Bich Na Shin

Neuronal damage in hippocampal subregions induced by various durations of transient cerebral ischemia in gerbils using Fluoro-Jade B histofluorescence

Although there are many studies on ischemic brain damage in the gerbil, which is a good model of transient cerebral ischemia, studies on neuronal damage according to the duration of ischemia-reperfusion (I-R) time are limited. We carried out neuronal damage in the gerbil hippocampus after various durations of I-R (5, 10, 15 and 20 min) using Fluoro-Jade B (F-J B, a maker for neuronal degeneration) histofluorescence as well as cresyl violet (CV) staining. The changes of CV positive ((+)) neurons were well detected in the hippocampal CA1 region, not in the other regions. F-J B histofluorescence staining showed apparent neuronal damage in all the hippocampal subregions. In the CA1, most of the pyramidal neurons of the stratum pyramidale (SP) were stained with F-J B (about 100/mm(2) in a section), and F-J B(+) neurons in the other ischemia-groups were not changed. In the CA2, a few F-J B(+) neurons were detected in the SP of the 5 min ischemia-group, and F-J B(+) neurons were gradually increased with the longer time of ischemia: in the 20 min ischemia-group, the mean number of F-J B(+) neurons was about 85/mm(2) in a section. In the CA3, some F-J B(+) neurons were observed only in the SP of the 20 min ischemia-group. In the dentate gyrus, some F-J B positive neurons were detected only in the polymorphic layer (PL) of the 5 min ischemia-group, and the number of F-J B(+) neurons were gradually increased with the longer ischemic time. Our findings indicate that F-J B histofluorescence showed a very high quality of neuronal damage in all the hippocampal subregions.

Neuronal Damage Using Fluoro-Jade B Histofluorescence and Gliosis in the Striatum After Various Durations of Transient Cerebral Ischemia in Gerbils

Ischemic damage occurs well in vulnerable regions of the brain, including the hippocampus and striatum. In the present study, we examined neuronal damage/death and glial changes in the striatum 4xa0days after 5, 10, 15 and 20xa0min of transient cerebral ischemia using the gerbil. Spontaneous motor activity was increased with the duration time of ischemia–reperfusion (I-R). To examine neuronal damage, we used Fluoro-Jade B (F-J B, a marker for neuronal degeneration) histofluorescence staining. F-J B positive cells were detected only in the 20xa0min ischemia-group, not in the other groups. In addition, we examined gliosis of astrocytes and microglia using anti-glial fibrillary acidic protein (GFAP) and anti- ionized calcium-binding adapter molecule 1 (Iba-1), respectively. In the 5xa0min ischemia-group, GFAP-immunoreactive astrocytes were distinctively increased in number, and the immunoreactivity was stronger than that in the sham-group. In the 10, 15 and 20xa0min ischemia-groups, GFAP-immunoreactivity was more increased with the duration of I-R. On the other hand, the immunoreactivity and the number of Iba-1-immunoreactive microglia were distinctively increased in the 5 and 10xa0min ischemia-groups. In the 15xa0min ischemia-group, cell bodies of microglia were largest, and the immunoreactivity was highest; however, in the 20xa0min ischemia-group, the immunoreactivity was low compared to the 15xa0min ischemia-group. The results of western blotting for GFAP and Iba-1 were similar to the immunohistochemical data. In brief, these findings showed that neuronal death could be detected only in the 20xa0min ischemia-group 4xa0days after I-R, and the change pattern of astrocytes and microglia were apparently different according to the duration time of I-R.

Melatonin Improves Cognitive Deficits via Restoration of Cholinergic Dysfunction in a Mouse Model of Scopolamine-Induced Amnesia

Melatonin is known to improve cognitive deficits, and its functions have been studied in various disease models, including Alzheimers 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.

Chronological changes in inflammatory cytokines immunoreactivities in the mouse hippocampus after systemic administration of high dosage of tetanus toxin.

Tetanus toxin (TeT) is an exotoxin and has a capacity for neuronal binding and internalization. In the present study, we compared changes in the immunoreactivities and protein levels of interleukin (IL-) 2 as a pro-inflammatory cytokine and IL-4 as an anti-inflammatory cytokine in the hippocampus proper (HP) and dentate gyrus (DG) after systemic treatment of 10 or 100xa0ng/kg TeT into mice. In this study, we could not find any neuronal damage or loss in any subregions of the hippocampus after TeT treatment. In the control groups, strong IL-2 immunoreactivity was shown in the stratum pyramidal (SP) of the HP and in the granule cell layer (GCL) of the DG. At 6xa0h post-treatment, IL-2 immunoreactivity was hardly detected in the SP and GCL; however, strong IL-2 immunoreactivity was shown in the stratum oriens of the HP in both the groups. Thereafter, intermediate IL-2 immunoreactivity was shown in the SP and GCL. On the other hand, intermediate IL-4 immunoreactivity was detected in the SP and GCL of the control groups. At 6xa0h post-treatment, IL-4 immunoreactivity in the SP and GCL was apparently increased. Thereafter, IL-4 immunoreactivity was lower than that at 6xa0h post-treatment. In brief, IL-2 and 4 immunoreactivities were easily detected in SP and GCL in the controls and dramatically decreased and increased at 6xa0h post-treatment, respectively.

Effects of chronic scopolamine treatment on cognitive impairment and neurofilament expression in the mouse hippocampus

Neurofilaments (NFs) including neurofilament-200 kDa (NF-H), neurofilament-165 kDa (NF-M) and neurofilament-68 kDa (NF-L) are major protein constituents of the brain, and serve important roles in the regulation of axonal transport. NF alteration is a key feature in the pathogenesis of neurological disorders involving cognitive dysfunction. In the present study, cognitive impairments were investigated, via assessments using the Morris water maze and passive avoidance tests, in mice following chronic systemic treatment with 1 mg/kg scopolamine (SCO) for 4 weeks. SCO-induced cognitive impairments were significantly observed 1 week following the SCO treatment, and these cognitive deficits were maintained for 4 weeks. However, the NF immunoreactivities and levels were altered differently according to the hippocampal subregion following SCO treatment. NF-H immunoreactivity and levels were markedly altered in all hippocampal subregions, and were significantly increased 1 week following the SCO treatment; thereafter, the immunoreactivity and levels significantly decreased with time. NF-M immunoreactivity and levels gradually decreased in the hippocampus and were significantly decreased 4 weeks following SCO treatment. NF-L immunoreactivity and levels gradually decreased in the hippocampus, and were significantly decreased 2 and 4 weeks following SCO treatment. In conclusion, the results of the present study demonstrated that chronic systemic treatment with SCO induced cognitive impairment from 1 week following SCO treatment, and NF expression was diversely altered according to the hippocampal subregion from 1 week following SCO treatment. These results suggest that SCO-induced changes in NF expression may be associated with cognitive impairment.

Early IV-injected human dermis-derived mesenchymal stem cells after transient global cerebral ischemia do not pass through damaged blood-brain barrier

There is lack of researches on effects of intravenously injected mesenchymal stem cells (MSCs) against transient cerebral ischemia (TCI). We investigated the disruption of the neurovascular unit (NVU), which comprises the blood–brain barrier and examined entry of human dermis‐derived MSCs (hDMSCs) into the damaged hippocampal CA1 area in a gerbil model of TCI and their subsequent effects on neuroprotection and cognitive function. Impairments of neurons and blood–brain barrier were examined by immunohistochemistry, electron microscopy, and Evans blue and immunoglobulin G leakage. Neuronal death was observed in pyramidal neurons 5‐day postischemia. NVU were structurally damaged; in particular, astrocyte end‐feet were severely damaged from 2‐day post‐TCI and immunoglobulin G leaked out of the CA1 area 2 days after 5 min of TCI; however, Evans blue extravasation was not observed. On the basis of the results of NVU damages, ischemic gerbils received PKH2‐transfected hDMSCs 3 times at early times (3 hr, 2, and 5 days) after TCI, and fluorescence imaging was used to detect hDMSCs in the tissue. PKH2‐transfected hDMSCs were not found in the CA1 from immediate time to 8 days after injection, although they were detected in the liver. Furthermore, hDMSCs transplantation did not protect CA1 pyramidal neurons and did not improve cognitive impairment. Intravenously transplanted hDMSCs did not migrate to the damaged CA1 area induced by TCI. These findings suggest no neuroprotection and cognitive improvement by intravenous hDMSCs transplantation after 5 min of TCI.

Intermittent fasting increases SOD2 and catalase immunoreactivities in the hippocampus but does not protect from neuronal death following transient ischemia in gerbils

Intermittent fasting has been shown to have neuroprotective effects against transient focal cerebral ischemic insults. However, the effects of intermittent fasting on transient global ischemic insult has not been studied much yet. The present study examined effects of intermittent fasting on endogenous antioxidant enzyme expression levels in the hippocampus and investigated whether the fasting protects neurons 5 days after 5 min of transient global cerebral ischemia. Gerbils were randomly subjected to either ad libitum or alternate-day intermittent fasting for two months and assigned to sham surgery or transient ischemia. Changes of antioxidant enzymes were examined using immunohistochemistry for cytoplasmic superoxide dismutase 1 (SOD1), mitochondrial (SOD2), catalase (CAT), and glutathione peroxidase (GPX). The effects of intermittent fasting on ischemia-induced antioxidant changes, neuronal damage/degeneration and glial activation were examined. The weight of fasting gerbils was not different from that of control gerbils. In controls, SOD1 and GPX immunoreactivities were strong in pyramidal neurons of filed cornu ammonis 1 (CA1). Transient ischemia in controls significantly decreased expressions of SOD1 and GPX in CA1 pyramidal neurons. Intermittent fasting resulted in increased expressions of SOD2 and CAT, not of SOD1 and GPX, in CA1 pyramidal neurons. Nevertheless, CA1 pyramidal neurons were not protected in gerbils subjected to fasting after transient ischemia, and inhibition of glial-cell activation was not observed in the gerbils. In summary, intermittent fasting for two months increased SOD2 and CAT immunoreactivities in hippocampal CA1 pyramidal neurons. However, fasting did not protect the CA1 pyramidal neurons from transient cerebral ischemia. The results of the present study indicate that intermittent fasting may increase certain antioxidants, but not protect neurons from transient global ischemic insult.

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

Selective neuronal death or loss in certain brain regions has been well characterized in animal models of transient global cerebral ischemia. However, selective neuronal death in transient focal cerebral ischemia needs more investigation. Therefore, in this study, we studied selective neuronal death in the striatum (caudate putamen) of rats subjected to 15 or 30xa0min middle cerebral artery occlusion (MCAO). Neuronal death occurred in the dorsolateral field, not in the medial field in 30xa0min, not 15xa0min, MCAO-operated rats 5xa0days after MCAO using neuronal nuclear antigen immunohistochemistry and Fluoro-Jade B histofluorescence staining. In this group, immunoreactivity of glial fibrillary acidic protein in astrocytes was hardly shown in the dorsolateral field, although the immunoreactivity increased in the medial field. In addition, immunoreactivity of ionized calcium binding adapter molecule 1 in microglia was dramatically increased in the dorsolateral, not in the medial, field only in 30xa0min MCAO-operated rats. Briefly, these results show that at least 30xa0min of MCAO can evoke selective neuronal death, astrocytic dysfunction and microglial activation in the dorsolateral field of the rat striatum and suggest that a rat model of 30xa0min MCAO can be used to investigate mechanisms of neuronal death and gliosis following brief transient focal cerebral ischemic events for acute transient ischemic attack.

Effects of long‑term scopolamine treatment on cognitive deficits and calcium binding proteins immunoreactivities in the mouse hippocampus

GABAergic projections terminate on numerous hippocampal interneurons containing calcium binding proteins (CBPs), including calbindin D-28k (CB), calretinin (CR) and parvalbumin (PV). Memory deficits and expression levels of CB, CR, and PV were examined in the hippocampal subregions following systemic scopolamine (Scop; 1 mg/kg) treatment for 4 weeks in mice. Scop treatment induced significant memory deficits from 1 week after Scop treatment. CB, CR and PV immunoreactivities distributions were in hippocampal subregions [CA1 and CA3 regions, and the dentate gyrus (DG)]. CB immunoreactivity (CB+) was gradually decreased in all subregions until 2 weeks after Scop treatment, and CB+ was decreased to the lowest level in all subregions at 3 and 4 weeks. CR+ in the CA1 region was gradually decreased until 2 weeks and hardly observed at 3 and 4 weeks; in the CA3 region, CR+ was not altered in all subregions at any time. In the DG, CR+ was gradually decreased until 2 weeks and lowest at 3 and 4 weeks. PV+ in the CA1 region was not altered at 1 week, and gradually decreased from 2 weeks. In the CA3 region, PV+ did not change in any subregions at any time. In the DG, PV+ was not altered at 1 week, decreased at 2 weeks, and lowest at 3 and 4 weeks. In brief, Scop significantly decreased CBPs expressions in the hippocampus ≥3 weeks after the treatment although memory deficits had developed at 1 week. Therefore, it is suggested that Scop (1 mg/kg) must be systemically treated for ≥3 weeks to investigate changes in expression levels of CBPs in the hippocampus.