelioma (presumably from the tunica vaginalis) was observed for one of ten rats while significant coexisting lesions were not observed for one rat. Discussion
Neuronal necrosis of the hippocampus has been associated with conditions or treatments which result in cerebral ischaemia. This may occur when the energy requirements of neurons exceeds the availability of oxygen and other nutrients. This m e c h a n i s m has been postulated to explain hippocampal necrosis occurring during epileptic seizures in man (Blennow, Brierley, Meldrum and Siesjo, 1978; D a m , 1980) and animals (Montgomery and Lee, 1983). Similar circumstances can occur after the administration of neuroexcitatory chemicals (Brown, Aldridge, Street and Verschoyle, 1979; Nadler, 1980; Wuerthele, Lovell, Jones and Moore, 1978) or organophosphate poisons (McLeod, et al., 1984). Alternatively, this imbalance in the supply and demand for oxygen and other nutrients can occur when the circulation is impaired, for example, after carotid artery ligation (BuNs, Fujimoto, Ito, Mrsulja, Spatz and Klatzo, 1976), or carotid artery ligation in combination with anoxic anoxia (Levine, 1960). Thus, the hippocampus serves as a sensitive indicator for cerebral ischaemia resulting from several different mechanisms. T h e most important coexisting lesions for the ten cases of hippocampal neuronal necrosis in the present review were left-sided atrial or valvular thrombosis observed in three cases and cerebral thrombosis observed in another case, which suggested a potential situation of cerebral ischaemia
340
T. A. Barbolt and R. M. Everett
resulting from obstruction of blood flow. A metastatic mesothelioma was observed in another case and, although metastases or micro-emboli of neoplastic cells were not observed in the brain, an impairment o f perfusion or ventilation as a result of extensive p u l m o n a r y metastases cannot be precluded as a possible cause of cerebral ischaemia. A c o m m o n neoplastic lesion occurring in F344 rats is m o n o n u c l e a r cell leukaemia, more recently classified as large lymphocyte leukaemia ( W a r d and Reynolds, 1983), which is associated with marked haemolytic a n a e m i a (Stromberg, Vogtsberger, Marsh and Wilson, 1983). Eight of the ten cases of h i p p o c a m p a l neuronal necrosis had concomitant mononuclear cell leukaemia, and four of these had atrial, valvular or cerebral thrombosis. A l t h o u g h the extent of anaemic anoxia associated with haemolytic a n a e m i a and the disturbance in oxygen transport related to the increased numbers ofleukaemic cells is not known for the rats with hippocampal lesions, these factors were considered to have been contributing mechanisms in predisposing these rats to cerebral ischaemia. However, since numerous cases of m o n o n u c l e a r cell leukaemia exist in the absence of hippocampal necrosis, there m a y be other mechanisms to explain the cause of these hippocampal lesions. The data from the current review describe for the first time, to our knowledge, hippocampal neuronal necrosis in control F344 rats and suggest that hippocampal neuronal necrosis is another example of a naturally occurring age-related lesion, principally in male rats, which can result from any factor or combination of factors which compromises cerebral perfusion. Acknowledgments
The authors thank Dr Mel Hamlin from the National Toxicology Program Archive for his assistance in providing the materials for review. The comments on the manuscript by Dr Darlene Dixon of the National Institute of Environmental Health Sciences are greatly appreciated. The expertise in photomicrography of Ms Joyce Shoffner and Mr Fred Talley is gratefully acknowledged. References
Blennow, A., Brierley, J. B., Meldrum, B. S. and Siesjo, B. K. (1978). Epileptic brain damage. Brain, 101, 687-700. Brown, A. W., Aldridge, W. N., Street, B. W. and Verschoyle, R. D. (1979). The behavioral and neuropathologic sequelae of intoxication by trimethyltin compounds in the rat. American Journal of Pathology, 97, 59-82. Bubis, J. J., Fujimoto, T., Ito, U., Mrsulja, B. J., Spatz, M. and Ktatzo, I. (1976). Experimental cerebral ischemia in Mongolian gerbils. V. Ultrastructural changes in the H3 sector of the hippocampus. Acta Neuropathologica, 36, 285-294. Darn, A. M. (1980). Epilepsy and neuron loss in the hippocampus. Epilepsia, 21, 617-629. Kluwe, W. M., Montgornery, C. A., Giles, H. D. and Prejean, J. D. (1983). Encephalopathy in rats and nephropathy in rats and mice after subchronic oral exposure to benzaldehyde. Food and Chemical Toxicology, 21, 245-250. Levine, S. (1960). Anoxic-ischemic encephalopathy in rats. American Journal of Pathology, 36, 1-17. McLeod, C. G. Jr, Singer, A. W. and Harrington, D. G. (l 984). Acute neuropathology in soman poisoned rats. Neurotoxicology, 5, 53-58.
N e u r o n a l N e c r o s i s in C o n t r o l R a t s
341
Montgomery, D. L. and Lee, A. C. (1983). Brain damage in the epileptic beagle dog. Veterinary Pathology, 20, 160-169. Nadler, J. V. (1980). Kainic acid neurotoxicity toward hippocampal formation: Dependence on specific excitatory pathways. Brain Research, 195~ 47-56. Robertson, D. G., Gray, R. H. and de la Iglesia, F. A. (1987). Quantitative assessment of trimethyltin induced pathology of the hippocampus. ToxicologicPathology, 15, 7-17. Stromberg, P. C., Vogtsberger, I. M., Marsh, L. R. and Wilson, F. D. (1983). Pathology of the mononuclear cell leukemia of Fischer rats. II. Hematology. Veterinary Pathology, 20, 709-717. Ward, J. M. and Reynolds, C. W. (1983). Large granular lymphocyte leukemia, a heterogenous lymphocytic leukemia in F344 rats. American Journal of Pathology, 111, 1-10. Wucrthele, S. M., Lovell, K. L., Jones, M. Z. and Moore, K. E. (1978). A histological study ofkainic acid-induced lesions in the rat brain. Brain Research, 149, 489-497.
I Received, January 3rd, 1990-] Accepled, May-19lh, 1990 ~J
H i p p o c a m p a l Neuronal Necrosis in Control Fischer 344 Rats T. A. B a r b o l t a n d R. M. E v e r e t t
Department of Toxicology, Sterling Research Group, Rensselaer, New York 12144, U.S.A. Summary Brain tissue sections from control male and female Fischer 344 (F344) rats from ten National Toxicology Program (NTP) Bioassays were histomorphologically reviewed for non-neoplastic lesions of the hippocampus. Unilateral segmental hippocampal neuronal necrosis, which has not been reported in normal rats, was observed in 9 of 433 (2"1 per cent) males and 1 of 454 (0"2 per cent) females. Significant coexisting lesions were left-sided atrial and/or valvular thrombosis, metastatic mesothelioma, and large lymphocyte leukaemia. These data suggest that this naturally occurring lesion of predominantly aged male rats may result from an impairment of cerebral perfusion secondary to vascular obstruction by thrombotic emboli or leukaemic cells and haemolytic anaemia concomitant with large lymphocyte leukaemia, which commonly occurs in F344 rats. Introduction Selective neuronal necrosis of the hippocampus of rats has been described following treatment with benzaldehyde (Kluwe, Montgomery, Giles and Prejean, 1983), trimethyltin (Robertson, Gray and de la Iglesia, 1987) and soman (McLeod, Singer and Harrington, 1984). It has been suggested that the mechanism for this toxicity is related to generalized cerebral ischaemia resulting from increased metabolic activity (neuroexcitation) which exceeds the delivery of oxygen and nutrients to the brain (consumptive anoxia). Similar hippocampal lesions have been produced experimentally in rats after combined anoxic anoxia and unilateral carotid ligation (Levine, 1960), further d~monstrating the sensitivity of the hippocampus to ischaemia. ,The purpose of this study was to determine whether hippocampal neuronal necrosis, which to our knowledge has not been reported in untreated rats, occurs naturally as a background lesion in control F344 rats. Carcinogenicity bioassays were chosen as a source of tissue for study because they provided access to ~ large pool of rats which were treated according to a standardized protocol.
Materials and Methods Ten NTP Bioassays using F344 rats were chosen for review at the NTP Archive (Research Triangle Park, NC) and included untreated control rats from five dietary feeding studies and vehicle (corn oil) control rats fi'om five gavage studies. Three 0021-9975/90/070335+ 07 $03.00/0
© 1990AcademicPressLimited
336
T. A. Barbolt and R. M . E v e r e t t
coronal sections of brain, including forebrain, midbrain and cerebellum, which had been fixed by immersion in 10 per cent neutral buffered formalin, embedded in paraffin wax and stained with haematoxylin and eosin (HE), were retrieved from archival storage of approximately 50 male and 50 female rats from each study. The histomorphological review was focused on non-neoplastic lesions of the hippocampus. The Individual Animal Data Records and the Table II Package (computerized data listings and incidence tables) and slides of other tissues from each animal were made available during the histomorphological review so that concomitant lesions for other tissues could be reviewed. Results
In most cases, the anterior and dorsal portions of the hippocampus were included in the standard coronal sections. Normally, the pyramidal neurons of the hippocampus and granular cells of the dentate gyrus form discrete continuous bands (Fig. 1). The hippocampal lesions observed during this review were characterized by unilateral segmental necrosis of pyramidal neurons, sometimes including an adjacent segment of granular cells of the dentate gyrus (Fig. 2). Affected neurons were shrunken and had nuclear and cytoplasmic eosinophilia, with or without a mild reactive gliosis (Figs 3 and 4). Generally, neuronal loss resulted from lysis of eosinophilic neurons without evidence of an inflammatory response. The lesions occurred at different sites in the dorsal hippocampus without a predilection for any specific region of the
Fig. 1, Normal dorsal hippocampus with continuous band of pyramidal neurons (large arrows) and granular cells of the dentate gyrus (small arrows). HE x 23.
Neuronal Necrosis in Control Rats
337
Fig, 2. Segmental necrosis of pyramidal neurons (between large arrows) and granular cells of the dentate gyrus (between small arrows). HE x 22. Fig. 3. Greater magn fication of Fig. 2. Segmental necrosis of pyramidal neurons with reactive gliosis. HE × 70. c o r n u a m m o n u s . O n e lesion was m o r e extensive t h a n the others a n d was c h a r a c t e r i z e d b y a large a r e a o f m a l a c i a with distortion of the c o r n u a m m o n u s (Fig. 5). T h e overall i n c i d e n c e o f these h i p p o c a m p a l lesions was 2"1 per cent (9 o f 433) for m a l e a n d 0"2 per c e n t (1 o f 454) for female F344 rats. F o r males, the r a n g e o f incidences a m o n g studies was 0 o f 49 to 2 o f 39, a n d there was no
338
Fig. 4.
T . A . B a r b o l t a n d R. M . E v e r e t t
Segmental necrosis of pyramidal neurons (between arrows) without reactive gliosis. HE x 92,
Fig. 5. Hippoeampal malacia with loss of the pyramidal band and granular cells of the dentate gyrus. HE x 68.
apparent difference in the incidences between untreated control rats and those given corn oil by garage. The time-on-test for the ten rats with hippocampal lesions ranged from 74 to 108 weeks (Table 1). Three rats were found dead, four were killed in extremis and three were killed at study termination. Significant coexisting lesions were observed in other tissues for nine of ten
Neuronal
N e c r o s i s in C o n t r o l R a t s
339
Table 1 D i s p o s i t i o n and Significant Coexisting Lesions for F344 Rats with H i p p o c a m p a l N e u r o n a l
Necrosis Case No.
Disposition
Weeks on test
1
Killed in extremis
97
2
Found dead
74
3
Killed in extremis
98
4 5 6 7 8 9 10
Killed Killed Killed Killed Killed Found Found
in extremis at termination in extremis at termination at termination dead dead
95 105 80 108 108 101 96
Coexisting lesions Cerebral thrombosis Mononuelear cell leukaemia Left atrial thrombosis Mononuelear cell leukaemia Left atrial thrombosis Mononuclear cell leukaemia Mononuclear cell leukaemia Mononuclear cell leukaemia Mononuclear cell leukaemia None Metastatic mesothelloma Mononuelear cell leukaemia A-V valve thrombosis Mononuclear cell leukaemia
rats with hippocampal lesions (Table 1). Mononuclear cell leukaemia (recorded by the original N T P pathologist) was observed for eight of ten rats, left atrial thrombosis was observed for two of ten rats, and thrombosis at the atrio-ventricular valve and" thrombosis of a superficial cerebral artery were each observed for one of ten rats. Metastatic mesothelioma (presumably from the tunica vaginalis) was observed for one of ten rats while significant coexisting lesions were not observed for one rat. Discussion
Neuronal necrosis of the hippocampus has been associated with conditions or treatments which result in cerebral ischaemia. This may occur when the energy requirements of neurons exceeds the availability of oxygen and other nutrients. This m e c h a n i s m has been postulated to explain hippocampal necrosis occurring during epileptic seizures in man (Blennow, Brierley, Meldrum and Siesjo, 1978; D a m , 1980) and animals (Montgomery and Lee, 1983). Similar circumstances can occur after the administration of neuroexcitatory chemicals (Brown, Aldridge, Street and Verschoyle, 1979; Nadler, 1980; Wuerthele, Lovell, Jones and Moore, 1978) or organophosphate poisons (McLeod, et al., 1984). Alternatively, this imbalance in the supply and demand for oxygen and other nutrients can occur when the circulation is impaired, for example, after carotid artery ligation (BuNs, Fujimoto, Ito, Mrsulja, Spatz and Klatzo, 1976), or carotid artery ligation in combination with anoxic anoxia (Levine, 1960). Thus, the hippocampus serves as a sensitive indicator for cerebral ischaemia resulting from several different mechanisms. T h e most important coexisting lesions for the ten cases of hippocampal neuronal necrosis in the present review were left-sided atrial or valvular thrombosis observed in three cases and cerebral thrombosis observed in another case, which suggested a potential situation of cerebral ischaemia
340
T. A. Barbolt and R. M. Everett
resulting from obstruction of blood flow. A metastatic mesothelioma was observed in another case and, although metastases or micro-emboli of neoplastic cells were not observed in the brain, an impairment o f perfusion or ventilation as a result of extensive p u l m o n a r y metastases cannot be precluded as a possible cause of cerebral ischaemia. A c o m m o n neoplastic lesion occurring in F344 rats is m o n o n u c l e a r cell leukaemia, more recently classified as large lymphocyte leukaemia ( W a r d and Reynolds, 1983), which is associated with marked haemolytic a n a e m i a (Stromberg, Vogtsberger, Marsh and Wilson, 1983). Eight of the ten cases of h i p p o c a m p a l neuronal necrosis had concomitant mononuclear cell leukaemia, and four of these had atrial, valvular or cerebral thrombosis. A l t h o u g h the extent of anaemic anoxia associated with haemolytic a n a e m i a and the disturbance in oxygen transport related to the increased numbers ofleukaemic cells is not known for the rats with hippocampal lesions, these factors were considered to have been contributing mechanisms in predisposing these rats to cerebral ischaemia. However, since numerous cases of m o n o n u c l e a r cell leukaemia exist in the absence of hippocampal necrosis, there m a y be other mechanisms to explain the cause of these hippocampal lesions. The data from the current review describe for the first time, to our knowledge, hippocampal neuronal necrosis in control F344 rats and suggest that hippocampal neuronal necrosis is another example of a naturally occurring age-related lesion, principally in male rats, which can result from any factor or combination of factors which compromises cerebral perfusion. Acknowledgments
The authors thank Dr Mel Hamlin from the National Toxicology Program Archive for his assistance in providing the materials for review. The comments on the manuscript by Dr Darlene Dixon of the National Institute of Environmental Health Sciences are greatly appreciated. The expertise in photomicrography of Ms Joyce Shoffner and Mr Fred Talley is gratefully acknowledged. References
Blennow, A., Brierley, J. B., Meldrum, B. S. and Siesjo, B. K. (1978). Epileptic brain damage. Brain, 101, 687-700. Brown, A. W., Aldridge, W. N., Street, B. W. and Verschoyle, R. D. (1979). The behavioral and neuropathologic sequelae of intoxication by trimethyltin compounds in the rat. American Journal of Pathology, 97, 59-82. Bubis, J. J., Fujimoto, T., Ito, U., Mrsulja, B. J., Spatz, M. and Ktatzo, I. (1976). Experimental cerebral ischemia in Mongolian gerbils. V. Ultrastructural changes in the H3 sector of the hippocampus. Acta Neuropathologica, 36, 285-294. Darn, A. M. (1980). Epilepsy and neuron loss in the hippocampus. Epilepsia, 21, 617-629. Kluwe, W. M., Montgornery, C. A., Giles, H. D. and Prejean, J. D. (1983). Encephalopathy in rats and nephropathy in rats and mice after subchronic oral exposure to benzaldehyde. Food and Chemical Toxicology, 21, 245-250. Levine, S. (1960). Anoxic-ischemic encephalopathy in rats. American Journal of Pathology, 36, 1-17. McLeod, C. G. Jr, Singer, A. W. and Harrington, D. G. (l 984). Acute neuropathology in soman poisoned rats. Neurotoxicology, 5, 53-58.
N e u r o n a l N e c r o s i s in C o n t r o l R a t s
341
Montgomery, D. L. and Lee, A. C. (1983). Brain damage in the epileptic beagle dog. Veterinary Pathology, 20, 160-169. Nadler, J. V. (1980). Kainic acid neurotoxicity toward hippocampal formation: Dependence on specific excitatory pathways. Brain Research, 195~ 47-56. Robertson, D. G., Gray, R. H. and de la Iglesia, F. A. (1987). Quantitative assessment of trimethyltin induced pathology of the hippocampus. ToxicologicPathology, 15, 7-17. Stromberg, P. C., Vogtsberger, I. M., Marsh, L. R. and Wilson, F. D. (1983). Pathology of the mononuclear cell leukemia of Fischer rats. II. Hematology. Veterinary Pathology, 20, 709-717. Ward, J. M. and Reynolds, C. W. (1983). Large granular lymphocyte leukemia, a heterogenous lymphocytic leukemia in F344 rats. American Journal of Pathology, 111, 1-10. Wucrthele, S. M., Lovell, K. L., Jones, M. Z. and Moore, K. E. (1978). A histological study ofkainic acid-induced lesions in the rat brain. Brain Research, 149, 489-497.
I Received, January 3rd, 1990-] Accepled, May-19lh, 1990 ~J
