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Background and Aim: Osteoarthritis (OA) is a chronic, painful, degenerative inflammatory disease of the synovial joints. Regular use of nonsteroidal anti-inflammatory drugs to decrease OA pain can have severe side effects, such as gastric irritation, ulcers, and heart problems. Natural products are extensively used to minimize OA-associated pain and inflammatory reactions. Lilium lancifolium is commonly used to alleviate several diseases through its anti-inflammatory effects. This study examined the impact of L. lancifolium extract on alleviating pain and inflammation associated with articular cartilage damage. Materials and Methods: Hydro-ethanol extracts of the L. lancifolium bulb were used. The experimental animals (adult beagle dogs) were divided into four groups: sham, which received neither treatment nor surgery; placebo, which received an empty gelatin capsule; glucosamine, which received glutamine (60 mg/kg); and L. lancifolium, which received an L. lancifolium extract-filled (60 mg/kg) gelatin capsule for 8 weeks. OA was induced by an expert orthopedic surgeon in 2-year-old dogs through resection of cranial cruciate ligament and lateral collateral ligament. Inflammatory cytokines, enzymes, lameness score, radiology, and histological changes were assessed. Results: Our experiments showed that long-term oral therapy with L. lancifolium alleviated inflammation and increased histological damage. L. lancifolium treatment effectively reduced cytokines, such as interleukin-6, metalloproteinase-9, leukotriene-4, prostaglandin, and cyclo-oxygenase in dogs with OA, suggesting the potential to minimize inflammatory reactions in OA. L. lancifolium showed anti-inflammatory qualities in dogs with OA. This effect was comparable with that of glucosamine OA treatment. Conclusion: L. lancifolium supplementation represents a possible therapeutic and management option in this model of OA.

Osteoarthritis (OA) is a chronic, painful, degenerative inflammatory disease of the synovial joints. Regular use of nonsteroidal anti-inflammatory drugs to decrease OA pain can have severe side effects, such as gastric irritation, ulcers, and heart problems. Natural products are extensively used to minimize OA-associated pain and inflammatory reactions.Hydro-ethanol extracts of theOur experiments showed that long-term oral therapy with

Background and Aim: Canine atopic dermatitis (CAD) is a hereditary susceptibility to the development of allergic symptoms in response to repeated exposure to generally innocuous substances known as "allergens." Allergens can be plants, animals, mold, mites, or milk. At present, serological enzyme-linked immunoassay (ELISA) kits are used for immunoglobulin E (IgE)-specific allergen detection due to their simplicity and accuracy. This study aimed to detect allergens in dogs with CAD and determine how they differ according to season, breed, age, and sex using a serological test in six provinces in South Korea for 12 months. This will allow practitioners to easily understand the risk factors related to CAD. Materials and Methods: In this study, IgE allergen-specific ELISA kits were used. The allergens were detected in serum samples collected from different regions considering season, sex, breed, and age. Allergens were divided into the following Ten categories: 1. Dairy, yeast, and egg, 2. grains, 3. vegetables, 4. meat, 5. seafood, 6. animals, 7. mold, 8. insects, 9. mites, and 10. trees. Results: The percentage of allergens detected in males (54.8%) was higher than that of females (45.2%); 54.2% of allergens occurred in 3-year-old dogs or older. Moreover, regarding frequency, 65.6% of overall allergens occur during autumn; Chungcheongnam-do and Jeollabuk-do showed 20.7% and 20.9%, respectively. Additionally, among allergens categories, notable allergen occurrence was as follows: 38.3% corn; 28.7% potatoes; 22.7% duck; 24.4%,codfish; 31.2% animal wool; 95.6% Aspergillus fumigatus; 31.9% flea; 41.8% oak; and 25.0% sheep's sorrel grass. Conclusion: This study showcases the frequency of 60 allergens in six provinces detected in dogs with CAD; most likely from food or the environment using serological ELISA kits. Environmental sensitizer results can be considered for humans suffering from allergies to avoid a similar environment. A large-scale study can be performed to evaluate the allergens in the state. However, neither a skin test nor feed analysis was conducted, which is a limitation of this study.

Ulcerative colitis (UC) is a severe inflammatory disease that has spread throughout the world. Cirsium japonicum (CJ) and Aralia elata (AE) are natural herbs with potent antioxidative antidiabetics and anti-inflammatory effects. In this investigation, we studied the defensive role of the combination of CJ and AE against LPS-induced inflammation in RAW 264.7 cells, dextran sulfate sodium (DSS)-induced colitis in mice, and acetic acid-induced colitis in dogs. MTT assay was performed to identify the toxic effect of CJ and AE extracts. NO, and MDA level was also measured by NO and MDA assay. To measure the pro-inflammatory protein expression, a western blot was performed. To induce colitis, 3% DSS was used for mice and 6% acetic acid was used for dogs. Histopathology and colonoscopy were executed to detect the effect of extracts. CJ and AE pretreatment reduced the level of NO, MDA, and the expression of pro-inflammatory proteins cyclooxygenase-2 (COX-2), tumor necrosis factor-α (TNF-α), and interleukin-1β (IL-1β) in RAW 264.7. Compared to the separate doses of CJ and AE, the combined dose of CJ and AE significantly reduced clinical symptoms induced by DSS in mice and acetic acid in dogs including weight loss, bloody stool, shortening of the colon, and the severity of colitis and degree of histological damage in the colon. Therefore, these results indicated that a combined dose of CJ and AE has a protective effect against LPS-induced RAW 264.7 cells, DSS-mediated colonic inflammation in mice, and acetic acid-induced colitis in dogs.

Although multi‑organ 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 Kaplan‑Meier 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 post‑CA. In addition, the serum creatinine level was significantly increased one day post‑CA. 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 (copper‑zinc 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.

The present study aimed to investigate the renoprotective effect of therapeutic hypothermia (TH) on renal ischemia-reperfusion injury (RI/RI) induced by asphyxial cardiac arrest (CA) in rats. A total of 48 male rats were randomly divided into five groups: i) Sham (n=6); ii) Normothermia + CA (Normo.) (n=14); iii) Normo. and 2 h of TH after return of spontaneous circulation (ROSC) (n=12); iv) Normo. and 4 h of TH after ROSC (n=9); and v) Normo. and 6 h of TH after ROSC (n=7). All rats except the Sham group underwent asphyxia CA and were sacrificed 1 day after ROSC. The survival rate increased from 42.8% in the Normo. group to 50, 66.6 and 85.7% in the groups with 2, 4 and 6 h of TH after CA, respectively. TH attenuated the histopathological changes of the renal tissues following ROSC and the levels of blood urea nitrogen, serum creatinine and malondialdehyde in renal tissues. On immunohistochemistry, the relative optical density of nuclear erythroid-related factor-2 (Nrf2) and heme oxygenase (HO-1) expression in renal tissues increased in the Normo. group compared with that in the Sham group and exhibited further significant increases at 6 h of TH after ROSC. In conclusion, TH attenuated renal injury and increased the expression of Nrf2 and HO-1 in a TH treatment time-dependent manner.

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.

It is now established that diethylstilbestrol (DES) has damaging effects on the male reproductive system. However, to date there have been no studies morphological analysis of adult rat testes upon treatment with DES. Here, we examined whether DES has any significant morphological effect on steroidogenesis and spermatogenesis. DES was injected subcutaneously at 3 μg/day and 30 μg/day in adult male Sprague-Dawley (SD) rats for two different treatment lengths (1 or 3 weeks), after which rats were necropsied. TUNEL labeling, cell counting, and morphological analysis were used to evaluate the effects of DES. A high dose of DES and longer exposure severely affected the cellular development of the testis. Specifically, DES treatment disrupted both steroidogenesis and spermatogenesis by decreasing the number of spermatogonia, Sertoli cells, and Leydig cells in a dose- and time-dependent manner. Thus, DES may account for decreases in the number of spermatogenic cells, Sertoli cells and Leydig cells, which in turn may lead to reduced fertility in males.

Recently, we have reported that Oenanthe javanica extract (OJE) displays strong neuroprotective effect against ischemic damage after transient global cerebral ischemia. However, neuroprotective mechanisms of OJE have not been fully identified. Thus, this study investigated the neuroprotection of OJE in the hippocampal CA1 area and its anti-inflammatory activity in gerbils subjected to 5 minutes of transient global cerebral ischemia. We treated the animals by intragastrical injection of OJE (100 and 200 mg/kg) once daily for 1 week prior to transient global cerebral ischemia. Neuroprotection of OJE was observed by immunohistochemistry for neuronal nuclear antigen and histofluorescence staining for Fluoro-Jade B. Immunohistochemistry of glial fibrillary acidic protein and ionized calcium-binding adapter molecule 1 was done for astrocytosis and microgliosis, respectively. To investigate the neuroprotective mechanisms of OJE, we performed immunohistochemistry of tumor necrosis factor-alpha and interleukin-2 for pro-inflammatory function and interleukin-4 and interleukin-13 for anti-inflammatory function. When we treated the animals by intragastrical administration of 200 mg/kg of OJE, hippocampal CA1 pyramidal neurons were protected from transient global cerebral ischemia and cerebral ischemia-induced gliosis was inhibited in the ischemic hippocampal CA1 area. We also found that interleukin-4 and -13 immunoreactivities were significantly increased in pyramidal neurons of the ischemic CA1 area after OJE pretreatment, and the increased immunoreactivities were sustained in the CA1 pyramidal neurons after transient global cerebral ischemia. However, OJE pretreatment did not increase interleukin-2 and tumor necrosis factor-alpha immunoreactivities in the CA1 pyramidal neurons. Our findings suggest that pretreatment with OJE can protect neurons and attenuate gliosis from transient global cerebral ischemia via increasing expressions of interleukin-4 and -13. The experimental plan of this study was reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) in Kangwon National University (approval No. KW-160802-1) on August 10, 2016.

Hypothermic treatment is known to protect against cardiac arrest (CA) and improve survival rate. However, few studies have evaluated the CA-induced liver damage and the effects of hypothermia on this damage. Therefore, the aim of the present study was to determine possible protective effects of hypothermia on the liver after asphyxial CA. Rats were subjected to a 5-min asphyxial CA followed by return of spontaneous circulation (ROSC). The body temperature was controlled at 37±0.5˚C (normothermia group) or 33±0.5˚C (hypothermia group) for 4 h after ROSC. Livers were examined at 6, 12 h, 1 and 2 days after ROSC. Histopathological examination was performed by H&E staining. Alterations in the expression levels of pro-inflammatory (TNF-α and interleukin IL-2) and anti-inflammatory cytokines (IL-4 and IL-13) were investigated by immunohistochemistry. Sinusoidal dilatation and vacuolization were observed after asphyxial CA by histopathological examination. However, these CA-induced structural alterations were prevented by hypothermia. In immunohistochemical examination, the expression levels of pro-inflammatory cytokines were reduced in the hypothermia group compared with those in the normothermia group while the expression levels of anti-inflammatory cytokines were increased in the hypothermia group compared with those in the normothermia group. In conclusion, hypothermic treatment for 4 h following asphyxial CA in rats inhibited the increase of pro-inflammatory cytokines and stimulated the expression of anti-inflammatory cytokines compared with the normothermic group. The results of the present study suggested that hypothermic treatment after asphyxial CA reduced liver damage via the regulation of inflammation.

Tumor Necrosis Factor-α (TNF-α) is a proinflammatory cytokine implicated in neuronal damage in response to cerebral ischemia. Ischemic preconditioning (IPC) provides neuroprotection against a subsequent severer or longer transient ischemia by ischemic tolerance. Here, we focused on the role of TNF-α in IPC-mediated neuroprotection against neuronal death following a subsequent longer transient cerebral ischemia (TCI). Gerbils used in this study were randomly assigned to eight groups; sham group, TCI operated group, IPC plus (+) sham group, IPC + TCI operated group, sham + etanercept (an inhibitor of TNF-a) group, TCI + etanercept group, IPC + sham + etanercept group, and IPC + TCI + etanercept group. IPC was induced by a 2-min sublethal transient ischemia, which was operated 1 day prior to a longer (5-min) TCI. A significant death of neurons was found in the stratum pyramidale (SP) in the CA1 area (CA1) of the hippocampus 5 days after TCI; however, IPC protected SP neurons from TCI. We found that TNF-α immunoreactivity was significantly increased in CA1 pyramidal neurons in the TCI and IPC + TCI groups compared to the sham group. TNF-R1 expression in CA1 pyramidal neurons of the TCI group was also increased 1 and 2 days after TCI; however, in the IPC + TCI group, TNF-R1 expression was significantly lower than that in the TCI group. On the other hand, we did not detect TNF-R2 immunoreactivity in CA1 pyramidal neurons 1 and 2 days after TCI; meanwhile, in the IPC + TCI group, TNF-R2 expression was significantly increased compared to TNF-R2 expression at 1 and 2 days after TCI. In addition, in this group, TNF-R2 was newly expressed in pericytes, which are important cells in the blood brain barrier, from 1 day after TCI. When we treated etanercept to the IPC + TCI group, IPC-induced neuroprotection was significantly weakened. In brief, this study indicates that IPC confers neuroprotection against TCI by TNF-α signaling through TNF-R2 and suggests that the enhancement of TNF-R2 expression by IPC may be a legitimate strategy for a therapeutic intervention of TCI.

Cardiac arrest (CA) is sudden loss of heart function and abrupt stop in effective blood flow to the body. The patients who initially achieve return of spontaneous circulation (RoSC) after CA have low survival rate. It has been known that multiorgan dysfunctions after RoSC are associated with high morbidity and mortality. Most previous studies have focused on the heart and brain in RoSC after CA. Therefore, the aim of this research was to perform serological, physiological, and histopathology study in the lung and to determine whether or how pulmonary dysfunction is associated with low survival rate after CA. Experimental animals were divided into sham-operated group (n=14 at each point in time), which was not subjected to CA operation, and CA-operated group (n=14 at each point in time), which was subjected to CA. The rats in each group were sacrificed at 6 hours, 12 hours, 24 hours, and 2 days, respectively, after RoSC. Then, pathological changes of the lungs were analyzed by hematoxylin and eosin staining, Western blot and immunohistochemistry for tumor necrosis factor α (TNF-α). The survival rate after CA was decreased with time past. We found that histopathological score and TNF-α immunoreactivity were significantly increased in the lung after CA. These results indicate that inflammation triggered by ischemia-reperfusion damage after CA leads to pulmonary injury/dysfunctions and contributes to low survival rate. In addition, the finding of increase in TNF-α via inflammation in the lung after CA would be able to utilize therapeutic or diagnostic measures in the future.

Background: Glehnia littoralis has been used for traditional Asian medicine, which has diverse therapeutic activities. However, studies regarding neurogenic effects of G. littoralis have not yet been considered. Therefore, in this study, we examined effects of G. littoralis extract on cell proliferation, neuroblast differentiation, and the maturation of newborn neurons in the hippocampus of adult mice. Methods: A total of 39 male ICR mice (12 weeks old) were randomly assigned to vehicle-treated and 100 and 200 mg/kg G. littoralis extract-treated groups (n = 13 in each group). Vehicle and G. littoralis extract were orally administrated for 28 days. To examine neurogenic effects of G. littoralis extract, we performed immunohistochemistry for 5-bromo-2-deoxyuridine (BrdU, an indicator for cell proliferation) and doublecortin (DCX, an immature neuronal marker) and double immunofluorescence staining for BrdU and neuronal nuclear antigen (NeuN, a mature neuronal marker). In addition, we examined expressional changes of brain-derived neurotrophic factor (BDNF) and its major receptor tropomyosin-related kinase B (TrkB) using Western blotting analysis. Results: Treatment with 200 mg/kg, not 100 mg/kg, significantly increased number of BrdU-immunoreactive (+) and DCX+ cells (48.0 ± 3.1 and 72.0 ± 3.8 cells/section, respectively) in the subgranular zone (SGZ) of the dentate gyrus (DG) and BrdU+/NeuN+ cells (17.0 ± 1.5 cells/section) in the granule cell layer as well as in the SGZ. In addition, protein levels of BDNF and TrkB (about 232% and 244% of the vehicle-treated group, respectively) were significantly increased in the DG of the mice treated with 200 mg/kg of G. littoralis extract. Conclusion: G. littoralis extract promots cell proliferation, neuroblast differentiation, and neuronal maturation in the hippocampal DG, and neurogenic effects might be closely related to increases of BDNF and TrkB proteins by G. littoralis extract treatment. Key words: Brain-Derived Neurotrophic Factor; Cell; Proliferation; Glehnia littoralis; Neuroblast Differentiation; Tropomyosin-Related Kinase B

Exercise improves cognitive impairments induced by transient cerebral ischemia, and modulates synaptic adhesion molecules. In this study, we investigated effects of long-term treadmill exercise on cognitive impairments and its relation to changes of synaptic cell adhesion molecule (SynCAM) 1/2/3 in the hippocampus after 5 min of transient cerebral ischemia in aged gerbils. Animals were assigned to sedentary and exercised groups, given treadmill exercise for 4 consecutive weeks from 5 days after transient ischemia, and evaluated cognitive function through passive avoidance test and Morris water maze test. SynCAM 2 protein levels were determined in the hippocampus by western blot. In addition, neuronal and synaptic changes were examined by NeuN immunohistochemistry, and SynCAM 1/2/3 and MAP2 double immunofluorescence, respectively. We found that transient cerebral ischemia led to neuronal death in the CA1 area and dentate gyrus, and impaired -memory function; however, 4 weeks of treadmill exercise improved ischemia-induced memory impairment. In addition, SynCAM 1/2/3 and SynCAM 2 expression in the hippocampus was significantly decreased in the sedentary group after transient cerebral ischemia; however, SynCAM 1/2/3 expressionand and SynCAM 2 protein level was significantly increased in the ischemic group with exercise. These results suggest that long-term treadmill exercise improves memory impairment through the restoration of decreased SynCAM 1/2/3 expression in the hippocampus induced by transient cerebral ischemia in the aged gerbil.

Hydroquinone (HQ), a major metabolite of benzene, exists in many plant-derived food and products. Although many studies have addressed biological properties of HQ including the regulation of immune responses and antioxidant activity, neuroprotective effects of HQ following ischemic insults have not yet been considered. Therefore, in this study, we examined neuroprotective effects of HQ against ischemic damage in the gerbil hippocampal cornu ammonis 1 (CA1) region following 5 min of transient cerebral ischemia. We found that pre- and post-treatments with 50 and 100 mg/kg of HQ protected CA1 pyramidal neurons from ischemic insult. Especially, pre- and post-treatments with 100 mg/kg of HQ showed strong neuroprotective effects against ischemic damage. In addition, pre- and post-treatments with 100 mg/kg of HQ significantly attenuated activations of astrocytes and microglia in the ischemic CA1 region compared to the vehicle-treated-ischemia-operated group. Briefly, these results show that pre- and post-treatments with HQ can protect neurons from transient cerebral ischemia and strongly attenuate ischemia-induced glial activation in the hippocampal CA1 region, and indicate that HQ can be used for both prevention and therapy of ischemic injury.

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.

Melatonin is known to improve cognitive deficits, and its functions have been studied in various disease models, including Alzheimer's disease. In this study, we investigated effects of melatonin on cognition and the cholinergic system of the septum and hippocampus in a mouse model of scopolamine-induced amnesia. Scopolamine (1 mg/kg) and melatonin (10 mg/kg) were administered intraperitoneally to mice for 2 and 4 weeks. The Morris water maze and passive avoidance tests revealed that both treatments of scopolamine significantly impaired spatial learning and memory; however, 2- and 4-week melatonin treatments significantly improved spatial learning and memory. In addition, scopolamine treatments significantly decreased protein levels and immunoreactivities of choline acetyltransferase (ChAT), high-affinity choline transporter (CHT), vesicular acetylcholine transporter (VAChT), and muscarinic acetylcholine receptor M1 (M1R) in the septum and hippocampus. However, the treatments with melatonin resulted in increased ChAT-, CHT-, VAChT-, and M1R-immunoreactivities and their protein levels in the septum and hippocampus. Our results demonstrate that melatonin treatment is effective in improving the cognitive deficits via restoration of the cholinergic system in the septum and hippocampus of a mouse model of scopolamine-induced amnesia.

Calbindin‑D28k (CB), calretinin (CR) and parvalbumin (PV), which regulate cytosolic free Ca2+ concentrations in neurons, are chemically expressed in γ‑aminobutyric acid (GABA)ergic neurons that regulate the degree of glutamatergic excitation and output of projection neurons. The present study investigated age‑associated 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 CB‑immunoreactive neurons were distributed in layers II and III, and age‑associated alterations in their number were different according to the species. CR‑immunoreactive neurons were not abundant in all layers; however, the number of CR‑immunoreactive neurons was the highest in all adult species. Many PV‑immunoreactive 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 PV‑containing 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.

Rufinamide, a voltage-gated sodium channel (VGSC) blocker, is widely used for the clinical treatment of seizures associated with Lennox-Gastaut syndrome. Previous studies have demonstrated that VGSC blockers have neuroprotective properties against ischemic damage following experimental cerebral ischemia. However, protective effects of rufinamide against cerebral ischemic insults have not been addressed. Therefore, in the present study, we firstly examined neuroprotective effects of rufinamide using a gerbil model of transient global cerebral ischemia.Gerbils were established by the occlusion of common carotid arteries for 5 min. The gerbils were divided into vehicle-treated sham-operated group, vehicle-treated ischemia-operated group, 50 and 100 mg/kg rufinamide-treated sham-operated groups, and 50 and 100 mg/kg rufinamide-treated ischemia-operated groups. Rufinamide was administrated intraperitoneally once daily for 3 days before ischemic surgery. To examine neuroprotective effects of rufinamide, we carried out cresyl violet staining, neuronal nuclear antigen immunohistochemistry and Fluoro-Jade B histofluorescence staining. In addition, we examined gliosis using immunohistochemistry for glial fibrillary acidic protein (a marker for astrocytes) and ionized calcium-binding adapter molecule 1 (a marker for microglia).We found that pre-treatment with 100 mg/kg of rufinamide effectively protected pyramidal neurons in the hippocampal cornus ammonis 1 (CA1) area after transient global cerebral ischemia. In addition, pre-treatment with 100 mg/kg of rufinamide significantly attenuated activations of astrocytes and microglia in the ischemic CA1 area.These findings suggest that rufinamide can display neuroprotective effect against cerebral ischemic insults and that its neuroprotective effect may involve the attenuation of ischemia-induced glial activation.

Background Blood–brain barrier (BBB) breakdown and inflammation are critical events in ischemic stroke, contributing to aggravated brain damage. The BBB mainly consists of microvascular endothelial cells sealed by tight junctions to protect the brain from blood-borne substances. Thus, the maintenance of BBB integrity may be a potential target for neuroprotection. Sac-1004, a pseudo-sugar derivative of cholesterol, enhances the endothelial barrier by the stabilization of the cortical actin ring. Results Here, we report on the protective effects of Sac-1004 on cerebral ischemia-reperfusion (I/R) injury. Treatment with Sac-1004 significantly blocked the interleukin-1β-induced monolayer hyperpermeability of human brain microvascular endothelial cells (HBMECs), loss of tight junctions, and formation of actin stress fiber. Sac-1004 suppressed the expression of adhesion molecules, adhesion of U937 cells, and activation of nuclear factor-κB in HBMECs. Using a rat model of transient focal cerebral ischemia, it was shown that Sac-1004 effectively ameliorated neurological deficits and ischemic damage. In addition, Sac-1004 decreased BBB leakage and rescued tight junction-related proteins. Moreover, the staining of CD11b and glial fibrillary acidic protein showed that Sac-1004 inhibited glial activation. Conclusions Taken together, these results demonstrate that Sac-1004 has neuroprotective activities through maintaining BBB integrity, suggesting that it is a great therapeutic candidate for stroke. Electronic supplementary material The online version of this article (doi:10.1186/s12974-017-0897-3) contains supplementary material, which is available to authorized users.

Therapeutic exercise is an integral component of the rehabilitation of patients who have suffered a stroke. The objective of the present study was to use immunohistochemistry to investigate the effects of post-ischemic exercise on neuronal damage or death and gliosis in the aged gerbil hippocampus following transient cerebral ischemia. Aged gerbils (male; age, 22–24 months) underwent ischemia and were subjected to treadmill exercise for 1 or 4 weeks. Neuronal death was detected in the stratum pyramidale of the hippocampal CA1 region and in the polymorphic layer of the dentate gyrus using cresyl violet and Fluoro-Jade B histofluorescence staining. No significant difference in neuronal death was identified following 1 or 4 weeks of post-ischemic treadmill exercise. However, post-ischemic treadmill exercise affected gliosis (the activation of astrocytes and microglia). Glial fibrillary acidic protein-immunoreactive astrocytes and ionized calcium binding adaptor molecule 1-immunoreactive microglia were activated in the CA1 and polymorphic layer of the dentate gyrus of the group without treadmill exercise. Conversely, 4 weeks of treadmill exercise significantly alleviated ischemia-induced astrocyte and microglial activation; however, 1 week of treadmill exercise did not alleviate gliosis. These findings suggest that long-term post-ischemic treadmill exercise following transient cerebral ischemia does not influence neuronal protection; however, it may effectively alleviate transient cerebral ischemia-induced astrocyte and microglial activation in the aged hippocampus.

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.

Quercetin (QE; 3,5,7,3',4'-pentahydroxyflavone), a well-known flavonoid, has been shown to prevent against neurodegenerative disorders and ischemic insults. However, few studies are reported regarding the neuroprotective mechanisms of QE after ischemic insults. Therefore, in this study, we investigated the effects of QE on ischemic injury and the expression of antioxidant enzymes in the hippocampal CA1 region of gerbils subjected to 5 minutes of transient cerebral ischemia. QE was pre-treated once daily for 15 days before ischemia. Pretreatment with QE protected hippocampal CA1 pyramidal neurons from ischemic injury, which was confirmed by neuronal nuclear antigen immunohistochemistry and Fluoro-Jade B histofluorescence staining. In addition, pretreatment with QE significantly increased the expression levels of endogenous antioxidant enzymes Cu/Zn superoxide dismutase, Mn superoxide dismutase, catalase and glutathione peroxidase in the hippocampal CA1 pyramidal neurons of animals with ischemic injury. These findings demonstrate that pretreated QE displayed strong neuroprotective effects against transient cerebral ischemia by increasing the expression of antioxidant enzymes.

Background: Glehnia littoralis, as a traditional herbal medicine to heal various health ailments in EastAsia, 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. Key words: Antioxidant; Glial Activation; Neurotrophic Factor; Neuroprotection; Pyramidal Neurons

Chrysanthemum indicum Linné extract (CIL) is used in herbal medicine in East Asia. In the present study, gerbils were orally pre‑treated with CIL, and changes of antioxidant enzymes including superoxide dismutase (SOD) 1 and SOD2, catalase (CAT) and glutathione peroxidase (GPX) in the hippocampal CA1 region following 5 min of transient cerebral ischemia were investigated and the neuroprotective effect of CIL in the ischemic CA1 region was examined. SOD1, SOD2, CAT and GPX immunoreactivities were observed in the pyramidal cells of the CA1 region and their immunoreactivities were gradually decreased following ischemia‑reperfusion and barely detectable at 5 days post‑ischemia. CIL pre‑treatment significantly increased immunoreactivities of SOD1, CAT and GPX, but not SOD2, in the CA1 pyramidal cells of the sham‑operated animals. In addition, SOD1, SOD2, CAT and GPX immunoreactivities in the CA1 pyramidal cells were significantly higher compared with the ischemia‑operated animals. Furthermore, it was identified that pre‑treatment with CIL protected the CA1 pyramidal cells in the CA1 region using neuronal nuclei immunohistochemistry and Fluoro‑Jade B histofluorescence staining; the protected CA1 pyramidal cells were 67.5% compared with the sham‑operated animals. In conclusion, oral CIL pre‑treatment increased endogenous antioxidant enzymes in CA1 pyramidal cells in the gerbil hippocampus and protected the cells from transient cerebral ischemic insult. This finding suggested that CIL is promising for the prevention of ischemia‑induced neuronal damage.

Oxidative stress plays a vital role in neurodegenerative diseases. Cornus officinalis (CC) has a wide range of pharmacological activities (e.g., antioxidant, neuroprotective, and anti-inflammatory). The present study was undertaken to elucidate the neuroprotective mechanism of CC and fermented CC (FCC) on stress and H2O2-induced oxidative stress damage in rats and SH-SY5Y cells. A dose of 100 mg/kg CC or FCC was orally administered to rats 1 h prior to immobilization 2 h per day for 14 days. CC, especially FCC administration decreased immobility time in forced swim test (FST), effectively alleviated the oxidative stress, and remarkably decreased corticosterone, &beta, endorphin and increased serotonin levels, respectively. In cells, CC and FCC significantly inhibited reactive oxygen species (ROS) generation, lactate dehydrogenase (LDH) release and significantly increased the genes expression of antioxidant and neuronal markers, such as superoxide dismutase (SOD), catalase (CAT), and brain-derived neurotrophic factor (BDNF). Moreover, the pro-apoptotic factor Bax and anti-apoptotic factor Bcl-2 (Bax/Bcl-2) ratio was regulated by CC and FCC pretreatment. Both in rats and cells, CC and FCC downregulated mitogen-activated protein kinase (MAPK) phosphorylation. Taken together, these results demonstrated that CC and particularly FCC ameliorated oxidative stress and may be used on the neuroprotection.

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&ndash, 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 &plusmn, 0.5 &deg, C for 4 h. Normothermia groups were maintained at 37 &plusmn, 0.2 &deg, 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.

Ischemic preconditioning (IPC) provides neuroprotection against subsequent severe ischemic insults by specific mechanisms. We tested the hypothesis that IPC attenuates post-ischemic neuronal death in the gerbil hippocampal CA1 region (CA1) throughout hypoxia inducible factor-1α (HIF-1α) and its associated factors such as vascular endothelial growth factor (VEGF) and nuclear factor-kappa B (NF-κB). Lethal ischemia (LI) without IPC increased expressions of HIF-1α, VEGF, and p-IκB-α (/and translocation of NF-κB p65 into nucleus) in CA1 pyramidal neurons at 12 h and/or 1-day post-LI; thereafter, their expressions were decreased in the CA1 pyramidal neurons with time and newly expressed in non-pyramidal cells (pericytes), and the CA1 pyramidal neurons were dead at 5-day post-LI, and, at this point in time, their immunoreactivities were newly expressed in pericytes. In animals with IPC subjected to LI (IPC/LI)-group), CA1 pyramidal neurons were well protected, and expressions of HIF-1α, VEGF, and p-IκB-α (/and translocation of NF-κB p65 into nucleus) were significantly increased compared to the sham-group and maintained after LI. Whereas, treatment with 2ME2 (a HIF-1α inhibitor) into the IPC/LI-group did not preserve the IPC-mediated increases of HIF-1α, VEGF, and p-IκB-α (/and translocation of NF-κB p65 into nucleus) expressions and did not show IPC-mediated neuroprotection. In brief, IPC protected CA1 pyramidal neurons from LI by upregulation of HIF-1α, VEGF, and p-IκB-α expressions. This study suggests that IPC increases HIF-1α expression in CA1 pyramidal neurons, which enhances VEGF expression and NF-κB activation and that IPC may be a strategy for a therapeutic intervention of cerebral ischemic injury.

Proto-oncogene c‑Fos (c‑Fos) is frequently used to detect a pathogenesis in central nervous system disorders. The present study examined changes in the immunoreactivity of c‑Fos in the paraventricular nucleus of the hypothalamus (PVNH) and paraventricular nucleus of the thalamus (PVNT) following myocardial infarction (MI) in rats. Infarction in the left ventricle was examined by Masson's trichrome staining. Neuronal degeneration was monitored for 56 days after MI using crystal violet and Fluoro‑Jade B histofluorescence staining. Changes in the immunoreactivity of c‑Fos were determined using immunohistochemistry for c‑Fos. The average infarct size of the left ventricle circumference was ~44% subsequent to MI. Neuronal degeneration was not detected in PVNH and PVNT following MI. c‑Fos immunoreactive (+) cells were infrequently observed in the nuclei of the sham‑group. However, the number of c‑Fos+ cells was increased in the nuclei following MI and peaked in the PVNH and PVNT at 3 and 14 days, respectively. The number of c‑Fos+ cells were comparable with the sham group at 56 days after MI. Therefore, MI may induce c‑Fos immunoreactivity in PVNH and PVNT, this increase of c‑Fos expression levels may be associated with the stress that occurs in the brain following MI.

Background. The positive correlation between therapeutic exercise and memory recovery in cases of ischemia has been extensively studied; however, long-term exercise begun after ischemic neuronal death as a chronic neurorestorative strategy has not yet been thoroughly examined. Objective. The purpose of this study is to investigate possible mechanisms by which exercise ameliorates ischemia-induced memory impairment in the aged gerbil hippocampus after transient cerebral ischemia. Methods. Treadmill exercise was begun 5 days after ischemia-reperfusion (I-R) and lasted for 1 or 4 weeks. The animals were sacrificed 31 days after the induction of ischemia. Changes in short-term memory, as well as the hippocampal expression of markers of cell proliferation, neuroblast differentiation, neurogenesis, myelin and microvessel repair, and growth factors were examined by immunohistochemistry and/or western blots. Results. Four weeks of exercise facilitated memory recovery despite neuronal damage in the stratum pyramidale (SP) of the hippocampal CA1 region and in the polymorphic layer (PoL) of the dentate gyrus (DG) after I-R. Long-term exercise enhanced cell proliferation and neuroblast differentiation in a time-dependent manner, and newly generated mature cells were found in the granule cell layer of the DG, but not in the SP of the CA1 region or in the PoL of the DG. In addition, long-term exercise ameliorated ischemia-induced damage of myelin and microvessels, which was correlated with increased BDNF expression in the CA1 region and the DG. Conclusions. These results suggest that long-term treadmill exercise after I-R can restore memory function through replacement of multiple damaged structures in the ischemic aged hippocampus.

Lacosamide is a new antiepileptic drug which is widely used to treat partial-onset seizures. In this study, we examined the neuroprotective effect of lacosamide against transient ischemic damage and expressions of antioxidant enzymes such as Zn-superoxide dismutase (SOD1), Mn-superoxide dismutase (SOD2), catalase (CAT) and glutathione peroxidase (GPX) in the hippocampal cornu ammonis 1 (CA1) region following 5 min of transient global cerebral ischemia in gerbils. We found that pre-treatment with 25 mg/kg lacosamide protected CA1 pyramidal neurons from transient global cerebral ischemic insult using hematoxylin-eosin staining and neuronal nuclear antigen immunohistochemistry. Transient ischemia dramatically changed expressions of SOD1, SOD2 and GPX, not CAT, in the CA1 pyramidal neurons. Lacosamide pre-treatment increased expressions of CAT and GPX, not SOD1 and 2, in the CA1 pyramidal neurons compared with controls, and their expressions induced by lacosamide pre-treatment were maintained after transient cerebral ischemia. In brief, pre-treatment with lacosamide protected hippocampal CA1 pyramidal neurons from ischemic damage induced by transient global cerebral ischemia, and the lacosamide-mediated neuroprotection may be closely related to increases of CAT and GPX expressions by lacosamide pre-treatment.

Catalase (CAT) is an important antioxidant enzyme and is crucial in modulating synaptic plasticity in the brain. In this study, CAT expression as well as neuronal distribution was compared in the hippocampus among young, adult and aged mice and rats. Male ICR mice and Sprague Dawley rats were used at postnatal month (PM) 1, PM 6 and PM 24 as the young, adult and aged groups, respectively (n=14/group). CAT expression was examined by immunohistochemistry and western blot analysis. In addition, neuronal distribution was examined by NeuN immunohistochemistry. In the present study, the mean number of NeuN‑immunoreactive neurons was marginally decreased in mouse and rat hippocampi during aging, although this change was not identified to be significantly different. However, CAT immunoreactivity was significantly increased in pyramidal and granule neurons in the adult mouse and rat hippocampi and was significantly decreased in the aged mouse and rat hippocampi compared with that in the young animals. CAT protein levels in the hippocampus were also lowest in the aged mouse and rat hippocampus. These results indicate that CAT expression is significantly decreased in the hippocampi of aged animals and decreased CAT expression may be closely associated with aging.

Objective(s): The alteration of glucose transporters is closely related with the pathogenesis of brain edema. We compared neuronal damage/death in the hippocampus between adult and young gerbils following transient cerebral ischemia/reperfusion and changes of glucose transporter-1(GLUT-1)-immunoreactive microvessels in their ischemic hippocampal CA1 region. Materials and Methods: Transient cerebral ischemia was developed by 5-min occlusion of both common carotid arteries. Neuronal damage was examined by cresyl violet staining, NeuN immunohistochemistry and Fluoro-Jade B histofluorescence staining and changes in GLUT-1 expression was carried out by immunohistochemistry. Results: About 90% of pyramidal neurons only in the adult CA1 region were damaged after ischemia/reperfusion; in the young, about 53 % of pyramidal neurons were damaged from 7 days after ischemia/reperfusion. The density of GLUT-1-immunoreactive microvessels was significantly higher in the young sham-group than that in the adult sham-group. In the ischemia-operated-groups, the density of GLUT-1-immunoreactive microvessels was significantly decreased in the adult and young at 1 and 4 days post-ischemia, respectively, thereafter, the density of GLUT-1-immunoreactive microvessels was gradually increased in both groups after ischemia/reperfusion. Conclusion: CA1 pyramidal neurons of the young gerbil were damaged much later than that in the adult and that GLUT-1-immunoreactive microvessels were significantly decreased later in the young. These data indicate that GLUT-1 might differently contribute to neuronal damage according to age after ischemic insults.

4-Hydroxy‑3-methoxybenzaldehyde (vanillin) and 4-hydroxybenzyl alcohol (4-HBA) are well‑known phenolic compounds, which possess various therapeutic properties and are widely found in a variety of plants. In the present study, the effects of vanillin and 4‑HBA were first investigated on cell proliferation, as well as neuronal differentiation and integration of granule cells in the dentate gyrus (DG) of adolescent mice using Ki‑67, doublecortin (DCX) immunohistochemistry and 5‑bromo‑2'‑deoxyuridine (BrdU)/feminizing Locus on X 3 (NeuN) double immunofluorescence. In both the vanillin and 4‑HBA groups, the number of Ki‑67+ cells, DCX+ neuroblasts and BrdU+/NeuN+ neurons were significantly increased in the subgranular zone of the DG, as compared with the vehicle group. In addition, the levels of brain‑derived neurotrophic factor (BDNF) and tropomyosin‑related kinase B (TrkB), a BDNF receptor, were significantly increased in the DG in the vanillin and 4‑HBA groups compared with the vehicle group. These results indicated that vanillin and 4‑HBA enhanced cell proliferation, neuroblast differentiation and integration of granule cells in the DG of adolescent mice . These neurogenic effects of vanillin and 4‑HBA may be closely associated with increases in BDNF and TrkB.

Objective(s): In the present study, we investigated the effect of ischemic preconditioning (IPC) on c-myb immunoreactivity as well as neuronal damage/death after a subsequent lethal transient ischemia in gerbils. Materials and Methods: IPC was subjected to a 2 min sublethal ischemia and a lethal transient ischemia was given 5 min transient ischemia. The animals in all of the groups were given recovery times of 1 day, 2 days and 5 days and we examined change in c-myb immunoreactivity as well as neuronal damage/death in the hippocampus induced by a lethal transient ischemia. Results: A lethal transient ischemia induced a significant loss of cells in the stratum pyramidale (SP) of the hippocampal CA1 region at 5 days post-ischemia, and this insult showed that c-myb immunoreactivity in cells of the SP of the CA1 region was significantly decreased at 2 days post-ischemia and disappeared at 5 days post-ischemia. However, IPC effectively prevented the neuronal loss in the SP and showed that c-myb immunoreactivity was constitutively maintained in the SP after a lethal transient ischemia. Conclusion: Our results show that a lethal transient ischemia significantly decreased c-myb immunoreactivity in the SP of the CA1 region and that IPC well preserved c-myb immunoreactivity in the SP of the CA1 region. We suggest that the maintenance of c-myb might be related with IPC-mediated neuroprotection after a lethal ischemic insult.

In this study, we tried to verify the neuroprotective effect of Chrysanthemum indicum Linne (CIL) extract, which has been used as a botanical drug in East Asia, against ischemic damage and to explore the underlying mechanism involving the anti-inflammatory approach. A gerbil was given CIL extract for 7 consecutive days followed by bilateral carotid artery occlusion to make a cerebral ischemia/reperfusion model. Then, we found that CIL extracts protected pyramidal neurons in the hippocampal CA1 region (CA1) from ischemic damage using neuronal nucleus immunohistochemistry and Fluoro-Jade B histofluorescence. Accordingly, interleukin-13 immunoreactivities in the CA1 pyramidal neurons of CIL-pretreated animals were maintained or increased after cerebral ischemia/reperfusion. These findings indicate that the pre-treatment of CIL can attenuate neuronal damage/death in the brain after cerebral ischemia/reperfusion via an anti-inflammatory approach.

Ischemic preconditioning (IPC) is a condition of sublethal transient global ischemia and exhibits neuroprotective effects against subsequent lethal ischemic insult. We, in this study, examined the neuroprotective effects of IPC and its effects on immunoreactive changes of antioxidant enzymes including superoxide dismutase (SOD) 1 and SOD2, catalase (CAT) and glutathione peroxidase (GPX) in the gerbil hippocampal CA1 region after transient forebrain ischemia. Pyramidal neurons of the stratum pyramidale (SP) in the hippocampal CA1 region of animals died 5 days after lethal transient ischemia without IPC (8.6% (ratio of remanent neurons) of the sham-operated group); however, IPC prevented the pyramidal neurons from subsequent lethal ischemic injury (92.3% (ratio of remanent neurons) of the sham-operated group). SOD1, SOD2, CAT and GPX immunoreactivities in the sham-operated animals were easily detected in pyramidal neurons in the stratum pyramidale (SP) of the hippocampal CA1 region, while all of these immunoreactivities were rarely detected in the stratum pyramidale at 5 days after lethal transient ischemia without IPC. Meanwhile, their immunoreactivities in the sham-operated animals with IPC were similar to (SOD1, SOD2 and CAT) or higher (GPX) than those in the sham-operated animals without IPC. Furthermore, their immunoreactivities in the stratum pyramidale of the ischemia-operated animals with IPC were steadily maintained after lethal ischemia/reperfusion. Results of western blot analysis for SOD1, SOD2, CAT and GPX were similar to immunohistochemical data. In conclusion, IPC maintained or increased the expression of antioxidant enzymes in the stratum pyramidale of the hippocampal CA1 region after subsequent lethal transient forebrain ischemia and IPC exhibited neuroprotective effects in the hippocampal CA1 region against transient forebrain ischemia.

To date, hypothermia has focused on improving rates of resuscitation to increase survival in patients sustaining cardiac arrest (CA). Towards this end, the role of body temperature in neuronal damage or death during CA needs to be determined. However, few studies have investigated the effect of regional temperature variation on survival rate and neurological outcomes. In this study, adult male rats (12 week-old) were used under the following four conditions: (i) whole-body normothermia (37 ± 0.5 °C) plus (+) no asphyxial CA, (ii) whole-body normothermia + CA, (iii) whole-body hypothermia (33 ± 0.5 °C)+CA, (iv) body hypothermia/brain normothermia + CA, and (v) brain hypothermia/body normothermia + CA. The survival rate after resuscitation was significantly elevated in groups exposed to whole-body hypothermia plus CA and body hypothermia/brain normothermia plus CA, but not in groups exposed to whole-body normothermia combined with CA and brain hypothermia/body normothermia plus CA. However, the group exposed to hypothermia/brain normothermia combined with CA exhibited higher neuroprotective effects against asphyxial CA injury, i.e. improved neurological deficit and neuronal death in the hippocampus compared with those involving whole-body normothermia combined with CA. In addition, neurological deficit and neuronal death in the group of rat exposed to brain hypothermia/body normothermia and CA were similar to those in the rats subjected to whole-body normothermia and CA. In brief, only brain hypothermia during CA was not associated with effective survival rate, neurological function or neuronal protection compared with those under body (but not brain) hypothermia during CA. Our present study suggests that regional temperature in patients during CA significantly affects the outcomes associated with survival rate and neurological recovery.

Although multiple reports using animal models have confirmed that melatonin appears to promote neuroprotective effects following ischemia/reperfusion-induced brain injury, the relationship between its protective effects and activation of autophagy in Purkinje cells following asphyxial cardiac arrest and cardiopulmonary resuscitation (CA/CPR) remains unclear. Rats used in this study were randomly assigned to 6 groups as follows; vehicle-treated sham operated group, vehicle-treated asphyxial CA/CPR operated group, melatonin-treated sham operated group, melatonin-treated asphyxial CA/CPR operated group, PDOT (a MT2 melatonin receptor antagonist) plus (+) melatonin-treated sham operated group and PDOT+melatonin-treated asphyxial CA/CPR operated group. Melatonin (20 mg/kg, i.p., 4 times before CA and 3 times after CA) treatment significantly improved survival rate and neurological deficit compared with the vehicle-treated asphyxial CA/CPR rats (survival rates ≥40% vs 10%), showing that melatonin treatment exhibited protective effect against asphyxial CA/CPR-induced Purkinje cell death. The protective effect of melatonin against CA/CPR-induced Purkinje cell death paralleled a remarkable attenuation of autophagy-like processes (Beclin-1, Atg7 and LC3), as well as a dramatic reduction in superoxide anion radical (O2·-), intense enhancements of CuZn superoxide dismutase (SOD1) and MnSOD (SOD2) expressions. Furthermore, the protective effect was notably reversed by treatment with PDOT, which is a selective MT2 antagonist. In brief, melatonin conferred neuroprotection against asphyxial CA/CPR-induced Purkinje cell death via inhibiting autophagic activation by reducing expressions of O2·- and increasing expressions of antioxidant enzymes, and suggests that MT2 is involved in neuroprotective effect of melatonin against Purkinje cell death caused by asphyxial CA/CPR.

4-Hydroxy-3-methoxybenzaldehyde (vanillin), contained in a number of species of plant, has been reported to display beneficial effects against brain injuries. In the present study, the impact of vanillin on scopolamine-induced alterations in cognition and the expression of DNA binding protein inhibitor ID-1 (ID1), one of the inhibitors of DNA binding/differentiation proteins that regulate gene transcription, in the mouse hippocampus. Mice were treated with 1 mg/kg scopolamine with or without 40 mg/kg vanillin once daily for 4 weeks. Scopolamine-induced cognitive impairment was observed from 1 week and was deemed to be severe 4 weeks following the administration of scopolamine. However, treatment with vanillin in scopolamine-treated mice markedly attenuated cognitive impairment 4 weeks following treatment with scopolamine. ID1-immunoreactive cells were revealed in the hippocampus of vehicle-treated mice, and were hardly detected 4 weeks following treatment with scopolamine. However, treatment with vanillin in scopolamine-treated mice markedly restored ID1-immunoreactive cells and expression 4 weeks subsequent to treatment. The results of the present study suggested that vanillin may be beneficial for cognitive impairment, by preventing the reduction of ID1 expression which may be associated with cognitive impairment.

We investigated the probability of newly generated neurons that could survive and mature in the ischemic hippocampal CA1 region (CA1) of a gerbil model of transient cerebral ischemia. Neuronal death was shown in the stratum pyramidale (SP) from 4 days post-ischemia, and a significant increase in NeuN-positive ((+) ) neurons was found in the SP at 180 days post-ischemia. 5-Bromo-2-deoxyuridine (BrdU)(+) cells were co-stained with NeuN and glutamic decarboxylase 67 (GAD67). Brain-derived neurotrophic factor (BDNF) immunoreactivity and protein level was shown in nonpyramidal cells from 4 days post-ischemia, and the immunoreactivity was strong at 30 days post-ischemia and not significantly changed until 180 days post-ischemia. Furthermore, TrkB immunoreactivity was co-stained with GAD67 when we examined at 180 days post-ischemia. Myelin basic protein (MBP)(+) nerve fibers were reduced at 4 days post-ischemia and maintained until 60 days post-ischemia, and MBP immunoreactivity and levels were significantly increased at 180 days post-ischemia. In the passive avoidance test, cognitive dysfunction was improved at 180 days post-ischemia. These results suggest that the differentiation of neural progenitor cells into new GABAergic neurons may be promoted via BDNF in the ischemic CA1 and that the neurogenesis may partially mediate the recovery of cognitive impairments via increasing myelinated nerve fibers.

Background: Water dropwort ( Oenanthe javanica ) as a popular traditional medicine in Asia shows various biological properties including antioxidant activity. In this study, we firstly examined the neuroprotective effect of Oenanthe javanica extract (OJE) in the hippocampal cornus ammonis 1 region (CA1 region) of the gerbil subjected to transient cerebral ischemia. Methods: Gerbils were established by the occlusion of common carotid arteries for 5 min. The neuroprotective effect of OJE was estimated by cresyl violet staining. In addition, 4 antioxidants (copper, zinc superoxide dismutase [SOD], manganese SOD, catalase, and glutathione peroxidase) immunoreactivities were investigated by immunohistochemistry. Results: Pyramidal neurons in the CA1 region showed neuronal death at 5 days postischemia; at this point in time, all antioxidants immunoreactivities disappeared in CA1 pyramidal neurons and showed in many nonpyramidal cells. Treatment with 200 mg/kg, not 100 mg/kg, OJE protected CA1 pyramidal neurons from ischemic damage. In addition, 200 mg/kg OJE treatment increased or maintained antioxidants immunoreactivities. Especially, among the antioxidants, glutathione peroxidase immunoreactivity was effectively increased in the CA1 pyramidal neurons of the OJE-treated sham-operated and ischemia-operated groups. Conclusion: Our present results indicate that treatment with OJE can protect neurons from transient ischemic damage and that the neuroprotective effect may be closely associated with increased or maintained intracellular antioxidant enzymes by OJE.

It is well known that neurons in the dentate gyrus (DG) of the hippocampus are resistant to short period of ischemia. Hyperthermia is a proven risk factor for cerebral ischemia and can produce more extensive brain damage related with mortality rates. The aim of this study was to examine the effect of hyperthermic conditioning (H) on neuronal death, gliosis and expressions of SODs as anti-oxidative enzymes in the gerbil DG following 5 min-transient cerebral ischemia. The animals were randomly assigned to 4 groups: 1) (N+sham)-group was given sham-operation with normothermia (N); 2) (N+ischemia)-group was given 5 min-transient ischemia with N; 3) (H+sham)-group was given sham-operation with H; and 4) (H+ischemia)-group was given 5 min-transient cerebral ischemia with H. H (39±0.5°C) was induced by subjecting the animals to a heating pad for 30 min before and during the operation. In the (N+ischemia)-groups, a significant neuronal death was observed in the polymorphic layer (PL) from 1 day after ischemia-reperfusion. In the (H+ischemia)-groups, neuronal death was also observed in the PL from 1day post-ischemia; the degree of the neuronal death was severer than that in the (N+ischemia)-groups. In addition, we examined the gliosis of astrocytes and microglia using anti-glial fibrillary acidic protein (GFAP) and anti- ionized calcium-binding adapter molecule 1 (Iba-1). GFAP(+) and Iba-1(+) glial cells were much more activated in the (H+ischemia)-groups than those in the (N+ischemia)-groups. On the other hand, immunoreactivities and levels of SOD1 rather than SOD2 were significantly lower in the (H+ischemia)-groups than those in the (N+ischemia)-groups. In brief, on the basis of our findings, we suggest that cerebral ischemic insult with hyperthermic conditioning brings up severer neuronal damage and gliosis in the polymorphic layer through reducing SOD1 expression rather than SOD2 expression in the DG.

Brain inflammation has a crucial role in various diseases of the central nervous system. The hippocampus in the mammalian brain exerts an important memory function, which is sensitive to various insults, including inflammation induced by exo/endotoxin stimuli. Tetanus toxin (TeT) is an exotoxin with the capacity for neuronal binding and internalization. The present study investigated changes in inflammatory mediators in the mouse hippocampus proper (CA1-3 regions) and dentate gyrus (DG) after TeT treatment. The experimental mice were intraperitoneally injected with TeT at a low dosage (100 ng/kg), while the control mice were injected with the same volume of saline. At 6, 12 and 24 h after TeT treatment, changes in the hippocampal levels of inflammatory mediators cyclooxygenase-2 (COX-2) and nuclear factor kappa-B (NF-κB/p65) were assessed using immunohistochemical and western blot analysis. In the control group, moderate COX-2 immunoreactivity was observed in the stratum pyramidal (SP) of the CA2-3 region, while almost no expression was identified in the CA1 region and the DG. COX-2 immunoreactivity was increased by TeT in the SP and granule cell layer (GCL) of the DG in a time-dependent manner. At 24 h post-treatment, COX-2 immunoreactivity in the SP of the CA1 region and in the GCL of the DG was high, and COX-2 immunoreactivity in the SP of the CA2/3 region was highest. Furthermore, the present study observed that NF-κB/p65 immunoreactivity was obviously increased in the SP and GCL at 6, 12 and 24 h after TeT treatment. In conclusion, the present study demonstrated that systemic treatment with TeT significantly increased the expression of COX-2 and NF-κB/p65 in the mouse hippo-campus, suggesting that increased COX-2 and NF-κB/65 expression may be associated with inflammation in the brain induced by exotoxins.

Lacosamide, which is a novel antiepileptic drug, has been reported to exert various additional therapeutic effects. The present study investigated the neuroprotective effects of lacosamide against transient cerebral ischemia-induced neuronal cell damage in the hippocampal cornu ammonis (CA)-1 region of a gerbil model. Neuronal Nuclei immunohistochemistry demonstrated that pre- and post-surgical treatment (5 min ischemia) with 25 mg/kg lacosamide protected CA1 pyramidal neurons in the lacosamide-treated-ischemia-operated group from ischemic injury 5 days post-ischemia, as compared with gerbils in the vehicle-treated-ischemia-operated group. Furthermore, treatment with 25 mg/kg lacosamide markedly attenuated the activation of astrocytes and microglia in the ischemic CA1 region at 5 days post-ischemia. The results of the present study suggested that pre- and post-surgical treatment of the gerbils with lacosamide was able to protect against transient cerebral ischemic injury-induced CA1 pyramidal neuronal cell death in the hippocampus. In addition, the neuroprotective effects of lacosamide may be associated with decreased activation of glial cells in the ischemic CA1 region.

Background: Oenanthe javanica ( O. javanica ) has been known to have high antioxidant properties via scavenging reactive oxygen species. We examined the effect of O. javanica extract (OJE) on antioxidant enzymes in the rat liver. Methods: We examined the effect of the OJE on copper, zinc-superoxide dismutase (SOD1), manganese superoxide dismutase (SOD2), catalase (CAT), and glutathione peroxidase (GPx) in the rat liver using immunohistochemistry and western blot analysis. Sprague-Dawley rats were randomly assigned to three groups; (1) normal diet fed group (normal-group), (2) diet containing ascorbic acid (AA)-fed group (AA-group) as a positive control, (3) diet containing OJE-fed group (OJE-group). Results: In this study, no histopathological finding in the rat liver was found in all the experimental groups. Numbers of SOD1, SOD2, CAT, and GPx immunoreactive cells and their protein levels were significantly increased in the AA-fed group compared with those in the normal-group. On the other hand, in the OJE-group, numbers of SOD1, SOD2, CAT, and GPx immunoreactive cells in the liver were significantly increased by about 190%, 478%, 685%, and 346%, respectively, compared with those in the AA-group. In addition, protein levels of SOD1, SOD2, CAT, and GPx in the OJE-group were also significantly much higher than those in the AA-group. Conclusion: OJE significantly increased expressions of SOD1 and SOD2, CAT, and GPx in the liver cells of the rat, and these suggests that significant enhancements of endogenous enzymatic antioxidants by OJE might be a legitimate strategy for decreasing oxidative stresses in the liver.

The thioredoxin (Trx) and peroxiredoxin (Prx) redox system is associated with neuronal damage and neuroprotective effects via the regulation of oxidative stress in brain ischemia. In the present study, ischemia-induced changes in the protein expression levels of Trx2 and Prx3 in the stratum pyramidale (SP) of the hippocampal CA1 region were investigated in adult and aged gerbils, subjected to 5 min of transient global cerebral ischemia, using immunohistochemistry and western blot analysis. In the adult ischemia-group, minimal Trx2 immunoreactivity was detected in the SP 2 days after ischemia-reperfusion. In the aged animals, the Trx2 immunoreactivity in the sham-group was marginally lower compared with that in the adult sham-group. In the aged ischemia-group, Trx2 immunoreactivity in the SP was significantly higher 1, 2 and 4 days post-ischemia, compared with that in the adult ischemia-group and, in the 5 days post-ischemia group, Trx2 immunoreactivity was significantly decreased in the SP. Prx3 immunoreactivity in the SP of the adult ischemia-group was significantly decreased from 4 days after ischemia-reperfusion. In the aged animals, Prx3 immunoreactivity in the sham-group was also marginally lower compared with that in the adult sham-group. Prx3 immunoreactivity in the aged ischemia-group was also significantly higher 1, 2 and 4 days post-ischemia, compared with the adult ischemia-group; however, the Prx3 immunoreactivity was significantly decreased 5 days post-ischemia. The western blot analyses revealed that the pattern of changes in the protein levels of Trx2 and Prx3 in the adult and aged hippocampal CA1 region following ischemic damage were similar to the results obtained in the immunohistochemical data. These findings indicated that cerebral ischemia lead to different protein expression levels of Trx2 and Prx3 in the hippocampal CA1 region between adult and aged gerbils, and these differences may be associated with more delayed neuronal death in the aged gerbil hippocampus following transient global cerebral ischemia.

Pyramidal neurons in region I of hippocampus proper (CA1) are particularly vulnerable to excitotoxic processes following transient forebrain ischemia. Kynurenic acid (KYNA) is a small molecule derived from tryptophan when this amino acid is metabolized through the kynurenine pathway. In the present study, we examined the effects of ischemic preconditioning (IPC) on the immunoreactivity and protein levels of KYNA following 5 min of transient forebrain ischemia in gerbils. The animals were randomly assigned to 4 groups (sham-operated group, ischemia-operated group, IPC + sham-operated group and IPC + ischemia-operated group). IPC was induced by subjecting the gerbils to 2 min of ischemia followed by 1 day of recovery. In the ischemia-operated group, we observed a significant loss of pyramidal neurons in the CA1 stratum pyramidale (SP) at 5 days post-ischemia; however, in the IPC + ischemia-operated group, the pyramidal neurons were well protected. KYNA immunoreactivity in the SP of the ischemia-operated group was significantly altered following ischemia-reperfusion and was very low 5 days following ischemia-reperfusion. In the IPC + ischemia-operated group, however, KYNA immunoreactivity was constitutively detected in the SP of the CA1 region after the ischemic insult. We also found that the alteration pattern of the KYNA protein level in the CA1 region following ischemia was generally similar to the immunohistochemical changes observed. In brief, our findings demonstrated that IPC maintained and even increased KYNA immunoreactivity in the SP of the CA1 region following ischemia-reperfusion. The data from the present study thus indicate that the enhancement of KYNA expression by IPC may be necessary for neuronal survival following transient ischemic injury.