OBJECTIVE

To assess the effect of a single intravitreal injection of brain-derived neurotrophic factor (BDNF) on the survival of mouse retinal ganglion cells (RGCs) and on phagocytic microglia after intraorbital optic nerve transection (IONT).

METHODS

One week before IONT or processing, RGCs from pigmented C57/BL6 and albino Swiss mice were traced by applying hydroxystilbamidine methanesulfonate (OHSt) to both superior colliculi. Right afterward unilateral IONT, BDNF or vehicle were intravitreally administered. At increasing time intervals postlesion retinas were dissected as flat-mounts and subjected to BRN3A and Iba1 immunodetection. BRN3A(+)RGCs were automatically quantified in all retinas and their distribution was assessed using isodensity maps. In all retinas, the Iba1-positive and OHSt-filled microglial cells present in the ganglion cell layer were manually quantified. Their distribution was observed by neighbor maps.

RESULTS

When vehicle was administered, IONT-induced RGC death was significant at 3 days, while BDNF treatment delayed it to 5 days. At 14 days after BDNF or vehicle injection, 45% and 18% of RGCs had survived, respectively. There was a significant increase in OHSt-filled microglial cells in the right (contralateral) retinas after both treatments, without concurring with quantifiable RGC death. In the injured eye, the number of OHSt-filled microglial cells increased as the population of RGCs decreased and spread from central to peripheral areas.

CONCLUSIONS

In axotomized mouse retinas, a single intravitreal injection of BDNF protects RGCs throughout the whole retina. There is a strong contralateral response that involves microglial activation and OHSt phagocytosis.

The aim of this study is to clarify the relationship of microglia to phosphorylated tau accumulation and the characteristics of microglial activation in brain lesions of human tauopathies in comparison to mutant tau transgenic (TG) mice. We performed immunocytochemical analyses of brains from six patients with tauopathies, and 24 mice (18 TG mice expressing mutant tau P301L and six non-TG control mice, 11 to 27 months of age) using anti-tau antibodies and various microglial markers. In the tau TG, both semiquantitative severity ratings of microglial activation and an ultrastructural study were performed. In human tauopathies, Iba1- and major histocompatibility complex (MHC) class II-positive activated microglia increased in regions of phosphorylated tau (AT8) accumulation. The immunoreactivity of scavenger receptor class A (SRA) was present in some activated microglia, including phagocytic microglia in Alzheimer's disease (AD). Double-immunofluorescent analysis under a confocal microscope showed activated microglia at the vicinity of AT8-positive cells. Semiquantitative data of the TG and control mice indicated that the immunopositivity of AT8 was closely associated with the number of Iba1-positive microglia in the cortical area. Tau-associated microglia showed rare immunoreactivity for MHC class II antigen and SRA in the TG mice. Ultrastructurally, activated microglia with enlarged cytoplasm were located near neurons containing abnormal cytoskeletons. This comparative study of human tauopathies and tau TG mice indicated that microglial activation was closely related to phosphorylated tau accumulation, and that activated microglia of the TG mice may have the low expression of MHC class II and SRA compared with those of human tauopathies.

Chronic implantation of neurotransmitter measuring devices is essential for awake, behavioral studies occurring over multiple days. Little is known regarding the effects of long term implantation on surrounding brain parenchyma and the resulting alterations in the functional properties of this tissue. We examined the extent of tissue damage produced by chronic implantation of either ceramic microelectrode arrays (MEAs) or microdialysis probes. Histological studies were carried out on fixed tissues using stains for neurons (cresyl violet), astrocytes (GFAP), microglia (Iba1), glutamatergic nerve fibers (VGLUT1), and the blood-brain barrier (SMI-71). Nissl staining showed pronounced tissue body loss with microdialysis implants compared to MEAs. The MEAs produced mild gliosis extending 50-100 microm from the tracks, with a significant change in the affected areas starting at 3 days. By contrast, the microdialysis probes produced gliosis extending 200-300 microm from the track, which was significant at 3 and 7 days. Markers for microglia and glutamatergic fibers supported that the MEAs produce minimal damage with significant changes occurring only at 3 and 7 days that return to control levels by 1 month. SMI-71 staining supported the integrity of the blood-brain barrier out to 1 week for both the microdialysis probes and the MEAs. This data support that the ceramic MEA's small size and biocompatibility are necessary to accurately measure neurotransmitter levels in the intact brain. The minimal invasiveness of the MEAs reduce tissue loss, allowing for long term (>6 month) electrochemical and electrophysiological monitoring of brain activity.

Although activation of spinal glia has been implicated in the development of pathological pain, the mechanisms underlying glial activation are not fully understood. One such mechanism may be triggered by reaction to neuroactive substances released from central axons of sensory afferents. The vanilloid receptor TRPV1, a nonselective cation channel in nociceptive sensory afferents, mediates the release of neurotransmitters, such as glutamate and CGRP in the dorsal horn, which can subsequently activate glia. To test the hypothesis that activation of spinal glia is mediated, at least in part, by TRPV1, we studied the expression of markers for microglia (ionized calcium-binding adapter molecule 1, Iba1) and astrocytes (glial fibrillary acidic protein, GFAP) in the spinal cord of TRPV1 knockout mice (KO) vs. wild-type mice (WT) in models of acute (intraplantar capsaicin), inflammatory (adjuvant-induced arthritis, AIA), and neuropathic pain (partial sciatic nerve ligation, PSNL). We found that (i) naïve KO mice had denser immunostaining for both Iba1 and GFAP than naive WT mice; (ii) the immunostaining for Iba1 increased significantly in treated mice, compared to naïve mice, 3 days after capsaicin and 7-14 days after AIA or PSNL, and was significantly greater in WT than in KO mice 3 days after capsaicin, 7-14 days after AIA, and 7 days after PSNL; and iii) the immunostaining for GFAP increased significantly in treated mice, compared to naïve mice, 3 days after capsaicin and 14-21 days after AIA or PSNL, and was significantly greater in WT than in KO mice 14 days after AIA or PSNL. Our results suggest that TRPV1 plays a role in the activation of spinal glia in mice with nociceptive, inflammatory, and neuropathic pain.

OBJECTIVE

To characterize a model of optic nerve axonal degeneration induced by tumor necrosis factor (TNF)-alpha and to determine the role of nuclear factor (NF)-kappaB p65 in axonal degeneration.

METHODS

Groups of rats were euthanatized at 1 day, 1 or 2 weeks, or 1 or 2 months after intravitreal injection of TNF-alpha. Morphometric analyses of neurofilament- or Thy-1-positive cells, retinal ganglion cells (flat preparations stained with cresyl violet or retrograde labeling with a neurotracer), the number of axons, immunostaining for myelin basic protein, and TUNEL assays were performed. Levels of NF-kappaB p65 protein in retina and optic nerve were determined by Western blot analysis and immunohistochemistry. The effects of antisense oligodeoxynucleotide (AS ODN) against NF-kappaB p65 and helenalin, an inhibitor of NF-kappaB p65 activation, on TNF-alpha-induced optic nerve degeneration were determined by counting the number of axons.

RESULTS

Intravitreal injections of TNF-alpha induced obvious axonal loss and extensive degeneration of the axons from 2 weeks to 2 months after injection, whereas significant retinal ganglion cell loss was noted only at 2 months after injection. NF-kappaB p65 was increased in the optic nerve but not in the retina and was found to colocalize with ED-1 and Iba1, markers of microglia. Inhibition of NF-kappaB p65 with AS ODN or helenalin significantly ameliorated the effects of TNF-alpha-mediated axonal loss.

CONCLUSIONS

TNF-alpha causes axonal degeneration with probable delayed loss of retinal ganglion cell bodies. NF-kappaB p65 may play a pivotal role in axonal degeneration, with the possible involvement of microglial cells.

OBJECTIVE

Diabetic retinopathy (DR) is associated with microglial activation and increased levels of inflammatory cytokines. Genistein, a tyrosine kinase inhibitor, has been shown to possess anti-inflammatory potential that so far untested in animal models of diabetes. The aims of this study are to evaluate the efficacy of genistein for alleviation of diabetes-induced retinal inflammation and also to gain insight into the molecular mechanisms involved therein by analyzing the effect of genistein on concomitant microglia activation in the diabetic retina and in isolated cells.

METHODS

Streptozotocin (STZ)-induced diabetic Sprague Dawley rats were used. After diabetes was established for two weeks a single intravitreal injection of genistein or vehicle was performed. Forty-eight hours later, rats were killed, their retinal and vitreal samples were processed for Quantitative Real Time-PCR (qRT-PCR) and Enzyme-linked immunosorbent assay (ELISA) analyses, respectively. For the in vitro study, isolated microglial cells from retinas of newborn rats were used.

RESULTS

mRNA as well as protein levels for tumor necrosis factor α (TNF-α), a robust marker of inflammation, were increased in the retina early in the course of diabetes. Moreover, diabetes resulted in elevation of ionized calcium binding adaptor molecule-1 (Iba1) mRNA, known to be upregulated in activated microglia. These effects of diabetes in retina were all reduced by intervention treatment with genistein. Using an in vitro bioassay, we demonstrated the release of TNF-α from microglia activated by glycated albumin, a risk factor for diabetic disorders. This inflammatory signal involves the activation of tyrosine kinase and its subsequent events, ERK and P38 MAPKs. Genistein represses the release of TNF-α and significantly inhibits ERK and P38 phosphorylation in activated microglial cells by acting as a tyrosine kinase inhibitor.

CONCLUSIONS

These findings show genistein to be effective in dampening diabetes-induced retinal inflammation by interfering with inflammatory signaling (ERK and P38 MAPKs) that occurs in activated microglia. This beneficial effect of genistein may represent a new intervention therapy to modulate early pathological pathways long before the occurrence of vision loss among diabetics.

Microglia and macrophages appear to be the most common cells in the GBM microenvironment. In the present study we investigated the status of macrophages/microglia activation in surgical specimens from 41 patients diagnosed with grade IV GBM. For each patient we analyzed both the center of tumor and the parenchyma surrounding the tumor. The specimens were stained for: i) IBA1, a 17-kDa EF hand protein specifically expressed in microglia/macrophages ii) CD163, a cell surface antigen associated with M2 phenotype; iii) iNOS, taken as a functional marker of M1 phenotype, and iv) ARG-I, taken as a functional marker of M2 phenotype. Staining was scored in a double-blinded score on a scale from 0 to 5. Our results suggest that CD163 expression is higher within the tumor than in surrounding periphery in both male and female patients; while iNOS is higher within the tumor in males, no significant difference was found for ARG-1. In addition, analyzing the data in TGCA database, we found that CD163 expression was significantly and inversely correlated with mean survival times, with average survival times ranging from 448days in patients having low expression, to 319 in mid, and 353 in patients with high CD163 expressing tumors. In contrast, no significant association was found between survival time and ARG-1 or iNOS expression.

BACKGROUND

An episode of peripheral immune response may create long-lasting alterations in the neural network. Recent studies indicate a glial involvement in synaptic remodeling. Therefore it is postulated that both synaptic and glial changes could occur under the peripheral inflammation.

RESULTS

We tested this possibility by in vivo two-photon microscopy of dendritic spines after induction of a peripheral immune response by lipopolysaccharide (LPS) treatment of mice.We observed that the spines were less stable in LPS-treated mice. The accumulation of spine changes gradually progressed and remained low over a week after LPS treatment but became significantly larger at four weeks. Over eight weeks after LPS treatment, the fraction of eliminated spines amounted to 20% of the initial population and this persistent destabilization resulted in a reduction of the total spine density.We next evaluated glial activation by LPS administration. Activation of microglia was confirmed by a persistent increase of Iba1 immunoreactivity. Morphological changes in microglia were observed two days after LPS administration and were partially recovered within one week but sustained over a long time period.

CONCLUSIONS

These results indicate long-lasting aggravating effects of a single transient peripheral immune response on both spines and microglia. The parallel persistent alterations of both spine turnover and the state of microglia in vivo suggest the presence of a pathological mechanism that sustains the enhanced remodeling of neural networks weeks after peripheral immune responses. This pathological mechanism may also underlie long-lasting cognitive dysfunctions after septic encephalopathy in human patients.

Besides the two main classical features of amyloid beta aggregation and tau-containing neurofibrillary tangle deposition, neuroinflammation plays an important yet unclear role in the pathophysiology of Alzheimer's disease (AD). Microglia are believed to be key mediators of neuroinflammation during AD and responsible for the regulation of brain homeostasis by balancing neurotoxicity and neuroprotective events. We have previously reported evidence that neuritic plaques are derived from dead neurons that have accumulated intraneuronal amyloid and further recruit Iba1-positive cells, which play a role in either neuronal demise or neuritic plaque maturation or both.

To study the impact of microglia on neuritic plaque development, we treated two-month-old 5XFAD mice with a selective colony stimulation factor 1 receptor (CSF1R) inhibitor, PLX3397, for a period of 3 months, resulting in a significant ablation of microglia. Directly after this treatment, we analyzed the amount of intraneuronal amyloid and neuritic plaques and performed behavioral studies including Y-maze, fear conditioning and elevated plus maze.

We found that early long-term PLX3397 administration results in a dramatic reduction of both intraneuronal amyloid as well as neuritic plaque deposition. PLX3397 treated young 5XFAD mice also displayed a significant decrease of soluble fibrillar amyloid oligomers in brain lysates, a depletion of soluble pre-fibrillar oligomers in plasma and an improvement in cognitive function measured by fear conditioning tests.

Our findings demonstrate that CSF1R signaling, either directly on neurons or mediated by microglia, is crucial for the accumulation of intraneuronal amyloid and formation of neuritic plaques, suggesting that these two events are serially linked in a causal pathway leading to neurodegeneration and neuritic plaque formation. CSF1R inhibitors represent potential preventative or therapeutic approach that target the very earliest stages of the formation of intraneuronal amyloid and neuritic plaques.

Subarachnoid hemorrhage (SAH) can lead to disabling motor, cognitive, and neuropsychological abnormalities. Part of the secondary injury to cerebral tissues associated with SAH is attributable to the neuroinflammatory response induced by blood. Heparin is a pleiotropic compound that reduces inflammatory responses in conditions outside the central nervous system. Using a model of SAH devoid of global insult, we evaluated the effect of delayed intravenous (IV) infusion of heparin, at a dose that does not produce therapeutic anticoagulation, on neuroinflammation, myelin preservation, and apoptosis. Adult male rats underwent bilateral stereotactic injections of autologous blood (50 μL) into the subarachnoid space of the entorhinal cortex. The rats were implanted with mini-osmotic pumps that delivered either vehicle or unfractionated heparin (10 U/kg/h IV) beginning 12 h after SAH. No mechanical or hemorrhagic injury was observed in the hippocampus. In vehicle controls assessed at 48 h, SAH was associated with robust neuroinflammation in the adjacent cortex [neutrophils, activated phagocytic microglia, nuclear factor-kappa B, tumor necrosis factor-alpha, and interleukin-1beta] and neurodegeneration (Fluoro-Jade C staining and loss of NeuN). In the hippocampus, a muted neuroinflammatory response was indicated by Iba1-positive, ED1-negative microglia exhibiting an activated morphology. The perforant pathway showed Fluoro-Jade C staining and demyelination, and granule cells of the dentate gyrus had pyknotic nuclei, labeled with Fluoro-Jade C and showed upregulation of cleaved caspase-3, consistent with transsynaptic apoptosis. Administration of heparin significantly reduced neuroinflammation, demyelination, and transsynaptic apoptosis. We conclude that delayed IV infusion of low-dose unfractionated heparin may attenuate adverse neuroinflammatory effects of SAH.

BACKGROUND

Multiple sclerosis (MS) is often accompanied by optic nerve inflammation. And some patients experience permanent vision loss. We examined if the grade of optic nerve infiltration and demyelination affects the severity of clinical signs in an experimental autoimmune encephalomyelitis (EAE) model. The loss of retinal ganglion cells (RGC) and alterations in glia activity were also investigated.

METHODS

C57BL/6 mice were immunized with peptide MOG35-55 in complete Freund's adjuvant (CFA) and controls received PBS in CFA. Then 23 days post immunization eyes were prepared for flatmounts and stained with Nissl to evaluated neuronal density. Clinical EAE symptoms as well as cell infiltration and demyelination in the optic nerve were examined. Retinal sections were stained with hematoxylin and eosin and silver stain. Immunohistochemistry was used to label RGCs (Brn-3a), apoptotic cells (caspase 3), macroglia (glial fibrillary acidic protein (GFAP)), microglia (Iba1), macrophages (F 4/80) and interleukin-6 (IL-6) secretion.

RESULTS

EAE symptoms started at day 8 and peaked at day 15. Cell infiltrations (P = 0.0047) and demyelination (P = 0.0018) of EAE nerves correlated with the clinical score (r>> 0.8). EAE led to a significant loss of RGCs (P< 0.0001). Significantly more caspase 3+ cells were noted in these animals (P = 0.0222). They showed an increased expression of GFAP (P< 0.0002) and a higher number of microglial cells (P< 0.0001). Also more macrophages and IL-6 secretion were observed in EAE mice.

CONCLUSIONS

MOG immunization leads to optic neuritis and RGC loss. EAE severity is related to the severity of optic nerve inflammation and demyelination. EAE not only affects activation of apoptotic signals, but also causes a glial response in the retina.

BACKGROUND

Schizophrenia is a highly disabling psychiatric disorder with a proposed neurodevelopmental basis. One mechanism through which genetic and environmental risk factors might act is by triggering persistent brain inflammation, as evidenced by long-lasting neuro-immunological disturbances in patients. Our goal was to investigate whether microglia activation is a neurobiological correlate to the altered behaviour in the maternal immune activation (MIA) model, a well-validated animal model with relevance to schizophrenia. A recent observation in the MIA model is the differential maternal body weight response to the immune stimulus, correlated with a different behavioural outcome in the offspring. Although it is generally assumed that the differences in maternal weight response reflect differences in cytokine response, this has not been investigated so far. Our aim was to investigate whether (i) the maternal weight response to MIA reflects differences in the maternal cytokine response, (ii) the differential behavioural phenotype of the offspring extends to depressive symptoms such as anhedonia and (iii) there are changes in chronic microglia activation dependent on the behavioural phenotype.

METHODS

Based on a dose-response study, MIA was induced in pregnant rats by injecting 4mg/kg Poly I:C at gestational day 15. Serum samples were collected to assess the amount of TNF-α in the maternal blood following MIA. MIA offspring were divided into weight loss (WL; n=14) and weight gain (WG; n=10) groups, depending on the maternal body weight response to Poly I:C. Adult offspring were behaviourally phenotyped for prepulse inhibition, locomotor activity with and without amphetamine and MK-801 challenge, and sucrose preference. Finally, microglia activation was scored on CD11b- and Iba1-immunohistochemically stained sections.

RESULTS

Pregnant dams that lost weight following MIA showed increased levels of TNF-α compared to controls, unlike dams that gained weight following MIA. Poly I:C WL offspring showed the most severe behavioural outcome. Poly I:C WG offspring, on the other hand, did not show clear behavioural deficits. Most interestingly a reduced sucrose preference indicative of anhedonia was found in Poly I:C WL but not Poly I:C WG offspring compared to controls. Finally, there were no significant differences in microglia activation scores between any of the investigated groups.

CONCLUSIONS

The individual maternal immune response to MIA is an important determinant of the behavioural outcome in offspring, including negative symptoms such as anhedonia. We failed to find any significant difference in the level of microglia activation between Poly I:C WL, Poly I:C WG and control offspring.

BACKGROUND

Retinitis pigmentosa is a heterogeneous group of inherited neurodegenerative retinal disorders characterized by a progressive peripheral vision loss and night vision difficulties, subsequently leading to central vision impairment. Chronic microglia activation is associated with various neurodegenerative diseases including retinitis pigmentosa. The objective of this study was to quantify microglia activation in the retina of P23H rats, an animal model of retinitis pigmentosa, and to evaluate the therapeutic effects of TUDCA (tauroursodeoxycholic acid), which has been described as a neuroprotective compound.

METHODS

For this study, homozygous P23H line 3 and Sprague-Dawley (SD) rats were injected weekly with TUDCA (500 mg/kg, ip) or vehicle (saline) from 20 days to 4 months old. Vertical retinal sections and whole-mount retinas were immunostained for specific markers of microglial cells (anti-CD11b, anti-Iba1 and anti-MHC-II). Microglial cell morphology was analyzed and the number of retinal microglial was quantified.

RESULTS

Microglial cells in the SD rat retinas were arranged in regular mosaics homogenously distributed within the plexiform and ganglion cell layers. In the P23H rat retina, microglial cells increased in number in all layers compared with control SD rat retinas, preserving the regular mosaic distribution. In addition, a large number of amoeboid CD11b-positive cells were observed in the P23H rat retina, even in the subretinal space. Retinas of TUDCA-treated P23H animals exhibited lower microglial cell number in all layers and absence of microglial cells in the subretinal space.

CONCLUSIONS

These results report novel TUDCA anti-inflammatory actions, with potential therapeutic implications for neurodegenerative diseases, including retinitis pigmentosa.

OBJECTIVE

Oligodendrogenesis is essential for white matter repair after stroke. Although agonists of peroxisome proliferator-activated receptors γ confer neuroprotection in models of cerebral ischemia, it is not known whether this effect extends to white matter protection. This study tested the hypothesis that the peroxisome proliferator-activated receptors γ agonist rosiglitazone enhances oligodendrogenesis and improves long-term white matter integrity after ischemia/reperfusion.

METHODS

Male adult C57/BL6 mice (25-30 g) were subjected to 60-minute middle cerebral artery occlusion and reperfusion. Rosiglitazone (3 mg/kg) was injected intraperitoneally once daily for 14 days beginning 2 hours after reperfusion. Sensorimotor and cognitive functions were evaluated ≤21 days after middle cerebral artery occlusion. Immunostaining was used to assess infarct volume, myelin loss, and microglial activation. Bromodeoxyuridine (BrdU) was injected for measurements of proliferating NG2(+) oligodendrocyte precursor cells (OPCs) and newly generated adenomatous polyposis coli(+) oligodendrocytes. Mixed glial cultures were used to confirm the effect of rosiglitazone on oligodendrocyte differentiation and microglial polarization.

RESULTS

Rosiglitazone significantly reduced brain tissue loss, ameliorated white matter injury, and improved sensorimotor and cognitive functions for at least 21 days after middle cerebral artery occlusion. Rosiglitazone enhanced OPC proliferation and increased the numbers of newly generated mature oligodendrocytes after middle cerebral artery occlusion. Rosiglitazone treatment also reduced the numbers of Iba1(+)/CD16(+) M1 microglia and increased the numbers of Iba1(+)/CD206(+) M2 microglia after stroke. Glial culture experiments confirmed that rosiglitazone promoted oligodendrocyte differentiation, perhaps by promoting microglial M2 polarization.

CONCLUSIONS

Rosiglitazone treatment improves long-term white matter integrity after cerebral ischemia, at least, in part, by promoting oligodendrogenesis and facilitating microglial polarization toward the beneficial M2 phenotype.

The purpose of this study was to identify the CNS cellular constituent immunoreactive for specific P2X7 receptor antiserum in the kainate-induced seizure and non-seizure rat brain. Analysis of P2X7 immunocytochemistry (ICC) revealed small immunoreactive cells with processes showing distinct morphological changes as seizures progressed in time. These morphological changes were reminiscent of reactive glia during CNS injury. In order to determine the identity of this non-neuronal cellular constituent, we employed dual ICC techniques using sequential antibody incubations and reacted the sections with contrasting chromagens. Specific glial markers tested in the series included Iba1 (microglia), COX-1 (microglia), and GFAP (astroglia). Results of this study revealed distinct colocalization when sections immunostained for P2X7 were dual immunostained with antisera specific for microglia (Iba1, COX-1). In contrast, no colocalization was evident when sections were dual immunostained with P2X7 and GFAP, an astrocytic marker. In the latter experiment, dual ICC revealed two distinct cell populations with contrasting color demonstrating a population of distinct GFAP immunopositive cells and a population of distinct P2X7 immunopositive cells. We conclude that P2X7 antiserum used in this study is specific for and identifies microglia in rat and that there exists a timeline of progressive changes in microglia morphology that can be demonstrated following kainate-induced seizures. In addition, the morphological changes in microglia following seizure induction that can be identified with P2X7 antisera or with antisera specific for microglia suggest a neuroinflammatory milieu in areas of CNS seizure activity.

Classical immunohistochemical studies in the Alzheimer disease (AD) brain reveal prominent glial reactions, but whether this pathological feature is due primarily to cell proliferation or to a phenotypic change of existing resting cells remains controversial. We performed double-fluorescence immunohistochemical studies of astrocytes and microglia, followed by unbiased stereology-based quantitation in temporal cortex of 40 AD patients and 32 age-matched nondemented subjects. Glial fibrillary acidic protein (GFAP) and major histocompatibility complex II (MHC2) were used as markers of astrocytic and microglial activation, respectively. Aldehyde dehydrogenase 1 L1 and glutamine synthetase were used as constitutive astrocytic markers, and ionized calcium-binding adaptor molecule 1 (IBA1) as a constitutive microglial marker. As expected, AD patients had higher numbers of GFAP(+) astrocytes and MHC2(+) microglia than the nondemented subjects. However, both groups had similar numbers of total astrocytes and microglia and, in the AD group, these total numbers remained essentially constant over the clinical course of the disease. The GFAP immunoreactivity of astrocytes, but not the MHC2 immunoreactivity of microglia, increased in parallel with the duration of the clinical illness in the AD group. Cortical atrophy contributed to the perception of increased glia density. We conclude that a phenotypic change of existing glial cells, rather than a marked proliferation of glial precursors, accounts for the majority of the glial responses observed in the AD brain.

IFN regulatory factor (IRF) 8 is a transcription factor that has a key role in the cellular response to IFN-γ and is pivotal in myeloid cell differentiation. Whether IRF8 plays a role in the development and function of microglia, the tissue-resident myeloid cells of the brain, is unknown. Here, we show IRF8 is a constitutively produced nuclear factor in microglia, which suggested that IRF8 might also be a key homeostatic transcriptional determinant of the microglial cell phenotype. In support of this, in mice with a targeted disruption of the IRF8 gene, microglia were increased in number and showed gross alterations in morphology and surface area. In situ analysis of some key myeloid markers revealed that IRF8-deficient microglia had significantly reduced levels of Iba1, but increased levels of CD206 (mannose receptor) and F4/80 as well as increased tomato lectin binding. Analysis of microglia ex vivo revealed IRF8-deficient microglia had significantly increased levels of CD45, CD11b and F4/80, but significantly decreased levels of the chemokine receptors CCR2, CCR5 and CX3CR1. The known involvement of some of these molecular markers in membrane dynamics and phagocytosis led us to examine the phagocytic capacity of cultured IRF8-deficient microglia, however, this was found to be similar to wild type microglia. We conclude IRF8 is a constitutively produced nuclear factor in resident microglia of the CNS being a crucial transcriptional determinant of the phenotype of these cells in the healthy brain.

Activation of α-7 nicotinic acetylcholine receptor (α-7 nAchR) has a neuro-protective effect on ischemic and hemorrhagic stroke. However, the underlying mechanism is not completely understood. We hypothesized that α-7 nAchR agonist protects brain injury after ischemic stroke through reduction of pro-inflammatory macrophages (M1) and oxidative stress. C57BL/6 mice were treated with PHA568487 (PHA, α-7 nAchR agonist), methyllycaconitine (MLA, nAchR antagonist), or saline immediately and 24 hours after permanent occlusion of the distal middle cerebral artery (pMCAO). Behavior test, lesion volume, CD68(+), M1 (CD11b(+)/Iba1(+)) and M2 (CD206/Iba1+) microglia/macrophages, and phosphorylated p65 component of NF-kB in microglia/macrophages were quantified using histological stained sections. The expression of M1 and M2 marker genes, anti-oxidant genes and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase were quantified using real-time RT-PCR. Compared to the saline-treated mice, PHA mice had fewer behavior deficits 3 and 7 days after pMCAO, and smaller lesion volume, fewer CD68(+) and M1 macrophages, and more M2 macrophages 3 and 14 days after pMCAO, whereas MLA's effects were mostly the opposite in several analyses. PHA increased anti-oxidant genes and NADPH oxidase expression associated with decreased phosphorylation of NF-kB p65 in microglia/macrophages. Thus, reduction of inflammatory response and oxidative stress play roles in α-7 nAchR neuro-protective effect.

Recent studies have indicated an important role of ATP receptors in spinal microglia, such as P2Y12 or P2Y13, in the development of chronic pain. However, intracellular signaling cascade of these receptors have not been clearly elucidated. We found that intrathecal injection of 2-(methylthio)adenosine 5'-diphosphate (2Me-SADP) induced mechanical hypersensitivity and p38 mitogen-activated protein kinase (MAPK) phosphorylation in the spinal cord. Intrathecal administration of P2Y12/P2Y13 antagonists and Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor H1152 suppressed not only p38 MAPK phosphorylation, but also mechanical hypersensitivity induced by 2Me-SADP. In the rat peripheral nerve injury model, intrathecal administration of antagonists for the P2Y12/P2Y13 receptor suppressed activation of p38 MAPK in the spinal cord. In addition, subarachnoidal injection of H1152 also attenuated nerve injury-induced spinal p38 MAPK phosphorylation and neuropathic pain behavior, suggesting an essential role of ROCK in nerve injury-induced p38 MAPK activation. We also found that the antagonists of the P2Y12/P2Y13 receptor and H1152 had inhibitory effects on the morphological changes of microglia such as retraction of processes in both 2Me-SADP and nerve injured rats. In contrast these treatments had no effect on the number of Iba1-positive cells in the nerve injury model. Collectively, our results have demonstrated roles of ROCK in the spinal microglia that is involved in p38 MAPK activation and the morphological changes. Inhibition of ROCK signaling may offer a novel target for the development of a neuropathic pain treatment.

We examined the cerebroprotective mechanism of cannabidiol, the non-psychoactive component of marijuana, against infarction in a 4-h mouse middle cerebral artery (MCA) occlusion model. Cannabidiol was intraperitoneally administrated immediately before and 3h after cerebral ischemia. Infarct size and myeloperoxidase (MPO) activity, a marker of neutrophil, monocyte/macropharge, were measured at 24h after cerebral ischemia. Activated microglia and astrocytes were evaluated by immunostaining. Moreover, high-mobility group box1 (HMGB1) was also evaluated at 1 and 3 days after MCA occlusion. In addition, neurological score and motor coordination on the rota-rod test were assessed at 1 and 3 days after cerebral ischemia. Cannabidiol significantly prevented infarction and MPO activity at 20h after reperfusion. These effects of cannabidiol were not inhibited by either SR141716 or AM630. Cannabidiol inhibited the MPO-positive cells expressing HMGB1 and also decreased the expression level of HMGB1 in plasma. In addition, cannabidiol decreased the number of Iba1- and GFAP-positive cells at 3 days after cerebral ischemia. Moreover, cannabidiol improved neurological score and motor coordination on the rota-rod test. Our results suggest that cannabidiol inhibits monocyte/macropharge expressing HMGB1 followed by preventing glial activation and neurological impairment induced by cerebral ischemia. Cannabidiol will open new therapeutic possibilities for post-ischemic injury via HMGB1-inhibiting mechanism.

Mesenchymal stem cells (MSCs) have been shown to ameliorate a variety of neurological dysfunctions. This effect is believed to be mediated by their paracrine functions, since these cells rarely differentiate into neuronal cells. It is of clinical interest whether neural induction of MSCs is beneficial for the replacement therapy of neurological diseases. Here we report that expression of Neurogenin1 (Ngn1), a proneural gene that directs neuronal differentiation of progenitor cells during development, is sufficient to convert the mesodermal cell fate of MSCs into a neuronal one. Ngn1-expressing MSCs expressed neuron-specific proteins, including NeuroD and voltage-gated Ca2+ and Na+ channels that were absent in parental MSCs. Most importantly, transplantation of Ngn1-expressing MSCs in the animal stroke model dramatically improved motor functions compared with the parental MSCs. MSCs with Ngn1 populated the ischemic brain, where they expressed mature neuronal markers, including microtubule associated protein 2, neurofilament 200, and vesicular glutamate transporter 2, and functionally connected to host neurons. MSCs with and without Ngn1 were indistinguishable in reducing the numbers of Iba1+, ED1+ inflammatory cells, and terminal deoxynucleotidyl transferase dUTP nick-end labeling(+) apoptotic cells and in increasing the numbers of proliferating Ki67+ cells. The data indicate that in addition to the intrinsic paracrine functions of MSCs, motor dysfunctions were remarkably improved by MSCs able to transdifferentiate into neuronal cells. Thus, neural induction of MSCs is advantageous for the treatment of neurological dysfunctions.

Neonatal stroke occurs in one in 4,000 live births and leads to significant morbidity and mortality. Approximately two thirds of the survivors have long-term sequelae including seizures and neurological deficits. However, the pathophysiological mechanisms of recovery after neonatal stroke are not clearly understood, and preventive measures and treatments are nonexistent in the clinical setting. In this study, we investigated the effect of vascular endothelial growth factor (VEGF) treatment on histological recovery and angiogenic response to the developing brain after an ischemic insult. Ten-day-old Sprague-Dawley rats underwent right middle cerebral arterial occlusion (MCAO) for 1.5 h. Diffusion-weighted MRI during occlusion confirmed focal ischemia that was then followed by reperfusion. On group of animals received 5-bromo-2-deoxyuridine and sacrificed at postnatal day (P)18 or P25. A second group of animals was treated with VEGF (1.5 µg/kg, icv) or phosphate-buffered saline (PBS) at P18 and perfusion fixed at P25. Based on Nissl and iron staining, a single VEGF injection reduced the injury score, compared to the animals that underwent MCAO and PBS injection. Furthermore, neurodegeneration represented by neuronal nuclei staining was markedly diminished. In addition, animals treated with VEGF revealed a positive trend in endothelial proliferation and a significant increase in total vessel volume in the peri-infarct region of the caudate. The number of Iba1-positive microglial cells was significantly reduced after a single VEGF injection, and myelin basic protein expression was enhanced in the caudate after ischemia without an effect of VEGF treatment. In conclusion, delayed treatment with VEGF ameliorates injury, promotes endothelial cell proliferation, and increases total vascular volume following neonatal stroke. These results suggest that VEGF has a neuroprotective effect, in part by enhancing endogenous angiogenesis. These data contribute to a better understanding of neonatal stroke.

Latent changes in trigeminal ganglion structure and function resembling inflammatory conditions may predispose to acute attacks of migraine pain. Here, we investigated whether, in trigeminal sensory ganglia, cytokines such as TNFα might contribute to a local inflammatory phenotype of a transgenic knock-in (KI) mouse model of familial hemiplegic migraine type-1 (FHM-1). To this end, macrophage occurrence and cytokine expression in trigeminal ganglia were compared between wild type (WT) and R192Q mutant Ca(V)2.1 Ca(2+) channel (R192Q KI) mice, a genetic model of FHM-1. Cellular and molecular characterization was performed using a combination of confocal immunohistochemistry and cytokine assays. With respect to WT, R192Q KI trigeminal ganglia were enriched in activated macrophages as suggested by their morphology and immunoreactivity to the markers Iba1, CD11b, and ED1. R192Q KI trigeminal ganglia constitutively expressed higher mRNA levels of IL1β, IL6, IL10 and TNFα cytokines and the MCP-1 chemokine. Consistent with the report that TNFα is a major factor to sensitize trigeminal ganglia, we observed that, following an inflammatory reaction evoked by LPS injection, TNFα expression and macrophage occurrence were significantly higher in R192Q KI ganglia with respect to WT ganglia. Our data suggest that, in KI trigeminal ganglia, the complex cellular and molecular environment could support a new tissue phenotype compatible with a neuroinflammatory profile. We propose that, in FHM patients, this condition might contribute to trigeminal pain pathophysiology through release of soluble mediators, including TNFα, that may modulate the crosstalk between sensory neurons and resident glia, underlying the process of neuronal sensitisation.

Progranulin (PGRN), a multifunctional growth factor, appears to play a role in neurodegenerative diseases accompanied by neuroinflammation. In this study, we investigated the role of PGRN in neuroinflammation, especially in the activation of microglia, by means of experimental traumatic brain injury (TBI) in the cerebral cortex of mice. The expression of GRN mRNA was increased in association with neuroinflammation after TBI. Double-immunohistochemical study showed that PGRN-immunoreactive (-IR) cells were mainly overlapped with CD68-IR cells, suggesting that the main source of PGRN was CD68-positive activated microglia. To investigate the role of PGRN in inflammatory responses related to activated microglia, we compared the immunoreactivity and expression of ionized calcium-binding adaptor molecule 1 (Iba1), CD68, and CD11b as markers for activated microglia between wild-type (WT) and GRN-deficient (KO) mice. The number of Iba1- and CD11b-IR cells and gene expression of Iba1 and CD11b were not significantly different between WT and KO mice, while the number of CD68-IR cells and CD68 expression in KO mice were significantly greater than those in WT mice. Double-immunohistochemical study showed that CD68-IR microglia were also IR for TGFβ1, and TGFβ1 expression and Smad3 phosphorylation in KO mice were elevated compared to WT mice. Moreover, double-immunostaining between phospho-Smad3 and glial fibrillary acidic protein suggested increased TGFβ1-Smad3 signal mainly by astrocytes. The levels of protein carbonyl groups, which reflect protein oxidation, and laminin immunoreactivity, which is associated with angiogenesis, were also significantly increased in KO mice compared to WT mice. These results suggest that PGRN is produced in CD68-positive microglia and suppresses excessive inflammatory responses related to activated microglia after TBI in mice.