Microglia are the resident immune cells in the spinal cord and brain. Mounting evidence suggests that activation of microglia plays an important role in the pathogenesis of chronic pain, including chronic orofacial pain. In particular, microglia contribute to the transition from acute pain to chronic pain, as inhibition of microglial signaling reduces pathologic pain after inflammation, nerve injury, and cancer but not baseline pain. As compared with inflammation, nerve injury induces much more robust morphologic activation of microglia, termed microgliosis, as shown by increased expression of microglial markers, such as CD11b and IBA1. However, microglial signaling inhibitors effectively reduce inflammatory pain and neuropathic pain, arguing against the importance of morphologic activation of microglia in chronic pain sensitization. Importantly, microglia enhance pain states via secretion of proinflammatory and pronociceptive mediators, such as tumor necrosis factor α, interleukins 1β and 18, and brain-derived growth factor. Mechanistically, these mediators have been shown to enhance excitatory synaptic transmission and suppress inhibitory synaptic transmission in the pain circuits. While early studies suggested a predominant role of microglia in the induction of chronic pain, further studies have supported a role of microglia in the maintenance of chronic pain. Intriguingly, recent studies show male-dominant microglial signaling in some neuropathic pain and inflammatory pain states, although both sexes show identical morphologic activation of microglia after nerve injury. In this critical review, we provide evidence to show that caspase 6-a secreted protease that is expressed in primary afferent axonal terminals surrounding microglia-is a robust activator of microglia and induces profound release of tumor necrosis factor α from microglia via activation of p38 MAP kinase. The authors also show that microglial caspase 6/p38 signaling is male dominant in some inflammatory and neuropathic pain conditions. Finally, the authors discuss the relevance of microglial signaling in chronic trigeminal and orofacial pain.

The substantia gelatinosa (SG) of the spinal cord processes incoming painful information to ascending projection neurons. Whole-cell patch clamp recordings from SG spinal cord slices documented that in a low Ca(2+) /no Mg(2+) (low X(2+) ) external medium adenosine triphosphate (ATP)/dibenzoyl-ATP, Bz-ATP) caused inward current responses, much larger in amplitude than those recorded in a normal X(2+) -containing bath medium. The effect of Bz-ATP was antagonized by the selective P2X7 receptor antagonist A-438079. Neuronal, but not astrocytic Bz-ATP currents were strongly inhibited by a combination of the ionotropic glutamate receptor antagonists AP-5 and CNQX. In fact, all neurons and some astrocytes responded to NMDA, AMPA, and muscimol with inward current, demonstrating the presence of the respective receptors. The reactive oxygen species H2 O2 potentiated the effect of Bz-ATP at neurons but not at astrocytes. Hippocampal CA1 neurons exhibited a behavior similar to, but not identical with SG neurons. Although a combination of AP-5 and CNQX almost abolished the effect of Bz-ATP, H2 O2 was inactive. A Bz-ATP-dependent and A-438079-antagonizable reactive oxygen species production in SG slices was proven by a microelectrode biosensor. Immunohistochemical investigations showed the colocalization of P2X7-immunoreactivity with microglial (Iba1), but not astrocytic (GFAP, S100β) or neuronal (MAP2) markers in the SG. It is concluded that SG astrocytes possess P2X7 receptors; their activation leads to the release of glutamate, which via NMDA- and AMPA receptor stimulation induces cationic current in the neighboring neurons. P2X7 receptors have a very low density under resting conditions but become functionally upregulated under pathological conditions.

Although significant effects of interferon-gamma (IFN-gamma) on glial cells are well documented, information on the expression level and localization of glial IFN-gamma receptors (IFN-gamma-R) in the human central nervous system (CNS) is sparse. To examine the glial expression of IFN-gamma-R in the human CNS, immunohistochemistry and quantitative analyses were performed on Alzheimer disease hippocampus, Parkinson disease substantia nigra, amyotrophic lateral sclerosis spinal cord and corresponding areas from non-neurological cases. Almost all IFN-gamma-R-positive (IFN-gamma-R(+)) cells corresponded to GFAP-positive (GFAP(+)) astrocytes, while none of IFN-gamma-R(+) cells corresponded to IBA1-positive (IBA1(+)) microglia or MBP-positive (MBP(+)) oligodendrocytes in these neurological cases. We observed a similar pattern of glial IFN-gamma-R expression in non-neurological cases. Also, we quantitatively analyzed the IFN-gamma-R expression by cultured human glial cells using immunocytochemistry and reverse transcription polymerase chain reaction (RT-PCR). In contrast to in vivo results, almost all IFN-gamma-R(+) cells were IBA1(+) in microglial cultures, GFAP(+) in astrocytic cultures and MBP(+) in oligodendrocytic cultures. Moreover, no significant difference in IFN-gamma-R mRNA expression was found for these glial cell types by RT-PCR. These results suggest that the microglial and oligodendrocytic expression levels of IFN-gamma-R are much lower than the astrocytic expression levels in the human CNS in vivo, whereas all three types of glial cells constitutively express IFN-gamma-R when cultured in vitro.

Excitotoxicity is well known in the neuronal death in the brain and is also linked to neuronal damages in the retina. Recent accumulating evidence show that microglia greatly affect excitotoxicity in the brain, but their roles in retina have received only limited attention. Here, we report that retinal excitotoxicity is mediated by microglia. To this end, we employed three discrete methods, that is, pharmacological inhibition of microglia by minocycline, pharmacological ablation by an antagonist for colony stimulating factor 1 receptor (PLX5622), and genetic ablation of microglia using Iba1-tTA::DTAtetO/tetO mice. Intravitreal injection of NMDA increased the number of apoptotic retinal ganglion cells (RGCs) followed by reduction in the number of RGCs. Although microglia did not respond to NMDA directly, they became reactive earlier than RGC damages. Inhibition or ablation of microglia protected RGCs against NMDA. We found up-regulation of proinflammatory cytokine genes including Il1b, Il6 and Tnfa, among which Tnfa was selectively blocked by minocycline. PLX5622 also suppressed Tnfa expression. Tumor necrosis factor α (TNFα) signals were restricted in microglia at very early followed by spreading into other cell types. TNFα up-regulation in microglia and other cells were significantly attenuated by minocycline and PLX5622, suggesting a central role of microglia for TNFα induction. Both inhibition of TNFα and knockdown of TNF receptor type 1 by siRNA protected RGCs against NMDA. Taken together, our data demonstrate that a phenotypic change of microglia into a neurotoxic one is a critical event for the NMDA-induced degeneration of RGCs, suggesting an importance of non-cell-autonomous mechanism in the retinal neuronal excitotoxicity.

The preferred fixative for whole eyes is Davidson's solution, which provides optimal tissue preservation while avoiding retinal detachment. Hitherto, the compatibility of Davidson's solution with immunohistochemistry has been largely untested. The goal of the present study was to compare the immunolabeling patterns of a wide-ranging panel of commercially available, previously validated antibodies in formalin- and Davidson's-fixed retinas. Immunohistochemistry was performed in normal pigmented rat eyes and, to facilitate localization of inducible proteins, eyes injected with the bacterial toxin lipopolysaccharide or subjected to laser-induced photoreceptor damage. Specificity of labeling was judged by the morphology and distribution of immunopositive cells, by the absence of signal in appropriate controls, and by comparison with expected staining patterns. Retinas fixed in formalin displayed only adequate morphological integrity but were highly compatible with all 39 antibodies evaluated. Retinas fixed in Davidson's solution displayed morphological integrity superior to those fixed in formalin. Generally, the cellular and subcellular patterns and intensities of immunoreactivities obtained with each fixative were identical; however, Davidson's fixative was less compatible with certain antibodies, such as the neurotransmitter γ-aminobutyric acid, the microglial marker iba1, the macroglial stress protein nestin, and the small heat shock proteins Hsp27 and αB-crystallin, shortfalls that somewhat temper enthusiasm concerning its use.

Growth arrest-specific protein 6 (GAS6) is a soluble agonist of the TYRO3, AXL, MERTK (TAM) family of receptor tyrosine kinases identified to have anti-inflammatory, neuroprotective, and promyelinating properties. During experimental autoimmune encephalomyelitis (EAE), wild-type (WT) mice demonstrate a significant induction of Gas6, Axl, and Mertk but not Pros1 or Tyro3 mRNA. We tested the hypothesis that intracerebroventricular delivery of GAS6 directly into the CNS of WT mice during myelin oligodendrocyte glycoprotein (MOG)-induced EAE would improve the clinical course of disease relative to artificial CSF (ACSF)-treated mice. GAS6 did not delay disease onset, but significantly reduced the clinical scores during peak and chronic EAE. Mice receiving GAS6 for 28 d had preserved SMI31(+) neurofilament immunoreactivity, significantly fewer SMI32(+) axonal swellings and spheroids and less demyelination relative to ACSF-treated mice. Alternate-day subcutaneous IFNβ injection did not enhance GAS6 treatment effectiveness. Gas6(-/-) mice sensitized with MOG35-55 peptide exhibit higher clinical scores during late peak to early chronic disease, with significantly increased SMI32(+) axonal swellings and Iba1(+) microglia/macrophages, enhanced expression of several proinflammatory mRNA molecules, and decreased expression of early oligodendrocyte maturation markers relative to WT mouse spinal cords with scores for 8 consecutive days. During acute EAE, flow cytometry showed significantly more macrophages but not T-cell infiltrates in Gas6(-/-) spinal cords than WT spinal cords. Our data are consistent with GAS6 being protective during EAE by dampening the inflammatory response, thereby preserving axonal integrity and myelination.

Toxoplasma gondii is a pathogen implicated in psychiatric disorders. As elevated antibodies to T. gondii are also present in non-symptomatic individuals, we hypothesized that the age during first exposure to the pathogen may affect symptom manifestation. We tested this hypothesis by evaluating neurobehavioral abnormalities and the immune response in mice following adolescent or adult T. gondii infection.

Mice were infected with T. gondii at postnatal day 33 (adolescent/juvenile) or 61 (adult). At 8weeks post-infection (wpi), pre-pulse inhibition of the acoustic startle (PPI) in mice administered MK-801 (0.1 and 0.3mg/kg) and amphetamine (5 and 10mg/kg) was assessed. Peripheral (anti-T. gondii, C1q-associated IgG and anti-GLUN2 antibodies) and central (C1q and Iba1) markers of the immune response were also evaluated. In addition, regional brain expression of N-methyl-d-aspartate receptor (NMDAR) subunits (GLUN1 and GLUN2A), glutamatergic (vGLUT1, PSD95) and GABAergic (GAD67) markers, and monoamines (DA, NE, 5-HT) and their metabolites were measured.

Juvenile and adult infected mice exhibited opposite effects of MK-801 on PPI, with decreased PPI in juveniles and increased PPI in adults. There was a significantly greater elevation of GLUN2 autoantibodies in juvenile-compared to adult-infected mice. In addition, age-dependent differences were found in regional expression of NMDAR subunits and markers of glutamatergic, GABAergic, and monoaminergic systems. Activated microglia and C1q elevations were found in both juvenile- and adult-T. gondii infected mice.

Our study demonstrates that the age at first exposure to T. gondii is an important factor in shaping distinct behavioral and neurobiological abnormalities. Elevation in GLUN2 autoantibodies or complement protein C1q may be a potential underlying mechanism. A better understanding of these age-related differences may lead to more efficient treatments of behavioral disorders associated with T. gondii infection.

OBJECTIVE

Obstructive sleep apnea (OSA) induces cognitive impairment that involves intermittent hypoxia (IH). Because OSA is recognized as a low-grade systemic inflammatory disease and only some patients develop cognitive deficits, we investigated whether IH-related brain consequences shared similar pathophysiology and required additional factors such as systemic inflammation to develop.

METHODS

Nine-week-old male C57BL/6J mice were exposed to 1 day, 6 or 24 w of IH (alternating 21-5% FiO2 every 30 sec, 8 h/day) or normoxia. Microglial changes were assessed in the functionally distinct dorsal (dH) and ventral (vH) regions of the hippocampus using Iba1 immunolabeling. Then the study concerned dH, as vH only tended to be lately affected. Seven proinflammatory and anti-inflammatory cytokine messenger RNA (mRNA) were assessed at all time points using semiquantitative real-time reverse transcription polymerase chain reaction (RT-PCR). Similar mRNA analysis was performed after 6 w IH or normoxia associated for the past 3 w with repeated intraperitoneal low-dose lipopolysaccharide or saline.

RESULTS

Chronic (6, 24 w) but not acute IH induced significant microglial changes in dH only, including increased density and morphological features of microglia priming. In dH, acute but not chronic IH increased IL-1β and RANTES/CCL5 mRNA, whereas the other cytokines remained unchanged. In contrast, chronic IH plus lipopolysaccharide increased interleukin (IL)-6 and IL10 mRNA whereas lipopolysaccharide alone did not affect these cytokines.

CONCLUSIONS

The obstructive sleep apnea component intermittent hypoxia (IH) causes low-grade neuroinflammation in the dorsal hippocampus of mice, including early but transient cytokine elevations, delayed but long-term microglial changes, and cytokine response alterations to lipopolysaccharide inflammatory challenge. These changes may contribute to IH-induced cognitive impairment and pathological brain aging.

Postoperative cognitive dysfunction (POCD) is a significant cause of morbidity after surgery, especially for the elderly. Accumulating evidence has demonstrated that neuroinflammation plays a key role in the pathogenesis of POCD. Thus, we hypothesized that berberine, an isoquinoline alkaloid with anti-inflammatory effects, could improve surgery-induced cognitive impairment. Twenty-month-old male C57BL/6 mice were subjected to exploratory laparotomy with isoflurane anesthesia to mimic the clinical human abdominal surgery. For the interventional studies, mice received berberine (10mg/kg) or vehicle intraperitoneally. For the in vitro study, we examined the effects of berberine on lipopolysaccharide (LPS)-induced inflammatory mediators by cultured BV2 cells. Behavioral tests, expressions of IBA1, tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and IL-6 were performed at the indicated time points. In the present study, we showed that surgery impaired the contextual fear memory, as evidenced by the significantly decreased freezing time to the context. This behavioral change coincided with marked increases in IBA1, TNF-α, IL-1β, and IL-6 in the prefrontal cortex and hippocampus only at 24h but not 7 d after surgery. In BV2 cells, LPS induced significantly increased TNF-α and IL-1β expressions. Notably, berberine treatment rescued surgery-induced cognitive impairment and inhibited the release of IBA1, IL-1β, and IL-6 in the hippocampus. In line with the in vivo study, berberine treatment suppressed LPS-stimulated production of TNF-α and IL-1β in BV2 cells. In conclusion, our study suggests that berberine could alleviate POCD by suppressing neuroinflammation in aged mice.

Neuroinflammation plays an important role in the progression of Parkinson's disease (PD) and hence may represent a target for treatment. The drugs used currently for PD only provide symptomatic relief and have adverse effects in addition to their inability in preventing degeneration of neurons. Flavonoids show potent antioxidant and anti-inflammatory activities which is very valuable for the health of human beings. Thus, in the present study, we have tried to explore the anti-inflammatory activity of orally given ursolic acid (UA) (25 mg/kg bwt), a pentacyclic triterpenoid in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-intoxicated mouse model. Significant severe oxidative stress and biochemical alterations have been seen in Parkinsonian mice after MPTP intoxication. Whereas, UA administration has significantly rescued the harmful consequence of MPTP intoxication. Ionized calcium-binding adaptor molecule 1 (Iba1), tumor necrosis factor-alpha (TNF-α), and nuclear transcription factor-κB (NF-κB) were seen to be altered in the substantia nigra pars compacta (SNpc) of MPTP-intoxicated mice through immunohistochemical studies. The changes in the expression level of these parameters primarily suggest increased inflammatory responses in MPTP-intoxicated mice as compared with the control. However, UA have significantly reduced these inflammatory parameters (Iba1 and TNF-α) along with transcription factor NF-κB, which regulates these inflammatory parameters and thus have inhibited MPTP-induced neuroinflammation. The immunoreactivity of tyrosine hydroxylase (TH) was considerably increased by UA treatment in the SNpc of Parkinsonian mice. The neuroinflammation and neurodegeneration along with impairments in biochemical and behavioral parameters were found to be reversed on treatment with UA. Thus, UA has shown potent anti-inflammatory activity by preventing the degeneration of dopaminergic neurons from MPTP-induced Parkinsonian mice.

UNASSIGNED

Inflammation is hypothesized to be a key event in the growth of sporadic vestibular schwannoma (VS). In this study we sought to investigate the relationship between inflammation and tumor growth in vivo using the PET tracer 11C-(R)-PK11195 and dynamic contrast enhanced (DCE) MRI derived vascular biomarkers.

UNASSIGNED

Nineteen patients with sporadic VS (8 static, 7 growing and 4 shrinking tumors), underwent prospective imaging with dynamic 11C-(R)-PK11195 PET and a comprehensive MR protocol; including high temporal resolution DCE-MRI in fifteen patients. An inter-tumor comparison of 11C-(R)-PK11195 binding potential (BPND) and DCE-MRI derived vascular biomarkers (Ktrans, vp, ve) across the three different tumor growth cohorts was undertaken. Tissue of eight tumors was examined with immunohistochemistry markers for inflammation (Iba1), neoplastic cells (S-100 protein), vessels (CD31), the PK11195 target Translocator protein (TSPO), fibrinogen for vascular permeability, and proliferation (Ki-67). Results were correlated with PET and DCE-MRI data.

UNASSIGNED

Compared to static tumors, growing VS displayed significantly higher mean 11C-(R)-PK11195 BPND (-0.07 vs 0.47, p=0.020), and higher mean tumor Ktrans (0.06 vs 0.14, p=0.004). Immunohistochemistry confirmed the imaging findings and demonstrated that TSPO is predominantly expressed in macrophages. Within growing VS, macrophages rather than tumor cells accounted for the majority of proliferating cells.

UNASSIGNED

We present the first in vivo imaging evidence of increased inflammation within growing sporadic VS. Our results demonstrate that 11C-(R)-PK11195 specific binding and DCE-MRI derived parameters can be used as imaging biomarkers of inflammation and vascular permeability in this tumor group.

Sporadic Alzheimer's disease (AD) is marked by a lengthy preclinical phase during which patients are nonsymptomatic but show pathology in variable manifestations. Whether or not neuroinflammation occurs in such nondemented individuals is unknown. We evaluated the medial temporal lobe of 66 nondemented subjects, aged 42-93, in terms of tau pathology, Aβ deposition, and microglial activation. We show that 100% of subjects had neurofibrillary degeneration (NFD), 35% had Aβ deposits, and 8% revealed microglial activation in individuals where early amyloid formation was apparent by Congo Red staining. Amyloid-induced neuroinflammatory clusters of Iba1, CD68, and ferritin-positive microglia were evident in the immediate vicinity of aggregated Aβ. Microglia in the adjacent neuropil were nonactivated. Thus, neuroinflammation in AD represents a highly localized phagocyte reaction, essentially a foreign body response, geared toward removal of insoluble Aβ. Because clustered microglia in some amyloid plaques were dystrophic and ferritin-positive, we hypothesize that these cells were exhausted by their attempts to remove the aggregated, insoluble Aβ. Our findings show that the sequence of pathologic events in AD begins with tau pathology, followed by Aβ deposition, and then by microglial activation. Because only 8% of our subjects revealed all three hallmark pathologic features, we propose that these nondemented individuals were near the threshold of transitioning from nonsymptomatic to symptomatic disease. The onset of neuroinflammation in AD may thus represent a tipping point in AD pathogenesis. Our study suggests that the role of microglia in AD pathogenesis entails primarily the attempted removal of potentially toxic, extracellular material.

In acute stage of ischemic stroke, the surrounding zone of fresh infarcts is termed penumbra, where microglia are activated in response to damaged cell-derived proinflammatory mediators. Rescuing penumbra by regulating inflammatory activity would minimize infarct volume, which positively correlates with functional outcome. To elucidate mechanisms by which inflammation occurs in penumbra, we performed immunohistochemical investigations using autopsied human brains affected by acute, subacute and chronic stages of cerebral infarction as well as cell culture experiments using a murine microglia-derived cell line (BV-2). In penumbra of fresh infarcts, immunoreactivity for secretory phospholipase A2 group X (sPLA2 -X), which is responsible for the production and release of the proinflammatory mediator lysophosphatidylcholine (LPC), was intensely detected in neurons and astrocytes. Furthermore, immunoreactivities for the LPC receptors G protein-coupled receptor 132 (G2A) and P2X purinoreceptor 7 (P2X7R), as well as the CC chemokine monocyte chemoattractant protein-1 (MCP-1) and its receptor CCR2, were detectable in activated microglia. Prior to cell culture experiments, it was confirmed that BV-2 cells were immunoreactive for ionized Ca(2+) -binding adaptor molecule 1 (Iba1), G2A, P2X7R, MCP-1 and CCR2. Reverse transcription-quantitative polymerase chain reaction analysis revealed that MCP-1 and CCR2 mRNA expression levels were significantly increased by LPC stimulation. The LPC-driven increase in MCP-1 transcripts was lowered by blockade of G2A or P2X7R or by inhibition of Rho-associated protein kinase (ROCK) or inhibitor of κBα kinase. The LPC-driven increase in CCR2 transcripts was lowered by blockade of G2A or P2X7R or by inhibition of ROCK, phosphatidylinositide 3-kinanse, extracellular signal-regulated kinase kinase, or p38 mitogen-activated protein kinase. The present results provide in vivo and in vitro evidence that in acute stage of ischemic stroke, the sPLA2 -X enzyme product LPC is released from neurons and astrocytes and stimulates penumbra microglia via G2A and P2X7R, thereby exerting the MCP-1/CCR2-mediated neurotoxicity through distinct cell-signaling pathways.

Ischemic stroke is a clinically common cerebrovascular disease whose main risks include necrosis, apoptosis and cerebral infarction, all caused by cerebral ischemia and reperfusion (I/R) injury. This process has particular significance for the treatment of stroke patients. Notoginseng leaf triterpenes (PNGL), as a valuable medicine, have been discovered to have neuroprotective effects. However, it was not confirmed that whether PNGL may possess neuroprotective effects against cerebral I/R injury. To explore the neuroprotective effects of PNGL and their underlying mechanisms, a middle cerebral artery occlusion/reperfusion (MCAO/R) model was established. In vivo results suggested that in MCAO/R model rats, PNGL pretreatment (73.0, 146, 292 mg/kg) remarkably decreased infarct volume, reduced brain water content, and improved neurological functions; moreover, PNGL (73.0, 146, 292 mg/kg) significantly alleviated blood-brain barrier (BBB) disruption and inhibited neuronal apoptosis and neuronal loss caused by cerebral I/R injury, while PNGL with a different concertation (146, 292 mg/kg) significantly reduced the concentrations of IL-6, TNF-α, IL-1 β, and HMGB1 in serums in a dose-dependent way, which indicated that inflammation inhibition could be involved in the neuroprotective effects of PNGL. The immunofluorescence and western blot analysis showed PNGL decreased HMGB1 expression, suppressed the HMGB1-triggered inflammation, and inhibited microglia activation (IBA1) in hippocampus and cortex, thus dose-dependently downregulating inflammatory cytokines including VCAM-1, MMP-9, MMP-2, and ICAM-1 concentrations in ischemic brains. Interestingly, PNGL administration (146 mg/kg) significantly downregulated the levels of p-P44/42, p-JNK1/2 and p-P38 MAPK, and also inhibited expressions of the total NF-κB and phosphorylated NF-κB in ischemic brains, which was the downstream pathway triggered by HMGB1. All of these results indicated that the protective effects of PNGL against cerebral I/R injury could be associated with inhibiting HMGB1-triggered inflammation, suppressing the activation of MAPKs and NF-κB, and thus improved cerebral I/R-induced neuropathological changes. This study may offer insight into discovering new active compounds for the treatment of ischemic stroke.

Reactive astrocytes and activated microglia are the key players in several pathophysiologic modifications of the central nervous system. We used the spared nerve injury (SNI) of the sciatic nerve to induce glial maladaptive response in the ventral horn of lumbar spinal cord and examine its role in the remodeling of the tripartite synapse plasticity. Imaging the ventral horn revealed that SNI was associated with both an early microglial and astrocytic activation, assessed, respectively, by analysis of Iba1 and GFAP expression. Microglia, in particular, localized peculiarly surrounding the motor neurons somata. Perineuronal astrocytes, which play a key role in maintaining the homeostasis of neuronal circuitry, underwent a substantial phenotypic change following peripheral axotomy, producing reactive gliosis. The gliosis was associated with the reduction of glial aminoacid transporters (GLT1 and GlyT1) and increase of neuronal glutamate transporter EAAC1. Although the expression of GABAergic neuronal marker GAD65/67 showed no change, glutamate increase, as demonstrated by HPLC analysis, shifted the excitatory/inhibitory balance as showed by the net increase of the glutamate/GABA ratio. Moreover, endogenous NGF levels were altered in SNI animals and not restored by the intrathecal NGF administration. This treatment reverted phenotypic changes associated with reactive astrocytosis, but failed to modify microglia activation. These findings on one hand confirm the correlation between gliopathy and maladaptive plasticity of the spinal synaptic circuitry, on the other hand add new data concerning the complex peculiar behavior of different glial cells in neuronal degenerative processes, defining a special role of microglia in sustaining the inflammatory response.

The human inner ear, which is segregated by a blood/labyrinth barrier, contains resident macrophages [CD163, ionized calcium-binding adaptor molecule 1 (IBA1)-, and CD68-positive cells] within the connective tissue, neurons, and supporting cells. In the lateral wall of the cochlea, these cells frequently lie close to blood vessels as perivascular macrophages. Macrophages are also shown to be recruited from blood-borne monocytes to damaged and dying hair cells induced by noise, ototoxic drugs, aging, and diphtheria toxin-induced hair cell degeneration. Precise monitoring may be crucial to avoid self-targeting. Macrophage biology has recently shown that populations of resident tissue macrophages may be fundamentally different from circulating macrophages. We removed uniquely preserved human cochleae during surgery for treating petroclival meningioma compressing the brain stem, after ethical consent. Molecular and cellular characterization using immunofluorescence with antibodies against IBA1, TUJ1, CX3CL1, and type IV collagen, and super-resolution structured illumination microscopy (SR-SIM) were made together with transmission electron microscopy. The super-resolution microscopy disclosed remarkable phenotypic variants of IBA1 cells closely associated with the spiral ganglion cells. Monitoring cells adhered to neurons with "synapse-like" specializations and protrusions. Active macrophages migrated occasionally nearby damaged hair cells. Results suggest that the human auditory nerve is under the surveillance and possible neurotrophic stimulation of a well-developed resident macrophage system. It may be alleviated by the non-myelinated nerve soma partly explaining why, in contrary to most mammals, the human's auditory nerve is conserved following deafferentiation. It makes cochlear implantation possible, for the advantage of the profoundly deaf. The IBA1 cells may serve additional purposes such as immune modulation, waste disposal, and nerve regeneration. Their role in future stem cell-based therapy needs further exploration.

Recent evidence suggests that transient receptor potential melastatin 2 (TRPM2) expressed in immune cells plays an important role in immune and inflammatory responses. We recently reported that TRPM2 expressed in macrophages and spinal microglia contributes to the pathogenesis of inflammatory and neuropathic pain aggravating peripheral and central pronociceptive inflammatory responses in mice. To further elucidate the contribution of TRPM2 expressed by peripheral immune cells to neuropathic pain, we examined the development of peripheral nerve injury-induced neuropathic pain and the infiltration of immune cells (particularly macrophages) into the injured nerve and spinal cord by using bone marrow (BM) chimeric mice by crossing wildtype (WT) and TRPM2-knockout (TRPM2-KO) mice. Four types of BM chimeric mice were prepared, in which irradiated WT or TRPM2-KO recipient mice were transplanted with either WT-or TRPM2-KO donor mouse-derived green fluorescence protein-positive (GFP(+)) BM cells (TRPM2(BM+/Rec+), TRPM2(BM-/Rec+), TRPM2(BM+/Rec-), and TRPM2(BM-/Rec-) mice). Mechanical allodynia induced by partial sciatic nerve ligation observed in TRPM2(BM+/Rec+) mice was attenuated in TRPM2(BM-/Rec+), TRPM2(BM+/Rec-), and TRPM2(BM-/Rec-) mice. The numbers of GFP(+) BM-derived cells and Iba1/GFP double-positive macrophages in the injured sciatic nerve did not differ among chimeric mice 14 days after the nerve injury. In the spinal cord, the number of GFP(+) BM-derived cells, particularly GFP/Iba1 double-positive macrophages, was significantly decreased in the three TRPM2-KO chimeric mouse groups compared with TRPM2(BM+/Rec+) mice. However, the numbers of GFP(-)/Iba1(+) resident microglia did not differ among chimeric mice. These results suggest that TRPM2 plays an important role in the infiltration of peripheral immune cells, particularly macrophages, into the spinal cord, rather than the infiltration of peripheral immune cells into the injured nerves and activation of spinal-resident microglia. The spinal infiltration of macrophages mediated by TRPM2 may contribute to the pathogenesis of neuropathic pain.

BACKGROUND

Although brain cooling has been reported to be effective in improving the outcome after traumatic brain injury (TBI) in rats, the mechanisms of brain cooling-induced neuroprotective actions remain unclear. This study was to test whether angiogenesis and neurogenesis attenuating TBI could be brain cooling stimulated.

METHODS

Anesthetized rats, immediately after the onset of TBI, were divided into two groups and given the brain cooling (infusing 5 mL of 4°C saline via the external jugular vein) or no brain cooling (infusing 5 mL of 37°C saline via the external jugular vein).

RESULTS

Brain cooling without interference with the core temperature in rats significantly attenuated TBI-induced cerebral infarction (90 mm³ vs. 250 mm³) and motor (61 degrees vs. 57 degrees maximal angle) and proprioceptive (14% vs. 42% maximal possible effect) function deficits, significantly reduced TBI-induced neuronal (24 vs. 62 neuronal-specific nuclear [NeuN]-TUNEL double-positive cells) and glial (5 vs. 35 GFAP-TUNEL double-positive cells) apoptosis (increased TUNEL-positive and caspase-3-positive cells), neuronal loss (102 vs. 66 NeuN-positive cells), and gliosis (40 vs. 66 GFAP-positive cells; 66 vs. 89 Iba1-positive cells), and significantly promoted angiogenesis (5-bromodeoxyuridine [BrdU]/endothelial cells vs. 1-BrdU/endothelial cell; 58 vs. 31 vascular endothelial growth factor-positive cells), and neurogenesis (33 vs. 14 BrdU/NeuN positive cells).

CONCLUSIONS

Brain cooling-stimulated angiogenesis and neurogenesis attenuated a fluid percussion TBI in rats.

BACKGROUND

Numerous studies have reported that increased expression of S100B, an intracellular Ca2+ receptor protein and secreted neuropeptide, exacerbates Alzheimer's disease (AD) pathology. However, the ability of S100B inhibitors to prevent/reverse AD histopathology remains controversial. This study examines the effect of S100B ablation on in vivo plaque load, gliosis and dystrophic neurons.

METHODS

Because S100B-specific inhibitors are not available, genetic ablation was used to inhibit S100B function in the PSAPP AD mouse model. The PSAPP/S100B-/- line was generated by crossing PSAPP double transgenic males with S100B-/- females and maintained as PSAPP/S100B+/- crosses. Congo red staining was used to quantify plaque load, plaque number and plaque size in 6 month old PSAPP and PSAPP/S100B-/- littermates. The microglial marker Iba1 and astrocytic marker glial fibrillary acidic protein (GFAP) were used to quantify gliosis. Dystrophic neurons were detected with the phospho-tau antibody AT8. S100B immunohistochemistry was used to assess the spatial distribution of S100B in the PSAPP line.

RESULTS

PSAPP/S100B-/- mice exhibited a regionally selective decrease in cortical but not hippocampal plaque load when compared to PSAPP littermates. This regionally selective reduction in plaque load was accompanied by decreases in plaque number, GFAP-positive astrocytes, Iba1-positive microglia and phospho-tau positive dystrophic neurons. These effects were not attributable to regional variability in the distribution of S100B. Hippocampal and cortical S100B immunoreactivity in PSAPP mice was associated with plaques and co-localized with astrocytes and microglia.

CONCLUSIONS

Collectively, these data support S100B inhibition as a novel strategy for reducing cortical plaque load, gliosis and neuronal dysfunction in AD and suggest that both extracellular as well as intracellular S100B contribute to AD histopathology.

OBJECTIVE

Minocycline, a second-generation tetracycline-class antibiotic, has been well established to exert a neuroprotective effect in animal models and neurodegenerative disease through the inhibition of microglia. Here, we investigated the effects of minocycline on motor recovery and neuropathic pain in a rat model of spinal cord injury.

METHODS

To simulate spinal cord injury, the rats' spinal cords were hemisected at the 10th thoracic level (T10). Minocycline was injected intraperitoneally, and was administered 30 minutes prior surgery and every second postoperative day until sacrifice 28 days after surgery. Motor recovery was assessed via the Basso-Beattie-Bresnahan test. Mechanical hyperalgesia was measured throughout the 28-day post-operative course via the von Frey test. Microglial and astrocyte activation was assessed by immunohistochemical staining for ionized calcium binding adaptor molecule 1 (Iba1) and glial fibrillary acidic protein (GFAP) at two sites: at the level of hemisection and at the 5th lumbar level (L5).

RESULTS

In rats, spinal cord hemisection reduced locomotor function and induced a mechanical hyperalgesia of the ipsilateral hind limb. The expression of Iba1 and GFAP was also increased in the dorsal and ventral horns of the spinal cord at the site of hemisection and at the L5 level. Intraperitoneal injection of minocycline facilitated overall motor recovery and attenuated mechanical hyperalgesia. The expression of Iba1 and GFAP in the spinal cord was also reduced in rats treated with minocycline.

CONCLUSIONS

By inhibiting microglia and astrocyte activation, minocycline may facilitate motor recovery and attenuate mechanical hyperalgesia in individuals with spinal cord injuries.

Cumulating evidence has demonstrated that μ opioid receptor (MOR) agonists promote spinal glial activation, lead to synthesis and release of proinflammatory cytokines and chemokines, and contribute to opioid-induced hyperalgesia and development of opioid tolerance and dependence. However, whether these MOR agonists directly or indirectly act on spinal cord astrocytes and microglial cells in vivo is unclear. In the present study, by combining the techniques of in-situ hybridization of MOR mRNA with immunohistochemistry of glial fibrillary acidic protein (GFAP; an astrocyte marker) and Iba1 (a microglial marker), we examined expression and distribution of GFAP, Iba1, and MOR mRNA in the spinal cord of rats under chronic morphine tolerance conditions. Intrathecal injections of morphine twice daily for 7 days reduced morphine analgesic effect and increased both GFAP and Iba1 immunostaining densities in the spinal cord. Surprisingly, neither GFAP nor Iba1 colocalized with MOR mRNA in spinal cord cells. Our findings indicate that MOR expression is absent from spinal cord astrocytes and microglia, suggesting that these cell types are indirectly activated by MOR agonists under chronic opioid tolerance conditions.

OBJECTIVE

This study was undertaken to determine the effect of an adenosine kinase inhibitor (AKI) in diabetic retinopathy (DR). We have shown previously that adenosine signaling via A2A receptors (A2AAR) is involved in retinal protection from diabetes-induced inflammation. Here we demonstrate that AKI-enhanced adenosine signaling provides protection from DR in mice.

METHODS

We targeted AK, the key enzyme in adenosine metabolism, using a treatment regime with the selective AKI, ABT-702 (1.5mg/kg intraperitoneally twice a week) commencing at the beginning of streptozotocin-induced diabetes at the age of eight weeks. This treatment, previously demonstrated to increase free adenosine levels in vivo, was maintained until the age of 16 weeks. Retinal inflammation was evaluated using Western blot, Real-Time PCR and immuno-staining analyses. Role of A2AAR signaling in the anti-inflammation effect of ABT-702 was analyzed in Amadori-glycated-albumin (AGA)-treated microglial cells.

RESULTS

At 16 weeks, when diabetic mice exhibit significant signs of retinal inflammation including up-regulation of oxidative/nitrosative stress, A2AAR, ENT1, Iba1, TNF-α, ICAM1, retinal cell death, and down-regulation of AK, the ABT-702 treated group showed lower signs of inflammation compared to control animals receiving the vehicle. The involvement of adenosine signaling in the anti-inflammation effect of ABT-702 was supported by the TNF-α release blocking effect of A2AAR antagonist in AGA-treated microglial cells.

CONCLUSIONS

These results suggest a role for AK in regulating adenosine receptor signaling in the retina. Inhibition of AK potentially amplifies the therapeutic effects of site- and event-specific accumulation of extracellular adenosine, which is of highly translational impact.

Microglia are believed to be the only resident immune cells in the CNS, originating from hematopoietic-derived myeloid cells and invading the CNS during development. However, the detailed mechanisms of differentiation and transformation of microglial cells are not fully understood. Here, we demonstrate that murine microglial cells show two morphological forms in vitro, namely, small round cells expressing CD11b, Iba1, triggering receptor expressing on myeloid cells-2 (TREM2), and weakly expressing major histocompatibility complex class II and large flat cells expressing only CD11b and Iba1. Moreover, lineage-negative bone marrow (LN) cells cultured with primary mixed glial culture cells could differentiate into only the small round microglia-like cells, despite the absence of CCR2 and Gr-1 expression. Addition of macrophage colony stimulating factor (M-CSF) to LN cell culture allowed the proliferation and expression of TREM2 in LN cells, and the addition of neutralizing anti-M-CSF antibodies suppressed the proliferation of LN cells despite the expression of TREM2. When LN cells were cultured with M-CSF, the number of small round cells in the culture was considerably low, indicating that the small round morphology of the immature cells is not maintained in the presence of only M-CSF. On the other hand, when LN cells were grown in the presence of astrocytes, the small round cells were maintained at a concentration of approximately 30% of the total population. Therefore, cell-cell contact with glial cells, especially astrocytes, may be necessary to maintain the small round shape of the immature cells expressing TREM2.

Calorie restriction (CR) increases longevity and elicits many health promoting benefits including delaying immunosenescence and reducing the incidence of age-related diseases. Although the mechanisms underlying the health-enhancing effects of CR are not known, a likely contributing factor is alterations in immune system functioning. CR suppresses lipopolysaccharide (LPS)-induced release of pro-inflammatory cytokines, blocks LPS-induced fever, and shifts hypothalamic signaling pathways to an anti-inflammatory bias. Furthermore, we have recently shown that CR attenuates LPS-stimulated microglial activation in the hypothalamic arcuate nucleus (ARC), a brain region containing neurons that synthesize neuropeptide Y (NPY), an orexigenic neuropeptide that is upregulated by a CR diet and has anti-inflammatory properties. To determine if increased NPY expression in the ARC following CR was associated with changes in microglial activation, a set of brain sections from mice that were exposed to 50% CR or ad libitum feeding for 28 days before being injected with LPS were immunostained for NPY. The density of NPY-immunolabeling was assessed across the rostrocaudal extent of the ARC and hypothalamic paraventricular nucleus (PVN). An adjacent set of sections were immunostained for ionized calcium-binding adapter molecule-1 (Iba1) and immunostained microglia in the ARC were digitally reconstructed to investigate the effects of CR on microglial morphology. We demonstrated that exposure to CR increased NPY expression in the ARC, but not the PVN. Digital reconstruction of microglia revealed that LPS increased Iba1 intensity in ad libitum fed mice but had no effect on Iba1 intensity in CR mice. CR also decreased the size of ARC microglial cells following LPS. Correlational analyses revealed strong associations between NPY and body temperature, and body temperature and microglia area. Together these results suggest that CR-induced changes in NPY are not directly involved in the suppression of LPS-induced microglial activation, however, NPY may indirectly affect microglial morphology through changes in body temperature.