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Effect of cerebrolysin on neural cell culture - morphologycal observation -
THIRD
INTERNATIONAL
CONFERENCE
ON ALZHEIMER’S
DISEASE
199 BRAIN-DERIVED LEVELS IN THE AGED MOUSE.
University AUSTRALIA
NEUROTROPHIC FACTOR AND Trk B mRNA COMPARISONS BETWEEN ADULT AND BRAIN:
K. Bailey. of Melbourne,
Department Parkville,
of Pathology, Victoria 3052,
Alzheimer's disease is a progressive dementia that elderly proportion of the affects a large There is evidence that some of the population. effects seen in Alzheimer's disease are due to loss of cholinergic function associated with degeneration It is known that, during of cholinergic neurons. development, neurons depend on the availability of specific neurotrophic factors for their survival and that these factors probably have a maintenance role later in life. Therefore, it is possible that at least some of the neuronal degeneration observed in Alzheimer's disease is due to decreased levels of either neurotrophic factors or their receptors. We the mRNA levels of brain-derived are studying neurotrophic factor (BDNF) and its receptor, Trk B, in the mouse brain to determine if the levels alter Total RNA was isolated from during normal aging. the whole brains of mice at various ages (6 week, 1 year and 2 year) and the levels of expression of BDNF and Trk B were studied using specific probes. Initial results suggest that the levels of both the factor and its receptor do not alter significantly during aging. Further studies are being undertaken to study specific areas of the brain using both Northern analysis and in situ hybridization.
disease, wehave examined the differentiationof the basal forebrain cholinergicneurons (BFCN) in chromosome 16 trisomic mice (Ts16), a murine genetic model for Down Syndrome. The Ts16 mouse exhibits impaired development of the basal forebrain cholinergic system. B-nerve growth factor (NGF) appears to be critical in the differentiationand maintenance of the cholinergic characteristicsexpression in the basal forebrain. This study shows the developmentof BFCN in primary cultures of Ts16 and littermateeuploid controls (Eupl), since Ts16 mice invariably die before birth. Basal forebrains obtained from Ts16 and Eupl fetuses at 15 days of gestation were dissociatedand cultured in a completelydefined medium, choline with cholinergic neurons identified by acetyltransferase (ChAT) immunoreactivity.The addition of NGF (50nglml) for 10 days raised the specific activity of CMT and neuritic extension for both Ts16 and Eupl cholinergicneurons. Furthermore, cell surface stimulationby NGF triggers rapid activation of a set of genes, c-fos, c-Jun, referred to as immediate early genes (IEG), coding for the transcription factors mediating a neuronal differentiation similar to the one induced by NGF. Immunocytochemical studies in vitro with antibodies to c-fos and c-Jun demonstrateintense staining in the nuclei of a subset of cholinergic neurons at 10 days in vitro. To assess whether this immunoreactivityis induced by spontaneous synaptic activity developing with a similar profile, we examined the effects of agents causing its reduction. Tetrodotoxin (TTI) basal suppresses the immunoreactivity to the IEG in both types of cultures while TTI + NGF restore their expression.This study suggests that the neuronal differentiation of BFCN is induced by NGFmediated activationof the IEG via multiple pathways.
202 200 EFFECT OF CEREBROLYSIN ON NEURAL CELL CULTURE - MORPHOLOGYCALOBSERVATION T. SATOH’. T. ITOH”. S. KOMATSU’. M. FUJIMOTO’. K. HOSOKAWA’,S. HIRUMA’and S.HASHlMOTO’ ‘Kinki University School of Medicine 377 -2 Ounohigasi.Osaka-sayama city.Osaka 589,Japan. ‘Habikino Laboratory.Fujimoto Diagnostics INC.l-3-40 Nisiotsuka.Matsubara city. Osaka 580. Japan. Cerebrolysin is a drug produced by mordern biotechnological methods.consisting of small peptides (10000 D) and free amino acids.lVe have reported that Cerebrolysin has like NGF reaction (group NTF).This time we tried examination to observe cells morphologycally. effects of Cerebrolysin on cultured The dorsal root ganglia (DRC) taken from lo-days-chicken embryos were put into PBS in plastic petridish ‘hese ganglia were moved to centrifuge tube and centrifuge 5 minutes 200 x g,After that,removed upper phase and 0.02 % EDTl - 0.05 % trypsin solution and incubate for 40 minutes at 37 a .Remove upper phase after centrifuge as same above, added Eagl’s MEM including 10 % fetal bovin serum and transfered to petridish through nyronmesh.As preculture,incubate for 130 minutes in CO2 incubator(5XC02. 37’ ).We cultured this solution on gelatin coated coverglass into petridish. We added Cerebrolysin 5~ 1-20~ l/ml into culture solution. As control we added PBS and NGF as same above volume. After lZhours,lday,Zday and (day we picked up coverglass and it was soaked in fix solution and washed by PBS. We tried HE stain, Bodian stain and imaunostain using antineurofirament and tublin and so on. We found neorofilament positive fibers in neural cells cultured by adding NGF and Cerebrolysin.But outgrowth speed was slower and fibers was finer in Cerebrolysin than in NGF. It was morphologically clear that the action of Cerebrolysin was different from the one of NCF,although Cerebrolysin induced neural fibers as well as NCF.
SUPEROXIDE DISHUTASE IN A6lN6 BRAIN AND RELATED DEGENERATIVE DISEASES. F Bracco, A P Burlina, M Scarpa, A Rlgo and L Battlstln Inst of Neurology and Dept of Biologmal Chemistry, Unlv of Padova, Padova, ITALY. Free radical reactlons have been reported to play a slgnlflcant role in the agtng braln and In age related degenerattve processes of the CNS An , Increase of free radical productTon and a reduction of normal tolerance not paralleled by an Increase of natural scavengers may partectpate in the neuronal cell degeneration Levels of Cu, Zn and Mn superoxlde dtsmutases (CuSOD and MnSOD respectively), spectfm scavengers of superoxlde radical, were assessed In the bratn cortex from rats of dtfferent ages by high-resolutton 19F NMR The concentrattons of Cu- and MnSOD were found to Increase ITnearly with the logarithm of the age of animals from 3 days before birth to 30 months Since cerebrosplnal fluid (CSF) can reflect the enzyme concentration in the CNS, we measured the SOD activity by the polarographic method of catalytic currents In the CSF from sublects of different groups a) 14 normal sublects of different ages (range 19-70 yrs) b) IO patients with Alzhelmer s Disease (AD) c) I1 patients with Amyotrophic Lateral Sclerosis (ALS) In the reference sub]ects the CSF SOD concentration Increased with age However, no slgntficant relationship was found between SOD concentratton and age in AD and ALS pattents The mean CSF SOD values tn ALS (mean 0 73+0.76 n t-l ~‘0 01) and In AD patlents (mean I30+0 99 nM p
554 THE CELL LOSS IN THE
201 lxx-lnDucm AlxIvATIon0Fn4maATEEARLY GElsEs In PRIMARY CUL-
OF
FoRmRun c!mLmeRGIC nEuRons, P. J.T. Coyle. Istituto di Fisiologia Umana, Universita di Bari: Dept. of Neuroscience, Johns Hopkins University,Baltimore, U.S.A. Given that by the fourth decade all patients with Down Syndrome develop the neuropathicalterations of Alrheimer's CWSi,
S.M.
Ts16 Cozza
EnsAL and
DORSAL HOTOR VAGAL AND ANBIG""S NUCLEI AFTER CHRONIC SECTION OF THE "AGUS NERVE : AN EXPERINENTAL NODEL TO EVALUATE "IN VIVO" THE EFFICACY OF POTENTIAL NEUROTROPHIC HOLECULES. E. Fernandez, R. Pallini, L. Lauretti. E. Narchese.
Dept of Neurosurgery,CatholicUniversity,Rome, Italy The exotomy-induced cell death in the brain stem motor nuclei at 90 days after section of tbe vagus nerve was studied in adult rats. The right vagus nerve was cut at the neck . To prevent regeneration, a 5 mm long segment of the "agus nerve was excised and the distal stump was displaced caudally. Ninety days postoperatively, the proxinal nerve stump of the vagus nerve was injected with horseradish peroxidase (HRP) to retrogradely label
INTERNATIONAL
CONFERENCE
ON ALZHEIMER’S
DISEASE
199 BRAIN-DERIVED LEVELS IN THE AGED MOUSE.
University AUSTRALIA
NEUROTROPHIC FACTOR AND Trk B mRNA COMPARISONS BETWEEN ADULT AND BRAIN:
K. Bailey. of Melbourne,
Department Parkville,
of Pathology, Victoria 3052,
Alzheimer's disease is a progressive dementia that elderly proportion of the affects a large There is evidence that some of the population. effects seen in Alzheimer's disease are due to loss of cholinergic function associated with degeneration It is known that, during of cholinergic neurons. development, neurons depend on the availability of specific neurotrophic factors for their survival and that these factors probably have a maintenance role later in life. Therefore, it is possible that at least some of the neuronal degeneration observed in Alzheimer's disease is due to decreased levels of either neurotrophic factors or their receptors. We the mRNA levels of brain-derived are studying neurotrophic factor (BDNF) and its receptor, Trk B, in the mouse brain to determine if the levels alter Total RNA was isolated from during normal aging. the whole brains of mice at various ages (6 week, 1 year and 2 year) and the levels of expression of BDNF and Trk B were studied using specific probes. Initial results suggest that the levels of both the factor and its receptor do not alter significantly during aging. Further studies are being undertaken to study specific areas of the brain using both Northern analysis and in situ hybridization.
disease, wehave examined the differentiationof the basal forebrain cholinergicneurons (BFCN) in chromosome 16 trisomic mice (Ts16), a murine genetic model for Down Syndrome. The Ts16 mouse exhibits impaired development of the basal forebrain cholinergic system. B-nerve growth factor (NGF) appears to be critical in the differentiationand maintenance of the cholinergic characteristicsexpression in the basal forebrain. This study shows the developmentof BFCN in primary cultures of Ts16 and littermateeuploid controls (Eupl), since Ts16 mice invariably die before birth. Basal forebrains obtained from Ts16 and Eupl fetuses at 15 days of gestation were dissociatedand cultured in a completelydefined medium, choline with cholinergic neurons identified by acetyltransferase (ChAT) immunoreactivity.The addition of NGF (50nglml) for 10 days raised the specific activity of CMT and neuritic extension for both Ts16 and Eupl cholinergicneurons. Furthermore, cell surface stimulationby NGF triggers rapid activation of a set of genes, c-fos, c-Jun, referred to as immediate early genes (IEG), coding for the transcription factors mediating a neuronal differentiation similar to the one induced by NGF. Immunocytochemical studies in vitro with antibodies to c-fos and c-Jun demonstrateintense staining in the nuclei of a subset of cholinergic neurons at 10 days in vitro. To assess whether this immunoreactivityis induced by spontaneous synaptic activity developing with a similar profile, we examined the effects of agents causing its reduction. Tetrodotoxin (TTI) basal suppresses the immunoreactivity to the IEG in both types of cultures while TTI + NGF restore their expression.This study suggests that the neuronal differentiation of BFCN is induced by NGFmediated activationof the IEG via multiple pathways.
202 200 EFFECT OF CEREBROLYSIN ON NEURAL CELL CULTURE - MORPHOLOGYCALOBSERVATION T. SATOH’. T. ITOH”. S. KOMATSU’. M. FUJIMOTO’. K. HOSOKAWA’,S. HIRUMA’and S.HASHlMOTO’ ‘Kinki University School of Medicine 377 -2 Ounohigasi.Osaka-sayama city.Osaka 589,Japan. ‘Habikino Laboratory.Fujimoto Diagnostics INC.l-3-40 Nisiotsuka.Matsubara city. Osaka 580. Japan. Cerebrolysin is a drug produced by mordern biotechnological methods.consisting of small peptides (10000 D) and free amino acids.lVe have reported that Cerebrolysin has like NGF reaction (group NTF).This time we tried examination to observe cells morphologycally. effects of Cerebrolysin on cultured The dorsal root ganglia (DRC) taken from lo-days-chicken embryos were put into PBS in plastic petridish ‘hese ganglia were moved to centrifuge tube and centrifuge 5 minutes 200 x g,After that,removed upper phase and 0.02 % EDTl - 0.05 % trypsin solution and incubate for 40 minutes at 37 a .Remove upper phase after centrifuge as same above, added Eagl’s MEM including 10 % fetal bovin serum and transfered to petridish through nyronmesh.As preculture,incubate for 130 minutes in CO2 incubator(5XC02. 37’ ).We cultured this solution on gelatin coated coverglass into petridish. We added Cerebrolysin 5~ 1-20~ l/ml into culture solution. As control we added PBS and NGF as same above volume. After lZhours,lday,Zday and (day we picked up coverglass and it was soaked in fix solution and washed by PBS. We tried HE stain, Bodian stain and imaunostain using antineurofirament and tublin and so on. We found neorofilament positive fibers in neural cells cultured by adding NGF and Cerebrolysin.But outgrowth speed was slower and fibers was finer in Cerebrolysin than in NGF. It was morphologically clear that the action of Cerebrolysin was different from the one of NCF,although Cerebrolysin induced neural fibers as well as NCF.
SUPEROXIDE DISHUTASE IN A6lN6 BRAIN AND RELATED DEGENERATIVE DISEASES. F Bracco, A P Burlina, M Scarpa, A Rlgo and L Battlstln Inst of Neurology and Dept of Biologmal Chemistry, Unlv of Padova, Padova, ITALY. Free radical reactlons have been reported to play a slgnlflcant role in the agtng braln and In age related degenerattve processes of the CNS An , Increase of free radical productTon and a reduction of normal tolerance not paralleled by an Increase of natural scavengers may partectpate in the neuronal cell degeneration Levels of Cu, Zn and Mn superoxlde dtsmutases (CuSOD and MnSOD respectively), spectfm scavengers of superoxlde radical, were assessed In the bratn cortex from rats of dtfferent ages by high-resolutton 19F NMR The concentrattons of Cu- and MnSOD were found to Increase ITnearly with the logarithm of the age of animals from 3 days before birth to 30 months Since cerebrosplnal fluid (CSF) can reflect the enzyme concentration in the CNS, we measured the SOD activity by the polarographic method of catalytic currents In the CSF from sublects of different groups a) 14 normal sublects of different ages (range 19-70 yrs) b) IO patients with Alzhelmer s Disease (AD) c) I1 patients with Amyotrophic Lateral Sclerosis (ALS) In the reference sub]ects the CSF SOD concentration Increased with age However, no slgntficant relationship was found between SOD concentratton and age in AD and ALS pattents The mean CSF SOD values tn ALS (mean 0 73+0.76 n t-l ~‘0 01) and In AD patlents (mean I30+0 99 nM p
554 THE CELL LOSS IN THE
201 lxx-lnDucm AlxIvATIon0Fn4maATEEARLY GElsEs In PRIMARY CUL-
OF
FoRmRun c!mLmeRGIC nEuRons, P. J.T. Coyle. Istituto di Fisiologia Umana, Universita di Bari: Dept. of Neuroscience, Johns Hopkins University,Baltimore, U.S.A. Given that by the fourth decade all patients with Down Syndrome develop the neuropathicalterations of Alrheimer's CWSi,
S.M.
Ts16 Cozza
EnsAL and
DORSAL HOTOR VAGAL AND ANBIG""S NUCLEI AFTER CHRONIC SECTION OF THE "AGUS NERVE : AN EXPERINENTAL NODEL TO EVALUATE "IN VIVO" THE EFFICACY OF POTENTIAL NEUROTROPHIC HOLECULES. E. Fernandez, R. Pallini, L. Lauretti. E. Narchese.
Dept of Neurosurgery,CatholicUniversity,Rome, Italy The exotomy-induced cell death in the brain stem motor nuclei at 90 days after section of tbe vagus nerve was studied in adult rats. The right vagus nerve was cut at the neck . To prevent regeneration, a 5 mm long segment of the "agus nerve was excised and the distal stump was displaced caudally. Ninety days postoperatively, the proxinal nerve stump of the vagus nerve was injected with horseradish peroxidase (HRP) to retrogradely label