Neuronal Damage Using Fluoro-Jade B Histofluorescence and Gliosis in the Gerbil Septum Submitted to Various Durations of Cerebral Ischemia

The extent of neuronal damage/death in some brain regions is highly correlated to duration time of transient ischemia. In the present study, we carried out neuronal degeneration/death and glial changes in the septum 4 days after 5, 10, 15, and 20 min of transient cerebral ischemia using gerbils. To examine neuronal damage, Fluoro-Jade B (F-J B, a marker for neuronal degeneration) histofluorescence staining was used. F-J B positive (⁺) cells were detected in the septo-hippocampal nucleus (SHN) of the septum only in the 20 min ischemia-group; the mean number of F-J B⁺ neurons was 14.9 ± 2.5/400 μm² in a section. Gliosis of astrocytes and microglia was examined using anti-glial fibrillary acidic protein (GFAP) and anti-ionized calcium-binding adapter molecule 1 (Iba-1), respectively. In all the ischemia-groups, GFAP- and Iba-1-immunoreactive astrocytes and microglia, respectively, were increased in number, and apparently tended to be increased in their immunoreactivity. Especially, in the 20 min ischemia-group, the number and immunoreactivity of Iba-immunoreactive microglia was highest and strongest in the ischemic SHN 4 days after ischemia–reperfusion. In brief, our findings showed that neuronal damage/death in the SHN occurred and gliosis was apparently increased in the 20 min ischemia-group at 4 days after ischemia–reperfusion.     

Transient Cerebral Ischemia Induces Active Astrocytosis Without Distinct Neuronal Death in the Gerbil Main Olfactory Bulb: A Long-Term Analysis

In the present study, we examined ischemia-induced neuronal and glial changes in the gerbil MOB at various time points during 60 days after 5 min of transient cerebral ischemia. The number of neuronal neuclei-immunoreactive neurons was not changed after ischemia/reperfusion (I/R). Myelin basic protein immunoreaction was well preserved after I/R. Five days after I/R, reactive form of GFAP-immunoreactive astrocytes began to increase in the external plexiform layer and granule cell layer: These reactive astrocytes peaked 10 days after I/R, thereafter, they decreased with time after I/R. Iba-1-immunoreactive microglia were ubiquitously distributed in all layers of the MOB. After I/R, significant changes in their morphology and immunoreactivity were not detected. The results of western blot analyses for GFAP, Iba-1 and MBP were similar to the immunohistochemical data. In addition, 8-hydroxy-2′-deoxyguanosine (a marker for DNA damage) immunoreactivity and SOD1, an antioxidant, protein levels were not changed in the ischemic MOB. These results indicate that neurons in the MOB are resistant to ischemic insult, showing that astrocytes are activated late in the ischemic MOB.     

Comparison of Alpha-Synuclein Immunoreactivity in the Hippocampus Between the Adult and Aged Beagle Dogs

Alpha-synuclein (α-syn), as a neuroprotein, is expressed in neural tissue, and it is related to a synaptic transmission and neuronal plasticity. In this study, we compared the distribution and immunoreactivity of α-syn and related gliosis in hippocampus between young adult (2–3 years) and aged (10–12 years) beagle dogs. In both groups, α-syn immunoreactivity was detected in neuropil of all the hippocampal sub-regions, but not in neuronal somata. In the aged hippocampus, α-syn immunoreactivity was apparently increased in mossy fibers compared to that in the adult dog. In addition, α-syn protein level was markedly increased in the aged hippocampus. On the other hand, GFAP and Iba-1 immunoreactivity in astrocytes and microglia, respectively, were increased in all the hippocampal sub-regions of the aged group compared to that in the adult group: especially, their immunoreactivity was apparently increased around mossy fibers. In addition, in this study, we could not find any expression of α-syn in astrocytes and microglia. These results indicate that α-syn immunoreactivity apparently increases in the aged hippocampus and that GFAP and Iba-1 immunoreactivity are also apparently increased at the regions with increased α-syn immunoreactivity. This increase in α-syn expression might be a feature of normal aging.     

Changes in Corticosteroid Hormone Receptors in the Ischemic Gerbil Hippocampal CA1 Region Following Repeated Restraint Stress

Restraint stress produces physiological changes including suppression of long-term potentiation in the brain. We observed the effects of repeated stress on ischemic damage associated with corticosteroid hormone receptors in gerbils. Animals were placed into restrainers for 5 h (between 09:30 h and 14:30 h) for 21 consecutive days prior to induction of transient cerebral ischemia. The animals were divided into 4 groups; (1) sham-operated-control-group (sham-group), (2) ischemia-operated-control-group (ischemia-group), (3) sham-operated-stress-group (stressed-sham-group), and (4) ischemia-operated-stress-group (stressed-ischemia-group). We found that serum corticosterone level in the ischemia-group was highest (374% of the sham-group) 12 h after ischemia/reperfusion and its level in the stressed-ischemia-group was significantly lower than the ischemia-group. Locomotor activity in the ischemia-group was significantly increased (295% of the sham-group) at 1 day post-ischemia; however, the locomotor activity in the stressed-ischemia-group was less increased compared to the ischemia-group. Cresyl violet positive (CV⁺) cells were significantly decreased in the stratum pyramidale (SP) of the hippocampal CA1 region (CA1) of the 4 days post-ischemia-group, while 79.4% of CV⁺ cells were detected in the CA1 of the stressed-ischemia-group. Also, a few NeuN (neuron-specific soluble nuclear antigen)⁺ cells were detected in the SP of the 4 days post-ischemia-group; however, in the 4 days stressed-post-ischemia-group, 77.2% of NeuN⁺ neurons were found in the SP. Glial fibrillary acidic protein⁺ astrocytes in the CA1 in the stressed-ischemia-groups were similar to those in the ischemia-groups; however, ionized calcium-binding adapter molecule 1⁺ microglia in the stressed-ischemia-groups were less activated compared to the ischemia-groups. Mineralocorticoid receptor (MCR) and glucocorticoid receptor (GR) immunoreactivity in the SP of the stressed-ischemia-group were higher than the ischemia-group; at 4 days post-ischemia, MCR and GR immunoreactivity were expressed in non-pyramidal cells. In brief, our results indicate that repeated restraint stress significantly increase levels of corticosteroid hormone receptors and attenuates neuronal damage in the ischemic hippocampal CA1 region.     

Comparison of the Immunoreactivity of Trx2/Prx3 Redox System in the Hippocampal CA1 Region Between the Young and Adult Gerbil Induced by Transient Cerebral Ischemia

In the present study, we compared the immunoreactivities and levels of Trx/prx redox system, thioredoxin 2 (Trx2), thioredoxin reductase 2 (TrxR2) and peroxiredoxin 3 (Prx3), as well as neuronal death in the hippocampal CA1 region between the adult and young gerbil after 5 min of transient cerebral ischemia. At 4 days post-ischemia, pyramidal neurons (about 90%) in the adult stratum pyramidale of the CA1 region showed "delayed neuronal death (DND)"; however, at this time point, few pyramidal neurons showed DND in the young stratum pyramidale. At 7 days post-ischemia, about 56% of pyramidal neurons showed DND in the young stratum pyramidale. The immunoreactivities of all the antioxidants in the young sham-group were similar to those in the adult sham-group. At 4 days post-ischemia, the immunoreactivity of TrxR2, not Trx2 and Prx3 in the adult ischemia-group was dramatically decreased in CA1 pyramidal neurons. At this time point, the immunoreactivities of all the antioxidants in the young ischemia-group were apparently increased compared to the adult ischemia-group. From 7 days pots-ischemia, non-pyramidal cells showed the immunoreactivities of all the antioxidants in the ischemic CA1 region; however, in the young ischemia-groups, the immunoreactivities were much lower than those in the adult ischemia-groups. In brief, our results showed that the immunoreactivities of Trx2, TrxR2 and Prx3 were dramatically increased in CA1 pyramidal neurons of the young ischemia-groups at 4 days post-ischemia compared to those in the adult ischemia-groups induced by transient cerebral ischemia.     

Comparison of Glial Activation in the Hippocampal CA1 Region Between The Young and Adult Gerbils After Transient Cerebral Ischemia

It has been reported that young animals are less vulnerable to brain ischemia. In the present study, we compared gliosis in the hippocampal CA1 region of the young gerbil with those in the adult gerbil induced by 5?min of transient cerebral ischemia by immunohistochemistry and western blot for glial cells. We used male gerbils of postnatal month 1 (PM 1) as the young and PM 6 as the adult. Neuronal death in CA1 pyramidal neurons in the adult gerbil occurred at 4?days posti-schemia; the neuronal death in the young gerbil occurred at 7?days post-ischemia. The findings of glial changes in the young gerbil after ischemic damage were distinctively different from those in the adult gerbil. Glial fibrillary acidic protein-immunoreactive astrocytes, ionized calcium-binding adapter molecule (Iba-1), and isolectin B4-immunoreactive microglia in the ischemic CA1 region were activated much later in the young gerbil than in the adult gerbil. In brief, very less gliosis occurred in the hippocampal CA1 region of the young gerbil than in the adult gerbil after transient cerebral ischemia.     

Gliosis in the Mice Hippocampus Without Neuronal Death After Systemic Administration of High Dosage of Tetanus Toxin

Tetanus toxin (TeT), an exotoxin, has been studied to cause tetanus in mammalian brains, and it can block the release of some neurotransmitters and affect seizure propagation. In the present study, we investigated neuronal damage/death and glial changes in the mouse hippocampus after systemic administration (intraperitoneal injection) of TeT 10 and 100 ng/kg. In both the 10 and 100 ng/kg TeT-treated groups, no neuronal death occurred in any subregions of the mouse hippocampus until 24 h post-treatment; however, there were changes in glia in the hippocampus depending on time course and dosage. The morphology of GFAP-immunoreactive astrocytes and Iba-1-immunoreactive microglia was apparently changed in the 100 ng/kg TeT treated-group compared to the 10 ng/kg TeT treated-group. In the 100 ng/kg TeT treated-group, they were increased in size and their immunoreactivity was distinctively increased from 12 h post-treatment. We also found that their protein levels were increased in the hippocampus at 12 h post-treatment of 100 ng/kg TeT. In conclusion, these results indicate that the systemic administration of 100 ng/kg TeT induced a distinctive microglia changes in the mouse hippocampus without any neuronal death/damage.     

Extract from Terminalia chebula Seeds Protect Against Experimental Ischemic Neuronal Damage Via Maintaining SODs and BDNF Levels

The fruit of Terminalia chebula Retz has been used as a traditional medicine in Asia and contains tannic acid, chebulagic acid, chebulinic acid and corilagin. Extract from T. chebula seeds (TCE) has various biological functions. We observed the neuroprotective effects of TCE against ischemic damage in the hippocampal C1 region (CA1) of the gerbil that had received oral administrations of TCE (100?mg/kg) once a day for 7?days before the induction of transient cerebral ischemia. In the TCE-treated ischemia group, neuronal neuclei (a marker for neurons)-positive neurons were distinctively abundant (62% of the sham group) in the CA1 4?days after ischemia-reperfusion (I-R) compared to those (12.2% of the sham group) in the vehicle-treated ischemia group. Four days after I-R TCE treatment markedly decreased the activation of astrocytes and microglia in the ischemic CA1 compared with the vehicle-treated ischemia group. In addition, immunoreactivities of Cu, Zn-superoxide dismutase (SOD1), Mn-superoxide dismutase (SOD2) and brain-derived neurotrophic factor (BDNF) in the CA1 of the TCE-treated ischemia group were much higher than those in the vehicle-ischemia group 4?days after I-R. Protein levels of SOD1, SOD2 and BDNF in the TCE-treated ischemia group were also much higher than those in the vehicle-ischemia group 4?days after I-R. These results indicate that the repeated supplement of TCE protected neurons from ischemic damage induced by transient cerebral ischemia by maintaining SODs and BDNF levels as well as decreasing glial activation.     

Characteristic Changes of Astrocyte and Microglia in Rat Striatum Induced by 3-NP and MCAO

Our previous studies had confirmed that both 3-NP and MCAO induced the behavioral defect as well as striatal neuronal injury and loss in experimental rats. This study aimed to examine different response forms of striatal astrocyte and microglia in 3-NP and MCAO rat models. The present results showed that the immunoreaction for GFAP was extremely weak in the lesioned core of striatum, but in the transition zone of 3-NP model and the penumbra zone of MCAO model, GFAP+ cells showed strong hypertrophic and proliferative changes. Statistical analysis for the number, size and integral optical density (IOD) of GFAP+ cells showed significant differences when compared with their controls and compared between the core and the transition zone or the penumbra zone, respectively, but no differences between the 3-NP and MCAO groups. However, Iba-1+ cells showed obvious hypertrophy and proliferation in the injured striatum in the 3-NP and the MCAO models, especially in the transition zone of 3-NP model and the penumbra zone of MCAO model. These Iba-1+ cells displayed two characteristic forms as branching cells with thick processes and amoeboid cells with thin processes. Statistical analysis showed that the number, size and IOD of Iba-1+ cells were significantly increased in the cores and the transition zone of 3-NP group and the penumbra zone of MCAO group than that of the controls, and the immune response of Iba-1 was stronger in the MCAO group than in the 3-NP group. The present results suggested that characteristic responses of astrocyte and microglia in the 3-NP and the MCAO models display their different effects on the pathological process of brain injury.     

Comparison of Glucocorticoid Receptor and Ionized Calcium-Binding Adapter Molecule 1 Immunoreactivity in the Adult and Aged Gerbil Hippocampus Following Repeated Restraint Stress

Stress leads to changes in homeostasis and internal balance of the body and is known to be one of important factors in the development of several diseases. In the present study, we investigated changes in glucocorticoid receptor (GR) and ionized calcium-binding adapter molecule 1 (Iba-1) immunoreactivity in the adult and aged gerbil hippocampus after chronic restraint stress. Serum corticosterone level was much higher in both the stress-groups than the control groups. No neuronal death was found in all hippocampal subregions of the adult and aged gerbil after chronic restraint stress. GR immunoreactivity was decreased in both the adult and aged groups after repeated restraint stress; however, GR immunoreactivity in the adult-stress-group was decreased much more than that in the aged-stress-group. Iba-1 immunoreactive microglia were hypertrophied and activated in the adult group after repeated restraint stress; in the aged-stress-group, there was no any significant change in Iba-1 immunoreactive microglia. In brief, level of GR, not Iba-1, in the hippocampus was much decreased in the adult gerbil compared to the aged gerbil following chronic restraint stress.     

Comparison of Ionized Calcium-binding Adapter Molecule 1 Immunoreactivity of the Hippocampal Dentate Gyrus and CA1 Region in Adult and Aged Dogs

Similarities between age-related changes in the canine and human brain have resulted in the general acceptance of the canine brain as a model of human brain aging. The hippocampus is essentially required for intact cognitive ability and appears to be particularly vulnerable to the aging process. We observed changes in ionized calcium-binding adapter molecule 1 (Iba-1, a microglial marker) immunoreactivity and protein levels in the hippocampal dentate gyrus and CA1 region of adult (2-3 years) and aged (10-12 years) dogs. We also observed the interferon-gamma (IFN-gamma), a pro-inflammatory cytokine, protein levels in these groups. In the dentate gyrus and CA1 region of the adult dog, Iba-1 immunoreactive microglia were well distributed and their processes were highly ramified. However, in the aged dog, the processes of Iba-1 immunoreactive microglia were hypertrophied in the dentate gyrus. Moreover, Iba-1 protein level in the dentate gyrus in the aged dog was higher than in the adult dog. IFN-gamma expression was increased in the dentate gyrus homogenates of aged dogs than adult dogs. In addition, we found that some neurons were positive to Fluoro-Jade B (a marker for neuronal degeneration) in the dentate polymorphic layer, but not in the hippocampal CA1 region in the aged dog. These results suggest that Iba-1 immunoreactive microglia are hypertrophied in the dentate gyrus in the aged dog.     

S100 immunoreactivity is increased in reactive astrocytes of the visual pathways following a mechanical lesion of the rat occipital cortex

After demonstration of the paracrine action of glial neurotrophic factors, gliosis has also been considered to be related to neuronal trophism and plasticity rather than solely a repair event following brain injury. S100 is a Ca2+ binding protein, present mainly in astrocytes, that exerts paracrine trophic effects on several neuronal populations. This study analyses the presence of S100 protein by means of immunohistochemistry combined with stereology in the reactive glial cells of the rat visual pathways following a lesion of the visual cortex. Adult male Wistar rats were submitted to a unilateral aspiration of the occipital cortex or to a sham operation. One week later the rats were killed and their brain processed for immunochemistry. Single antibody immunohistochemistry was performed for the visualization of glial fibrillary acidic protein (GFAP, a marker for astrocytes), OX-42 (a marker for microglia) and S100 protein. Double immunofluorescence procedures were applied for co-localization of the S100/GFAP and S100/OX-42. An optical dissector, point interceptors and rotators were used to quantify the degree of glial activation and the changes in the S100 immunoreactivity. We observed an intense microglial and astroglial reaction in addition to an increased S100 immunoreactivity in the occipital cerebral cortex, geniculate nucleus and hippocampus ipsilateral to the lesion. In the ipsilateral superior colliculus, an intense astroglial activation was accompanied by an up-regulation of S100 immunoreactivity. Double-immunofluoresence revealed an increased S100 immunoreactivity in reactive astrocytes, but not in the reactive microglia. Evidence has therefore been obtained that after mechanical trauma, the astroglial S100 protein participates in the trophism and plasticity of the injured visual pathways.     

The use of sodium sulphide-fixed brain tissue for immunocytochemical staining of activated microglia and reactive astrocytes

The use of sodium sulphide-perfused material for the immunocytochemical demonstration of microglia and astrocytes is described. An intracerebroventricular injection of kainic acid (KA) was used to induce neuronal degeneration and subsequent axonal sprouting in the hippocampus. Animals under deep anaesthesia were killed by perfusion with either 4% paraformaldehyde alone or with 1% sodium sulphide followed by 4% paraformaldehyde solution. Microglial cells were identified with OX-42, a monoclonal antibody towards CR3 complement receptors, and astrocytes with a polyclonal antibody to glial fibrillary acidic protein (GFAP). The present study reveals a marked enhancement in the immunoreactivity of activated microglial cells in sodium sulphide perfused tissues compared to those observed in tissues fixed in paraformaldehyde alone. GFAP immunoreactivity of the astrocytes was not compromised by the use of sodium sulphide. The results clearly show the suitability of sodium sulphide perfused tissues for immunocytochemical procedures and should provide a useful tool for investigation of the role of neuroglial cells in axonal sprouting.     

Time course of nitric oxide synthase activity in neuronal,glial, and endothelial cells of rat striatum following focal cerebral ischemia

Summary 1. The time course of nitric oxide synthase (NOS) activity in neuronal, endothelial, and glial cells in the rat striatum after middle cerebral artery (MCA) occlusion and reperfusion was examined using a histochemical NADPH-diaphorase staining method.2. In sham-operated rats, neuronal cells of the striatum exhibited strong NADPH-diaphorase activities. When rats were subjected to MCA occlusion for 1 hr, neuronal damage, including neurons with positive NADPH-diaphorase activities, appeared in the striatum at 3 hr after and extended to all areas of the striatum 3–4 days after reperfusion.3. NADPH-diaphorase activities in the endothelial cells increased in the damaged part of striatum from 3 hr after, peaked at 1–2 days after MCA occlusion/reperfusion, then gradually decreased.4. In parallel with the development of neuronal damage, some astrocytes and a high proportion of microglia/macrophages located in the perisite and in the center of the damaged striatum, respectively, exhibited a moderate to high level of NADPH-diaphorase activities. Most of these activities disappeared at 4 days after MCA occlusion.5. These findings provided evidence that an inappropriate activation of NOS in endothelial cells and microglia/macrophages, in response to MCA occlusion/reperfusion, is closely associated with initiation and progression of ischemic neuronal injury in the striatum.     

Effect of Transient Cerebral Ischemia on the Expression of Receptor for Advanced Glycation End Products (RAGE) in the Gerbil Hippocampus Proper

The receptor for advanced glycation end products (RAGE) is a multi-ligand receptor of the immunoglobulin superfamily that has been implicated in multiple neuronal and inflammatory stress processes. In this study, we examined changes in RAGE immunoreactivity and its protein levels in the gerbil hippocampus (CA1-3 regions) after 5 min of transient global cerebral ischemia. The ischemic hippocampus was stained with cresyl violet, neuronal nuclei (a neuron-specific soluble nuclear antigen) antibody and Fluoro-Jade B (a marker for neuronal degeneration). 5 days after ischemia–reperfusion, delayed neuronal death occurred in the stratum pyramidale of the CA1 region. RAGE immunoreactivity was not detected in any regions of the CA1-3 regions of the sham-group; the immunoreactivity was markedly increased only in the CA1 region from 3 days after ischemia–reperfusion. On the other hand, RAGE immunoreactivity was newly expressed in astrocytes, not in microglia. Western blot analysis showed that RAGE protein level was highest at 5 days post-ischemia. In brief, both the RAGE immunoreactivity and protein level were distinctively increased in astrocytes in the ischemic CA1 region from 3 days after transient cerebral ischemia. These results indicate that the increase of RAGE expression in astrocytes after ischemia–reperfusion may be related to the ischemia-caused activation of astrocytes in the ischemic CA1 region.     

原代培养星形胶质细胞和小胶质细胞的特性与鉴定

本研究旨在明确原代培养的星形胶质细胞和小胶质细胞不同代次的生长特性,优化高效获取状态一致细胞的技术方法。将新生乳鼠的脑组织进行原代分离培养胶质细胞,通过细胞增殖检测试剂盒（cell counting kit-8,CCK-8）测定混合胶质细胞增殖曲线,使用流式细胞术检测两类细胞比例,并通过免疫荧光染色鉴定两类胶质细胞分型情况。生长曲线显示P0和P1代混合胶质细胞增殖活力最好;通过170 r/min机械振摇30 min可获得97.3%的高纯度小胶质细胞,该纯化方法得到的P0、P1、P2代离子钙接头蛋白-1（ionized calcium-binding adapter molecule 1,Iba-1）阳性小胶质细胞的形态及其M1、M2表型比例无代次差别;通过星形胶质细胞表面抗原-2（astrocyte cell surface antigen-2,ACSA-2）磁珠抗体分选的方法可获得纯度达到95.7%的星形胶质细胞,该纯化方法得到的P0、P1、P2代胶质纤维酸性蛋白（glial fibrillary acidic protein,GFAP）阳性星形胶质细胞的形态及其A1、A2表型比例无代次差别。本研究详述了原代分离培养的小胶质细胞和星形胶质细胞的生长特点,证明了获取两类胶质细胞的最佳代次,优化了获取两类胶质细胞的技术方法,验证了连续培养两代不会影响其功能表型。本结果为研究神经系统炎症相关疾病的分子机制提供了技术支撑。     

Time-course alterations of Toll-like receptor 4 and NF-κB p65, and their co-expression in the gerbil hippocampal CA1 region after transient cerebral ischemia

Innate immune system is very important to modulate the host defense against a large variety of pathogens. Toll-like receptors (TLRs) play a key role in controlling innate immune response. Among TLRs, TLR4 is a specific receptor for lipopolysaccharide and associated with the release of pro-inflammatory cytokines. In the present study, we investigated ischemia-related changes of TLR4 immunoreactivity and its protein level, and nuclear factor κB (NF-κB) p65 immunoreactivity regarding inflammatory responses in the hippocampal CA1 region after 5 min of transient cerebral ischemia to identify the correlation between transient ischemia and inflammation. In the sham-operated group, TLR4 immunoreactivity was easily detected in pyramidal neurons of the hippocampal CA1 region (CA1). TLR4 immunoreactivity in pyramidal neurons was distinctively decreased after ischemia/reperfusion (I/R); instead, based on double immunofluorescence study, TLR4 immunoreactivity was expressed in non-pyramidal neurons and astrocytes from 2 days postischemia. In addition, TLR4 protein level was lowest at 1 day postischemia and highest 4 days after I/R. On the other hand, NF-κB p65 immunoreactivity was not detected in the CA1 of the sham-operated group, and NF-κB p65 immunoreactivity was not observed until 1 day after I/R. However, NF-κB p65 immunoreactivity began to be expressed in astrocytes at 2 days postischemia, and the immunoreactivity was strong 4 days postischemia. Our results indicate that TLR4 and NF-κB p65 immunoreactivity are changed in CA1 pyramidal neurons and newly expressed in astrocytes, not in microglia, in the CA1 region after transient cerebral ischemia.