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.

Neuronal injury and tumor necrosis factor-alpha immunoreactivity in the rat hippocampus in the early period of asphyxia-induced cardiac arrest under normothermia

Low survival rate occurs in patients who initially experience a spontaneous return of circulation after cardiac arrest（CA）. In this study, we induced asphyxial CA in adult male Sprague-Daley rats, maintained their body temperature at 37 ± 0.5°C, and then observed the survival rate during the post-resuscitation phase. We examined neuronal damage in the hippocampus using cresyl violet（CV） and Fluore-Jade B（F-J B） staining, and pro-inflammatory response using ionized calcium-binding adapter molecule 1（Iba-1）, glial fibrillary acidic protein（GFAP）, and tumor necrosis factor-alpha（TNF-α） immunohistochemistry in the hippocampus after asphyxial CA in rats under normothermia. Our results show that the survival rate decreased gradually post-CA（about 63% at 6 hours, 37% at 1 day, and 8% at 2 days post-CA）. Rats were sacrificed at these points in time post-CA, and no neuronal damage was found in the hippocampus until 1 day post-CA. However, some neurons in the stratum pyramidale of the CA region in the hippocampus were dead 2 days post-CA. Iba-1 immunoreactive microglia in the CA1 region did not change until 1 day postCA, and they were activated（enlarged cell bodies with short and thicken processes） in all layers 2 days postCA. Meanwhile, GFAP-immunoreactive astrocytes did not change significantly until 2 days post-CA. TNF-α immunoreactivity decreased significantly in neurons of the stratum pyramidale in the CA1 region 6 hours post-CA, decreased gradually until 1 day post-CA, and increased significantly again 2 days post-CA. These findings suggest that low survival rate of normothermic rats in the early period of asphyxia-induced CA is related to increased TNF-α immunoreactivity, but not to neuronal damage in the hippocampal CA1 region.

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, Neu N 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.

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.

Effect of ischemic preconditioning on antioxidant status in the gerbil hippocampal CA1 region after transient forebrain ischemia

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.

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.

Effect of Oenanthe Javanica Extract on Antioxidant Enzyme in the Rat Liver

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 SOD 1,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 SOD 1 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.

Oenanthe javanica extract increases immunoreactivities of antioxidant enzymes in the rat kidney

Background Oenanthe javanica is an aquatic perennial herb originated from East Asia.Nowadays,the effects of Oenanthe javanica have been proven in various disease models.Studies regarding the antioxidant effect of Oenanthe javanica in the kidney are still unclear.Methods This study was therefore performed to investigate the effect of the Oenanthe javanica extract （OJE） in the rat kidney using immunohistochemistry for antioxidant enzymes,copper,zinc-superoxide dismutase （SOD1）,manganese superoxide dismutase （SOD2）,catalase （CAT） and glutathione peroxidase （GPx）.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）.AA and OJE were supplied during 28 days.Results The side-effects were not observed in all the groups.Immunoreactivities of SOD1,SOD2,CAT and GPx were easily detected in the distal tubules of the kidney,and their immunoreactivities in the AA-and OJE-groups were increased to about 1.4-1.5 times and 2 times,respectively,compared with those in the normal-group.Conclusion OJE significantly increased expressions of SOD1 ＆ 2,CAT and GPx immunoreactivities in the distal tubules of the rat kidney,and this finding suggests that significant enhancements of endogenous enzymatic antioxidants by OJE treatment may be a legitimate strategy for decreasing oxidative stresses in the kidney.

Oenanthe Javanica Extract Protects Against Experimentally Induced Ischemic Neuronal Damage via its Antioxidant Effects

Background： Water dropwort （Oenanthejavanica） as a popular traditional medicine in Asia shows various biological properties including antioxidant activity. In this study, we firstly examined the neuroprotective effect of Oenanthejavanica extract （OJE） in the hippocampal comus ammonis 1 region （CA 1 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 100 mg/kg, OJE protected CA 1 pyramidal neurons from ischemic damage in many nonpyramidal cells. Treatment with 200 mg/kg, not In addition, 200 mg/kg OJE treatment increased or maintained antioxidants immunoreactivities. Especially, among the antioxidants, glutathione peroxidase immunoreactivity was effectively increased in the CA 1 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.