Gender differences in long-term beneficial effects of erythropoietin given after neonatal stroke in postnatal day-7 rats.

Recently, we reported that erythropoietin attenuates neonatal brain injury caused by focal cerebral ischemia. The long-term effects of erythropoietin on focal cerebral ischemia-induced injury to the developing brain and the potential gender differences in these long-term effects have not been studied in detail. The current study demonstrated a similarity in the mean infarct volume in both the vehicle-treated male and female rats at 6 and 12 weeks after focal cerebral ischemia. On the other hand, erythropoietin treatment (1000 U/kg x three doses after focal cerebral ischemia) caused a significant reduction in the mean infarct volume in both males and females at 6 weeks after focal cerebral ischemia when compared with the corresponding vehicle-treated animals (males: 141.4+/-48.2 mm3 vs. 194.0+/-59.2 mm3, P<0.05; females: 85.4+/-31.6 mm3 vs. 183.4+/-46.3 mm3, P<0.05). Interestingly, the reduction in the mean infarct volume in the erythropoietin-treated males was significantly less than that in the erythropoietin-treated females at 6 weeks after focal cerebral ischemia (141.4+/-48.2 mm3 vs. 85.4+/-31.6 mm3, P<0.05). At 12 weeks after focal cerebral ischemia, the mean infarct volume in the erythropoietin-treated males significantly increased to 181.0+/-50.4 mm3 (P<0.05). In contrast, the mean infarct volume in the erythropoietin-treated females remained stable (87.0+/-41.7 mm3). Additionally, erythropoietin treatment significantly improved sensorimotor function recovery with a misstep number similar to the sham-operation group at 6 and 12 weeks after focal cerebral ischemia. Moreover, the mean number of missteps in the erythropoietin-treated females was less than that in males at 6 (13.5+/-2.0 vs. 24.5+/-2.5, P<0.05) and 12 (12.5+/-2.0 vs. 20.0+/-2.0, P<0.05) weeks after focal cerebral ischemia. These results indicate that erythropoietin administration after focal cerebral ischemia produces a significant long-term neuroprotective benefit on the developing brain, and that this effect is more beneficial in the female rats.

In situ UV-visible spectroelectrochemical evidences for conducting copolymer formation between diphenylamine and m-methoxyaniline.

Electrochemical copolymerization of diphenylamine (DPA) with m-methoxy aniline (MA) was carried out in 4 M H(2)SO(4) by cyclic voltammetry (CV). Cyclic voltammograms (CVs) of the copolymer films were recorded in monomer-free background electrolyte. In situ sepectroelectrochemical studies were carried out on an optically transparent electrode (Indium tin oxide (ITO) coated glass) in 4 M H(2)SO(4) for different feed ratios of the comonomers. Constant potential and potential sweep methods were employed for performing polymerization. UV-visible absorption spectra were collected continuously and concurrently during the copolymerization in both the cases. The results from constant potential electropolymerisation indicated the formation of an intermediate with an absorption peak at 576 nm. Derivative cyclic voltabsorptogram (DCVA) was deduced from the results of cyclic spectrovoltammetry. The DCVA derived at 576 nm confirms the intermediates formed during the electrochemical copolymerization. The compositional changes of the two monomers in the copolymers with changes in feed composition of two monomers as predicted from in situ spectro electrochemical studies are evident from elemental analysis. A plausible copolymerization mechanism is suggested.

Protective effect of a prosaposin-derived, 18-mer peptide on slowly progressive neuronal degeneration after brief ischemia.

SUMMARY: Slowly progressive degeneration of the hippocampal CA1 neurons was induced by 3-minute transient global ischemia in gerbils. Sustained degeneration of hippocampal CA1 neurons was evident 1 month after ischemia. To investigate the effects of an 18-mer peptide comprising the hydrophilic sequence of the rat saposin C domain (18MP) on this sustained neuronal degeneration, an intracerebroventricular 18MP infusion was initiated 3 days after ischemia. Histopathologic and behavior evaluations were conducted 1 week and 1 month after induction of ischemia. When compared with the vehicle infusion, 18MP treatment significantly increased the response latency time in a passive avoidance task. Increased neuronal density was also evident, as was the number of intact synapses in the hippocampal CA1 region at 1 week and 1 month after ischemia. 18MP treatment also significantly decreased the number of TUNEL-positive CA1 neurons 1 week after ischemia. Subsequent in vitro experiments using cultured neurons demonstrated that the 18MP at optimal extracellular concentrations of 1 to 100 fg/mL prevented nitric oxide-induced neuronal damage as expected and significantly up-regulated the expressions of bcl-x(L) mRNA and its translated protein. These results suggest that the gerbil model of 3-minute ischemia is useful in studying the pathogenesis of slowly progressive neuronal degeneration after stroke and in evaluating effects of novel therapeutic agents. It is likely that the 18MP at low extracellular concentrations prevents neuronal apoptosis possibly through up-regulation of the mitochondrial antiapoptotic factor Bcl-x(L).

Induction of phosphorylated-Stat3 following focal cerebral ischemia in mice.

It has been shown that Stat3 is induced following transient cerebral ischemia in rat. However there is no evidence that cerebral ischemia stimulates the expression of phosphorylated-Stat3 (p-Stat3), which can activate cytokine-mediated signal transduction from the membrane to the nucleus. In the present study, we investigated the changes in p-Stat3 expression following middle cerebral artery occlusion in mice. Western blot analysis revealed a significant increase in the p-Stat3 protein in the peripheral part of the ischemic area, starting from 6 h after ischemia. p-Stat3 immunoreactivity was detected only in neurons, but not in astrocytes or microglia, and p-Stat3-positive neurons were increased in number in the peripheral part of the ischemic area at 24 h after ischemia. Double staining with aTdT-mediated biotinylated UTP nick end labeling (TUNEL) kit and the p-Stat3 antibody indicated that p-Stat3-positive neurons were also TUNEL-positive. Subsequent immuno-electron microscopic observations showed that p-Stat3-positive neurons were at different stages of degeneration. The present findings suggest that the increased expression of p-Stat3 after cerebral ischemia could play a crucial role in ischemia-induced neuron death.

10 years of experience with penile prosthesis implantation in Taiwanese patients.

PURPOSE: We evaluated the long-term results of penile prosthesis implantation during the last 10 years. Special considerations were examined concerning Asian corporeal diameter, device survival and sociocultural factors influencing patient-partner satisfaction. MATERIALS AND METHODS: We followed 331 patients who underwent penile prosthesis implantation from December 1985 to May 1996. Surgical data, postoperative complications and information from a survey on satisfaction are reported. RESULTS: The most prevalent corporeal diameter in our patients was 11.0 to 11.5 cm. Postoperatively wound infection was the most serious surgical complication. The 5-year device survival was similar in the malleable and inflatable types except for a poorer outcome with the self-contained inflatable Hydroflex penile prosthesis. Overall patient satisfaction with surgery was 86.6%. CONCLUSIONS: Penile prosthesis implantation still has its place as definitive therapy in some patients with uncorrectable erectile dysfunction. In Taiwanese society more consideration of prosthesis selection, surgical preparation and patient-partner counseling is the basis for further success in the future.

Titration study of MUSE (Medicated Urethral System for Erection) in erectile dysfunction.

PURPOSE: We assessed the erectile responses and adverse reactions to prostaglandin E1 delivered via the medicated urethral system for erection (MUSE) in a titration study to determine optimal dose. METHODS: Sixty-four patients with erectile dysfunction underwent in-office testing with MUSE to determine erectile response. Following thorough instructions, patients were taught how to use MUSE themselves. The titration study started with a dose of 125 micrograms, and increased to 250 micrograms, 500 micrograms, and finally 1,000 micrograms. In patients who had an erectile response, erection scoring, color duplex Doppler study, blood pressure monitoring, recording of subjective complaints, and repeated laboratory examinations were performed to evaluate the positive and adverse effects of MUSE. RESULTS: Forty-three of the 64 patients achieved a maximal erection with MUSE, as shown by systolic peak flow velocities in the deep penile arteries of greater than 27 cm/s on Doppler ultrasonography. One patient was unsatisfied with his erectile response and dropped out of the study. Of the 42 patients who completed the study, most (35) were satisfied with the response achieved at the higher doses of 500 micrograms and 1,000 micrograms. A modified soft rubber band was needed to achieve a satisfactory erectile response in 18 patients. Only four patients experienced serious urethral burning during MUSE application. CONCLUSIONS: MUSE was effective and safe in most patients with erectile dysfunction in this study. For patients not satisfied with the extent of penile rigidity achieved with MUSE, the use of a modified soft rubber band for erectile assistance, as well as a program for improved patient education, may help to achieve a better erectile response and facilitate compliance with treatment regimens at home.

Differential expressions of glycine transporter 1 and three glutamate transporter mRNA in the hippocampus of gerbils with transient forebrain ischemia.

The extracellular concentrations of glutamate and its co-agonist for the N-methyl-d-aspartate (NMDA) receptor, glycine, may be under the control of amino acid transporters in the ischemic brain. However, there is little information on changes in glycine and glutamate transporters in the hippocampal CA1 field of gerbils with transient forebrain ischemia. This study investigated the spatial and temporal expressions of glycine transporter 1 (GLYT1) and three glutamate transporter (excitatory amino acid carrier 1, EAAC1; glutamate/aspartate transporter, GLAST; glutamate transporter 1, GLT1) mRNA in the gerbil hippocampus after 3 minutes of ischemia. The GLYT1 mRNA was transiently upregulated by the second day after ischemia in astrocytelike cells in close vicinity to hippocampal CA1 pyramidal neurons, possibly to reduce glycine concentration in the local extracellular spaces. The EAAC1 mRNA was abundantly expressed in almost all pyramidal neurons and dentate granule cells in the control gerbil hippocampus, whereas the expression level in CA1 pyramidal neurons started to decrease by the fourth day after ischemia in synchrony with degeneration of the CA1 neurons. The GLAST and GLT1 mRNA were rather intensely expressed in the dentate gyrus and CA3 field of the control hippocampus, respectively, but they were weakly expressed in the CA1 field before and after ischemia. As GLAST and GLT1 play a major role in the control of extracellular glutamate concentration, the paucity of these transporters in the CA1 field may account for the vulnerability of CA1 neurons to ischemia, provided that the functional GLAST and GLT1 proteins are also less in the CA1 field than in the CA3 field. This study suggests that the amino acid transporters play pivotal roles in the process of delayed neuronal death in the hippocampal CA1 field.

Interleukin 3 prevents delayed neuronal death in the hippocampal CA1 field.

In the central nervous system, interleukin (IL)-3 has been shown to exert a trophic action only on septal cholinergic neurons in vitro and in vivo, but a widespread distribution of IL-3 receptor (IL-3R) in the brain does not conform to such a selective central action of the ligand. Moreover, the mechanism(s) underlying the neurotrophic action of IL-3 has not been elucidated, although an erythroleukemic cell line is known to enter apoptosis after IL-3 starvation possibly due to a rapid decrease in Bcl-2 expression. This in vivo study focused on whether IL-3 rescued noncholinergic hippocampal neurons from lethal ischemic damage by modulating the expression of Bcl-xL, a Bcl-2 family protein produced in the mature brain. 7-d IL-3 infusion into the lateral ventricle of gerbils with transient forebrain ischemia prevented significantly hippocampal CA1 neuron death and ischemia-induced learning disability. TUNEL (terminal deoxynucleotidyltransferase-mediated 2'-deoxyuridine 5'-triphosphate-biotin nick end labeling) staining revealed that IL-3 infusion caused a significant reduction in the number of CA1 neurons exhibiting DNA fragmentation 7 d after ischemia. The neuroprotective action of IL-3 appeared to be mediated by a postischemic transient upregulation of the IL-3R alpha subunit in the hippocampal CA1 field where IL-3Ralpha was barely detectable under normal conditions. In situ hybridization histochemistry and immunoblot analysis demonstrated that Bcl-xL mRNA expression, even though upregulated transiently in CA1 pyramidal neurons after ischemia, did not lead to the production of Bcl-xL protein in ischemic gerbils infused with vehicle. However, IL-3 infusion prevented the decrease in Bcl-xL protein expression in the CA1 field of ischemic gerbils. Subsequent in vitro experiments showed that IL-3 induced the expression of Bcl-xL mRNA and protein in cultured neurons with IL-3Ralpha and attenuated neuronal damage caused by a free radical-producing agent FeSO4. These findings suggest that IL-3 prevents delayed neuronal death in the hippocampal CA1 field through a receptor-mediated expression of Bcl-xL protein, which is known to facilitate neuron survival. Since IL-3Ralpha in the hippocampal CA1 region, even though upregulated in response to ischemic insult, is much less intensely expressed than that in the CA3 region tolerant to ischemia, the paucity of IL-3R interacting with the ligand may account for the vulnerability of CA1 neurons to ischemia.

In vivo evidence that erythropoietin protects neurons from ischemic damage.

Erythropoietin (EPO) produced by the kidney and the liver (in fetuses) stimulates erythropoiesis. In the central nervous system, neurons express EPO receptor (EPOR) and astrocytes produce EPO. EPO has been shown to protect primary cultured neurons from N-methyl-D-aspartate (NMDA) receptor-mediated glutamate toxicity. Here we report in vivo evidence that EPO protects neurons against ischemia-induced cell death. Infusion of EPO into the lateral ventricles of gerbils prevented ischemia-induced learning disability and rescued hippocampal CA1 neurons from lethal ischemic damage. The neuroprotective action of exogenous EPO was also confirmed by counting synapses in the hippocampal CA1 region. Infusion of soluble EPOR (an extracellular domain capable of binding with the ligand) into animals given a mild ischemic treatment that did not produce neuronal damage, caused neuronal degeneration and impaired learning ability, whereas infusion of the heat-denatured soluble EPOR was not detrimental, demonstrating that the endogenous brain EPO is crucial for neuronal survival. The presence of EPO in neuron cultures did not repress a NMDA receptor-mediated increase in intracellular Ca2+, but rescued the neurons from NO-induced death. Taken together EPO may exert its neuroprotective effect by reducing the NO-mediated formation of free radicals or antagonizing their toxicity.

Epidermal growth factor protects neuronal cells in vivo and in vitro against transient forebrain ischemia- and free radical-induced injuries.

Epidermal growth factor (EGF) has been considered to be a candidate for neurotrophic factors on the basis of the results of several in vitro studies. However, the in vivo effect of EGF on ischemic neurons as well as its mechanism of action have not been fully understood. In the present in vivo study using a gerbil ischemia-model, we examined the effects of EGF on ischemia-induced learning disability and hippocampal CA1 neuron damage. Cerebroventricular infusion of EGF (24 or 120 ng/d) for 7 days to gerbils starting 2 hours before or immediately after transient forebrain ischemia caused a significant prolongation of response latency time in a passive avoidance task in comparison with the response latency of vehicle-treated ischemic animals. Subsequent histologic examinations showed that EGF effectively prevented delayed neuronal death of CA1 neurons in the stratum pyramidale and preserved synapses intact within the strata moleculare, radiatum, and oriens of the hippocampal CA1 region. In situ detection of DNA fragmentation (TUNEL staining) revealed that ischemic animals infused with EGF contained fewer TUNEL-positive neurons in the hippocampal CA1 field than those infused with vehicle alone at the seventh day after ischemia. In primary hippocampal cultures, EGF (0.048 to 6.0 ng/mL) extended the survival of cultured neurons, facilitated neurite outgrowth, and prevented neuronal damage caused by the hydroxyl radical-producing agent FeSO4 and by the peroxynitrite-producing agent 3-morpholinosydnonimine in a dose-dependent manner. Moreover, EGF significantly attenuated FeSO4-induced lipid peroxidation of cultured neurons. These findings suggest that EGF has a neuroprotective effect on ischemic hippocampal neurons in vivo possibly through inhibition of free radical neurotoxicity and lipid peroxidation.

Ginsenoside Rb(1) prevents image navigation disability, cortical infarction, and thalamic degeneration in rats with focal cerebral ischemia.

Oral administration of red ginseng powder before but not after transient forebrain ischemia prevents delayed neuronal death in gerbils. One neuroprotective molecule within red ginseng powder is ginsenoside Rb(1). The mechanism of action(s) of ginsenoside Rb(1) remains to be determined. We performed intracerebroventricular infusion of 0.6 microg/d ginsenoside Rb(1) before or after permanent occlusion of the left middle cerebral artery in stroke-prone spontaneous hypertensive rats. Ginsenoside Rb(1) significantly decreased escape latency on repeated trials of the Morris water maze test, throughout the first to fourth trial days at 2 and 4 weeks after MCA occlusion (P<.05, P<.01 or P<.001). The ratio of the infarcted area to the left hemispheric area in the groups treated with 0.6 microg/d of ginsenoside Rb(1) was significantly smaller than that in the saline-treated ischemic group (P<.05 or P<.001). The continuous infusion of ginsenoside Rb(1) (0.06 microg/d) was less effective and the other doses examined were ineffective in ameliorating ischemia-induced image navigation disability and reducing cortical infarct size. There were significant differences in neuron numbers in the ventroposterior thalamic nucleus and in the left-to-right ratio of the thalamic area between the saline-infused ischemic group and the ginsenoside Rb(1)-treated ischemic group (P<.05 or P<.01). Moreover, ginsenoside Rb(1) at concentrations of 0.1 to 100 fg/mL (0.09 to 90 fM), facilitated neurite extension and rescued cortical neurons from lethal damage caused by the free radical-promoting agent FeSO(4), in vitro (P<.05 or P<.01). These findings suggest that ginsenoside Rb(1) protects the cerebral cortex against lethal ischemic damage possibly by acting as a neurotrophic factor-like agent and by scavenging free radicals, which are overproduced in situ during and after brain ischemia. The final link between the in vivo neuroprotective action and the in vitro neurotrophic and antioxidant activities of ginsenoside Rb(1) remains to be determined.

Protective effect of a prostaglandin I2 analog, TEI-7165, on ischemic neuronal damage in gerbils.

TTC-909 (Clinprost), a chemically stable PGI2 analog, isocarbacyclin methyl ester (TEI-9090 or Clinprost) incorporated in lipid microspheres, when administered intravenously after brain ischemia, prevents ischemic neuronal damage possibly by modulating cerebral blood flow and platelet aggregation. However, the possibility exists that TEI-7165, which is the free acid form and a central metabolite of TEI-9090, has direct neurotrophic action in vivo, since TEI-7165 has been shown to block neuronal voltage-dependent Ca2+ channels in vitro, and a novel prostacyclin receptor showing high affinity with TEI-7165 has been detected in a variety of brain regions including the hippocampus. In the present study, we infused TEI-7165 for 7 days into the lateral ventricle of gerbils starting 2 h before or just after 3-min forebrain ischemia. TEI-7165 infusion prevented significantly the ischemia-induced shortening of response latency time as revealed by a step-down passive avoidance task. Subsequent light and electron microscopic examinations showed that pyramidal neurons in the hippocampal CA1 region, as well as synapses within the strata moleculare, radiatum and oriens of the region, were significantly more numerous in gerbils infused with TEI-7165 than in those receiving vehicle infusion. TEI-7165 infusion did not affect hippocampal blood flow or temperature. These findings, together with the previously depicted accumulation of centrally administered [3H]TEI-7165 around hippocampal neurons, suggest that TEI-7165 has a direct neuroprotective action in brain ischemia.

Protection of ischemic hippocampal neurons by ginsenoside Rb1, a main ingredient of ginseng root.

Our previous study showed that the oral administration of red ginseng powder before but not after transient forebrain ischemia prevented delayed neuronal death in gerbils, and that a neuroprotective molecule within red ginseng powder was ginsenoside Rb1. However, it remains to be clarified whether or not ginsenoside Rb1 acts directly on the ischemic brain, and the mechanism by which ginsenoside Rb1 protects the ischemic CA1 neurons is not determined. Without elucidation of the pharmacological property of ginsenoside Rb1, the drug would not be accepted as a neuroprotective agent. The present study demonstrated that the intracerebroventricular infusion of ginsenoside Rb1 after 3.5 min or 3 min forebrain ischemia, precluded significantly the ischemia-induced shortening of response latency in a step-down passive avoidance task and rescued a significant number of hippocampal CA1 neurons from lethal ischemic damage. The intracerebroventricular infusion of ginsenoside Rb1 did not affect hippocampal blood flow or hippocampal temperature except that it caused a slight increase in hippocampal blood flow at 5 min after transient forebrain ischemia. Furthermore, ginsenoside Rb1 at concentrations of 0.1-100 fg/ml (0.09-90 fM) rescued hippocampal neurons from lethal damage caused by the hydroxyl radical-promoting agent FeSO4 in vitro, and the Fenton reaction system containing p-nitrosodimethylaniline confirmed the hydroxyl radical-scavenging activity of ginsenoside Rb1. These findings suggest that the central infusion of ginsenoside Rb1 after forebrain ischemia protects hippocampal CA1 neurons against lethal ischemic damage possibly by scavenging free radicals which are overproduced in situ after brain ischemia and reperfusion. The present study may validate the empirical usage of ginseng root over thousands of years for the prevention of cerebrovascular diseases.

Suppression by platelet factor 4 of the myogenic activity of basic fibroblast growth factor.

The effect of platelet factor 4 (PF4) on myoblast cultures with or without basic fibroblast growth factor (bFGF) or other growth factors was investigated in the present in vitro experiments, with reference to bFGF binding to myoblast membrane fraction. When PF4 was added to the culture medium 1 day after myoblast cultivation, the nuclei of both myoblasts and myotubes were markedly reduced in number in a dose-dependent manner, whereas the inhibitory effect of PF4 on myoblast development was not observed when PF4 was added to the culture medium 3, 7, or 14 days after myoblast cultivation. In contrast, bFGF significantly increased the numbers of myoblast and myotube nuclei. When bFGF and PF4 were simultaneously added to the culture medium, PF4 abolished the facilitatory effects of bFGF on myogenesis. The real-time biospecific interaction analysis (BLA) core system showed that the myoblast membrane fraction at 1 day after cultivation contains bFGF-binding elements which are blocked by PF4 in a dose-dependent manner. Moreover, [126I]-bFGF binding experiments indicated the existence of both high and low affinity binding sites on myoblast membranes, although the high affinity binding sites decreased in number and the dissociation constant increased in value as the culture period was prolonged. Among the six other growth factors examined, acidic fibroblast growth factor and platelet-derived growth factor-BB stimulated myogenesis, and their effects were blocked by PF4 treatment. These findings suggest that: 1) PF4 inhibits myoblast proliferation and myotube formation only for a limited initial period of cultivation, possibly because of the time-dependent down-regulation of high affinity bFGF receptors: and 2) PF4 may be used as a tool to investigate the function of endogenous heparin-binding growth factors upregulated transiently at a certain developmental stage or in case of tissue damage and repair, even though it is not monospecific to bFGF.

Platelet-derived growth factor prevents ischemia-induced neuronal injuries in vivo.

Platelet-derived growth factor (PDGF) has been considered to be a neuroprotective factor candidate on the basis of several in vitro studies. However, the in vivo effect of PDGF on ischemic neurons has not been determined. In the present study, the effect of PDGF-BB on the ischemia-induced disability of passive avoidance task and hippocampal CA1 neuron death in normothermic gerbils, whose the brain temperature was kept at 37.0 +/- 0.2 degrees C during 3-min occlusion of the common carotid arteries was investigated. When PDGF-BB was continuously infused for 7 days into the cerebral ventricles of gerbils with transient forebrain ischemia, response latency time in a passive avoidance task was significantly prolonged. Subsequent histological examinations showed that PDGF-BB effectively increased the number of viable pyramidal neurons in the hippocampal CA1 region as well as synapses within the strata moleculare, radiatum and oriens of the region in comparison with the numbers of neurons and synapses in vehicle-treated ischemic gerbils. In situ detection of DNA fragmentation (TUNEL staining) revealed that TUNEL-positive neurons in the hippocampal CA1 field of vehicle-treated ischemic gerbils were much more numerous than those in the field of PDGF-BB-treated ischemic animals after 7 days ischemia. These findings suggest that the present ischemic animal model exhibits a more delayed neuronal degeneration of the hippocampal CA1 field than the conventional 5-min ischemic model and that the 7-day infusion of PDGF-BB, starting 2 h before ischemic insult, not only prevents delayed neuronal death in the hippocampal CA1 field at 7 days after forebrain ischemia but also inhibits a slowly progressive neuronal degeneration occurring thereafter.

Beta-estradiol protects hippocampal CA1 neurons against transient forebrain ischemia in gerbil.

Beta-estradiol has been considered to be a neurotrophic agent, but its in vivo effect on gerbils with transient forebrain ischemia has not yet been demonstrated. In the first set of the present experiments, we infused beta-estradiol at a dose of 0.05 or 0.25 microg/day for 7 days into the lateral ventricles of normothermic gerbils starting 2 h before 3-min forebrain ischemia. Beta-estradiol infusion at a dose of 0.25 microg/day prevented significantly the ischemia-induced reduction of response latency time as revealed by a step-down passive avoidance task. Subsequent light and electron microscopic examinations showed that pyramidal neurons in the hippocampal CA1 region as well as synapses within the strata moleculare, radiatum and oriens of the region were significantly more numerous in gerbils infused with beta-estradiol than in those receiving saline infusion. Beta-estradiol at a dose of 1.25 microg/day was ineffective and occasionally increased the mortality of experimental animals. Since the total brain content of exogenous beta-estradiol at 12 h after forebrain ischemia was estimated to be less than 145 ng, the second set of experiments focused on the neurotrophic action of beta-estradiol at concentrations around 100 ng/ml in vitro. Beta-estradiol at concentrations of 1-100 ng/ml facilitated the survival and process extension of cultured hippocampal neurons, but it did not exhibit any significant radical-scavenging effects at the concentration range. On the other hand, 100 microg/ml of beta-estradiol, even though failing to support hippocampal neurons in vitro, effectively scavenged free radicals in subsequent in vitro studies, as demonstrated elsewhere. These findings suggest that beta-estradiol at a dose of 0.25 microg/day prevents ischemia-induced learning disability and neuronal loss at early stages after transient forebrain ischemia, possibly via a receptor-mediated pathway without attenuating free radical neurotoxicity.

Persistent c-fos expression in the brains of mice with chronic social stress.

The present study was conducted to demonstrate immunohistochemically, the sites of c-fos protein expression in the brains of mice subjected to acute and chronic social defeat stress. To induce social stress, mice were placed in situations of species-specific intermale aggression either only once or five times at 24 h intervals. Two hours after the single or fifth defeat stress, many c-fos immunoreactive neurons were observed in a variety of brain regions including the limbic system and sensory relay nuclei. The c-fos immunoreactive neurons in the brains of acute defeat mice decreased in number with time and the c-fos staining pattern of acute defeat mice became indistinguishable from that of normal control mice by 24 h after the single defeat stress. In contrast, chronic defeat stress induced persistent c-fos expression in the forebrain and brainstem even 24 h after the fifth defeat stress. In the forebrain of chronic defeat mice, the olfactory bulb, cingulate cortex, hippocampus, entire hypothalamus, septal nuclei and the amygdaloid complex, except for the central nucleus, were labeled intensely with c-fos antiserum. In the lower brainstem, nerve cells with c-fos immunoreactivity were seen mainly in ascending and descending sensory relay nuclei relevant to auditory and vestibular transmission, epicritic sensation (gracile and external cuneate nuclei), pain inhibition (central gray and raphe nuclei), and viscerosensory transmission (solitary tract nucleus). The differences in c-fos expression among the normal control, acute and chronic defeat mice were evaluated by an enumeration of the immunopositive neurons within each brain nucleus examined, and they were confirmed subsequently by statistical analysis. There was little c-fos expression in the nucleus putamen, lateral, ventral and posterior thalamic nuclei, pretectal nuclei, medial geniculate nucleus, red nucleus, substantia nigra, cerebellum, spinal cord, or cranial nerve nuclei. These findings suggest that chronic but not acute defeat stress causes persistent c-fos expression in more widespread brain regions than do any other stresses so far investigated. The present study may shed light on the central mechanisms by which behavioral abnormalities and/or chronic sociopsychological stress leads to the occurrence of abnormal behavior and/or psychosomatic disorders in experimental animals and humans.

A hydrophilic peptide comprising 18 amino acid residues of the prosaposin sequence has neurotrophic activity in vitro and in vivo.

Prosaposin, a 517-amino-acid glycoprotein, not only acts as the precursor of saposin A, B, C, and D but also possesses neurotrophic activity to rescue hippocampal CA1 neurons from ischemic damage in vivo and to promote neurite extension of neuroblastoma cells in vitro. Recently, the trophic activity of prosaposin on human neuroblastoma cells has been shown to reside in the NH2-terminal hydrophilic sequence (LIDNNRTEEILY) of the human saposin C. Here we show that prosaposin, saposin C, and a peptide comprising the 18-amino-acid sequence (18-mer peptide; LSELIINNATEELLIKGL) located in the NH2-terminal hydrophilic sequence of the rat saposin C-domain promoted survival and neurite outgrowth of cultured rat hippocampal neurons in a dose-dependent manner. Moreover, infusion for 7 days of the 18-mer peptide into the lateral ventricle of gerbils, starting either 2 h before or immediately after 3 min of forebrain ischemia, protected ischemia-induced learning disability and hippocampal CA1 neuronal loss. Thus, we ascribe the in vitro and in vivo trophic actions of prosaposin on hippocampal neurons to the linear 18-mer sequence and raise the possibility that this peptide can be used as an agent for the treatment of forebrain ischemic damage.

Interleukin-6 prevents ischemia-induced learning disability and neuronal and synaptic loss in gerbils.

Interleukin-6 (IL-6) has been shown to have potent neurotrophic activity on peripheral and central neurons in vitro. However, it remains to be determined whether or not IL-6 rescues hippocampal CA1 neurons from lethal ischemia and prevents ischemia-induced learning disability. In the present in vivo study, we infused IL-6 continuously for 7 days into the lateral ventricle of gerbil starting 2 h before 3-min forebrain ischemia. IL-6 infusion prevented the occurrence of ischemia-induced learning disability in a dose-dependent manner as revealed by a step-down passive avoidance task. Subsequent light and electron microscopic examinations showed that pyramidal neurons in the CA1 region of the hippocampus as well as synapses within the strata moleculare, radiatum and oriens of the region were significantly more numerous in gerbils infused with IL-6 than in those receiving vehicle infusion. These findings suggest that IL-6 has a trophic effect on ischemic hippocampal neurons.

Ginseng root prevents learning disability and neuronal loss in gerbils with 5-minute forebrain ischemia.

The present study was designed to investigate the possible neuroprotective activity of ginseng roots in 5-min ischemic gerbils using a step-down passive avoidance task and subsequent neuron and synapse counts in the hippocampal CA1 region. The following drugs were administered for 7 days before the induced ischemia: red ginseng powder (RGP), crude ginseng saponin (CGS), crude ginseng non-saponin (CGNS), and pure ginsenosides Rb1, Rg1 and Ro. Oral administration of RGP significantly prevented the ischemia-induced decrease in response latency, as determined by the passive avoidance test, and rescued a significant number of ischemic hippocampal CA1 pyramidal neurons in a dose-dependent manner. Intraperitoneal injections of CGS exhibited a similar neuroprotective effect. CGNS had a significant but less potent protective effect against impaired passive avoidance task and degeneration of hippocampal CA1 neurons. Ginsenoside Rb1 significantly prolonged the response latency of ischemic gerbils and rescued a significant number of ischemic CA1 pyramidal neurons, whereas ginsenosides Rg1 and Ro were ineffective. Postischemic treatment with RGP, CGS or ginsenoside Rb1 was ineffective. The neuroprotective activities of RGP, CGS and ginsenoside Rb1 were confirmed by electron microscopy counts of synapses in individual strata of the CA1 field of ischemic gerbils pretreated with the drugs. These findings suggest that RGP and CGS are effective in the prevention of delayed neuronal death, and that ginsenoside Rb1 is one of the neuroprotective molecules within ginseng root.