Chronic social defeat stress (CSDS) is an ethologically relevant psychosocial stress animal model and has been widely used in depression studies. Ginsenoside Rg1 (Rg1) is the major active ingredients of ginseng with low toxicity and neuroprotective effects. The present study aims to investigate the antidepressant effects of Rg1 in CSDS mice and explore its molecular mechanism. We found that Rg1 (20 or 40 mg/kg, i.g.) administration significantly alleviated depressive-like behaviors caused by 4-week CSDS exposure, as measured by social interaction test and sucrose preference test, tail suspension test and forced swim test. Additionally, Rg1 treatment inhibited CSDS-induced production of IL-6, TNF-α and IL-1β, decreased the expression of iNOS, COX2, and caspase-9 and -3, and inhibited microglial activation (Iba1) in the hippocampus. Rg1 was found to significantly downregulate p-JNK1/2 and p-P38 MAPK levels, upregulate p-ERK1/2 levels and inhibit the expression of phosphorylated NF-κB in the hippocampus. Meanwhile, Rg1 regulated SIRT1 and decreased the levels of acetylated p65 (ac-p65) in the hippocampus. Moreover, the reduction in adult hippocampal neurogenesis in CSDS mice was reversed by Rg1 treatment. In conclusion, our findings suggest that Rg1 prevents depressive-like behavior in CSDS-exposed mice, partially through the downregulation of hippocampal neuroinflammation and the upregulation of adult hippocampal neurogenesis and that these changes presumably occur through increased anti-inflammatory effects and the inhibition of proinflammatory cytokine and neurotoxic mediator expression and microglial activation, which is partly mediated by the regulation of the MAPK and SIRT1 signaling pathways and results in the inhibition of NF-κB transcriptional activity.

Intrastriatal administration of mesenchymal stem cells (MSCs) has shown beneficial effects in rodent models of Huntington disease (HD). However, the invasive nature of surgical procedure and its potential to trigger the host immune response may limit its clinical use. Hence, we sought to evaluate the non-invasive intranasal administration (INA) of MSC delivery as an effective alternative route in HD. GFP-expressing MSCs derived from bone marrow were intranasally administered to 4-week-old R6/2 HD transgenic mice. MSCs were detected in the olfactory bulb, midbrain and striatum five days post-delivery. Compared to phosphate-buffered saline (PBS)-treated littermates, MSC-treated R6/2 mice showed an increased survival rate and attenuated circadian activity disruption assessed by locomotor activity. MSCs increased the protein expression of DARPP-32 and tyrosine hydroxylase (TH) and downregulated gene expression of inflammatory modulators in the brain 7.5 weeks after INA. While vehicle treated R6/2 mice displayed decreased Iba1 expression and altered microglial morphology in comparison to the wild type littermates, MSCs restored both, Iba1 level and the thickness of microglial processes in the striatum of R6/2 mice. Our results demonstrate significantly ameliorated phenotypes of R6/2 mice after MSCs administration via INA, suggesting this method as an effective delivering route of cells to the brain for HD therapy.

BACKGROUND

We have uncovered a caspase-dependent (caspase-8/caspase-3/7) signaling governing microglia activation and associated neurotoxicity. Importantly, a profuse non-nuclear activation of cleaved caspases 8 and 3 was found in reactive microglia in the ventral mesencephalon from subjects with Parkinson's disease, thus supporting the existence of endogenous factors activating microglia through a caspase-dependent mechanism. One obvious candidate is neuromelanin, which is an efficient proinflammogen in vivo and in vitro and has been shown to have a role in the pathogenesis of Parkinson's disease. Consequently, the goal of this study is to test whether synthetic neuromelanin activates microglia in a caspase-dependent manner.

RESULTS

We found an in-vivo upregulation of CD16/32 (M1 marker) in Iba1-immunolabeled microglia in the ventral mesencephalon after neuromelanin injection. In vitro experiments using BV2 cells, a microglia-derived cell line, demonstrated that synthetic neuromelanin induced a significant chemotactic response to BV2 microglial cells, along with typical morphological features of microglia activation, increased oxidative stress and induction of pattern-recognition receptors including Toll-like receptor 2, NOD2, and CD14. Analysis of IETDase (caspase-8) and DEVDase (caspase-3/7) activities in BV2 cells demonstrated a modest but significant increase of both activities in response to neuromelanin treatment, in the absence of cell death.

CONCLUSIONS

Caspase-8 inhibition prevented typical features of microglia activation, including morphological changes, a high rate of oxidative stress and expression of key proinflammatory cytokines and iNOS.

Glucose is a major fuel for the central nervous system and hypoglycemia is a significant homeostatic stressor, which elicits counterregulatory reactions. Hypothalamic metabolic- and stress-related neurons initiate these actions, however recruitment of glia in control such adaptive circuit remain unknown. Groups of fed- and fasted-, vehicle-injected, and fasted + insulin-injected male mice were compared in this study. Bolus insulin administration to fasted mice resulted in hypoglycemia, which increased hypothalamo-pituitary-adrenal (HPA) axis- and sympathetic activity, increased transcription of neuropeptide Y (Npy) and agouti-related peptide (Agrp) in the hypothalamic arcuate nucleus and activated IBA1+ microglia in the hypothalamus. Activated microglia were found in close apposition to hypoglycemia-responsive NPY neurons. Inhibition of microglia by minocycline increased counterregulatory sympathetic response to hypoglycemia. Fractalkine-CX3CR1 signaling plays a role in control of microglia during hypoglycemia, because density and solidity of IBA1-ir profiles was attenuated in fasted, insulin-treated, CX3CR1 KO mice, which was parallel with exaggerated neuropeptide responses and higher blood glucose levels following insulin administration. Hypoglycemia increased Il-1b expression in the arcuate nucleus, while IL-1a/b knockout mice display improved glycemic control to insulin administration. In conclusion, activated microglia in the arcuate nucleus interferes with central counterregulatory responses to hypoglycemia. These results underscore involvement of microglia in hypothalamic regulation of glucose homeostasis.

Ischemia cerebral stroke is one of the common neurological diseases with severe inflammatory response and neuron death. The inhibition of colony-stimulating factor 1 receptor (CSF1R) which especially expressed in microglia/macrophage exerted neuroprotection in stroke. However, the underlying neuroinflammatory regulation effects of CSF1R in ischemia stroke are not clear. In this study, cerebral ischemia stroke mice model was established. The C57/B6J mice were administered with Ki20227, a CSF1R inhibitor, by gavage for 7 consecutive days (0.002 mg/kg/day) before modeling. The Rota-Rod test and neurobehavioral score test were investigated to assess neurobehavioral functions. The area of infarction was assessed by 2, 3, 5-triphenyltetrazolium chloride (TTC) staining. The mRNA expressions of M1/M2 microglia markers were evaluated by real-time PCR. Immunofluorescence and Western blot were utilized to detect the changes of Iba1 and NLRP3 pathway proteins. Results showed that neurobehavioral function improvement was demonstrated by an increased stay time on the Rota-Rod test and a decreased neurobehavioral score in the Ki20227 treatment group. The area of infarction reduced in Ki20227 group when compared to the stroke group. Moreover, the mRNA expression of M1 microglia markers (TNF-α and iNOS) decreased while M2 microglia markers (IL-10 and Arg-1) increased. Meanwhile, compared to the stroke and stroke+PBS group, Ki20227 administration downregulated the expression of NLRP3, active caspase 1, and NF-κB protein in the ischemia penumbra of Ki20227 treatment group mice. In short, the CSF1R inhibitor, Ki20227, played vital neuroprotective roles in ischemia cerebral stroke mice, and the mechanisms may be via inhibiting microglia M1 polarization and NLRP3 inflammasome pathway activation. Our study provides a potential new target for the treatment of ischemic stroke injury.

Neurosteroids are involved in the pathogenesis of hepatic encephalopathy (HE). This study evaluated the effects of finasteride, inhibitor of neurosteroid synthesis, on motor, EEG, and cellular changes in rat brain in thioacetamide-induced HE. Male Wistar rats were divided into the following groups: 1) control; 2) thioacetamide-treated group, TAA (300 mg·kg(-1)·day(-1)); 3) finasteride-treated group, FIN (50 mg·kg(-1)·day(-1)); and 4) group treated with FIN and TAA (FIN + TAA). Daily doses of TAA and FIN were administered in three subsequent days intraperitoneally, and in the FIN + TAA group FIN was administered 2 h before every dose of TAA. Motor and reflex activity was determined at 0, 2, 4, 6, and 24 h, whereas EEG activity was registered about 24 h after treatment. The expressions of neuronal (NeuN), astrocytic [glial fibrilary acidic protein (GFAP)], microglial (Iba1), and oligodendrocyte (myelin oligodendrocyte glycoprotein) marker were determined 24 h after treatment. While TAA decreased all tests, FIN pretreatment (FIN + TAA) significantly improved equilibrium, placement test, auditory startle, head shake reflex, motor activity, and exploratory behavior vs. the TAA group. Vital reflexes (withdrawal, grasping, righting and corneal reflex) together with mean EEG voltage were significantly higher (P < 0.01) in the FIN + TAA vs. the TAA group. Hippocampal NeuN expression was significantly lower in TAA vs. control (P < 0.05). Cortical Iba1 expression was significantly higher in experimental groups vs. control (P < 0.05), whereas hippocampal GFAP expression was increased in TAA and decreased in the FIN + TAA group vs. control (P < 0.05). Finasteride improves motor and EEG changes in TAA-induced HE and completely prevents the development of hepatic coma.

The purpose of this study was to evaluate the cerebral protection of salvianolic acid B (Sal B) against cerebral I/R injury and investigate the underlying mechanism. As shown by 2,3,5-Triphenyltetrazolium chloride (TTC) staining and magnetic resonance imaging (MRI) analyses, Sal B significantly reduced cerebral infarct size, and accompanied with improved neurobehavioral functions as indicated by the modified Bederson score and Longa five-point scale. Sal B decreased the production of reactive oxygen species (p < .05, n = 10). The data of Western blotting and reverse transcription quantitative real time polymerase chain reaction (qRT-PCR) analyses showed that the expression of GFAP, Iba1, IL-1β, IL-6, TNF-α and Cleaved-caspase 3 was significantly reduced by Sal B in I/R injured brain tissues as compared to corresponding controls (p < .05, n = 10). Over activation of astrocytes and microglia were inhibited by Sal B as shown by immunostaining of GFAP and Iba 1. These data suggest that Sal B has neural protective effects against I/R-induced cerebral injury and could be an effective candidate for further development of clinical therapy.

Trimethyltin (TMT) is an organotin neurotoxicant with effects that are selectively localized to the limbic system (especially the hippocampus), which produces memory deficits and temporal lobe seizures. Galectin-3 (Gal-3) is a beta-galactoside-binding lectin that is important in cell proliferation and regulation of apoptosis. The present study evaluated the temporal expression of Gal-3 in the hippocampus of adult BALB/c mice after TMT treatment (i.p., 2.5mg/kg). Western blotting analyses showed that Gal-3 immunoreactivity began to increase days after treatment; the immunoreactivity peaked significantly within days after treatment but significantly declined between days 4 and 8. Immunohistochemical analysis indicated that Gal-3 expression was very rare in the hippocampi of vehicle-treated controls. However, Gal-3 immunoreactivity appeared between 2 and 8 days after TMT treatment and was primarily localized to the hippocampal dentate gyrus (DG), in which neuronal degeneration occurred. The immunoreactivity was detected predominantly in most of the Iba1-positive microglia and in some GFAP-positive astrocytes of the hippocampal DG. Furthermore, Gal-3 expression co-localized with the pro-inflammatory enzymes cyclooxygenase-2 and inducible nitric oxide synthase in the hippocampal DG. Therefore, we suggest that Gal-3 is involved in the inflammatory process of neurodegenerative disorder induced by organotin intoxication.

A monoclonal antibody (mAb) against the blood-brain barrier (BBB) transferrin receptor (TfR) is a potential agent for delivery of biologic drugs to the brain across the BBB. However, to date, no TfRMAb has been tested with chronic dosing in a primate model. A humanized TfRMAb against the human (h) TfR1, which cross reacts with the primate TfR, was genetically engineered with high affinity (ED50 = 0.18 ± 0.04 nM) for the human TfR type 1 (TfR1). For acute dosing, the hTfRMAb was tritiated and injected intravenously (IV) in the Rhesus monkey, which confirmed rapid delivery of the humanized hTfRMAb into both brain parenchyma, via transport across the BBB, and into cerebrospinal fluid (CSF), via transport across the choroid plexus. For chronic dosing, a total of 8 adult Rhesus monkeys (4 males, 4 females) were treated twice weekly for 4 weeks with 0, 3, 10, or 30 mg/kg of the humanized hTfRMAb via a 60 min IV infusion for a total of 8 doses prior to euthanasia and microscopic examination of brain and peripheral organs. A pharmacokinetics analysis showed the plasma clearance of the hTfRMAb in the primate was nonlinear, and plasma clearance was increased over 20-fold with chronic treatment of the low dose, 3 mg/kg, of the antibody. Chronic treatment of the primates with the 30 mg/kg dose caused anemia associated with suppressed blood reticulocytes. Immunohistochemistry of terminal brain tissue showed microglia activation, based on enhanced IBA1 immuno-staining, in conjunction with astrogliosis, based on increased GFAP immuno-staining. Moderate axonal/myelin degeneration was observed in the sciatic nerve. Further studies need to be conducted to determine if this neuropathology is induced by the antibody effector function, or is an intrinsic property of targeting the TfR in brain. The results indicate that chronic treatment of Rhesus monkeys with a humanized hTfRMAb may have a narrow therapeutic index, with associated toxicity related to microglial activation and astrogliosis of the brain.

Transient ischemia in the whole brain leads to neuronal loss/death in vulnerable brain regions. The striatum, neocortex and hippocampus selectively loose specific neurons after transient ischemia. Just 5 minutes of transient ischemia can cause pyramidal neuronal death in the hippocampal cornu ammonis (CA) 1 field at 4 days after transient ischemia. In this study, we investigated the effects of 5-minute (mild), 15-minute (severe), and 20-minute (lethal) transient ischemia by bilateral common carotid artery occlusion (BCCAO) on behavioral change and neuronal death and gliosis (astrocytosis and microgliosis) in gerbil hippocampal subregions (CA1-3 region and dentate gyrus). We performed spontaneous motor activity test to evaluate gerbil locomotor activity, cresyl violet staining to detect cellular distribution, neuronal nuclei immunohistochemistry to detect neuronal distribution, and Fluoro-Jade B histofluorescence to evaluate neuronal death. We also conducted immunohistochemical staining for glial fibrillary acidic protein and ionized calcium-binding adapter molecule 1 (Iba1) to evaluate astrocytosis and microgliosis, respectively. Animals subjected to 20-minute BCCAO died in at least 2 days. BCCAO for 15 minutes led to pyramidal cell death in hippocampal CA1-3 region 2 days later and granule cell death in hippocampal dentate gyrus 5 days later. Similar results were not found in animals subjected to 5-minute BCCAO. Gliosis was much more rapidly and severely progressed in animals subjected to 15-minute BCCAO than in those subjected to 5-minute BCCAO. Our results indicate that neuronal loss in the hippocampal formation following transient ischemia is significantly different according to regions and severity of transient ischemia. The experimental protocol was approved by Institutional Animal Care and Use Committee (AICUC) of Kangwon National University (approval No. KW-180124-1) on May 22, 2018.

Overwhelming evidence suggests that microglia play an important role in ischemic injury and they polarize into two different phenotypes with distinct functions after ischemic stroke. We performed the present study to investigate whether L-3-n butylphthalide (NBP) has an effect on microglial polarization. Mice were subjected to transient middle cerebral artery occlusion (MCAO) for 45 min, and then immediately after reperfusion were treated with NBP or vehicle via the caudal vein for 7 consecutive days. 2,3,5-Triphenyltetrazolium chloride (TTC) staining showed that NBP treatment resulted in a tendency to decrease cerebral infarct volume at 1 day after MCAO, and significant decreased infarct volume at 3 days after MCAO. Sensorimotor function was evaluated by the adhesive removal test and balance beam test, which were superior in NBP-treated mice compared with vehicle-treated mice at 1 and 3 days after MCAO. Immunofluorescent staining further indicated that NBP treatment significantly increased the number of CD206+/Iba1+ M2 microglia/macrophages and reduced the number of CD16+/Iba1+ M1 cells at 3 and 7 days after MCAO reperfusion. Western blot also showed an elevation of M2 marker (arginase-1) in NBP-treated brains at 7 days after MCAO. In conclusion, our results clearly show that NBP treatment significantly mitigates ischemic brain damage and promotes recovery of neurological function in early phase after ischemic stroke, probably by skewing M1 microglia/macrophages polarization towards M2 phenotype. Thus, our study provides new evidence that NBP might be a promising candidate for ameliorating injury caused by ischemic stroke.

Functional magnetic resonance imaging (fMRI) techniques can be used to assess cerebrovascular dysfunction in Alzheimer's disease, an important and early contributor to pathology. We hypothesized that bradykinin receptor inhibition alleviates the vascular dysfunction in a transgenic arcAβ mouse model of cerebral amyloidosis and that fMRI techniques can be used to monitor the treatment response. Transgenic arcAβ mice, and non-transgenic littermates of 14 months-of-age were either treated with the bradykinin receptors 1 and 2 blocker noscapine or received normal drinking water as control over 3 months (n = 8-11/group) and all mice were assessed using fMRI at the end of the treatment period. Perfusion MRI using an arterial spin labeling technique showed regional hypoperfusion in arcAβ compared to non-transgenic controls, which was alleviated by noscapine treatment. Similarly, measuring cerebral blood volume changes upon pharmacological stimulation using vessel dilator acetazolamide revealed recovery of regional impairment of cerebral vascular reactivity in arcAβ mice upon noscapine treatment. In addition, we assessed with immunohistochemistry beta-amyloid (Aβ) and inflammation levels in brain sections. Immunohistological stainings for Aβ deposition (6E10) and related microgliosis (Iba1) in the cortex and hippocampus were found comparable between noscapine-treated and untreated arcAβ mice. In addition, levels of soluble and insoluble Aβ₃₈, Aβ₄₀, Aβ₄₂ were found to be similar in brain tissue homogenates of noscapine-treated and untreated arcAβ mice using electro-chemiluminescent based immunoassay. In summary, bradykinin receptors blockade recovered cerebral vascular dysfunction in a mouse model of cerebral amyloidosis. fMRI methods revealed the functional deficit in disease condition and were useful tools to monitor the treatment response.

BACKGROUND

Cerebrovascular dysfunction and inflammation occur in epilepsy. Here we asked whether pericytes, a pivotal cellular component of brain capillaries, undergo pathological modifications during experimental epileptogenesis and in human epilepsy. We evaluated whether pro-inflammatory cytokines, present in the brain during seizures, contribute to pericyte morphological modifications.

METHODS

In vivo, unilateral intra-hippocampal kainic acid (KA) injections were performed in NG2DsRed/C57BL6 mice to induce status epilepticus (SE), epileptogenesis, and spontaneous recurrent seizures (SRS). NG2DsRed mice were used to visualize pericytes during seizure progression. The effect triggered by recombinant IL-1β, TNFα, or IL-6 on pericytes was evaluated in NG2DsRed hippocampal slices and in human-derived cell culture. Human brain specimens obtained from temporal lobe epilepsy (TLE) with or without sclerosis (HS) and focal cortical dysplasia (FCD-IIb) were evaluated for pericyte-microglial cerebrovascular assembly.

RESULTS

A disarray of NG2DsRed+ pericyte soma and ramifications was found 72 h post-SE and 1 week post-SE (epileptogenesis) in the hippocampus. Pericyte modifications topographically overlapped with IBA1+ microglia clustering around the capillaries with cases of pericytes lodged within the microglial cells. Microglial clustering around the NG2DsRed pericytes lingered at SRS. Pericyte proliferation (Ki67+) occurred 72 h post-SE and during epileptogenesis and returned towards control levels at SRS. Human epileptic brain tissues showed pericyte-microglia assemblies with IBA1/HLA microglial cells outlining the capillary wall in TLE-HS and FCD-IIb specimens. Inflammatory mediators contributed to pericyte modifications, in particular IL-1β elicited pericyte morphological changes and pericyte-microglia clustering in NG2DsRed hippocampal slices. Modifications also occurred when pro-inflammatory cytokines were added to an in vitro culture of pericytes.

CONCLUSIONS

These results indicate the occurrence of pericytosis during seizures and introduce a pericyte-microglial mediated mechanism of blood-brain barrier dysfunction in epilepsy.

Microglial activation (neuroinflammation) is often cited as a pathogenic factor in the development of neurodegenerative diseases. However, there are significant caveats associated with the idea that inflammation directly causes either α-synuclein pathology or neurofibrillary degeneration (NFD). We have performed immunohistochemical studies on microglial cells in five cases of dementia with Lewy bodies (DLB), median age 87, and nine cases of non-demented (ND) controls, median age 74, using tissue samples from the temporal lobe and the superior frontal gyrus. Three different antibodies known to label microglia and macrophages were employed: iba1, anti-CD68, and anti-ferritin. All DLB cases showed both α-synuclein pathology (Lewy bodies and neurites) and NFD ranging from Braak stage II to IV. In contrast, all controls were devoid of α-synuclein pathology but did show NFD ranging from Braak stage I to III. Using iba1 labeling, our current results show a notable absence of activated microglia in all cases with the exception of two controls that showed small focal areas of microglial activation and macrophage formation. Both iba1 and ferritin antibodies revealed a mixture of ramified and dystrophic microglial cells throughout the regions examined, and there were no measurable differences in the prevalence of dystrophic microglial cells between DLB and controls. Double-labeling for α-synuclein and iba1-positive microglia showed that cortical Lewy bodies were surrounded by both ramified and dystrophic microglial cells. We found an increase in CD68 expression in DLB cases relative to controls. Since microglial dystrophy has been linked to NFD and since it did not appear to be worse in DLB cases over controls, our findings support the idea that the additional Lewy body pathology in DLB is not the result of intensified microglial dystrophy. CD68 is likely associated with lipofuscin deposits in microglial cells which may be increased in DLB cases because of impaired proteostasis. Overall, we conclude that neurodegenerative changes in DLB are unlikely to result directly from activated microglia but rather from dysfunctional ones.

The complement system is involved in the pathogenesis of age-related macular degeneration (AMD). Because activated microglia are also associated with AMD, we studied the relationship between complement anaphylatoxin receptors and microglial recruitment.

We assessed the effect of anaphylatoxin C3a receptor (C3aR) and C5a receptor (C5aR) knockout (KO) on light damage-induced migration of microglia/macrophages into the mouse outer retina via immunofluorescence and real-time quantitative PCR.

We found that the mRNA levels of C3, C5, C3aR, C5aR, and two activators of the complement alternative pathway, Cfb and Cfd, were all upregulated after light exposure. Retinal Iba1-positive microglia/macrophages express receptors for C3a and C5a. Light damage increased the number of retinal Iba1-positive cells and the mRNA levels of Iba1. Compared with the wild-type (WT) mice, these increases were attenuated in the C5aR KO mice but not in the C3aR KO mice.

C5aR but not C3aR promoted the recruitment of microglia/macrophages. These divergent properties of complement anaphylatoxins in the light damage model provide a rationale for testing the differential effects of these receptors in additional retinal and neurodegeneration models.

BACKGROUND

Many brain areas participate to supraspinal control of nociception. In these regions, few studies have investigated the role of glial cells in supraspinal plasticity and the effect of 7-day intrathecal nerve growth factor-like (BB14®, Blueprint Biotech, Milano, Italy) treatment.

METHODS

In male Sprague-Dawley rats, we evaluated by immunohistochemistry the morphological and molecular rearrangement of neuroglial network occurring in several supraspinal brain regions involved in pain processing following spared nerve injury (SNI) of the sciatic nerve. In particular, the medial prefrontal cortex, the amygdala (Amy), the nucleus accumbens (Acb), the thalamus and the periaqueductal gray were analysed.

RESULTS

Despite the modifications occurring in the dorsal horn of spinal cord following SNI, no significant changes in the Iba1 and glial fibrillary acidic protein (GFAP) expression were detected in all the analysed supraspinal regions, except for the Amy, showing a remarkable GFAP increase. Interestingly, neuropathic rats also displayed a significant increase of glial transporters (GTs) in all the supraspinal regions. Finally, the analysis of vesicular glutamate transporter 1 (vGLUT1) and vesicular gamma-aminobutyric acid (GABA) transporter (vGAT) expression revealed a significant enhancement of glutamatergic/GABAergic ratio in all selected brain regions of SNI animals, except for Acb. Both glial activation in the Amy and alteration of GTs and vGLUT/vGAT levels observed in neuropathic animals were largely reversed by BB14® treatment.

CONCLUSIONS

All together, these data strengthen the role of supraspinal neuroglial network plasticity in the establishment of neuropathic pain syndrome. The hallmark is represented by the divergence between glial reaction confined to Amy and the widespread changes in the GT distribution and glutamate/GABA ratio detected in the other supraspinal region.

Occupational exposure to contaminants in agriculture and other industries is known to cause significant respiratory ailments. The effect of organic dust on lung inflammation and tissue remodeling has been actively investigated over many years but the adverse effect of organic dust-exposure on the central vital organ brain is beginning to emerge. Brain microglial cells are a major driver of neuroinflammation upon exposure to danger signals. Therefore, we tested a hypothesis that organic dust-exposure of microglial cells induces microglial cell activation and inflammation through HMGB1-RAGE signaling. Mouse microglial cells were exposed to organic dust extract showed a time-dependent increase in cytoplasmic translocation of high-mobility group box 1 (HMGB1) from the nucleus, increased expression of receptor for advanced glycation end products (RAGE) and activation of Iba1 as compared to control cells. Organic dust also induced reactive oxygen species generation, NF-κB activation, and proinflammatory cytokine release. To establish a functional relevance of HMGB1-RAGE activation in microglia-mediated neuroinflammation, we used both pharmacological and genetic approaches involving HMGB1 translocation inhibitor ethyl pyruvate (EP), anti-HMGB1 siRNA, and NOX-inhibitor mitoapocynin. Interestingly, EP effectively reduced HMGB1 nucleocytoplasmic translocation and RAGE expression along with reactive oxygen species (ROS) generation and TNF-α and IL-6 production but not NF-κB activation. HMGB1 knockdown by siRNA also reduced both ROS and reactive nitrogen species (RNS) and IL-6 levels but not TNF-α. NOX2 inhibitor mitoapocynin significantly reduced RNS levels. Collectively, our results demonstrate that organic dust activates HMGB1-RAGE signaling axis to induce a neuroinflammatory response in microglia and that attenuation of HMGB1-RAGE activation by EP and mitoapocynin treatments or genetic knockdown can dampen the neuroinflammation.

Peripheral nerve injury induces neuropathic pain which is characterized by tactile allodynia and thermal hyperalgesia. N-type voltage-dependent Ca(2+) channel (VDCC) plays pivotal roles in the development of neuropathic pain, since mice lacking Cav2.2, the pore-forming subunit of N-type VDCC, show greatly reduced symptoms of both tactile allodynia and thermal hyperalgesia. Our study on gene expression profiles of the Cav2.2 knockout (KO) spinal cord after spinal nerve ligation (SNL)-injury revealed altered expression of genes known to be expressed in microglia, raising an odd idea that N-type VDCC may function in not only excitable (neurons) but also non-excitable (microglia) cells in neuropathic pain state. In the present study, we have tested this idea by using a transgenic mouse line, in which suppression of Cav2.2 expression can be achieved specifically in microglia/macrophage by the application of tamoxifen. We found SNL-operated transgenic mice exhibited greatly reduced signs of tactile allodynia, whereas the degree of thermal hyperalgesia was almost the same as that of control. Immunohistochemical analysis of the transgenic lumbar spinal cord revealed reduced accumulation of Iba1-positive cells (microglia/macrophage) around the injured neurons, indicating microglial N-type VDCC is important for accumulation of microglia at the lesion sites. Although the mechanism of its activation is not clear at present, activation of N-type VDCC expressed in non-excitable microglial cells contributes to the pathophysiology of neuropathic pain.

Developing new therapies for stroke is urgently needed, as this disease is the leading cause of death and disability worldwide, and the existing treatment is only available for a small subset of patients. The interruption of blood flow to the brain during ischemic stroke launches multiple immune responses, characterized by infiltration of peripheral immune cells, the activation of brain microglial cells, and the accumulation of immune mediators. Copper is an essential trace element that is required for many critical processes in the brain. Copper homeostasis is disturbed in chronic neurodegenerative diseases and altered in stroke patients, and targeted copper delivery has been shown to be protective against chronic neurodegeneration. This study was undertaken to assess whether the copper bis(thiosemicarbazone) complex, CuII(atsm), is beneficial in acute brain injury, in preclinical mouse models of ischemic stroke. We demonstrate that the copper complex CuII(atsm) protects neurons from excitotoxicity and N2a cells from OGD in vitro, and is protective in permanent and transient ischemia models in mice as measured by functional outcome and lesion size. Copper delivery in the ischemic brains modulates the inflammatory response, specifically affecting the myeloid cells. It reduces CD45 and Iba1 immunoreactivity, and alters the morphology of Iba1 positive cells in the ischemic brain. CuII(atsm) also protects endogenous microglia against ischemic insult and reduces the proportion of invading monocytes. These results demonstrate that the copper complex CuII(atsm) is an inflammation-modulating compound with high therapeutic potential in stroke and is a strong candidate for the development of therapies for acute brain injury.

Extracellular superoxide dismutase (EC-SOD) is an isoform of SOD normally found both intra- and extra-cellularly and accounting for most SOD activity in blood vessels. Here we explored the role of EC-SOD in protecting against brain damage induced by chronic hypoxia. EC-SOD Transgenic mice, were exposed to hypoxia (FiO2.1%) for 10 days (H-KI) and compared to transgenic animals housed in room air (RA-KI), wild type animals exposed to hypoxia (H-WT or wild type mice housed in room air (RA-WT). Overall brain metabolism evaluated by positron emission tomography (PET) showed that H-WT mice had significantly higher uptake of 18FDG in the brain particularly the hippocampus, hypothalamus, and cerebellum. H-KI mice had comparable uptake to the RA-KI and RA-WT groups. To investigate the functional state of the hippocampus, electrophysiological techniques in ex vivo hippocampal slices were performed and showed that H-KI had normal synaptic plasticity, whereas H-WT were severely affected. Markers of oxidative stress, GFAP, IBA1, MIF, and pAMPK showed similar values in the H-KI and RA-WT groups, but were significantly increased in the H-WT group. Caspase-3 assay and histopathological studies showed significant apoptosis/cell damage in the H-WT group, but no significant difference in the H-KI group compared to the RA groups. The data suggest that EC-SOD has potential prophylactic and therapeutic roles in diseases with compromised brain oxygenation.

Glial cells regulate some neural functions which depend on the homeostatic maintenance of extracellular calcium within narrow physiological ranges. In this study, the presence of microglia-specific ionized calcium binding adaptor molecule 1 (Iba1) was examined in the mouse olfactory bulb. A heterogenous pattern of Iba1-positive cells expression was observed between the main and accessory olfactory bulbs (MOB and AOB, respectively). While Iba1 was almost uniformly expressed among the laminae of the MOB, its expression showed spatial variations from the anterior to the posterior regions of the AOB. Double immunofluorescence was used to confirm that Iba1 is not expressed in astrocytes which stained for glial fibrillary acidic protein. Since Iba1 may mediate calcium signals in microglia, the observations suggest a potential involvement of Iba1 and hence microglia in olfactory bulb function and/or homeostasis. Together with our previous observation, this provides further support of a bulbar neuron-glia system of potential physiological significance.

Since the brain is naturally inefficient in regenerating functional tissue after injury or disease, novel restorative strategies including stem cell transplantation and tissue engineering have to be considered. We have investigated the use of such strategies in order to achieve better functional repair outcomes. One of the fundamental challenges of successful transplantation is the delivery of cells to the injured site while maintaining cell viability. Classical cell delivery methods of intravenous or intraparenchymal injections are plagued by low engraftment and poor survival of transplanted stem cells. Novel implantable devices such as 3D bioactive scaffolds can provide the physical and metabolic support required for successful progenitor cell engraftment, proliferation, and maturation. In this study, we performed in situ analysis of laminin-linked dextran and gelatin macroporous scaffolds. We revealed the protective action of gelatin-laminin (GL) scaffolds seeded with mesenchymal stem cells derived from donated human Wharton's jelly (hUCMSCs) against neuroinflammatory reactions of injured mammalian brain tissue. These bioscaffolds have been implanted into (i) intact and (ii) ischemic rat hippocampal organotypic slices and into the striatum of (iii) normal and (iv) focally injured brains of adult Wistar rats. We found that transplantation of hUCMSCs encapsulated in GL scaffolds had a significant impact on the prevention of glial scar formation (low glial acidic fibrillary protein) and in the reduction of neuroinflammation (low interleukin-6 and the microglial markers ED1 and Iba1) in the recipient tissue. Moreover, implantation of hUCMSCs encapsulated within GL scaffolds induced matrix metalloproteinase-2 and -9 proteolytic activities in the surrounding brain tissue. This facilitated scaffold biodegradation while leaving the remaining grafted hUCMSCs untouched. In conclusion, transplanting GL scaffolds preseeded with hUCMSCs into mammalian brain tissue escaped the host's immune system and protected neural tissue from neuroinflammatory injury. This manuscript is published as part of the International Association of Neurorestoratology (IANR) supplement issue of Cell Transplantation.

Accelerated ovarian failure (AOF) can be induced in young mice with low doses of 4-vinylcyclohexene diepoxide (VCD), modeling the hormone changes observed across menopause. We assessed markers of synaptic plasticity in the hippocampus, anxiety-like behavior, and spatial learning longitudinally at 4 time points across the AOF model: premenopause, early perimenopause, late perimenopause, and postmenopause (POST). As others have shown, VCD administration decreased ovarian follicle counts and increased acyclicity as the model progressed to POST but with no impact on organ or body weights. The morphology of Iba1 immunoreactive microglia did not differ between vehicle- and VCD-administered mice. Hippocampal postsynaptic density 95 levels were minimally altered across the AOF model but decreased at POST in CA3b 24 hours after exogenous estradiol benzoate (EB). In contrast, hippocampal phosphorylated AKT levels transiently decreased in premenopause but increased at POST after 24 hours of EB in select subregions. Electron microscopy revealed fewer estrogen receptor α containing dendritic spines and terminals in CA1 stratum radiatum at POST. mRNA levels of most brain-derived neurotrophic factor exons (except V and VI) were lower in POST compared with ovariectomized mice. Exon V was sensitive to 24 hours of EB administration in POST-VCD. Anxiety-like behavior was unaffected at any menopause phase. Spatial learning was unaffected in all groups, but POST-VCD mice performed below chance. Our results suggest that the AOF model is suitable for longitudinal studies of neurobiological changes across the menopause transition in mice. Our findings also point to complex interactions between estrogen receptors and pathways involved in synaptic plasticity.