Risperidone Administration Attenuates Renal Ischemia and Reperfusion Injury following Cardiac Arrest by Antiinflammatory Effects in Rats.

Multi-organ dysfunction following cardiac arrest is associated with poor outcome as well as high mortality. The kidney, one of major organs in the body, is susceptible to ischemia and reperfusion; however, there are few studies on renal ischemia and reperfusion injury (IRI) following the return of spontaneous circulation (ROSC) after cardiac arrest. Risperidone, an atypical antipsychotic drug, has been discovered to have some beneficial effects beyond its original effectiveness. Therefore, the aim of the present study was to investigate possible therapeutic effects of risperidone on renal IRI following cardiac arrest. Rats were subjected to cardiac arrest induced by asphyxia for five minutes followed by ROSC. When serum biochemical analyses were examined, the levels of serum blood urea nitrogen, creatinine, and lactate dehydrogenase were dramatically increased after cardiac arrest, but they were significantly reduced by risperidone administration. Histopathology was examined using hematoxylin and eosin staining. Histopathological injury induced by cardiac arrest was apparently attenuated by risperidone administration. Furthermore, alterations in pro-inflammatory cytokines (interleukin-6 and tumor necrosis factor-α) and anti-inflammatory cytokines (interleukin-4 and interleukin-13) were examined by immunohistochemistry. Pro-inflammatory and anti-inflammatory cytokine immunoreactivities were gradually and markedly increased and decreased, respectively, in the kidneys following cardiac arrest; however, risperidone administration after cardiac arrest significantly attenuated the increased pro-inflammatory cytokine immunoreactivities and the decreased anti-inflammatory cytokine immunoreactivities. Collectively, our current results revealed that, in rats, risperidone administration after cardiac arrest protected kidneys from IRI induced by cardiac arrest and ROSC through anti-inflammatory effects.

Therapeutic Treatment with Pycnogenol® Attenuates Ischemic Brain Injury in Gerbils Focusing on Cognitive Impairment, Neuronal Death, BBB Leakage and Neuroinflammation in the Hippocampus.

BACKGROUND: A gerbil model of ischemia and reperfusion (IR) injury in the forebrain has been developed for studies on mechanisms, prevention and therapeutic strategies of IR injury in the forebrain. Pycnogenol® (PYC), a standardized extract of French maritime pine tree (Pinus pinaster Aiton) has been exploited as an additive for dietary supplement. In the present study, we investigated the neuroprotective effects of post-treatment with PYC and its therapeutic mechanisms in gerbils. METHODS: The gerbils were given sham and IR operation and intraperitoneally injected with vehicle and Pycnogenol® (25, 50 and 100 mg/kg, respectively) immediately, at 24 hours and 48 hours after sham and IR operation. Through 8-arm radial maze test and passive avoidance test, each spatial memory and short-term memory function was assessed. To examine the neuroprotection of Pycnogenol®, we conducted cresyl violet staining, immunohistochemistry for neuronal nuclei, and Fluoro-Jade B histofluorescence. Moreover, we carried out immunohistochemistry for immunoglobulin G (IgG) to investigate blood-brain barrier (BBB) leakage and interleukin-1ß (IL-1ß) to examine change in pro-inflammatory cytokine. RESULTS: We found that IR-induced memory deficits were significantly ameliorated when 100 mg/kg Pycnogenol® was treated. In addition, treatment with 100 mg/kg Pycnogenol®, not 25 mg/kg nor 50 mg/kg, conferred neuroprotective effect against IR injury. For its mechanisms, we found that 100 mg/kg Pycnogenol® significantly reduced BBB leakage and inhibited the expression of IL-1ß. CONCLUSIONS: Therapeutic treatment (post-treatment) with Pycnogenol® after IR effectively attenuated ischemic brain injury in gerbils. Based on these results, we suggest that PYC can be employed as an important material for ischemic drugs.

Therapeutic Hypothermia after Cardiac Arrest Attenuates Hindlimb Paralysis and Damage of Spinal Motor Neurons and Astrocytes through Modulating Nrf2/HO-1 Signaling Pathway in Rats.

Cardiac arrest (CA) and return of spontaneous circulation (ROSC), a global ischemia and reperfusion event, lead to neuronal damage and/or death in the spinal cord as well as the brain. Hypothermic therapy is reported to protect neurons from damage and improve hindlimb paralysis after resuscitation in a rat model of CA induced by asphyxia. In this study, we investigated roles of nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) in the lumbar spinal cord protected by therapeutic hypothermia in a rat model of asphyxial CA. Male Sprague-Dawley rats were subjected to seven minutes of asphyxial CA (induced by injection of 2 mg/kg vecuronium bromide) and hypothermia (four hours of cooling, 33 ± 0.5 °C). Survival rate, hindlimb motor function, histopathology, western blotting, and immunohistochemistry were examined at 12, 24, and 48 h after CA/ROSC. The rats of the CA/ROSC and hypothermia-treated groups had an increased survival rate and showed an attenuated hindlimb paralysis and a mild damage/death of motor neurons located in the anterior horn of the lumbar spinal cord compared with those of the CA/ROSC and normothermia-treated groups. In the CA/ROSC and hypothermia-treated groups, expressions of cytoplasmic and nuclear Nrf2 and HO-1 were significantly higher in the anterior horn compared with those of the CA/ROSC and normothermia-treated groups, showing that cytoplasmic and nuclear Nrf2 was expressed in both motor neurons and astrocytes. Moreover, in the CA/ROSC and hypothermia-treated group, interleukin-1ß (IL-1ß, a pro-inflammatory cytokine) expressed in the motor neurons was significantly reduced, and astrocyte damage was apparently attenuated compared with those found in the CA/ROSC and normothermia group. Taken together, our results indicate that hypothermic therapy after CA/ROSC attenuates CA-induced hindlimb paralysis by protecting motor neurons in the lumbar spinal cord via activating the Nrf2/HO-1 signaling pathway and attenuating pro-inflammation and astrocyte damage (reactive astrogliosis).

Therapeutic Administration of Oxcarbazepine Saves Cerebellar Purkinje Cells from Ischemia and Reperfusion Injury Induced by Cardiac Arrest through Attenuation of Oxidative Stress.

Research reports using animal models of ischemic insults have demonstrated that oxcarbazepine (a carbamazepine analog: one of the anticonvulsant compounds) extends neuroprotective effects against cerebral or forebrain injury induced by ischemia and reperfusion. However, research on protective effects against ischemia and reperfusion cerebellar injury induced by cardiac arrest (CA) and the return of spontaneous circulation has been poor. Rats were assigned to four groups as follows: (Groups 1 and 2) sham asphyxial CA and vehicle- or oxcarbazepine-treated, and (Groups 3 and 4) CA and vehicle- or oxcarbazepine-treated. Vehicle (0.3% dimethyl sulfoxide/saline) or oxcarbazepine (200 mg/kg) was administered intravenously ten minutes after the return of spontaneous circulation. In this study, CA was induced by asphyxia using vecuronium bromide (2 mg/kg). We conducted immunohistochemistry for calbindin D-28kDa and Fluoro-Jade B histofluorescence to examine Purkinje cell death induced by CA. In addition, immunohistochemistry for 4-hydroxy-2-nonenal (4HNE) was carried out to investigate CA-induced oxidative stress, and immunohistochemistry for Cu, Zn-superoxide dismutase (SOD1) and Mn-superoxide dismutase (SOD2) was performed to examine changes in endogenous antioxidant enzymes. Oxcarbazepine treatment after CA significantly increased the survival rate and improved neurological deficit when compared with vehicle-treated rats with CA (survival rates ≥ 63.6 versus 6.5%), showing that oxcarbazepine treatment dramatically protected cerebellar Purkinje cells from ischemia and reperfusion injury induced by CA. The salvation of the Purkinje cells from ischemic injury by oxcarbazepine treatment paralleled a dramatic reduction in 4HNE (an end-product of lipid peroxidation) and increased or maintained the endogenous antioxidant enzymes (SOD1 and SOD2). In brief, this study shows that therapeutic treatment with oxcarbazepine after CA apparently saved cerebellar neurons (Purkinje cells) from CA-induced neuronal death by attenuating oxidative stress and suggests that oxcarbazepine can be utilized as a therapeutic medicine for ischemia and reperfusion brain (cerebellar) injury induced by CA.

Protective Effects of Topical Administration of Laminarin in Oxazolone-Induced Atopic Dermatitis-like Skin Lesions.

Laminarin is a polysaccharide isolated from brown marine algae and has a wide range of bioactivities, including immunoregulatory and anti-inflammatory properties. However, the effects of laminarin on atopic dermatitis have not been demonstrated. This study investigated the potential effects of topical administration of laminarin using a Balb/c mouse model of oxazolone-induced atopic dermatitis-like skin lesions. Our results showed that topical administration of laminarin to the ear of the mice improved the severity of the dermatitis, including swelling. Histological analysis revealed that topical laminarin significantly decreased the thickening of the epidermis and dermis and the infiltration of mast cells in the skin lesion. Serum immunoglobulin E levels were also significantly decreased by topical laminarin. Additionally, topical laminarin significantly suppressed protein levels of oxazolone-induced proinflammatory cytokines, such as interleukin-1ß, tumor necrosis factor-α, monocyte chemoattractant protein-1, and macrophage inflammatory protein-1α in the skin lesion. These results indicate that topical administration of laminarin can alleviate oxazolone-induced atopic dermatitis by inhibiting hyperproduction of IgE, mast cell infiltration, and expressions of proinflammatory cytokines. Based on these findings, we propose that laminarin can be a useful candidate for the treatment of atopic dermatitis.

Effect of therapeutic hypothermia against renal injury in a rat model of asphyxial cardiac arrest: Α focus on the survival rate, pathophysiology and antioxidant enzymes.

Although multi­organ dysfunction is associated with the survival rate following cardiac arrest (CA), the majority of studies to date have focused on hearts and brains, and few studies have considered renal failure. The objective of the present study, therefore, was to examine the effects of therapeutic hypothermia on the survival rate, pathophysiology and antioxidant enzymes in rat kidneys following asphyxial CA. Rats were sacrificed one day following CA. The survival rate, which was estimated using Kaplan­Meier analysis, was 42.9% one day following CA. However, hypothermia, which was induced following CA, significantly increased the survival rate (71.4%). In normothermia rats with CA, the serum blood urea nitrogen level was significantly increased one day post­CA. In addition, the serum creatinine level was significantly increased one day post­CA. However, in CA rats exposed to hypothermia, the levels of urea nitrogen and creatinine significantly decreased following CA. Histochemical staining revealed a significant temporal increase in renal injury after the normothermia group was subjected to CA. However, renal injury was significantly decreased in the hypothermia group. Immunohistochemical analysis of the kidney revealed a significant decrease in antioxidant enzymes (copper­zinc superoxide dismutase, manganese superoxide dismutase, glutathione peroxidase and catalase) with time in the normothermia group. However, in the hypothermia group, these enzymes were significantly elevated following CA. Collectively, the results revealed that renal dysfunction following asphyxial CA was strongly associated with the early survival rate and therapeutic hypothermia reduced renal injury via effective antioxidant mechanisms.

Brain Factor-7® Improves Cognitive Impairment Following Transient Ischemia and Reperfusion Injury in Gerbil Forebrain through Promoting Remyelination and Restoring Cholinergic and Glutamatergic Neurotransmission in the Hippocampus.

BACKGROUND: Ischemia and reperfusion injury in the brain triggers cognitive impairment which are accompanied by neuronal death, loss of myelin sheath and decline in neurotransmission. In this study, we investigated whether therapeutic administration of Brain Factor-7® (BF-7®; a silk peptide) in ischemic gerbils which were developed by transient (five minutes) ischemia and reperfusion in the forebrain (tFI/R) improved cognitive impairment. METHODS: Short-term memory and spatial memory functions were assessed by passive avoidance test and Barnes maze test, respectively. To examine neuronal change in the hippocampus, cresyl violet staining, immunohistochemistry for neuronal nuclei and fluoro Jade B histofluorescence were performed. We carried out immunohistochemistry for myelin basic protein (a marker for myelin) and receptor interacting protein (a marker for oligodendrocytes). Furthermore, immunohistochemistry for vesicular acetylcholine transporter (as a cholinergic transporter) and vesicular glutamate transporter 1 (as a glutamatergic synapse) was done. RESULTS: Administration of BF-7® significantly improved tFI/R-induced cognitive impairment. tFI/R-induced neuronal death was found in the Cornu Ammonis 1 (CA1) subfield of the hippocampus from five days after tFI/R. Treatment with BF-7® following tFI/R did not restore the death (loss) of CA1 neurons following tFI/R. However, BF-7® treatment to the ischemic gerbils significantly improved remyelination and proliferation of oligodendrocytes in the hippocampus with ischemic injury. Treatment with BF-7® to the ischemic gerbils significantly restored vesicular acetylcholine transporter-immunoreactive and vesicular glutamate transporter 1-immunoreactive structures in the hippocampus with ischemic injury. CONCLUSIONS: Based on these results, we suggest that BF-7® can be utilized for improving cognitive impairments induced by ischemic injury as an additive for health/functional foods and/or medicines.

Therapeutic Effects of Risperidone against Spinal Cord Injury in a Rat Model of Asphyxial Cardiac Arrest: A Focus on Body Temperature, Paraplegia, Motor Neuron Damage, and Neuroinflammation.

Cardiac arrest (CA) causes severe spinal cord injury and evokes spinal cord disorders including paraplegia. It has been reported that risperidone, an antipsychotic drug, effectively protects neuronal cell death from transient ischemia injury in gerbil brains. However, until now, studies on the effects of risperidone on spinal cord injury after asphyxial CA (ACA) and cardiopulmonary resuscitation (CPR) are not sufficient. Therefore, this study investigated the effect of risperidone on hind limb motor deficits and neuronal damage/death in the lumbar part of the spinal cord following ACA in rats. Mortality, severe motor deficits in the hind limbs, and the damage/death (loss) of motor neurons located in the anterior horn were observed two days after ACA/CPR. These symptoms were significantly alleviated by risperidone (an atypical antipsychotic) treatment after ACA. In vehicle-treated rats, the immunoreactivities of tumor necrosis factor-alpha (TNF-α) and interleukin 1-beta (IL-1ß), as pro-inflammatory cytokines, were increased, and the immunoreactivities of IL-4 and IL-13, as anti-inflammatory cytokines, were reduced with time after ACA/CPR. In contrast, in risperidone-treated rats, the immunoreactivity of the pro-inflammatory cytokines was significantly decreased, and the anti-inflammatory cytokines were enhanced compared to vehicle-treated rats. In brief, risperidone treatment after ACA/CPR in rats significantly improved the survival rate and attenuated paralysis, the damage/death (loss) of motor neurons, and inflammation in the lumbar anterior horn. Thus, risperidone might be a therapeutic agent for paraplegia by attenuation of the damage/death (loss) of spinal motor neurons and neuroinflammation after ACA/CPR.

Therapeutic Hypothermia Improves Hind Limb Motor Outcome and Attenuates Oxidative Stress and Neuronal Damage in the Lumbar Spinal Cord Following Cardiac Arrest.

: Hypothermia enhances outcomes of patients after resuscitation after cardiac arrest (CA). However, the underlying mechanism is not fully understood. In this study, we investigated effects of hypothermic therapy on neuronal damage/death, microglial activation, and changes of endogenous antioxidants in the anterior horn in the lumbar spinal cord in a rat model of asphyxial CA (ACA). A total of 77 adult male Sprague-Dawley rats were randomized into five groups: normal, sham ACA plus (+) normothermia, ACA + normothermia, sham ACA + hypothermia, and ACA + hypothermia. ACA was induced for 5 min by injecting vecuronium bromide. Therapeutic hypothermia was applied after return of spontaneous circulation (ROSC) via rapid cooling with isopropyl alcohol wipes, which was maintained at 33 ± 0.5 °C for 4 h. Normothermia groups were maintained at 37 ± 0.2 °C for 4 h. Neuronal protection, microgliosis, oxidative stress, and changes of endogenous antioxidants were evaluated at 12 h, 1 day, and 2 days after ROSC following ACA. ACA resulted in neuronal damage from 12 h after ROSC and evoked obvious degeneration/loss of spinal neurons in the ventral horn at 1 day after ACA, showing motor deficit of the hind limb. In addition, ACA resulted in a gradual increase in microgliosis with time after ACA. Therapeutic hypothermia significantly reduced neuronal loss and attenuated hind limb dysfunction, showing that hypothermia significantly attenuated microgliosis. Furthermore, hypothermia significantly suppressed ACA-induced increases of superoxide anion production and 8-hydroxyguanine expression, and significantly increased superoxide dismutase 1 (SOD1), SOD2, catalase, and glutathione peroxidase. Taken together, hypothermic therapy was found to have a substantial impact on changes in ACA-induced microglia activation, oxidative stress factors, and antioxidant enzymes in the ventral horn of the lumbar spinal cord, which closely correlate with neuronal protection and neurological performance after ACA.

Therapeutic hypothermia attenuates paraplegia and neuronal damage in the lumbar spinal cord in a rat model of asphyxial cardiac arrest.

Spinal cord ischemia can result from cardiac arrest. It is an important cause of severe spinal cord injury that can lead to serious spinal cord disorders such as paraplegia. Hypothermia is widely acknowledged as an effective neuroprotective intervention following cardiac arrest injury. However, studies on effects of hypothermia on spinal cord injury following asphyxial cardiac arrest and cardiopulmonary resuscitation (CA/CPR) are insufficient. The objective of this study was to examine effects of hypothermia on motor deficit of hind limbs of rats and vulnerability of their spinal cords following asphyxial CA/CPR. Experimental groups included a sham group, a group subjected to CA/CPR, and a therapeutic hypothermia group. Severe motor deficit of hind limbs was observed in the control group at 1 day after asphyxial CA/CPR. In the hypothermia group, motor deficit of hind limbs was significantly attenuated compared to that in the control group. Damage/death of motor neurons in the lumbar spinal cord was detected in the ventral horn at 1 day after asphyxial CA/CPR. Neuronal damage was significantly attenuated in the hypothermia group compared to that in the control group. These results indicated that therapeutic hypothermia after asphyxial CA/CPR significantly reduced hind limb motor dysfunction and motoneuronal damage/death in the ventral horn of the lumbar spinal cord following asphyxial CA/CPR. Thus, hypothermia might be a therapeutic strategy to decrease motor dysfunction by attenuating damage/death of spinal motor neurons following asphyxial CA/CPR.

Neuronal loss and gliosis in the rat striatum subjected to 15 and 30 minutes of middle cerebral artery occlusion.

Selective neuronal death or loss in certain brain regions has been well characterized in animal models of transient global cerebral ischemia. However, selective neuronal death in transient focal cerebral ischemia needs more investigation. Therefore, in this study, we studied selective neuronal death in the striatum (caudate putamen) of rats subjected to 15 or 30 min middle cerebral artery occlusion (MCAO). Neuronal death occurred in the dorsolateral field, not in the medial field in 30 min, not 15 min, MCAO-operated rats 5 days after MCAO using neuronal nuclear antigen immunohistochemistry and Fluoro-Jade B histofluorescence staining. In this group, immunoreactivity of glial fibrillary acidic protein in astrocytes was hardly shown in the dorsolateral field, although the immunoreactivity increased in the medial field. In addition, immunoreactivity of ionized calcium binding adapter molecule 1 in microglia was dramatically increased in the dorsolateral, not in the medial, field only in 30 min MCAO-operated rats. Briefly, these results show that at least 30 min of MCAO can evoke selective neuronal death, astrocytic dysfunction and microglial activation in the dorsolateral field of the rat striatum and suggest that a rat model of 30 min MCAO can be used to investigate mechanisms of neuronal death and gliosis following brief transient focal cerebral ischemic events for acute transient ischemic attack.

Effects of Scopolamine and Melatonin Cotreatment on Cognition, Neuronal Damage, and Neurogenesis in the Mouse Dentate Gyrus.

It has been demonstrated that melatonin plays important roles in memory improvement and promotes neurogenesis in experimental animals. We examined effects of melatonin on cognitive deficits, neuronal damage, cell proliferation, neuroblast differentiation and neuronal maturation in the mouse dentate gyrus after cotreatment of scopolamine (anticholinergic agent) and melatonin. Scopolamine (1 mg/kg) and melatonin (10 mg/kg) were intraperitoneally injected for 2 and/or 4 weeks to 8-week-old mice. Scopolamine treatment induced significant cognitive deficits 2 and 4 weeks after scopolamine treatment, however, cotreatment of scopolamine and melatonin significantly improved spatial learning and short-term memory impairments. Two and 4 weeks after scopolamine treatment, neurons were not damaged/dead in the dentate gyrus, in addition, no neuronal damage/death was shown after cotreatment of scopolamine and melatonin. Ki67 (a marker for cell proliferation)- and doublecortin (a marker for neuroblast differentiation)-positive cells were significantly decreased in the dentate gyrus 2 and 4 weeks after scopolamine treatment, however, cotreatment of scopolamine and melatonin significantly increased Ki67- and doublecortin-positive cells compared with scopolamine-treated group. However, double immunofluorescence for NeuN/BrdU, which indicates newly-generated mature neurons, did not show double-labeled cells (adult neurogenesis) in the dentate gyrus 2 and 4 weeks after cotreatment of scopolamine and melatonin. Our results suggest that melatonin treatment recovers scopolamine-induced spatial learning and short-term memory impairments and restores or increases scopolamine-induced decrease of cell proliferation and neuroblast differentiation, but does not lead to adult neurogenesis (maturation of neurons) in the mouse dentate gyrus following scopolamine treatment.

Effects of long­term scopolamine treatment on cognitive deficits and calcium binding proteins immunoreactivities in the mouse hippocampus.

GABAergic projections terminate on numerous hippocampal interneurons containing calcium binding proteins (CBPs), including calbindin D­28k (CB), calretinin (CR) and parvalbumin (PV). Memory deficits and expression levels of CB, CR, and PV were examined in the hippocampal subregions following systemic scopolamine (Scop; 1 mg/kg) treatment for 4 weeks in mice. Scop treatment induced significant memory deficits from 1 week after Scop treatment. CB, CR and PV immunoreactivities distributions were in hippocampal subregions [CA1 and CA3 regions, and the dentate gyrus (DG)]. CB immunoreactivity (CB+) was gradually decreased in all subregions until 2 weeks after Scop treatment, and CB+ was decreased to the lowest level in all subregions at 3 and 4 weeks. CR+ in the CA1 region was gradually decreased until 2 weeks and hardly observed at 3 and 4 weeks; in the CA3 region, CR+ was not altered in all subregions at any time. In the DG, CR+ was gradually decreased until 2 weeks and lowest at 3 and 4 weeks. PV+ in the CA1 region was not altered at 1 week, and gradually decreased from 2 weeks. In the CA3 region, PV+ did not change in any subregions at any time. In the DG, PV+ was not altered at 1 week, decreased at 2 weeks, and lowest at 3 and 4 weeks. In brief, Scop significantly decreased CBPs expressions in the hippocampus ≥3 weeks after the treatment although memory deficits had developed at 1 week. Therefore, it is suggested that Scop (1 mg/kg) must be systemically treated for ≥3 weeks to investigate changes in expression levels of CBPs in the hippocampus.

Age-dependent differences in myelin basic protein expression in the hippocampus of young, adult and aged gerbils.

Myelin degeneration is one of the characteristics of aging and degenerative diseases. This study investigated age-related alterations in expression of myelin basic protein (MBP) in the hippocampal subregions (dentate gyrus, CA2/3 and CA1 areas) of gerbils of various ages; young (1 month), adult (6 months) and aged (24 months), using western blot and immunohistochemistry. Western blot results showed tendencies of age-related reductions of MBP levels. MBP immunoreactivity was significantly decreased with age in synaptic sites of trisynaptic loops, perforant paths, mossy fibers, and Schaffer collaterals. In particular, MBP immunoreactive fibers in the dentate molecular cell layer (perforant path) was significantly reduced in adult and aged subjects. In addition, MBP immunoreactive mossy fibers in the dentate polymorphic layer and in the CA3 striatum radiatum was significantly decreased in the aged group. Furthermore, we observed similar age-related alterations in the CA1 stratum radiatum (Schaffer collaterals). However, the density of MBP immunoreactive fibers in the dentate granular cell layer and CA stratum pyramidale was decreased with aging. These findings indicate that expression of MBP is age-dependent and tissue specific according to hippocampal layers.

Immunoreactivities of calbindin­D28k, calretinin and parvalbumin in the somatosensory cortex of rodents during normal aging.

Calbindin­D28k (CB), calretinin (CR) and parvalbumin (PV), which regulate cytosolic free Ca2+ concentrations in neurons, are chemically expressed in γ­aminobutyric acid (GABA)ergic neurons that regulate the degree of glutamatergic excitation and output of projection neurons. The present study investigated age­associated differences in CB, CR and PV immunoreactivities in the somatosensory cortex in three species (mice, rats and gerbils) of young (1 month), adult (6 months) and aged (24 months) rodents, using immunohistochemistry and western blotting. Abundant CB­immunoreactive neurons were distributed in layers II and III, and age­associated alterations in their number were different according to the species. CR­immunoreactive neurons were not abundant in all layers; however, the number of CR­immunoreactive neurons was the highest in all adult species. Many PV­immunoreactive neurons were identified in all layers, particularly in layers II and III, and they increased in all layers with age in all species. The present study demonstrated that the distribution pattern of CB­, CR­ and PV­containing neurons in the somatosensory cortex were apparently altered in number with normal aging, and that CB and CR exhibited a tendency to decrease in aged rodents, whereas PV tended to increase with age. These results indicate that CB, CR and PV are markedly altered in the somatosensory cortex, and this change may be associated with normal aging. These findings may aid the elucidation of the mechanisms of aging and geriatric disease.

Neuroprotective effects of ischemic preconditioning on hippocampal CA1 pyramidal neurons through maintaining calbindin D28k immunoreactivity following subsequent transient cerebral ischemia.

Ischemic preconditioning elicited by a non-fatal brief occlusion of blood flow has been applied for an experimental therapeutic strategy against a subsequent fatal ischemic insult. In this study, we investigated the neuroprotective effects of ischemic preconditioning (2-minute transient cerebral ischemia) on calbindin D28k immunoreactivity in the gerbil hippocampal CA1 area following a subsequent fatal transient ischemic insult (5-minute transient cerebral ischemia). A large number of pyramidal neurons in the hippocampal CA1 area died 4 days after 5-minute transient cerebral ischemia. Ischemic preconditioning reduced the death of pyramidal neurons in the hippocampal CA1 area. Calbindin D28k immunoreactivity was greatly attenuated at 2 days after 5-minute transient cerebral ischemia and it was hardly detected at 5 days post-ischemia. Ischemic preconditioning maintained calbindin D28k immunoreactivity after transient cerebral ischemia. These findings suggest that ischemic preconditioning can attenuate transient cerebral ischemia-caused damage to the pyramidal neurons in the hippocampal CA1 area through maintaining calbindin D28k immunoreactivity.

Pretreated Glehnia littoralis Extract Prevents Neuronal Death Following Transient Global Cerebral Ischemia through Increases of Superoxide Dismutase 1 and Brain-derived Neurotrophic Factor Expressions in the Gerbil Hippocampal Cornu Ammonis 1 Area.

BACKGROUND: Glehnia littoralis, as a traditional herbal medicine to heal various health ailments in East Asia, displays various therapeutic properties including antioxidant effects. However, neuroprotective effects of G. littoralis against cerebral ischemic insults have not yet been addressed. Therefore, in this study, we first examined its neuroprotective effects in the hippocampus using a gerbil model of transient global cerebral ischemia (TGCI). METHODS: Gerbils were subjected to TGCI for 5 min. G. littoralis extract (GLE; 100 and 200 mg/kg) was administrated orally once daily for 7 days before ischemic surgery. Neuroprotection was examined by neuronal nuclear antigen immunohistochemistry and Fluoro-Jade B histofluorescence staining. Gliosis was observed by immunohistochemistry for glial fibrillary acidic protein and ionized calcium-binding adapter molecule 1. For neuroprotective mechanisms, immunohistochemistry for superoxide dismutase (SOD) 1 and brain-derived neurotrophic factor (BDNF) was done. RESULTS: Pretreatment with 200 mg/kg of GLE protected pyramidal neurons in the cornu ammonis 1 (CA1) area from ischemic insult area (F = 29.770, P < 0.05) and significantly inhibited activations of astrocytes (F = 22.959, P < 0.05) and microglia (F = 44.135, P < 0.05) in the ischemic CA1 area. In addition, pretreatment with GLE significantly increased expressions of SOD1 (F = 28.561, P < 0.05) and BDNF (F = 55.298, P < 0.05) in CA1 pyramidal neurons of the sham- and ischemia-operated groups. CONCLUSIONS: Our findings indicate that pretreatment with GLE can protect neurons from ischemic insults, and we suggest that its neuroprotective mechanism may be closely associated with increases of SOD1 and BDNF expressions as well as attenuation of glial activation.

Atomoxetine Protects Against NMDA Receptor-mediated Hippocampal Neuronal Death Following Transient Global Cerebral Ischemia.

BACKGROUND: Atomoxetine has been widely used for the treatment of attention-deficit/ hyperactivity disorder. ATX has additional abilities such as antagonistic effects on the N-methyl-Daspartate receptors (NMDARs) and benefit effects in some animal models of neurological disorders. However, there were few studies regarding protective effects and related mechanisms of ATX against cerebral ischemic insults. OBJECTIVE: The objective of this study is to investigate neuroprotection of ATX pretreatment and its mechanisms in the hippocampal cornu ammonis 1 (CA1) region following transient global cerebral ischemia in gerbils. METHOD: Gerbils were subjected to transient global cerebral ischemia induced by the occlusion of common carotid arteries for 5 min. Thirty mg/kg of ATX was administrated intraperitoneally once daily for 3 days before ischemic surgery. To examine neuroprotective effects of ATX, we carried out neuronal nuclear antigen immunohistochemistry and Fluoro-Jade B histofluorescence staining. In addition, immunoreactivities of NMDAR1, NMDAR2A/B, B-cell lymphoma 2 (Bcl-2) and Bcl-2- associated X protein (Bax) are closely related with neuroexcitotoxicity. RESULTS: ATX pretreatment reduced ischemia-induced hyperactivity and protected CA1 pyramidal neurons from ischemia. Pretreatment with ATX significantly attenuated ischemia-induced increases of NMDAR1 and NMDAR2A/B immunoreactivities in the CA1 pyramidal neurons at early time following ischemia. In addition, significant ischemia-induced alterations of Bcl-2 and Bax immunoreactivities were not observed in the ATX-treated group following ischemia. CONCLUSION: These results show that pretreatment with ATX protected against ischemic neuronal via inhibition of ischemia-induced excitotoxicity at early time following transient global cerebral ischemia.

Transient Cerebral Ischemia Alters GSK-3ß and p-GSK-3ß Immunoreactivity in Pyramidal Neurons and Induces p-GSK-3ß Expression in Astrocytes in the Gerbil Hippocampal CA1 Area.

Glycogen synthase kinase 3ß (GSK-3ß) is a key downstream protein in the PI3K/Akt pathway. Phosphorylation of serine 9 of GSK-3ß (GSK-3ß activity inhibition) promotes cell survival. In this study, we examined changes in expressions of GSK-3ß and phosphorylation of GSK-3ß (p-GSK-3ß) in the gerbil hippocampal CA1 area after 5 min of transient cerebral ischemia. GSK-3ß immunoreactivity in the CA1 area was increased in pyramidal cells at 6 h after ischemia-reperfusion. It was decreased in CA1 pyramidal cells from 12 h after ischemia-reperfusion, and hardly detected in the CA1 pyramidal cells at 5 days after ischemia-reperfusion. p-GSK-3ß immunoreactivity was slightly decreased in CA1 pyramidal cells at 6 and 12 h after ischemia-reperfusion. It was significantly increased in these cells at 1 and 2 days after ischemia-reperfusion. Five days after ischemia-reperfusion, p-GSK-3ß immunoreactivity was hardly found in CA1 pyramidal cells. However, p-GSK-3ß immunoreactivity was strongly expressed in astrocytes primarily distributed in strata oriens and radiatum. In conclusion, GSK-3ß and p-GSK-3ß were significantly changed in pyramidal cells and/or astrocytes in the gerbil hippocampal CA1 area following 5 min of transient cerebral ischemia. This finding indicates that GSK-3ß and p-GSK-3ß are closely related to delayed neuronal death.

Pre-treated Populus tomentiglandulosa extract inhibits neuronal loss and alleviates gliosis in the gerbil hippocampal CA1 area induced by transient global cerebral ischemia.

The genus Populus (poplar) belonging to the Salicaceae family has been used in traditional medicine, and its several species show various pharmacological properties including antioxidant and anti-inflammatory effects. No study regarding protective effects of Populus species against cerebral ischemia has been reported. Therefore, in the present study, we examined neuroprotective effects of ethanol extract from Populus tomentiglandulosa (Korea poplar) in the hippocampal cornu ammonis (CA1) area of gerbils subjected to 5 minutes of transient global cerebral ischemia. Pretreatment with 200 mg/kg of P. tomentiglandulosa extract effectively protected CA1 pyramidal neurons from transient global cerebral ischemia. In addition, glial fibrillary acidic protein immunoreactive astrocytes and ionized calcium binding adapter molecule 1 immunoreactive microglia were significantly diminished in the ischemic CA1 area by pretreatment with 200 mg/kg of P. tomentiglandulosa extract. Briefly, our results indicate that pretreatment with P. tomentiglandulosa extract protects neurons from transient cerebral ischemic injury and diminish cerebral ischemia-induced reactive gliosis in ischemic CA1 area. Based on these results, we suggest that P. tomentiglandulosa can be used as a potential candidate for prevention of ischemic injury.