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

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

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-1β (IL-1β) 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-1β. 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

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-1β, 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

Delayed hippocampal neuronal death in young gerbil following transient global cerebral ischemia is related to higher and longer-term expression of p63 in the ischemic hippocampus

The tumor suppressor p63 is one of p53 family members and plays a vital role as a regulator of neuronal apoptosis in the development of the nervous system. However, the role of p63 in mature neuronal death has not been addressed yet. In this study, we first compared ischemia-induced effects on p63 expression in the hippocampal regions (CA1- 3) between the young and adult gerbils subjected to 5 minutes of transient global cerebral ischemia. Neuronal death in the hippocampal CA1 region of young gerbils was significantly slow compared with that in the adult gerbils after transient global cerebral ischemia. p63 immunoreactivity in the hippocampal CA1 pyramidal neurons in the sham-operated young group was significantly low compared with that in the sham-operated adult group. p63 immunoreactivity was apparently changed in ischemic hippocampal CA1 pyramidal neurons in both ischemia-operated young and adult groups. In the ischemia-operated adult groups, p63 immunoreactivity in the hippocampal CA1 pyramidal neurons was significantly decreased at 4 days post-ischemia; however, p63 immunoreactivity in the ischemia-operated young group was significantly higher than that in the ischemia-operated adult group. At 7 days post-ischemia, p63 immunoreactivity was decreased in the hippocampal CA1 pyramidal neurons in both ischemia-operated young and adult groups. Change patterns of p63 level in the hippocampal CA1 region of adult and young gerbils after ischemic damage were similar to those observed in the immunohistochemical results. These findings indicate that higher and longer-term expression of p63 in the hippocampal CA1 region of the young gerbils after ischemia/reperfusion may be related to more delayed neuronal death compared to that in the adults

Neuroprotection of Chrysanthemum indicum Linne against cerebral ischemia/reperfusion injury by anti-inflammatory effect in gerbils

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

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-1β (IL-1β, 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)

Monocarboxylate transporter 4 plays a significant role in the neuroprotective mechanism of ischemic preconditioning in transient cerebral ischemia

Monocarboxylate transporters (MCTs), which carry monocarboxylates such as lactate across biological membranes, have been associated with cerebral ischemia/reperfusion process. In this study, we studied the effect of ischemic preconditioning (IPC) on MCT4 immunoreactivity after 5 minutes of transient cerebral ischemia in the gerbil. Animals were randomly designated to four groups (sham-operated group, ischemia only group, IPC + sham-operated group and IPC + ischemia group). A serious loss of neuron was found in the stratum pyramidale of the hippocampal CA1 region (CA1), not CA2/3, of the ischemia-only group at 5 days post-ischemia; however, in the IPC + ischemia groups, neurons in the stratum pyramidale of the CA1 were well protected. Weak MCT4 immunoreactivity was found in the stratum pyramidale of the CA1 in the sham-operated group. MCT4 immunoreactivity in the stratum pyramidale began to decrease at 2 days post-ischemia and was hardly detected at 5 days post-ischemia; at this time point, MCT4 immunoreactivity was newly expressed in astrocytes. In the IPC + sham-operated group, MCT4 immunoreactivity in the stratum pyramidale of the CA1 was increased compared with the sham-operated group, and, in the IPC + ischemia group, MCT4 immunoreactivity was also increased in the stratum pyramidale compared with the ischemia only group. Briefly, present findings show that IPC apparently protected CA1 pyramidal neurons and increased or maintained MCT4 expression in the stratum pyramidale of the CA1 after transient cerebral ischemia. Our findings suggest that MCT4 appears to play a significant role in the neuroprotective mechanism of IPC in the gerbil with transient cerebral ischemia

Activation of immediate-early response gene c-Fos protein in the rat paralimbic cortices after myocardial infarction

c-Fos is a good biological marker for detecting the pathogenesis of central nervous system disorders. Few studies are reported on the change in myocardial infarction-induced c-Fos expression in the paralimbic regions. Thus, in this study, we investigated the changes in c-Fos expression in the rat cingulate and piriform cortices after myocardial infarction. Neuronal degeneration in cingulate and piriform cortices after myocardial infarction was detected using cresyl violet staining, NeuN immunohistochemistry and Fluoro-Jade B histofluorescence staining. c-Fos-immunoreactive cells were observed in cingulate and piriform cortices at 3 days after myocardial infarction and peaked at 7 and 14 days after myocardial infarction. But they were hardly observed at 56 days after myocardial infarction. The chronological change of c-Fos expression determined by western blot analysis was basically the same as that of c-Fos immunoreactivity. These results indicate that myocardial infarction can cause the chronological change of immediate-early response gene c-Fos protein expression, which might be associated with the neural activity induced by myocardial infarction

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