Current therapy of a number of neuropsychiatric maladies has only symptomatic modality. Effective treatment of these neuro-degenerative diseases, including amyotrophic lateral sclerosis (ALS), may benefit from combined gene/stem-cell approaches. In this report, mononuclear fraction of human umbilical cord blood cells (hUCBCs) were transfected by electroporation with dual plasmid constructs, simultaneously expressing vascular endothelial growth factor 165 (VEGF(165)) and human fibroblast growth factor 2 (FGF(2)) (pBud-VEGF-FGF(2)). These genetically modified hUCBCs were injected retro-orbitally into presymptomatic ALS transgenic animal models ((G)93(A) mice). Lumbar spinal cords of rodents were processed for immunofluoresent staining with antibodies against human nuclear antigen (HNA), oligodendrocyte-specific protein, S100, iba1, neuronal β(3)-tubulin and CD34. Co-localization of HNA and S100 was found in the spinal cord of mice after transplantation of genetically modified hUCBCs over-expressing VEGF-FGF(2). Double staining in control animals treated with unmodified hUCBCs, however, revealed HNA+ cells expressing iba1 and CD34. Neuron-specific β(3)-tubulin or oligodendrocyte-specific protein were not expressed in hUCBCs in either control or experimental mice. These results demonstrate that genetically naïve hUCBCs may differentiate into endothelial (CD34+) and microglial (iba1+) cells; however when over-expressing VEGF-FGF(2), hUCBCs transform into astrocytes (S100+). Autocrine regulation of VEGF and FGF(2) on hUCBCs, signal molecules from dying motor neurons in spinal cord, as well as self-differentiating potential may provide a unique microenvironment for the transformation of hUCBCs into astrocytes that eventually serve as a source of growth factors to enhance the survive potential of surrounding cells in the diseased regions.

An estrogen receptor (ER) β ligand (LY3201) with a preference for ERβ over ERα was administered in s.c. pellets releasing 0.04 mg/d. The brains of these mice were examined 3 d after treatment had begun. Although estradiol-17β is known to increase spine density and glutaminergic signaling, as measured by Golgi staining, a clear reduction in spines was evident on the dendritic branches in LY3201-treated mice but no morphological alteration and no difference in the number of dendritic spines on dendritic stems were observed. In the LY3201-treatment group, there was higher expression of glutamic acid decarboxylase (GAD) in layer V of cortex and in the CA1 of hippocampus, more GAD(+) terminals surrounding the pyramidal neurons and less glutamate receptor (NMDAR) on the neurons in layer V. There were no alterations in expression of Iba1 or in Olig2 or CNPase. However, GFAP(+) astrocytes were increased in the LY3201-treatment group. There were also more projections characteristic of activated astrocytes and increased expression of glutamine synthetase (GS). No expression of ERβ was detectable in the nuclei of astrocytes. Clearly, LY3201 caused a shift in the balance between excitatory and inhibitory neurotransmission in favor of inhibition. This shift was due in part to increased synthesis of GABA and increased removal of glutamate from the synaptic cleft by astrocytes. The data reveal that treatment with a selective ERβ agonist results in changes opposite to those reported in estradiol-17β-treated mice and suggests that ERα and ERβ play opposing roles in the brain.

Given the involvement of post-mitotic neurons, long axonal tracts and incompletely elucidated injury and repair pathways, spinal cord injury (SCI) presents a particular challenge for the creation of preclinical models to robustly evaluate longitudinal changes in neuromotor function in the setting in the presence and absence of intervention. While rodent models exhibit high degrees of spontaneous recovery from SCI injury, animal care concerns preclude complete cord transections in non-human primates and other larger vertebrate models. To overcome such limitations a segmental thoracic (T9-T10) spinal cord hemisection was created and characterized in the African green monkey. Physiological tolerance of the model permitted behavioral analyses for a prolonged period post-injury, extending to predefined study termination points at which histological and immunohistochemical analyses were performed. Four monkeys were evaluated (one receiving no implant at the lesion site, one receiving a poly(lactide-co-glycolide) (PLGA) scaffold, and two receiving PLGA scaffolds seeded with human neural stem cells (hNSC)). All subjects exhibited Brown-Séquard syndrome 2 days post-injury consisting of ipsilateral hindlimb paralysis and contralateral hindlimb hypesthesia with preservation of bowel and bladder function. A 20-point observational behavioral scoring system allowed quantitative characterization of the levels of functional recovery. Histological endpoints including silver degenerative staining and Iba1 immunohistochemistry, for microglial and macrophage activation, were determined to reliably define lesion extent and correlate with neurobehavioral data, and justify invasive telemetered electromyographic and kinematic studies to more definitively address efficacy and mechanism.

BACKGROUND

Activation of the mTOR pathway has been linked to the cytopathology and epileptogenicity of malformations, specifically Focal Cortical Dysplasia (FCD) and Tuberous Sclerosis (TSC). Experimental and clinical trials have shown than mTOR inhibitors have anti-epileptogenic effects in TS. Dysmorphic neurones and balloon cells are hallmarks of FCDIIb and TSC, but similar cells are also occasionally observed in other acquired epileptogenic pathologies, including hippocampal sclerosis (HS) and Rasmussen's encephalitis (RE). Our aim was to explore mTOR pathway activation in a range of epilepsy-associated pathologies and in lesion-negative cases.

RESULTS

50 epilepsy surgical pathologies were selected including HS ILAE type 1 with (5) and without dysmorphic neurones (4), FCDIIa (1), FCDIIb (5), FCDIIIa (5), FCDIIIb (3), FCDIIId (3), RE (5) and cortex adjacent to cavernoma (1). We also included pathology-negative epilepsy cases; temporal cortex (7), frontal cortex (2), paired frontal cortical samples with different ictal activity according to intracranial EEG recordings (4), cortex with acute injuries from electrode tracks (5) and additionally non-epilepsy surgical controls (3). Immunohistochemistry for phospho-S6 (pS6) ser240/244 and ser235/236 and double-labelling for Iba1, neurofilament, GFAP, GFAPdelta, doublecortin, and nestin were performed. Predominant neuronal labelling was observed with pS6 ser240/244 and glial labelling with pS6 ser235/236 in all pathology types but with evidence for co-expression in a proportion of cells in all pathologies. Intense labelling of dysmorphic neurones and balloon cells was observed in FCDIIb, but dysmorphic neurones were also labelled in RE and HS. There was no difference in pS6 labelling in paired samples according to ictal activity. Double-labelling immunofluorescent studies further demonstrated the co-localisation of pS6 with nestin, doublecortin, GFAPdelta in populations of small, immature neuroglial cells in a range of epilepsy pathologies.

CONCLUSIONS

Although mTOR activation has been more studied in the FCDIIb and TSC, our observations suggest this pathway is activated in a variety of epilepsy-associated pathologies, and in varied cell types including dysmorphic neurones, microglia and immature cell types. There was no definite evidence from our studies to suggest that pS6 expression is directly related to disease activity.

OBJECTIVE

Bone morphogenetic proteins and their receptors are expressed in adult brains, and their expression levels increase after cerebral ischemia. The brain also expresses an inhibitor of bone morphogenetic protein signaling, noggin, but the role of noggin in ischemic disease outcome has not been studied.

METHODS

We used transgenic mice overexpressing noggin to assess whether inhibition of bone morphogenetic protein signaling affects ischemic injury responses after permanent middle cerebral artery occlusion.

RESULTS

Transgenic mice overexpressing noggin mice had significantly smaller infarct volumes and lower motor deficits compared to wild-type mice. CD11b(+) and IBA1(+) microglia along with oligodendroglial progenitors were significantly increased in transgenic mice overexpressing noggin mice at 14 days after permanent middle cerebral artery occlusion.

CONCLUSIONS

These results provide genetic evidence that overexpression of noggin reduces ischemic brain injury after permanent middle cerebral artery occlusion via enhanced activation of microglia and oligodendrogenesis.

BACKGROUND

Resident and exudate macrophages play an important role in the development of liver cirrhosis. Ionized calcium binding adaptor molecule 1(+) (Iba1(+)) and galectin-3(+) (Gal-3(+)) macrophages regulate liver fibrosis probably through pro-inflammatory and pro-fibrotic factors. Macrophages show polarized functions in liver fibrosis; however, M1-/M2-polarization of Iba1(+) and Gal-3(+) macrophages remains obscured. This study investigated the M1-/M2-polarized properties of Iba1(+) and Gal-3(+) macrophages in chemical-induced liver cirrhosis.

METHODS

Cirrhosis was induced in F344 rats by repeated injections of thioacetamide (100mg/kg BW, twice a week for 25 weeks). Liver samples were collected from post-first-injection (PFI) week 5 to 25. Macrophage immunophenotypes and myofibroblasts in the fibrous bridges (FBs) and pseudolobules (PLs) were analyzed by immunohistochemistry. Expressions of M1- and M2-related factors were analyzed with RT-PCR, separately in FBs and PLs.

RESULTS

Activation of myofibroblasts was most pronounced in livers at week 15. CD68(+) (M1), CD204(+) (M2), Iba1(+) and Gal-3(+) macrophages in the FBs increased gradually and peaked at week 15, consistent with the upregulation of both M1-(MCP-1, IFN-γ, IL-1β, IL-6, and TNF-α) and M2-(TGF-β1, IL-4, and IL-10) related factors. Iba1(+) and Gal-3(+) macrophages showed both M1- and M2-immunophenotypes. CD163(+) macrophages showed a persistent increase, consistent with TGF-β1 upregulation. MHC class II(+) macrophages increased in the developing fibrotic lesions, and then reduced in the advanced stage cirrhosis.

CONCLUSIONS

Both M1- and M2-macrophage polarizations occur during development of liver cirrhosis. Iba1(+) and Gal-3(+) macrophages participate in liver cirrhosis through production of both M1- and M2-related factors.

Sex is an important factor in the response to ischemic insults in both the laboratory and the clinic. Inflammation and cell death are points where sex-specific pathways diverge in stroke, and serum estrogen level status affect the response to inflammation. The cytokine macrophage migration inhibitory factor (MIF) is detrimental in experimental stroke models in male animals. However MIF is known to have sex-specific actions on inflammation and wound healing. The role of MIF in the ischemic female brain has not been evaluated. A transient middle cerebral artery occlusion (MCAO/90min) model was used to induce stroke in male, intact female, and ovariectomized female wildtype (WT) and MIF knockout (KO) mice. Infarct size was quantified 72h after stroke. Protein and cytokine levels were assessed post stroke. Female MIF KO mice had significantly larger strokes compared to WT females (mean hemispheric infarct±SEM: 63%±2% versus 29%±3%; n=8; p<0.05). Ovariectomized female MIF KO mice also had larger infarcts than ovariectomized WT littermates (70%±3% versus 47%±4%; n=11; p<0.05). In males, however, infarct size was equivalent between MIF KO and WT mice (63%±2% versus 67%±3%; n=9; p=0.25). There were no significant differences in cytokine levels at 6h post-infarct between mice of either genotype in brain. MIF KO females displayed more microglial activation (ionized calcium binding adaptor molecule 1 (Iba1) immunofluorescence) after stroke than did WT mice or MIF KO males. The larger infarcts in MIF KO females were associated with an early increase in mitochondrial localization of Jun activation domain-binding protein 1 (JAB1). Loss of MIF exacerbated injury in the female brain after experimental stroke, which was independent of changes in pro-inflammatory cytokine levels. This response is sex-specific, and is in part independent of physiological serum levels of estrogen.

It is unclear whether a single, brief, 15-minute episode of background anesthesia already modulates delayed secondary processes after experimental brain injury. Therefore, this study was designed to characterize three anesthesia protocols for their effect on molecular and histological study endpoints. Mice were randomly separated into groups that received sevoflurane (sevo), isoflurane (iso) or an intraperitoneal anesthetic combination (midazolam, fentanyl and medetomidine; comb) prior to traumatic brain injury (controlled cortical impact, CCI; 8 m/s, 1 mm impact depth, 3 mm diameter). Twenty-four hours after insult, histological brain damage, neurological function (via neurological severity score), cerebral inflammation (via real-time RT-PCR for IL6, COX-2, iNOS) and microglia (via immunohistochemical staining for Iba1) were determined. Fifteen minutes after CCI, the brain contusion volume did not differ between the anesthetic regimens (sevo = 17.9±5.5 mm(3); iso = 20.5±3.7 mm(3); comb = 19.5±4.6 mm(3)). Within 24 hours after injury, lesion size increased in all groups (sevo = 45.3±9.0 mm(3); iso = 31.5±4.0 mm(3); comb = 44.2±6.2 mm(3)). Sevo and comb anesthesia resulted in a significantly larger contusion compared to iso, which was in line with the significantly better neurological function with iso (sevo = 4.6±1.3 pts.; iso = 3.9±0.8 pts.; comb = 5.1±1.6 pts.). The expression of inflammatory marker genes was not significantly different at 15 minutes and 24 hours after CCI. In contrast, significantly more Iba1-positive cells were present in the pericontusional region after sevo compared to comb anesthesia (sevo = 181±48/mm(3); iso = 150±36/mm(3); comb = 113±40/mm(3)). A brief episode of anesthesia, which is sufficient for surgical preparations of mice for procedures such as delivering traumatic brain injury, already has a significant impact on the extent of secondary brain damage.

Patients with major depressive disorder have elevated peripheral inflammation; the degree of this increase correlates with the severity of the disorder. Chronic psychological stress increases pro-inflammatory cytokines and promotes microglial activation, leading to stress vulnerability. Epigenetics, including DNA methylation and histone modification, are also related to the pathophysiology of major depressive disorder. Sodium butyrate (SB), a histone deacetylase inhibitor, exerts an antidepressant effect by altering gene expression in the hippocampus. In this study, we investigated whether lipopolysaccharide (LPS)-induced depressive-like behaviors in mice are affected by the repeated treatment with SB. Intraperitoneal injection of LPS (5 mg/kg) induced cytokines and ionized calcium-binding adaptor molecule 1(Iba1), a marker of microglial activation, in the hippocampus. It also increased the immobility time in a forced swim test, without changing locomotion. Repeated treatment with SB reduced LPS-induced alterations. These findings suggested that epigenetic regulation exist in hippocampal microglial activation, and is involved in depressive-like behaviors associated with neuro-inflammation. Further, using cDNA microarray analyses, we examined whether LPS and SB treatment affected the microglial gene profiles. Our results indicated 64 overlapping genes, between LPS-increased genes and SB-decreased genes. Among these genes, EF hand calcium binding domain 1 was a particularly distinct candidate gene. Altogether, our findings indicated that microglial activation mediated through epigenetic regulation may be involved in depressive-like behaviors. In addition, we demonstrated the effect of SB on gene information in hippocampal microglia under neuroinflammatory conditions.

Subventricular zone (SVZ) neural stem cells (NSCs) are the cornerstone of the perinatal neurogenic niche. Microglia are immune cells of the nervous system that are enriched in the neonatal SVZ. Although microglia regulate NSCs, the extent to which this interaction is bi-directional is unclear. Extracellular vesicles (EVs) are cell-derived particles that encase miRNA and proteins. Here, we demonstrate that SVZ NSCs generate and release EVs. Neonatal electroporated fluorescent EV fusion proteins were released by NSCs and subsequently cleared from the SVZ. EVs were preferentially targeted to microglia. Small RNA sequencing identified miRNAs within the EVs that regulate microglia physiology and morphology. EVs induced a transition to a CD11b/Iba1 non-stellate microglial morphology. The transition accompanied a microglial transcriptional state characterized by Let-7-regulated cytokine release and a negative feedback loop that controlled NSC proliferation. These findings implicate an NSC-EV-microglia axis and provide insight to normal and pathophysiological brain development.

Currently, there are no effective therapies to restore lost brain neurons, although rapid progress in stem cell biology and biomaterials development provides new tools for regeneration of central nervous system. Here we describe neurogenic properties of bioactive scaffolds generated by cryogelation of dextran or gelatin linked to laminin - the main component of brain extracellular matrix. We showed that such scaffolds promoted differentiation of human cord blood-derived stem cells into artificial neural tissue in vitro. Our experiments revealed that optimal range of scaffolds' pore size for neural tissue engineering was 80-100 microns. We found that scaffold seeded with undifferentiated, but not neutrally committed stem cells, gave optimal cell adhesion and proliferation in "niche"-like structures. Subsequent differentiation resulted in generation of mature 3D networks of neurons (MAP2+) and glia (S100beta+) cells. We showed that cryogel scaffolds could be transplanted into the brain tissue or organotypic hippocampal slices in a rat models. The scaffolds did not induced inflammation mediated by microglial cells (ED1-, Ox43-, Iba1-) and prevented formation of glial scar (GFAP-). Contrary, laminin-rich scaffolds attracted infiltration of host's neuroblasts (NF200+, Nestin+) indicating high neuroregeneration properties.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease of unknown cause. Absence of specific targets and biomarkers compromise the development of new therapeutic strategies and of innovative tools to stratify patients and assess their responses to treatment. Here, we investigate changes in neuroprotective-neuroinflammatory actions in the spinal cord of SOD1 G93A mice, at presymptomatic and symptomatic stages to identify stage-specific biomarkers and potential targets. Results showed that in the presymptomatic stage, there are alterations in both astrocytes and microglia, which comprise decreased expression of GFAP and S100B and upregulation of GLT-1, as well as reduced expression of CD11b, M2-phenotype markers, and a set of inflammatory mediators. Reduced levels of Connexin-43, Pannexin-1, CCL21, and CX3CL1 further indicate the existence of a compromised intercellular communication. In contrast, in the symptomatic stage, increased markers of inflammation became evident, such as NF-κB/Nlrp3-inflammasome, Iba1, pro-inflammatory cytokines, and M1-polarizion markers, together with a decreased expression of M2-phenotypic markers. We also observed upregulation of the CX3CL1-CX3CR1 axis, Connexin-43, Pannexin-1, and of microRNAs (miR)-124, miR-125b, miR-146a and miR-21. Reduced motor neuron number and presence of reactive astrocytes with decreased GFAP, GLT-1, and GLAST further characterized this inflammatory stage. Interestingly, upregulation of miR-155 and downregulation of MFG-E8 appear as consistent biomarkers of both presymptomatic and symptomatic stages. We hypothesize that downregulated cellular interplay at the early stages may represent neuroprotective mechanisms against inflammation, SOD1 aggregation, and ALS onset. The present study identified a set of inflamma-miRNAs, NLRP3-inflammasome, HMGB1, CX3CL1-CX3CR1, Connexin-43, and Pannexin-1 as emerging candidates and promising pharmacological targets that may represent potential neuroprotective strategies in ALS therapy.

Hepatic encephalopathy (HE) is a common complication of chronic alcoholism and patients show neurological symptoms ranging from mild cognitive dysfunction to coma and death. The HE brain is characterized by glial changes, including microglial activation, but the exact pathogenesis of HE is poorly understood. During a study investigating cell proliferation in the subventricular zone of chronic alcoholics, a single case with widespread proliferation throughout their adjacent grey and white matter was noted. This case also had concomitant HE raising the possibility that glial proliferation might be a pathological feature of the disease. In order to explore this possibility fixed postmortem human brain tissue from chronic alcoholics with cirrhosis and HE (n = 9), alcoholics without HE (n = 4) and controls (n = 4) were examined using immunohistochemistry and cytokine assays. In total, 4/9 HE cases had PCNA- and a second proliferative marker, Ki-67-positive cells throughout their brain and these cells co-stained with the microglial marker, Iba1. These cases were termed 'proliferative HE' (pHE). The microglia in pHEs displayed an activated morphology with hypertrophied cell bodies and short, thickened processes. In contrast, the microglia in white matter regions of the non-proliferative HE cases were less activated and appeared dystrophic. pHEs were also characterized by higher interleukin-6 levels and a slightly higher neuronal density . These findings suggest that microglial proliferation may form part of an early neuroprotective response in HE that ultimately fails to halt the course of the disease because underlying etiological factors such as high cerebral ammonia and systemic inflammation remain.

BACKGROUND

The steroid hormone estrogen (17-β-estradiol, E2) provides neuroprotection against cerebral ischemic injury by activating estrogen receptors. The novel estrogen receptor G protein-coupled receptor 30 (GPR30) is highly expressed in the brain and provides acute neuroprotection against stroke. However, the underlying mechanisms remain unclear.

METHODS

In this study, ovariectomized female mice were subjected to middle cerebral artery occlusion (MCAO), and E2, G1, and ICI182780 were administered immediately upon reperfusion. The infarction volume, neurological scores, and neuronal injuries were examined. Primary microglial cells were subjected to oxygen-glucose deprivation (OGD), and the drugs were administered immediately upon reintroduction. The pro-inflammatory cytokines TNF-α, IL-1β, and IL-6 in penumbra and microglia were assessed by ELISA. The cell viability and lactose dehydrogenase (LDH) release of neurons co-cultured with microglia were analyzed using cell counting kit-8 (CCK8) and LDH release assays. Microglial activation as well as GPR30, Iba1, and Toll-like receptor 4 (TLR4) protein expression and TLR4 mRNA expression were detected. Additionally, NF-κB activity was detected in lipopolysaccharide (LPS)-activated microglia after the activation of GPR30.

RESULTS

GPR30 was highly expressed in microglia and significantly increased after ischemic injury. The activation of GPR30 significantly reduced the infarction volume, improved the neurological deficit, and alleviated neuronal injuries. Moreover, GPR30 activation significantly reduced the release of TNF-α, IL-1β, and IL-6 from ischemic penumbra and microglia subjected to OGD and alleviated neuronal injury as assessed using the CCK8 and LDH assays. Finally, the activation of GPR30 relieved microglial activation, reduced Iba1 and TLR4 protein expression and TLR4 mRNA levels, and inhibited NF-κB activity.

CONCLUSIONS

Microglial GPR30 exerts acute neuroprotective effects by inhibiting TLR4-mediated microglial inflammation, which indicates that GPR30 may be a potential target for the treatment of ischemic stroke.

Extremely low birth weight (ELBW) infants, who died at 12 hours to 7 days after birth, with periventricular leukomalacia (PVL), were examined by means of neuropathological and immunohistochemical methods. Fourteen infants without PVL were used as controls. Anti-beta-amyloid precursor protein (APP), glial fibrillary acidic protein (GFAP), and ionized calcium-binding adaptor molecule 1 (Iba1) antibodies were used as markers for axonal damage, reactive astrocytes and activated microglia, respectively. Thirteen of 14 ELBW infants with PVL showed a widespread distribution of leukomalacia and 10 showed postnatal-onset of leukomalacia. In 12 of the 14 infants with PVL, regions of APP-reactive axons were found multifocally in the cerebral white matter, but 8 of them did not show coagulation necrosis on HE staining. GFAP-positive cells and Iba1-positive cells were markedly found in the white matter of all cases with PVL and slightly in all 14 controls. These results indicated that in ELBW infants, the distribution and formation of PVL foci are widespread and characteristic and so may involve motor and intellectual abilities in ELBW infants. Therefore, the perinatal management to maintain an appropriate cerebral circulation and oxygenation may be very important.

Macrophages are closely related to various diseases and it is therefore important that the properties of macrophages are adequately evaluated in human diseases and mouse disease models. Immunohistochemistry (IHC) of formalin fixed paraffin-embedded (FFPE) samples is a very useful tool for examination of macrophages; however, an adequate IHC protocol is required for the examination of macrophage states. In this study, we assessed various antigen retrieval methods in order to devise the optimal protocols for staining of macrophages with a range of antibodies. Optimum combinations of primary antibodies and antigen retrieval protocols were determined; for example, heat treatment with ethylenediamine tetraacetic acid solution, pH 8.0, was the best procedure for IHC using mouse anti-Iba1 and human anti-CD11b, -CD163, -CD169, -CD204, and -CD206 antibodies. Moreover, we found that the immunoreactivity of sliced tissue sections decreased gradually over time in long term storage but that this immunoreactivity was preserved in storage at -80 °C in a deep freezer. The optimal IHC protocols and storage procedures that were determined in this study should be a useful tool for macrophage research.

BACKGROUND

Elevated plasma homocysteine (Hcy) levels have been indicated as a strong and modifiable risk factor of ischemic stroke; the previous studies have shown that exposure to Hcy activates cultured microglia. However, whether neurotoxicity of Hcy involves microglia activation following brain ischemia and the underlying mechanisms remains incompletely understood.

METHODS

The cerebral damage was evaluated by staining with 2,3,5-triphenyltetrazolium chloride, hematoxylin-eosin, and Fluoro Jade B. The activation state of microglia was assessed via immunoreaction using the microglial markers Iba1 and OX-42. Then, the inflammatory factors such as tumor necrosis factor α (TNF-α), interleukin 6 (IL-6), and phosphorylated signal transducer and activator of transcription 3 (pSTAT3) were examined by Western blot analysis and fluorescence immunohistochemistry.

RESULTS

Elevated Hcy level augmented brain damage and neural cell toxicity in the brain cortex and the dentate gyrus region of the hippocampus after cerebral ischemia/reperfusion. Meanwhile, Hcy activated microglia and induced the expression of the inflammatory factors such as TNF-α and IL-6. Moreover, Hcy caused an increase in pSTAT3 expression which occurs in microglial cells. AG490, a JAK2-STAT3 inhibitor, effectively inhibited the phosphorylation of STAT3, microglial cell activation and the secretion of IL-6, TNF-α raised by Hcy treatment.

CONCLUSIONS

STAT3 signaling pathway located in microglia plays a critical role in mediating Hcy-induced activation of microglia and neuroinflammation in rat MCAO model. This suggests the feasibility of targeting the JAK2/STAT3 pathway as an effective therapeutic strategy to alleviate the progression of Hcy-associated ischemia stroke.

Selected morphological, molecular and functional aspects of various microglial cell populations were characterized in cell cultures established from the forebrains of E18 rat embryos. The mixed primary cortical cultures were maintained for up to 28days using routine culturing techniques when the microglial cells in the culture were not stimulated or immunologically challenged. During culturing, expansion of the microglial cell populations was observed, as evidenced by quantitative assessment of selected monocyte/macrophage/microglial cell-specific markers (human leukocyte antigen (HLA) DP, DQ, DR, CD11b/c and Iba1) via immunocyto- and histochemistry and Western blot analysis. The Iba1 immunoreactivity in Western blots steadily increased about 750-fold, and the number of Iba1-immunoreactive cells rose at least 67-fold between one day in vitro (DIV1) and DIV28. Morphometric analysis on binary (digital) silhouettes of the microglia revealed their evolving morphology during culturing. Microglial cells were mainly ameboid in the early stages of in vitro differentiation, while mixed populations of ameboid and ramified cell morphologies were characteristic of older cultures as the average transformation index (TI) increased from 1.96 (DIV1) to 15.17 (DIV28). Multiple immunofluorescence labeling of selected biomarkers revealed different microglial phenotypes during culturing. For example, while HLA DP, DQ, DR immunoreactivity was present exclusively in ameboid microglia (TI<3) between DIV1 and DIV10, CD11b/c- and Iba1-positive microglial cells were moderately (TI<13) and progressively (TI<81) more ramified, respectively, and always present throughout culturing. Regardless of the age of the cultures, proliferating microglia were Ki67-positive and characterized by low TI values (TI<3). The microglial function was assessed by an in vitro phagocytosis assay. Unstimulated microglia with low TI values were significantly more active in phagocytosing fluorescent microspheres than the ramified forms. In vitro studies on microglial population dynamics combined with phenotypic characterization can be of importance when different in vivo pathophysiological situations are modeled in vitro.

Peptidomimetics hold a great promise as therapeutic agents for neurodegenerative disorders. We previously described a Nerve Growth Factor (NGF)-like peptide, now named BB14, which was found to act as a strong TrkA agonist and to be effective in the sciatic nerve injury model of neuropathic pain. In this report we present the effects of BB14 in reducing reactive astrocytosis and reverting neuroplastic changes of the glutamate/GABAergic circuitry in the lumbar spinal cord following spared nerve injury (SNI) of the sciatic nerve. Immunohistochemical analysis of spinal cord sections revealed that SNI was associated with increased microglial (Iba1) and astrocytic (GFAP) responses, indicative of reactive gliosis. These changes were paralleled by (i) decreased glial aminoacid transporters (GLT1 and GlyT1) and increased levels of (ii) neuronal glutamate transporter EAAC1, (iii) neuronal vesicular GABA transporter (vGAT) and (iv) the GABAergic neuron marker GAD65/67. A remarkable increase of the Glutamate/GABA ratio and the reduction of glutathione (GSH) levels were also indicative of modifications of glial function in neuroprotection. All these molecular changes were found to be linked to an alteration of endogenous NGF metabolism, as demonstrated by decreased levels of mature NGF, increase of proNGF and increased activity of NGF-degrading methallo-proteinases (MMPs). Biochemical alterations and SNI-related neuropathic behavior, characterized by allodynia and hyperalgesia, were reversed by 7-days i.t. administration of the NGF-like peptide BB14, as well as by increasing endogenous NGF levels by i.t. infusion of GM6001, a MMPs inhibitor. All together, while confirming the correlation between reactive astrogliosis and perturbation of synaptic circuitry in the SNI model of peripheral nerve injury, these data strongly support the beneficial effect of BB14 in reducing reactive astrogliosis and restoring synaptic homeostasis under pathological conditions linked to alteration of NGF availability and signaling, thereby suggesting a potential role of BB14 as a therapeutic agent.

Normobaric oxygen (NBO) reduces infarction at 24-48 h in experimental models of focal cerebral ischemia. However, to be clinically relevant, longer term safety and efficacy must be explored. Here, we assessed the effects of NBO on glial activation, neurovascular recovery, and behavioral outcomes at 2 weeks after transient focal ischemia in rats. 100 min transient focal ischemia was induced by intraluminal occlusion of the middle cerebral artery in adult male Sprague-Dawley rats. Animals were randomized into sham, controls or 85'NBO started 15 min after ischemic onset. Infarct volumes and behavioral outcomes were blindly quantified. Immunohistochemistry was used to examine the effects of NBO on glial activation and neurovascular responses. After 2 weeks of reperfusion the infarct volume was marked reduced in animals subjected to NBO. They also had better outcomes in forelimb placement test and in body-swing test and weight loss reduction. After 14 days, NBO decreased expression of Iba1, a marker of activated microglia, and GFAP, a marker of activated astrocytes. NBO treatment had no detectable effect on angiogenesis. These results suggest that protective effects of NBO may persist for up to 2 weeks post-stroke.

OBJECTIVE

The dorsal root ganglion is an attractive target for implanting neural electrode arrays that restore sensory function or provide therapy via stimulation. However, penetrating microelectrodes designed for these applications are small and deliver low currents. For long-term performance of microstimulation devices, novel coating materials are needed in part to decrease impedance values at the electrode-tissue interface and to increase charge storage capacity.

METHODS

Conductive polymer poly(3,4-ethylenedioxythiophene) (PEDOT) and multi-wall carbon nanotubes (CNTs) were coated on the electrode surface and doped with the anti-inflammatory drug, dexamethasone. Electrode characteristics and the tissue reaction around neural electrodes as a result of stimulation, coating and drug release were characterized. Hematoxylin and eosin staining along with antibodies recognizing Iba1 (microglia/macrophages), NF200 (neuronal axons), NeuN (neurons), vimentin (fibroblasts), caspase-3 (cell death) and L1 (neural cell adhesion molecule) were used. Quantitative image analyses were performed using MATLAB.

RESULTS

Our results indicate that coated microelectrodes have lower in vitro and in vivo impedance values. Significantly less neuronal death/damage was observed with coated electrodes as compared to non-coated controls. The inflammatory response with the PEDOT/CNT-coated electrodes was also reduced.

CONCLUSIONS

This study is the first to report on the utility of these coatings in stimulation applications. Our results indicate PEDOT/CNT coatings may be valuable additions to implantable electrodes used as therapeutic modalities.

Microglia are resident mononuclear phagocytes that play a principal role in the maintenance of normal tissue homeostasis in the central nervous system (CNS). Microglia, rapidly activated in response to proinflammatory stimuli, are accumulated in brain lesions of neurodegenerative diseases, such as Alzheimer's disease and Parkinson's disease. The E26 transformation-specific (ETS) family transcription factor PU.1/Spi1 acts as a master regulator of myeloid and lymphoid development. PU.1-deficient mice show a complete loss of microglia, indicating that PU.1 plays a pivotal role in microgliogenesis. However, the comprehensive profile of PU.1/Spi1 target genes in microglia remains unknown. By analyzing a chromatin immunoprecipitation followed by deep sequencing (ChIP-Seq) dataset numbered SRP036026 with the Strand NGS program, we identified 5,264 Spi1 target protein-coding genes in BV2 mouse microglial cells. They included Spi1, Irf8, Runx1, Csf1r, Csf1, Il34, Aif1 (Iba1), Cx3cr1, Trem2, and Tyrobp. By motif analysis, we found that the PU-box consensus sequences were accumulated in the genomic regions surrounding ChIP-Seq peaks. By using pathway analysis tools of bioinformatics, we found that ChIP-Seq-based Spi1 target genes show a significant relationship with diverse pathways essential for normal function of monocytes/macrophages, such as endocytosis, Fc receptor-mediated phagocytosis, and lysosomal degradation. These results suggest that PU.1/Spi1 plays a crucial role in regulation of the genes relevant to specialized functions of microglia. Therefore, aberrant regulation of PU.1 target genes might contribute to the development of neurodegenerative diseases with accumulation of activated microglia.

We report here in adult rat viral vector mediate-gene knockdown in the primary sensory neurons and the associated cellular and behavior consequences. Self-complementary adeno-associated virus serotype 5 (AAV5) was constructed to express green fluorescent protein (GFP) and a small interfering RNA (siRNA) targeting mammalian target of rapamycin (mTOR). The AAV vectors were injected via an intrathecal catheter. We observed profound GFP expression in lumbar DRG neurons beginning at 2-week post-injection. Of those neurons, over 85% were large to medium-diameter and co-labeled with NF200, a marker for myelinated fibers. Western blotting of mTOR revealed an 80% reduction in the lumbar DRGs (L4-L6) of rats treated with the active siRNA vectors compared to the control siRNA vector. Gene knockdown became apparent as early as 7-day post-injection and lasted for at least 5 weeks. Importantly, mTOR knockdown occurred in large (NF200) and small-diameter neurons (nociceptors). The viral administration induced an increase of Iba1 immunoreactivity in the DRGs, which was likely attributed to the expression of GFP but not siRNA. Rats with mTOR knockdown in DRG neurons showed normal general behavior and unaltered responses to noxious stimuli. In conclusion, intrathecal AAV5 is a highly efficient vehicle to deliver siRNA and generate gene knockdown in DRG neurons. This will be valuable for both basic research and clinic intervention of diseases involving primary sensory neurons.

Inflammation is a pathophysiological hallmark of many diseases of the brain. Specific imaging of cells and molecules that contribute to cerebral inflammation is therefore highly desirable, both for research and in clinical application. The 18 kDa translocator protein (TSPO) has been established as a suitable target for the detection of activated microglia/macrophages. A number of novel TSPO ligands have been developed recently. Here, we evaluated the high affinity TSPO ligand DPA-714 as a marker of brain inflammation in two independent animal models. For the first time, the specificity of radiolabeled DPA-714 for activated microglia/macrophages was studied in a rat model of epilepsy (induced using Kainic acid) and in a mouse model of stroke (transient middle cerebral artery occlusion, tMCAO) using high-resolution autoradiography and immunohistochemistry. Additionally, cold-compound blocking experiments were performed and changes in blood-brain barrier (BBB) permeability were determined. Target-to-background ratios of 2 and 3 were achieved in lesioned vs. unaffected brain tissue in the epilepsy and tMCAO models, respectively. In both models, ligand uptake into the lesion corresponded well with the extent of Ox42- or Iba1-immunoreactive activated microglia/macrophages. In the epilepsy model, ligand uptake was almost completely blocked by pre-injection of DPA-714 and FEDAA1106, another high-affinity TSPO ligand. Ligand uptake was independent of the degree of BBB opening and lesion size in the stroke model. We provide further strong evidence that DPA-714 is a specific ligand to image activated microglia/macrophages in experimental models of brain inflammation.