- Email: [email protected]
Expression of c-src gene product in the gerbil model of transient forebrain ischemia
S188 1 I-
10
EXPRESSION OF C-SRC GENE PRODUCT IN THE GERBIL MODEL OF TRANSIENT FOREBRAIN ISCHEMIA. MASAFUMI TAGAYA. TOMOHIRO MATSUYAMA. HITOSHI NAKAMURA, SOUICHIRO SHIMIZU, DAIZO IHAKU, MASAYASU MATSUMOTO’ AND MINORU SUGITA, Fifth Department of Internal Medicine, Hvopo College of Medicine -9 j-1 Mukoaawacho. Nishinomiva 663, Japan, first Department of Internal Medicine, Osaka Universitv Medical School The involvement of proto-oncogenes in regulation of cell growth and differentiation has been the focus of much interest. The c-src gene product is highly expressed in neural tissues. In the present study, we tried to localize c-src gene product in the selective vulnerable regions to ischemia using gerbil model of transient forebrain ischemia. Cerebral ischemia was produced by occluding bilateral common carotids for 5 min. Animals were allowed reperfusion for 1, 2, 3 or 7 days and their brains were subjected to both immunocytochemistry and Western blot analysis for c-src protein, We also perfomed immunocytochemistry for synaptophysin which is a marker protein of presynaptic terminals. In normal brains, immunocytochemistry showed that mossy fibers in the hippocampus were highly positive for c-src protein. The molecular layer of the dentate gyrus showed higher staining compared to the stratum radiatum in the CA1 region, In ischemic brains, neurons in the cerebral cortex had an increase in the expression of c-src protein, and the stratum radiatum in the CA1 region gained higher staining than that obtained from the normal brains. Brain ischemia had no effects on the synaptophysin
immunoreactivity
throughout the hippocampus. Western blot analysis showed that ischemia
for 5 min induced the expression of c-src protein. These results suggest that c-src gene may play an important role in regulation of synaptic maintenance and reconstruction in the ischemic brain lesion.
11-j
1
Chronic
maintenance
by ischemia.Chiaki University
School
of :Iedicine,
of hippocampal
noradrenergic
fibers
Tomioka, l-1 Seirqomachi.
after
loss
of CA-1 pyramidal
Department Sendai
of Neurology
neurons Tohoku
980,Japan
The hypothesis that target-derived trophic factors should maintain the connected fibers and neurons in the central nervous system (CNS) was proposed after discovery of nerve growth factor (NGF). The hypothesis proposes that the degenerative disorders are caused by a lack of NGF. The effect of NGF to CNS and peripheral catecholaminergic systems were almost comfilmed by the experimental facts. However, the existence of target-derived trophic factors from target neurons to connected fibers and neurons in the central nervous system (CNS) has not been confilmed. Thus, we examined the maintenance of the catecholaminergic fibers in rats and gerbils immunohistochemically I. 3. and 30 days after ischemia. The immunoreactivity of tyrosine hydroxylase immunoreactive (THl) fibers was maintained after ischemia except in the stratum oriens in the rat hippocampus. This study was demonstrated chronic maintenance of THI fibers in the hippocampus in rodents after loss of target neurons. We elucidated one factor that loss of target neurons does not followed degeneration of connected central catecholaminergic fibers and neurons.
11-12
MECHANICAL BRAIN MICROINJURY MODIFIES INTRACELLULAR Ca*‘RESPONSE TO HYPOXIA PLUS GLUCOSE DEPLETION IN MOUSE HIPPOCAMPAL SLICES. NAOSHI FUJIWARA, TADAYUKI HONDA, TAKASHI ABE AND KOKI SHIMOJI, Department of Anesthesiology, Niigata University School of Medicine, I-757 Asahimachi-dori, Niigata 9.51. Japan
The effect of mechanical brain injury on intracellular Ca’+ ([Ca”],) in response to hypoxia plus glucose depletion V,ZS studied in mouse hippocampal slices in order to characterize the mechanism of brain injury-induced anti-ischemic z.cti\?y reported previously (Takahata & Shimoji, Brain Res 381: 361-371, 1986). Male dd-Y mice were divided into two grr?tlj\_‘and anesthetized with pentobarbital (80 mg/kg, i.p.). The brain-injured group was surgically incised on the scalp and inc :cted with a 25G needle into the brain from the skull to make a lesion in the hippocampal CA 1 area. The sham-operated group was given a surgical incision on the scalp without brain injury. Three, 7 and 14 days after the brain injury, the mice were decapitated and 350 pm-thick hippocampal slices were prepared. Slices were dyed with fura- and superfused with normoxic Krebs solution at 36.37°C. Exposure to hypoxia plus glucose depletion was performed for 10 min with glucosedepleted Krebs solution gassed with 95%N,-5%CO,. Changes in [Ca”], of the slices were analyzed using fluorescence ratio images. The increase in [Ca2’], induced by hypoxia plus glucose depletion was inhibited in the vicinity of lesion site in the CA1 area, especially in the opposite side to the CA3 across the lesion, in the slices of 3 and 7 days following the brain injury. Such regional differences in [Ca”], increase were not found in the slices of 14 days after the injury and of the shamoperated mice. The results suggest that an anti-ischemic activity generated in the lesion site inhibits the [Ca*‘], increase in response to hypoxia plus glucose depletion in 3 and 7 days after the brain injury but that the activity disappears in 14 days.
10
EXPRESSION OF C-SRC GENE PRODUCT IN THE GERBIL MODEL OF TRANSIENT FOREBRAIN ISCHEMIA. MASAFUMI TAGAYA. TOMOHIRO MATSUYAMA. HITOSHI NAKAMURA, SOUICHIRO SHIMIZU, DAIZO IHAKU, MASAYASU MATSUMOTO’ AND MINORU SUGITA, Fifth Department of Internal Medicine, Hvopo College of Medicine -9 j-1 Mukoaawacho. Nishinomiva 663, Japan, first Department of Internal Medicine, Osaka Universitv Medical School The involvement of proto-oncogenes in regulation of cell growth and differentiation has been the focus of much interest. The c-src gene product is highly expressed in neural tissues. In the present study, we tried to localize c-src gene product in the selective vulnerable regions to ischemia using gerbil model of transient forebrain ischemia. Cerebral ischemia was produced by occluding bilateral common carotids for 5 min. Animals were allowed reperfusion for 1, 2, 3 or 7 days and their brains were subjected to both immunocytochemistry and Western blot analysis for c-src protein, We also perfomed immunocytochemistry for synaptophysin which is a marker protein of presynaptic terminals. In normal brains, immunocytochemistry showed that mossy fibers in the hippocampus were highly positive for c-src protein. The molecular layer of the dentate gyrus showed higher staining compared to the stratum radiatum in the CA1 region, In ischemic brains, neurons in the cerebral cortex had an increase in the expression of c-src protein, and the stratum radiatum in the CA1 region gained higher staining than that obtained from the normal brains. Brain ischemia had no effects on the synaptophysin
immunoreactivity
throughout the hippocampus. Western blot analysis showed that ischemia
for 5 min induced the expression of c-src protein. These results suggest that c-src gene may play an important role in regulation of synaptic maintenance and reconstruction in the ischemic brain lesion.
11-j
1
Chronic
maintenance
by ischemia.Chiaki University
School
of :Iedicine,
of hippocampal
noradrenergic
fibers
Tomioka, l-1 Seirqomachi.
after
loss
of CA-1 pyramidal
Department Sendai
of Neurology
neurons Tohoku
980,Japan
The hypothesis that target-derived trophic factors should maintain the connected fibers and neurons in the central nervous system (CNS) was proposed after discovery of nerve growth factor (NGF). The hypothesis proposes that the degenerative disorders are caused by a lack of NGF. The effect of NGF to CNS and peripheral catecholaminergic systems were almost comfilmed by the experimental facts. However, the existence of target-derived trophic factors from target neurons to connected fibers and neurons in the central nervous system (CNS) has not been confilmed. Thus, we examined the maintenance of the catecholaminergic fibers in rats and gerbils immunohistochemically I. 3. and 30 days after ischemia. The immunoreactivity of tyrosine hydroxylase immunoreactive (THl) fibers was maintained after ischemia except in the stratum oriens in the rat hippocampus. This study was demonstrated chronic maintenance of THI fibers in the hippocampus in rodents after loss of target neurons. We elucidated one factor that loss of target neurons does not followed degeneration of connected central catecholaminergic fibers and neurons.
11-12
MECHANICAL BRAIN MICROINJURY MODIFIES INTRACELLULAR Ca*‘RESPONSE TO HYPOXIA PLUS GLUCOSE DEPLETION IN MOUSE HIPPOCAMPAL SLICES. NAOSHI FUJIWARA, TADAYUKI HONDA, TAKASHI ABE AND KOKI SHIMOJI, Department of Anesthesiology, Niigata University School of Medicine, I-757 Asahimachi-dori, Niigata 9.51. Japan
The effect of mechanical brain injury on intracellular Ca’+ ([Ca”],) in response to hypoxia plus glucose depletion V,ZS studied in mouse hippocampal slices in order to characterize the mechanism of brain injury-induced anti-ischemic z.cti\?y reported previously (Takahata & Shimoji, Brain Res 381: 361-371, 1986). Male dd-Y mice were divided into two grr?tlj\_‘and anesthetized with pentobarbital (80 mg/kg, i.p.). The brain-injured group was surgically incised on the scalp and inc :cted with a 25G needle into the brain from the skull to make a lesion in the hippocampal CA 1 area. The sham-operated group was given a surgical incision on the scalp without brain injury. Three, 7 and 14 days after the brain injury, the mice were decapitated and 350 pm-thick hippocampal slices were prepared. Slices were dyed with fura- and superfused with normoxic Krebs solution at 36.37°C. Exposure to hypoxia plus glucose depletion was performed for 10 min with glucosedepleted Krebs solution gassed with 95%N,-5%CO,. Changes in [Ca”], of the slices were analyzed using fluorescence ratio images. The increase in [Ca2’], induced by hypoxia plus glucose depletion was inhibited in the vicinity of lesion site in the CA1 area, especially in the opposite side to the CA3 across the lesion, in the slices of 3 and 7 days following the brain injury. Such regional differences in [Ca”], increase were not found in the slices of 14 days after the injury and of the shamoperated mice. The results suggest that an anti-ischemic activity generated in the lesion site inhibits the [Ca*‘], increase in response to hypoxia plus glucose depletion in 3 and 7 days after the brain injury but that the activity disappears in 14 days.