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262 Phosphotyrosine immunoreactivity of activated microglia in an in vivo model of inflammation in the rat brain
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FIFTH INTERNATIONAL CONFERENCE ON A L Z H E I M E R ' S DISEASE
Currently available animal models fail to provide adequate assessment of the cognitive deficits seen in Alzheimer's disease (AD). The hippocampus is involved in learning and memory processes but difficulty in producing selective hippocampal lesions so far provided no model for understanding the role of hippocampus in learning and memory. Long-term adrenalectomy produces selective neuronal loss in the hippocampus. Our results show neuronal loss in the dentate gyrus and the different hippocampal fields of adrenalectomized (ADX) rats. Immunohistochemical staining of brain sections from ADX but not sham operated rats showed intense staining of CD4 and Major histocompatibility complex class II antigens in hippocampal areas. The demonstration of induction of these antigens in the hippocampus after long-term adrenalectomy provides evidence of neuronal damage mediated by inflammatory processes. Preliminary results showed amyloid precursor protein (APP) inmaunoreactivity in the hippocampal neurons and associated with particularly dystrophic appearing cells and neudtes. This was more clearly evident with carboxyl terminal antibodies of APP rather than the amino terminal ones. Electron microscopic studies show that the neuron loss could be possibly mediated by apoptosis. The ADX rats show increased latency in the Morris water maze and impaired retention when compared to the sham operated rats. The latter data indicate that long-term adrenalectomy causes impairment in spatial learning and explorative behaviour in the rat. Selective lesioning of the hippocampus by long-term adrenalectomy may provide a good model to assess the cognitive deficits seen in AD as well as shed_light on the mechanisms which might be involved in neurodegenerative processes involved in AD.
262 Phosphotyrosine Immunoreactivityof Activated Microglia in an In Vivo Model of Inflammation in the Rat Brain G. Noy, S.J. Newman, F. Brown* and G.W. Roberts SmithKline Beecham, New Frontiers Science Park, Third Avenue, Harlow, Essex, CM19 5AW, UK CNS diseases or traumas, e.g. Alzheimer's disease (AD), multiple sclerosis (MS) and head injury, involve CNS inflammatory reactions. Evidence is increasing that some inflammatory factors (e.g. IL-1, TNFa) promote pathology in these disorders whereas anti-inflammatory drug therapy is associated with reduced incidence of AD, and inhibitors of TNFa appear beneficial in models of MS. However, the therapeutic potential for drugs that ameliorate CNS inflammation requires further study. We have developed an animal model for investigating CNS inflammatory responses based on the visualisation of microglia in vivo using an antibody to phosphorylated tyrosine residues (Sigma, P3300). A standardised protocol was developed to test compounds for inhibitory activity against microglia activation. Bacterial lipopolysaccharide endotoxin (LPS) 2.5 mg/kg ip was given to male Sprague Dawley rats and induced a consistent, selective, time- and dose-dependent increase in microglial phosphotyrosine immunoreactivity (P-TYR-IR). Animals were sacrificed and the brains were perfused-fixed with buffered 4% formaldehyde. Paraffin embedded brain sections 10 [am thick were processed for P-TYR-IR using microwave enhancement. The stained microglia of the lateral neocortex were quantified using an image analysis protocol. This model is sensitive to cyclooxygenase inhibitors (e.g. indomethacin) and phosphodiesterase inhibitors (e.g. rolipram), where dose-dependent inhibition of P-TYR-IR was produced. These results and those of current studies on the nature of the P-TYR substrate(s) and its relation to the inflammatory cascade in the CNS will be presented.
stages of plaque maturation. Small cloudy plaques (Type I or preamyloid stage) were detected only by PAM. The three other stages were evidenced both by PAM and immunocytochemistry. Type II was characterized by diffuse fibrillar amyloid deposits forming plaques and infiltrations enclosing microvessels. In type III, clusters of denser amyloid were scattered among the amyloid fibrils. Type IV consisted of a densely stained core of 13A4 amyloid surrounded by a halo of granular amyloid. Of the 76 microcebes studies, 60% of the animals of I to 13 years old showed vascular amyloid deposits in pia and leptomeninges. Fifty six percent of animals over 6 years were affected by parenchymal deposits, 15% of them presented very numerous plaques of the various types including the compact type. The temporal and the parietal lobes were the most affected areas. The evidence of parenchymal amyloid deposits in microcebes of various ages led to the conclusion that these lesions occured not only in the old animals but also in some young ones. However, the small number of plaques encountered in the later seems to indicate that these animals did not develop a pathology whereas old adults have shown a characterized pathology. These results show that Microcebus murinus could provide valuable informations on the genesis and the maturation of the amyloid plaques
264 How can the lemurian primate Microcebus murinus contribute to our understanding of cerebral aging and Alzheimer's disease. N. Bons*, V. Jallageas, N. Privat, N. Mestre-Francrs, S. Silhol Neuromorphologie Fonctionnelle, Ecole Pratique des Hautes Etudes, Place Bataillon, 34095 Montpellier cedex 5, France. Previously, cytological and biochemical investigations performed on the Mwrocebus murinus brains have shown in this little lemurian primate the characteristic neuropathological hallmarks of the Alzheimer's disease specific to the humans. In particular, vascular and parenchymal 13A4 amyloid (13) deposits and abnormally phosphorylated Tau proteins in the pyramidal neurons as well as changes in the basal cholinergic systems were immunocytochemicaUy and -biochemically evidenced. On the other hand, whereas the 13A4 precursor protein (APP) and apolipoprotein E (APOE) have been revealed by immunocytochemistry in different cortical structures, vessels walls, amyloid plaques, pyramidal neurons and glial cells, the APOE and APP genes were sequenced in this species by PCR. Additional behavioral alterations to those related to the age were seen in animals showing very numerous cerebral lesions. The proteins such as 13, Tan, APP, ApoE, were investigated in 76 adult microcebes of I to 13 years old. The mapping of these immunolabelled proteins avere obtained by image analysis on well-defined level brain sections. Parenchymal 13 deposits that showed 4 different stages Of maturation were pathological in 15% of animals. In pyramidal cortical neurons abnormally aggregated and PHF-immunoreactive Tau proteins, 13, APP and APOE were evidenced and the number of affected neurons was related to the age and to the presence of 13plaques; the same remark must be done for the 13, APP, ApoE in the vascular walls and the glial cells. These investigations evidence for the first time an animal which develops spontaneously all the lesions observed in AD. The examination of numerous microcebes lead to the conclusion that this pathology occurs in animals over 6 years old and when the different proteins involved are in large quantities. Considering its size (body weight around 100g), its life expectancy (9 to 13 years in captivity) and its ability in breeding, Mwrocebus murinus seems to be a precious model not only for studies on the mechanisms involved in cerebral aging and in the genesis and the development of neurodegenerative pathologies, particularly of Alzheimer's type, but also for various therapeutical experiments.
263 Evolution of B-amyloid deposits in the cerebral cortex of the primate Microcebus murinus.
265
N. Mestre-Francrs*, S. Silhol and N. Bons Neuromorphologie Fonctionnelle, Ecole Pratique des Hautes Etudes, Place Bataillon, 34095 Montpellier cedex 5, France The distribution of 13A4 amyloid protein has been studied in 76 microcebe brains to identify the different types of amyloid deposits and their frequency in order to better understand the evolutionary sequences of evehts leading to 13A4 deposition and to the genesis of amyloid plaques in primates. 13A4 amyloid was evidenced in paraffin sections both by amyloid specific silver method (periodic acid methenamine silver (PAM) and immunocytochemistty using a polyclonal antibody raised to the (1-40) aminoacid sequence of the 13A4 and the avidin-biotin method. 13A4 amyloid deposition was observed both in meninges, blood vessel walls and neuropil of the Microcebus murinus cerebral cortex. Amyloid parenchymal plaques (AP) were identified in four forms which may characterize different
Dynorphin A (1-13) Improves Carbachol-lnduced Impairment of Learning Accompanied by Blockage of Reduction in Acetylcholine Release in Rat M. Hiramatsu*, H. Murasawa, H. Mori and T. Kameyama Department of Chemical Pharmacology, Faculty of Pharmaceutical Sciences, Meijo University, 150 Yagotoyama, Tenpaku-ku, Nagoya 468, Japan Although the role of dynorphin A (1-13), an endogenous kappa-opioid receptor agonist, on learning and memory is controversial, we reported that dynorphin A (1-13) improves galanin-induced impairment of memory. Therefore, we have investigated the effects of dynorphin A (1-13) on carbachol-induced learning impairment and decrease in extracellular acetylcholine concentration using an in vivo microdialysis technique. Male Sprague-Dawley rats (250-300 g) were implanted the guide earmula to the hippocampus or the frontal cortex. After injection of carbachol (1.7 pmol) into the hippocampus 30 min before acquisition trial, significant learning impairment was observed in a step-through type passive avoidance performance. Dynorphin A (1-13) (0.5 nmol/rat, i.c.v.) administered 5
FIFTH INTERNATIONAL CONFERENCE ON A L Z H E I M E R ' S DISEASE
Currently available animal models fail to provide adequate assessment of the cognitive deficits seen in Alzheimer's disease (AD). The hippocampus is involved in learning and memory processes but difficulty in producing selective hippocampal lesions so far provided no model for understanding the role of hippocampus in learning and memory. Long-term adrenalectomy produces selective neuronal loss in the hippocampus. Our results show neuronal loss in the dentate gyrus and the different hippocampal fields of adrenalectomized (ADX) rats. Immunohistochemical staining of brain sections from ADX but not sham operated rats showed intense staining of CD4 and Major histocompatibility complex class II antigens in hippocampal areas. The demonstration of induction of these antigens in the hippocampus after long-term adrenalectomy provides evidence of neuronal damage mediated by inflammatory processes. Preliminary results showed amyloid precursor protein (APP) inmaunoreactivity in the hippocampal neurons and associated with particularly dystrophic appearing cells and neudtes. This was more clearly evident with carboxyl terminal antibodies of APP rather than the amino terminal ones. Electron microscopic studies show that the neuron loss could be possibly mediated by apoptosis. The ADX rats show increased latency in the Morris water maze and impaired retention when compared to the sham operated rats. The latter data indicate that long-term adrenalectomy causes impairment in spatial learning and explorative behaviour in the rat. Selective lesioning of the hippocampus by long-term adrenalectomy may provide a good model to assess the cognitive deficits seen in AD as well as shed_light on the mechanisms which might be involved in neurodegenerative processes involved in AD.
262 Phosphotyrosine Immunoreactivityof Activated Microglia in an In Vivo Model of Inflammation in the Rat Brain G. Noy, S.J. Newman, F. Brown* and G.W. Roberts SmithKline Beecham, New Frontiers Science Park, Third Avenue, Harlow, Essex, CM19 5AW, UK CNS diseases or traumas, e.g. Alzheimer's disease (AD), multiple sclerosis (MS) and head injury, involve CNS inflammatory reactions. Evidence is increasing that some inflammatory factors (e.g. IL-1, TNFa) promote pathology in these disorders whereas anti-inflammatory drug therapy is associated with reduced incidence of AD, and inhibitors of TNFa appear beneficial in models of MS. However, the therapeutic potential for drugs that ameliorate CNS inflammation requires further study. We have developed an animal model for investigating CNS inflammatory responses based on the visualisation of microglia in vivo using an antibody to phosphorylated tyrosine residues (Sigma, P3300). A standardised protocol was developed to test compounds for inhibitory activity against microglia activation. Bacterial lipopolysaccharide endotoxin (LPS) 2.5 mg/kg ip was given to male Sprague Dawley rats and induced a consistent, selective, time- and dose-dependent increase in microglial phosphotyrosine immunoreactivity (P-TYR-IR). Animals were sacrificed and the brains were perfused-fixed with buffered 4% formaldehyde. Paraffin embedded brain sections 10 [am thick were processed for P-TYR-IR using microwave enhancement. The stained microglia of the lateral neocortex were quantified using an image analysis protocol. This model is sensitive to cyclooxygenase inhibitors (e.g. indomethacin) and phosphodiesterase inhibitors (e.g. rolipram), where dose-dependent inhibition of P-TYR-IR was produced. These results and those of current studies on the nature of the P-TYR substrate(s) and its relation to the inflammatory cascade in the CNS will be presented.
stages of plaque maturation. Small cloudy plaques (Type I or preamyloid stage) were detected only by PAM. The three other stages were evidenced both by PAM and immunocytochemistry. Type II was characterized by diffuse fibrillar amyloid deposits forming plaques and infiltrations enclosing microvessels. In type III, clusters of denser amyloid were scattered among the amyloid fibrils. Type IV consisted of a densely stained core of 13A4 amyloid surrounded by a halo of granular amyloid. Of the 76 microcebes studies, 60% of the animals of I to 13 years old showed vascular amyloid deposits in pia and leptomeninges. Fifty six percent of animals over 6 years were affected by parenchymal deposits, 15% of them presented very numerous plaques of the various types including the compact type. The temporal and the parietal lobes were the most affected areas. The evidence of parenchymal amyloid deposits in microcebes of various ages led to the conclusion that these lesions occured not only in the old animals but also in some young ones. However, the small number of plaques encountered in the later seems to indicate that these animals did not develop a pathology whereas old adults have shown a characterized pathology. These results show that Microcebus murinus could provide valuable informations on the genesis and the maturation of the amyloid plaques
264 How can the lemurian primate Microcebus murinus contribute to our understanding of cerebral aging and Alzheimer's disease. N. Bons*, V. Jallageas, N. Privat, N. Mestre-Francrs, S. Silhol Neuromorphologie Fonctionnelle, Ecole Pratique des Hautes Etudes, Place Bataillon, 34095 Montpellier cedex 5, France. Previously, cytological and biochemical investigations performed on the Mwrocebus murinus brains have shown in this little lemurian primate the characteristic neuropathological hallmarks of the Alzheimer's disease specific to the humans. In particular, vascular and parenchymal 13A4 amyloid (13) deposits and abnormally phosphorylated Tau proteins in the pyramidal neurons as well as changes in the basal cholinergic systems were immunocytochemicaUy and -biochemically evidenced. On the other hand, whereas the 13A4 precursor protein (APP) and apolipoprotein E (APOE) have been revealed by immunocytochemistry in different cortical structures, vessels walls, amyloid plaques, pyramidal neurons and glial cells, the APOE and APP genes were sequenced in this species by PCR. Additional behavioral alterations to those related to the age were seen in animals showing very numerous cerebral lesions. The proteins such as 13, Tan, APP, ApoE, were investigated in 76 adult microcebes of I to 13 years old. The mapping of these immunolabelled proteins avere obtained by image analysis on well-defined level brain sections. Parenchymal 13 deposits that showed 4 different stages Of maturation were pathological in 15% of animals. In pyramidal cortical neurons abnormally aggregated and PHF-immunoreactive Tau proteins, 13, APP and APOE were evidenced and the number of affected neurons was related to the age and to the presence of 13plaques; the same remark must be done for the 13, APP, ApoE in the vascular walls and the glial cells. These investigations evidence for the first time an animal which develops spontaneously all the lesions observed in AD. The examination of numerous microcebes lead to the conclusion that this pathology occurs in animals over 6 years old and when the different proteins involved are in large quantities. Considering its size (body weight around 100g), its life expectancy (9 to 13 years in captivity) and its ability in breeding, Mwrocebus murinus seems to be a precious model not only for studies on the mechanisms involved in cerebral aging and in the genesis and the development of neurodegenerative pathologies, particularly of Alzheimer's type, but also for various therapeutical experiments.
263 Evolution of B-amyloid deposits in the cerebral cortex of the primate Microcebus murinus.
265
N. Mestre-Francrs*, S. Silhol and N. Bons Neuromorphologie Fonctionnelle, Ecole Pratique des Hautes Etudes, Place Bataillon, 34095 Montpellier cedex 5, France The distribution of 13A4 amyloid protein has been studied in 76 microcebe brains to identify the different types of amyloid deposits and their frequency in order to better understand the evolutionary sequences of evehts leading to 13A4 deposition and to the genesis of amyloid plaques in primates. 13A4 amyloid was evidenced in paraffin sections both by amyloid specific silver method (periodic acid methenamine silver (PAM) and immunocytochemistty using a polyclonal antibody raised to the (1-40) aminoacid sequence of the 13A4 and the avidin-biotin method. 13A4 amyloid deposition was observed both in meninges, blood vessel walls and neuropil of the Microcebus murinus cerebral cortex. Amyloid parenchymal plaques (AP) were identified in four forms which may characterize different
Dynorphin A (1-13) Improves Carbachol-lnduced Impairment of Learning Accompanied by Blockage of Reduction in Acetylcholine Release in Rat M. Hiramatsu*, H. Murasawa, H. Mori and T. Kameyama Department of Chemical Pharmacology, Faculty of Pharmaceutical Sciences, Meijo University, 150 Yagotoyama, Tenpaku-ku, Nagoya 468, Japan Although the role of dynorphin A (1-13), an endogenous kappa-opioid receptor agonist, on learning and memory is controversial, we reported that dynorphin A (1-13) improves galanin-induced impairment of memory. Therefore, we have investigated the effects of dynorphin A (1-13) on carbachol-induced learning impairment and decrease in extracellular acetylcholine concentration using an in vivo microdialysis technique. Male Sprague-Dawley rats (250-300 g) were implanted the guide earmula to the hippocampus or the frontal cortex. After injection of carbachol (1.7 pmol) into the hippocampus 30 min before acquisition trial, significant learning impairment was observed in a step-through type passive avoidance performance. Dynorphin A (1-13) (0.5 nmol/rat, i.c.v.) administered 5