Pain patients who are nicotine dependent report a significantly increased incidence and severity of pain intensity. The goal of this study was to determine the effects of prolonged nicotine administration on inflammatory proteins implicated in the development of peripheral and central sensitization of the trigeminal system. Behavioral, immunohistochemical, and microarray studies were utilized to investigate the effects of nicotine administered daily for 14 days via an Alzet® osmotic pump in Sprague Dawley rats. Systemic nicotine administration caused a significant increase in nocifensive withdrawals to mechanical stimulation of trigeminal neurons. Nicotine stimulated expression of the pro-inflammatory signal transduction proteins phosphorylated-extracellular signal-regulated kinase (p-ERK), phosphorylated-c-Jun N-terminal kinase (p-JNK), and protein kinase A (PKA) in the spinal trigeminal nucleus. Nicotine also promoted elevations in the expression of glial fibrillary acidic protein (GFAP), a biomarker of activated astrocytes, and the microglia biomarker ionized calcium-binding adapter molecule 1 (Iba1). Similarly, levels of eleven cytokines were significantly elevated with the largest increase in expression of TNF-α. Levels of PKA, p-ERK, and p-JNK in trigeminal ganglion neurons were increased by nicotine. Our findings demonstrate that prolonged systemic administration of nicotine promotes sustained behavioral and cellular changes in the expression of key proteins in the spinal trigeminal nucleus and trigeminal ganglion implicated in the development and maintenance of peripheral and central sensitization.

Microgliosis, the activation of microglial cells, is thought to contribute to synaptic transmission in the dorsal horn and thereby promote chronic pain. The primary aim of this study was to document the temporal profile of dorsal horn microgliosis after destabilization of the medial meniscus (DMM) in wild type (WT) and Adamts5 null mice. Since neuronal fractalkine (CX3CL1) contributes to microgliosis, we assessed its release from dorsal root ganglia (DRG) cultures after DMM.

DMM or sham surgery was performed in the right knee of 10-week old male WT, CX3CR1-green fluorescent protein (GFP), or Adamts5 null C57BL/6 mice. Hind paw mechanical allodynia was monitored using von Frey fibers. L4 dorsal horn microgliosis was assessed 4, 8 and 16 weeks after surgery, based on the morphology of Iba1-immunoreactive microglia. DRG cells (L3-L5) were cultured and supernatants collected for fractalkine (FKN) ELISA.

In WT mice, numbers of activated microglia were increased 8 and 16 weeks, but not 4 weeks, after DMM but not sham surgery. DRG cultures showed increased basal FKN release at 8 and 16 weeks. Adamts5 null mice did not develop mechanical allodynia up to 16 weeks after DMM. Accordingly, DRG cultures from these mice did not exhibit increased FKN release and dorsal horn microgliosis did not occur.

DMM surgery leads to late stage dorsal horn microgliosis. The temporal correlation with DRG FKN release suggests it may contribute to microgliosis. Reduced microgliosis in Adamts5 null mice, which are protected from joint damage and associated mechanical allodynia after DMM, suggests that microgliosis is associated with joint damage and accompanying persistent pain.

Alzheimer disease (AD) is a chronic neurodegenerative disease with a multitude of contributing genetic factors, many of which are related to inflammation. The apolipoprotein E (APOE) ε4 allele is the most common genetic risk factor for AD and is related to a pro-inflammatory state. To test the hypothesis that microglia and AD-implicated cytokines were differentially associated with AD pathology based on the presence of APOE ε4, we examined the dorsolateral frontal cortex from deceased participants within a community-based aging cohort (n = 154). Cellular density of Iba1, a marker of microglia, was positively associated with tau pathology only in APOE ε4 positive participants (p = 0.001). The cytokines IL-10, IL-13, IL-4, and IL-1α were negatively associated with tau pathology, independent of Aβ_1-42 levels, only in APOE ε4 negative participants. Overall, the association of mostly anti-inflammatory cytokines with less tau pathology suggests a protective effect in APOE ε4 negative participants. These associations are largely absent in the presence of APOE ε4 where tau pathology was significantly associated with increased microglial cell density. Taken together, these results suggest that APOE ε4 mediates an altered inflammatory response and increased tau pathology independent of Aβ_1-42 pathology.

Neurological dysfunction after traumatic brain injury (TBI) is associated with pathology in cortical, subcortical, and brainstem nuclei. Our laboratory has reported neuropathology and microglial activation in the somatosensory barrel cortex (S1BF) and ventral posterior medial thalamus (VPM) after diffuse TBI in the rat, which correlated with post-injury whisker sensory sensitivity. The present study extends our previous work by evaluating pathology in whisking-associated sensory and motor brainstem nuclei. Brains from adult, male rats were recovered over 1 month after midline fluid percussion or sham injury. The principal trigeminal nucleus (PrV, sensory nucleus) and facial nucleus (VIIN, motor nucleus) were examined for neuropathology (silver histochemistry) and microglial activation (Iba1). Significant neuropathology in PrV was evident at 2 and 7 days post-injury compared to sham. Iba1-labeled microglia showed swollen somata and thickened processes over 1 month post-injury. In contrast, the VIIN showed non-significant neuropathology and reduced labeling of activated Iba1 microglia over 1 month post-injury. Together with our previous data, neuropathology and neuroinflammation in the whisker somatosensory pathway may contribute to post-injury sensory sensitivity more than the motor pathway. Whether these findings are direct results of the mechanical injury or consequences of progressive degeneration remains to be determined.

Radiation necrosis (RN) in brain tumor patients is often symptomatic, persistent without immediate resolution, and confused with tumor recurrence. Cerebral vascular pericytes are essential for endothelial function, vascular integrity, and angiogenesis. In this study, we showed that the loss of pericytes is involved in the pathogenesis of RN. From a brain tumor tissue repository, we identified three patients since 2011 with pathologically confirmed RN after the standard treatment for glioblastoma (GBM). The RN and their preradiation GBM tissues were serially processed for Western blotting using cell-type-specific antibodies against endothelial (CD31, active RhoA), pericyte [platelet-derived growth factor receptor-beta (PDGFR-β)], alpha-smooth muscle actin (α-SMA), astrocyte (GFAP), myelin sheath protein (MBP), and microglial markers (Iba1). Normal brain tissues from a brain bank were used as normal controls. The expressions of PDGFR-β and α-SMA were remarkably reduced in the RN, compared to those of GBM. However, the levels of CD31 or RhoA were not different between the two groups, which suggest that there was no change in the number of endothelial cells or their cytoskeletal assembly. The RN tissues showed a decreased ratio of pericyte/endothelial markers and an increased level of Iba1 compared to the GBM and even to the normal brain. The levels of GFAP and MBP were not changed in the RN. In the histopathology, the RN tissues showed a loss of markers (PDGFR-β), whereas the GBM tissues had abundant expression of the markers. The loss of pericytes and vascular smooth muscle cells, and the unsupported endothelial cells might be the cause of the leaky blood-brain barrier and tissue necrosis.

Background: Muramyl dipeptide (MDP) is a component derived from minimal peptidoglycan motif from bacteria, and it is a ligand for the NOD2 receptor. Peripheral administration of MDP converts Ly6C^high into Ly6C^low monocytes. Previously, we have shown that Ly6C^low monocytes play crucial roles in the pathology of a mouse model of Alzheimer's disease (AD). However, medications with mild immunomodulatory effects that solely target specific monocyte subsets, without triggering microglial activation, are rare.

Methods: Three months old APP_swe/PS1 transgenic male mice and age-matched C57BL/6 J mice were used for high frequency (2 times/week) over 6 months and low frequency (once a week) over 3 months of intraperitoneally MDP (10 mg/kg) administrations. Flow cytometry analysis of monocyte subsets in blood, and behavioral and postmortem analyses were performed.

Results: Memory tests showed mild to a strong improvement in memory function, increased expression levels of postsynaptic density protein 95 (PSD95), and low-density lipoprotein receptor-related protein 1 (LRP1), which are involved in synaptic plasticity and amyloid-beta (Aβ) elimination, respectively. In addition, we found monocyte chemoattractant protein-1(MCP-1) levels significantly increased, whereas intercellular adhesion molecule-1(ICAM-1) significantly decreased, and microglial marker (Iba1) did not change in the treatment group compared to the control. In parallel, we discovered elevated cyclooxygenase-2 (COX2) expression levels in the treated group, which might be a positive factor for synaptic activity.

Conclusions: Our results demonstrate that MDP is beneficial in both the early phase and, to some extent, later phases of the pathology in the mouse model of AD. These data open the way for potential MDP-based medications for AD.

Keywords: Alzheimer’s disease; Brain blood vessels; Cerebral amyloid angiopathy; Immunotherapy; Macrophages; Microglia; Monocytes; NOD2 receptor; Synapse.

X-linked Charcot-Marie-Tooth disease (CMT1X) is a common form of inherited neuropathy resulting from different mutations affecting the gap junction (GJ) protein connexin32 (Cx32). A subset of CMT1X patients may additionally present with acute fulminant CNS dysfunction, typically triggered by conditions of systemic inflammation and metabolic stress. To clarify the underlying mechanisms of CNS phenotypes in CMT1X we studied a mouse model of systemic inflammation induced by lipopolysaccharide (LPS) injection to compare wild type (WT), connexin32 (Cx32) knockout (KO), and KO T55I mice expressing the T55I Cx32 mutation associated with CNS phenotypes. Following a single intraperitoneal LPS or saline (controls) injection at the age of 40-60 days systemic inflammatory response was documented by elevated TNF-α and IL-6 levels in peripheral blood and mice were evaluated 1 week after injection. Behavioral analysis showed graded impairment of motor performance in LPS treated mice, worse in KO T55I than in Cx32 KO and in Cx32 KO worse than WT. Iba1 immunostaining revealed widespread inflammation in LPS treated mice with diffusely activated microglia throughout the CNS. Immunostaining for the remaining major oligodendrocyte connexin Cx47 and for its astrocytic partner Cx43 revealed widely reduced expression of Cx43 and loss of Cx47 GJs in oligodendrocytes. Real-time PCR and immunoblot analysis indicated primarily a down regulation of Cx43 expression with secondary loss of Cx47 membrane localization. Inflammatory changes and connexin alterations were most severe in the KO T55I group. To examine why the presence of the T55I mutant exacerbates pathology even more than in Cx32 KO mice, we analyzed the expression of ER-stress markers BiP, Fas and CHOP by immunostaining, immunoblot and Real-time PCR. All markers were increased in LPS treated KO T55I mice more than in other genotypes. In conclusion, LPS induced neuroinflammation causes disruption of the main astrocyte-oligodendrocyte GJs, which may contribute to the increased sensitivity of Cx32 KO mice to LPS and of patients with CMT1X to various stressors. Moreover the presence of an intracellularly retained, misfolded CMT1X mutant such as T55I induces ER stress under inflammatory conditions, further exacerbating oligodendrocyte dysfunction and pathological changes in the CNS.

To characterize the cellular density and proliferative activity of GFAP-negative cells in pilocytic astrocytoma (PA), surgically excised tissues of PAs (n=37) and diffuse astrocytomas (DAs) (n=11) were examined morphologically and immunohistochemically using antibodies against GFAP, Olig2, Iba1 and Ki-67 (MIB-1). In PA, Olig2 immunoreactivity was significantly expressed in protoplasmic astrocytes in microcystic, loose areas and cells in oligodendroglioma-like areas. Iba1-positive, activated microglia/macrophages were also commonly observed in microcystic areas. In compact areas, a prominent reaction for GFAP was observed, but for Olig2 and Iba1 to a lesser degree. On semiquantitative analysis, the number of Olig2-positive cells was significantly higher in PAs (mean labeling index (LI) +/- standard deviation (SD): 46.8+/-15.4%) than in DAs (13.3+/-7.8%) (P<0.001). Many Iba1-positive, microglia/macrophages were observed in PAs (19.9+/-6.5%), similarly to DAs (20.9+/-9.9%). Re-immunostaining of PA demonstrated that most Ki-67-positive, proliferating cells expressed Olig2, whereas GFAP or Iba1 expression in Ki-67-positive cells was less frequent (14.7+/-13.7%, and 8.8+/-13.6%) in a double immunostaining study. Conversely, the percentage of Olig2-positive, proliferating cells in total Olig2-positive cells (7.2+/-3.9%) was higher than that of Iba1-positive, proliferating cells in total Iba1-positive cells (0.9+/-0.6%). In conclusion, the present study found that PA consisted of numerous GFAP-negative cells, including Olig2-positive cells with high proliferation. Semiquantitative analysis of Olig2 immunohistochemistry in microcystic areas might therefore be useful for the differential diagnosis of PA and DA.

Methamphetamine (Meth) use is common among HIV-infected persons. It remains unclear whether Meth dependence is associated with long-lasting degenerative changes in the brain parenchyma and microvasculature of HIV-infected individuals. We examined the postmortem brains of 78 HIV-infected adults, twenty of whom were diagnosed with lifetime Meth dependence (18 past and two current at the final follow-up visit). Using logistic regression models, we analyzed associations of Meth with cerebral gliosis (immunohistochemistry for ionized calcium-binding adapter molecule-1 (Iba1) and glial fibrillary acidic protein (GFAP) in frontal, temporo-parietal, and putamen-internal capsule regions), synaptodendritic loss (confocal microscopy for synaptophysin (SYP) and microtubule-associated protein-2 (MAP2) in frontal cortex), β-amyloid plaque deposition (immunohistochemistry in frontal and temporo-parietal cortex and putamen), and arteriolosclerosis (histopathology in forebrain white matter). We found that Meth was associated with marked Iba1 gliosis in the temporo-parietal region (odds ratio, 4.42 (95 % confidence interval, 1.36, 14.39), p = 0.014, n = 62), which remained statistically significant after adjusting for HIV encephalitis, white matter lesions, and opportunistic diseases (n = 61); hepatitis C virus seropositivity (n = 54); and lifetime dependence on alcohol, opiates, and cannabis (n = 62). There was no significant association of Meth with GFAP gliosis, SYP or MAP2 loss, β-amyloid plaque deposition, or arteriolosclerosis. In conclusion, we found lifetime Meth dependence to be associated with focal cerebral microgliosis among HIV-infected adults, but not with other brain degenerative changes examined. Some of the changes in select brain regions might be reversible following extended Meth abstinence or, alternatively, might have not been induced by Meth initially.

Ba-DupGreen (BDG) is a highly attenuated, Bartha-derived pseudorabies virus (PRV) expressing green fluorescent protein (GFP) with immediate-early kinetics. Innate immune mechanisms underlying the low infectivity of the virus and the disappearance of infected neurons from the brain were studied at cellular level following injection of BDG into the spleen. The temporal shift in the expression between GFP and viral structural proteins allowed us to discriminate three stages of viral infection in the compromised neurons in correlation with the ongoing local inflammatory response. Iba1/lectin/OX42-positive microglia were recruited to infected neurons within 4-6 h following the initiation of virus replication, incorporated BrdU, isolated the infected cells before the disintegration of their membranes and phagocytosed collapsed neurons. Ex vivo-labeled blood and bone marrow-derived leukocytes, including ED-1-positive macrophages were involved in the immune cell assembly around compromised neurons, which resulted in the complete clearance of infected neurons from the early-infected brain regions.

Lumbar disc herniation is commonly encountered in clinical practice and can induce sciatica due to mechanical and/or chemical irritation and the release of proinflammatory cytokines. However, symptoms are not confined to the affected spinal cord segment. The purpose of this study was to determine whether multisegmental molecular changes exist between adjacent lumbar spinal segments using a rat model of lumbar disc herniation. Twenty-nine male Sprague-Dawley rats were randomly assigned to either a sham-operated group (n=10) or a nucleus pulposus (NP)-exposed group (n=19). Rats in the NP-exposed group were further subdivided into a significant pain subgroup (n=12) and a no significant pain subgroup (n=7) using mechanical pain thresholds determined von Frey filaments. Immunohistochemical stainings of microglia (ionized calcium-binding adapter molecule 1; Iba1), astrocytes (glial fibrillary acidic protein; GFAP), calcitonin gene-related peptide (CGRP), and transient receptor potential vanilloid 1 (TRPV1) was performed in spinal dorsal horns and dorsal root ganglions (DRGs) at 10 days after surgery. It was found immunoreactivity for Iba1-positive microglia was higher in the L5 (P=0.004) dorsal horn and in the ipsilateral L4 (P=0.009), L6 (P=0.002), and S1 (P=0.002) dorsal horns in the NP-exposed group than in the sham-operated group. The expression of CGRP was also significantly higher in ipsilateral L3, L4, L6, and S1 segments and in L5 DRGs at 10 days after surgery in the NP-exposed group than in the sham-operated group (P<0.001). Our results indicate that lumbar disc herniation upregulates microglial activity and CGRP expression in many adjacent and ipsilateral lumbar spinal segments.

BACKGROUND

HIV-associated neuroinflammation is believed to be a major contributing factor in the development of HIV-associated neurocognitive disorders (HAND). In this study, we used micropositron emission tomography (PET) imaging to quantify neuroinflammation in HIV-1 transgenic rat (Tg), a small animal model of HIV, known to develop neurological and behavioral problems.

METHODS

Dynamic [(18)F]DPA-714 PET imaging was performed in Tg and age-matched wild-type (WT) rats in three age groups: 3-, 9-, and 16-month-old animals. As a positive control for neuroinflammation, we performed unilateral intrastriatal injection of quinolinic acid (QA) in a separate group of WT rats. To confirm our findings, we performed multiplex immunofluorescent staining for Iba1 and we measured cytokine/chemokine levels in brain lysates of Tg and WT rats at different ages.

RESULTS

[(18)F]DPA-714 uptake in HIV-1 Tg rat brains was generally higher than in age-matched WT rats but this was not statistically significant in any age group. [(18)F]DPA-714 uptake in the QA-lesioned rats was significantly higher ipsilateral to the lesion compared to contralateral side indicating neuroinflammatory changes. Iba1 immunofluorescence showed no significant differences in microglial activation between the Tg and WT rats, while the QA-lesioned rats showed significant activation. Finally, cytokine/chemokine levels in brain lysates of the Tg rats and WT rats were not significantly different.

CONCLUSIONS

Microglial activation might not be the primary mechanism for neuropathology in the HIV-1 Tg rats. Although [(18)F]DPA-714 is a good biomarker of neuroinflammation, it cannot be reliably used as an in vivo biomarker of neurodegeneration in the HIV-1 Tg rat.

OBJECTIVE

Brachial plexus pain is thought to be generated not by avulsed roots but rather by nonavulsed roots, because avulsed roots could not transmit action potentials to central nerves. The aim of this study was to evaluate pain-related behavior and the extent of glial activation in a model of brachial plexus avulsion (BPA).

METHODS

We used 24 male Wistar rats. For rats in the BPA group, the C8-T1 roots were avulsed from the spinal cord at the level of the lower trunk (n = 10). Rats in a sham-surgery group had a similar surgery without the root avulsion (n = 7). Rats in an untreated group had no surgery (n = 7). Mechanical hyperalgesia of the forelimb plantar surfaces corresponding to C6 and C7 dermatomes was evaluated using a Semmes-Weinstein monofilament test every third day for 3 weeks (n = 15). Activation of astrocytes and microglia was examined immunohistochemically using anti-glia fibrillary acidic protein and anti-Iba1 antibodies 3 days after surgery (n = 9).

RESULTS

When compared with rats in the sham-surgery and naive control groups, rats in the BPA group displayed significant mechanical hyperalgesia in the dermatome innervated by uninjured nerves both ipsilaterally and contralaterally and continuing through day 21. Iba1-immunoreactive microglia and glia fibrillary acidic protein-immunoreactive astrocytes were significantly activated on the ipsilateral side in the BPA group from levels C3 to T3 compared with the sham-surgery and untreated groups of rats.

CONCLUSIONS

Activation of glia at uninjured levels of the dorsal horn may facilitate pain transmission following BPA injury. Consequently, spared spinal glial cells may represent therapeutic targets for treatment of pain related to BPA injury.

CONCLUSIONS

Our findings may indicate why neuropathic pain is so frequent and intense following BPA injury.

The possible role of minocycline in microglial activation and neuronal death after cardiac arrest (CA) and cardiopulmonary resuscitation (CPR) in mice was investigated in this study. The mice were given potassium chloride to stop the heart beating for 8 min to achieve CA, and they were subsequently resuscitated with epinephrine and chest compressions. Forty adult C57BL/6 male mice were divided into 4 groups (n=10 each): sham-operated group, CA/CPR group, CA/CPR+minocycline group, and CA/CPR+vehicle group. Animals in the latter two groups were intraperitoneally injected with minocycline (50 mg/kg) or vehicle (normal saline) 30 min after recovery of spontaneous circulation (ROSC). Twenty-four h after CA/CPR, the brains were removed for histological evaluation of the hippocampus. Microglial activation was evaluated by detecting the expression of ionized calcium-binding adapter molecule-1 (Iba1) by immunohistochemistry. Neuronal death was analyzed by hematoxylin and eosin (H&E) staining and the levels of tumor necrosis factor-alpha (TNF-α) in the hippocampus were measured by enzyme-linked immunosorbent assay (ELISA). The results showed that the neuronal death was aggravated, most microglia were activated and TNF-α levels were enhanced in the hippocampus CA1 region of mice subjected to CA/CPR as compared with those in the sham-operated group (P<0.05). Administration with minocycline 30 min after ROSC could significantly decrease the microglial response, TNF-α levels and neuronal death (P<0.05). It was concluded that early administration with minocycline has a strong therapeutic potential for CA/CPR-induced brain injury.

The phagocyte pattern recognition receptor Toll-like receptor 2 (TLR2) and the multi-receptor adaptor MyD88 contribute to the reduction of bacterial load in infections with intra- and extra-cellular Gram-positive bacteria. Their mechanism of antibacterial action is mostly unresolved but evident in vivo by an increased pathogen burden in infected TLR2-/- and MyD88-/- compared to C57BL/6 wild type (wt) mice. We had previously observed higher bacterial numbers in brains of TLR2-/- than of wt mice with meningitis. Here we study bacteria-phagocyte interaction by comparing S. pneumoniae distribution and localization in wt and TLR2-/- brain by confocal microscopy using a green fluorescent protein-transformed encapsulated S. pneumoniae (C5017). Colony-forming units were similarly distributed in TLR2-/- and wt mice and exclusively localized in meninges and ventricles. Bacteria were more abundant in ventricles, in and around TLR2-/- than wt GLT1v+ plexus choroideus epithelial cells. S. pneumoniae were also found in and around Gr-1+ granulocytes, but never in F4/80+ macrophages, Iba1+ microglia, GFAP+ astrocytes, Meca-31+ endothelial cells or Neun+ neurons of either mouse strain. The results indicate that TLR2 does not change bacterial distribution, but may contribute to antibacterial defense by modulating S. pneumoniae adherence and uptake in plexus epithelia.

BACKGROUND

One of the more profound features of systemic lupus erythematosus (SLE) is that females have a 9:1 prevalence of this disease over males. Up to 80% of SLE patients have cognitive defects or affective disorders. The mechanism of CNS injury responsible for cognitive impairment is unknown. We previously showed that ERα deficiency significantly reduced renal disease and increased survival in lupus-prone mice. We hypothesized that ERα deficiency would be similarly protective in the brain, and that ERα may play a role in modulating blood-brain barrier (BBB) integrity and/or neuroinflammation in lupus-prone mice.

METHODS

MRL/lpr ERα+/+ and ERαKO mice (n = 46) were ovariectomized, received 17β-estradiol pellets, and underwent radial arm water maze (WRAM) and novel object recognition (NOR) testing starting at eight weeks of age. Mice were sacrificed and brains were hemisected and processed for either immunohistochemistry, or hippocampus and parietal cortex dissection for Western blotting.

RESULTS

MRL/lpr ERαKO mice (n = 21) performed significantly better in WRAM testing than wild-type MRL/lpr mice (n = 25). There was a significant reduction in reference memory errors (P <0.007), working memory errors (P <0.05), and start arm errors (P <0.02) in ERαKO mice. There were significant differences in NOR testing, particularly total exploration time, with ERα deficiency normalizing behavior. No significant differences were seen in markers of tight junction, astrogliosis, or microgliosis in the hippocampus or cortex by Western blot, however, there was a significant reduction in numbers of Iba1+ activated microglia in the hippocampus of ERαKO mice, as evidenced by immunohistochemietry (IHC).

CONCLUSIONS

ERα deficiency provides significant protection against cognitive deficits in MRL/lpr mice as early as eight weeks of age. Additionally, the significant reduction in Iba1+ activated microglia in the MRL/lpr ERαKO mice was consistent with reduced inflammation, and may represent a biological mechanism for the cognitive improvement observed.

The cellular prion protein (PrPC) is an evolutionary conserved protein abundantly expressed not only in the central nervous system but also peripherally including the immune system. A line of Norwegian dairy goats naturally devoid of PrPC (PRNP Ter/Ter) provides a novel model for studying PrPC physiology.

In order to explore putative roles for PrPC in acute inflammatory responses, we performed a lipopolysaccharide (LPS, Escherichia coli O26:B6) challenge of 16 goats (8 PRNP +/+ and 8 PRNP Ter/Ter) and included 10 saline-treated controls (5 of each PRNP genotype). Clinical examinations were performed continuously, and blood samples were collected throughout the trial. Genome-wide transcription profiles of the choroid plexus, which is at the blood-brain interface, and the hippocampus were analyzed by RNA sequencing, and the same tissues were histologically evaluated.

All LPS-treated goats displayed clinical signs of sickness behavior, which were of significantly (p < 0.01) longer duration in animals without PrPC. In the choroid plexus, a substantial alteration of the transcriptome and activation of Iba1-positive cells were observed. This response included genotype-dependent differential expression of several genes associated with the immune response, such as ISG15, CXCL12, CXCL14, and acute phase proteins, among others. Activation of cytokine-responsive genes was skewed towards a more profound type I interferon response, and a less obvious type II response, in PrPC-deficient goats. The magnitude of gene expression in response to LPS was smaller in the hippocampus than in the choroid plexus. Resting state expression profiles revealed a few differences between the PRNP genotypes.

Our data suggest that PrPC acts as a modulator of certain pathways of innate immunity signaling, particularly downstream of interferons, and probably contributes to protection of vulnerable tissues against inflammatory damage.

We developed a well reproducible method for detection of brain microglia on paraffin sections on the basis of modern immunohistochemical methods. Polyclonal antibodies to Iba1 antigen obtained after immunization with a synthetic peptide corresponding to 81-93 amino acid fragment of Iba1 antigen selectively interact with microglia cells on paraffin sections of rat brain. Detailed protocol of preparation treatment was drawn up ensuring complete detection of the microglia expressing Iba1 protein.

The contribution of the Ca(2+) sensor S100A1 to in vivo Alzheimer's disease (AD) pathobiology has not been elucidated although S100A1 regulates numerous cellular processes linked to AD. This study uses genetic ablation to ascertain the effects of S100A1 on neuroinflammation, beta-amyloid (Aβ) plaque deposition and Akt activity in the PSAPP AD mouse model. PSAPP/S100A1(-/-) mice exhibited decreases in astrocytosis (GFAP burden), microgliosis (Iba1 burden) and plaque load/number when compared to PSAPP/S100A1(+/+) mice at six and twelve months of age. The presence of detectable S100A1 staining in human AD specimens is consistent with a detrimental gain of S100A1 function in AD. S100A1 ablation also reduced plaque associated and increased non-plaque associated PO4-Akt and PO4-GSK3β staining. S100A1·Akt complexes were undetectable in PC12 cells and AD brain tissue suggesting that S100A1 indirectly modulates Akt activity. In contrast, S100A1·RyR (ryanodine receptor) complexes were present in human/mouse AD brain and exhibited Ca(2+)-dependent formation in neuronal cells. This is the first direct demonstration of an S100A1· target protein complex in tissue/cells and identifies the RyR as a primary S100A1 target protein in the brain. Collectively, these data suggest that S100A1 inhibition may be a novel strategy for normalizing aberrant Ca(2+) signaling in AD.

BACKGROUND

The mechanisms of agmatine-induced neuroprotective effects in traumatic brain injury (TBI) remain unclear. This study was to test whether inhibition of gliosis, angiogenesis, and neurogenesis attenuating TBI could be agmatine stimulated.

METHODS

Anesthetized rats were randomly assigned to sham-operated group, TBI rats treated with saline (1 mL/kg, intraperitoneally), or TBI rats treated with agmatine (50 mg/kg, intraperitoneally). Saline or agmatine was injected 5 minutes after TBI and again once daily for the next 3 postoperative days.

RESULTS

Agmatine therapy in rats significantly attenuated TBI-induced motor function deficits (62° vs. 52° maximal angle) and cerebral infarction (88 mm vs. 216 mm), significantly reduced TBI-induced neuronal (9 NeuN-TUNEL double positive cells vs. 60 NeuN-TUNEL double positive cells) and glial (2 GFAP-TUNEL double positive cells vs. 20 GFAP-TUNEL double positive cells) apoptosis (increased TUNEL-positive and caspase-3-positive cells), neuronal loss (82 NeuN-positive cells vs. 60 NeuN-positive cells), gliosis (35 GFAP-positive cells vs. 72 GFAP-positive cells; 60 Iba1-positive cells vs. 90 Iba1-positive cells), and neurotoxicity (30 n-NOS-positive cells vs. 90 n-NOS-positive cells; 35 3-NT-positive cells vs. 90 3-NT-positive cells), and significantly promoted angiogenesis (3 BrdU/endothelial cells vs. 0.5 BrdU/endothelial cells; 50 vascular endothelial growth factor positive cells vs. 20 vascular endothelial growth factor-positive cells) and neurogenesis (27 BrdU/NeuN positive cells vs. 15 BrdU/NeuN positive cells).

CONCLUSIONS

Resultantly, agmatine therapy may attenuate TBI in rats via promoting angiogenesis, neurogenesis, and inhibition of gliosis.

Modulation of extracellular matrix (ECM) remodeling after peripheral nerve injury (PNI) could represent a valid therapeutic strategy to prevent maladaptive synaptic plasticity in central nervous system (CNS). Inhibition of matrix metalloproteinases (MMPs) and maintaining a neurotrophic support could represent two approaches to prevent or reduce the maladaptive plastic changes in the ventral horn of spinal cord following PNI. The purpose of our study was to analyze changes in the ventral horn produced by gliopathy determined by the suffering of motor neurons following spared nerve injury (SNI) of the sciatic nerve and how the intrathecal (i.t.) administration of GM6001 (a MMPs inhibitor) or the NGF mimetic peptide BB14 modulate these events. Immunohistochemical analysis of spinal cord sections revealed that motor neuron disease following SNI was associated with increased microglial (Iba1) and astrocytic (GFAP) response in the ventral horn of the spinal cord, indicative of reactive gliosis. These changes were paralleled by decreased glial aminoacid transporters (glutamate GLT1 and glycine GlyT1), increased levels of the neuronal glutamate transporter EAAC1, and a net increase of the Glutamate/GABA ratio, as measured by HPLC analysis. These molecular changes correlated to a significant reduction of mature NGF levels in the ventral horn. Continuous i.t. infusion of both GM6001 and BB14 reduced reactive astrogliosis, recovered the expression of neuronal and glial transporters, lowering the Glutamate/GABA ratio. Inhibition of MMPs by GM6001 significantly increased mature NGF levels, but it was absolutely ineffective in modifying the reactivity of microglia cells. Therefore, MMPs inhibition, although supplies neurotrophic support to ECM components and restores neuro-glial transporters expression, differently modulates astrocytic and microglial response after PNI.

OBJECTIVE

To investigate the glial response of the rat retina to single or repeated intravitreal injections (IVI).

METHODS

Albino Sprague-Dawley rats received one or three (one every 7 days) IVI of anti-rat VEGF (5 μL; 0.015 μg/μL), triamcinolone (2.5 or 5 μL; 40 μg/μL; Trigón Depot), bevacizumab (5 μL; 25 μg/μL; Avastin), or their vehicles (PBS and balanced salt solution) and were processed 7 days after the last injection. Retinas were dissected as whole mounts and incubated with antibodies against: Iba1 (Ionized Calcium-Binding Adapter Molecule 1) to label retinal microglia, GFAP (Glial Fibrillary Acidic Protein) to label macroglial cells, and vimentin to label Müller cells. The retinas were examined with fluorescence and confocal microscopy, and the numbers of microglial cells in the inner retinal layers were quantified using a semiautomatic method.

RESULTS

All the injected substances caused an important micro- and macroglial response locally at the injection site and all throughout the injected retina that was exacerbated by repeated injections. The microglial response was also observed but was milder in the contralateral noninjected eyes. The IVI of the humanized antibody bevacizumab caused a very strong microglial reaction in the ipsilateral retina. Two types of macroglial response were observed: astrocyte hypertrophy and Müller end-foot hypertrophy. While astrocyte hypertrophy was widespread throughout the injected retina, Müller end-foot hypertrophy was localized and more extensive with triamcinolone use or after repeated injections.

CONCLUSIONS

Intravitreal injections cause micro- and macroglial responses that vary depending on the injected agent but increase with repeated injections. This inflammatory glial response may influence the effects of the injected substances on the retina.

The neuronal pathology caused by neonatal infection of rats with the PVC-211 murine leukemia virus (PVC-211 MuLV) and its underlying mechanisms are not well defined even though a loss of neurons and spongiform neurodegeneration has been reported to accompany the disease. Here we sought to identify sites of neurodegeneration using microglial reactivity as an indirect marker and to characterize microglial activation during disease progression. Using a panel of microglial antibodies including Iba1, OX-42, ED1, and anti-ferritin, we have studied the response of microglial cells to neonatal CNS infection with PVC-211 at post-infection survival times 7, 14, 21, and 28 days. We found that microglial activation occurred primarily in the spinal cord and brainstem where it gradually increased in intensity over the time course of this study. Other brain areas were relatively unremarkable in their microglial reaction to viral infection within this time frame. However, the presence of activated microglial cells was not correlated directly with the presence of viral glycoprotein (gp70), which was expressed in endothelial cells throughout the CNS. Although double-labeling of microglia with Iba1 and ED1 revealed numerous actively phagocytic microglia during disease progression, not all activated microglia were ED1-positive. In addition to the intense microglial activation, we found increased ferritin expression sporadically throughout the virus-infected brain. The ferritin-positive cells were mostly microglia that exhibited dystrophic changes and likely represented a degenerating subpopulation of microglial cells. Thus, activated microglia can co-exist with degenerating microglia in the same brain region. We attempted to localize degenerating neurons or neurites using Fluoro-Jade, anti-tau, and anti-alpha synuclein staining, but none of these procedures yielded results to indicate obvious neuronal pathology. We conclude that the visualization of microglial activation is a more sensitive measure of neuronal perturbations than direct detection of neuronal pathology which may be subtle and not produce overt degenerative changes.

Spinal cord injury (SCI) among people over age 40 has been steadily increasing since the 1980s and is associated with worsened outcome than injuries in young people. Age-related increases in reactive oxygen species (ROS) are suggested to lead to chronic inflammation. The NADPH oxidase 2 (NOX2) enzyme is expressed by microglia and is a primary source of ROS. This study aimed to determine the effect of age on inflammation, oxidative damage, NOX2 gene expression, and functional performance with and without SCI in young adult (3 months) and middle-aged (12 months) male rats.

Young adult and middle-aged rats were assessed in two groups-naïve and moderate contusion SCI. Functional recovery was determined by weekly assessment with the Basso, Beattie, and Breshnahan general motor score (analyzed two-way ANOVA) and footprint analysis (analyzed by Chi-square analysis). Tissue was analyzed for markers of oxidative damage (8-OHdG, Oxyblot, and 3-NT), microglial-related inflammation (Iba1), NOX2 component (p47PHOX, p22PHOX, and gp91PHOX), and inflammatory (CD86, CD206, TNFα, and NFκB) gene expression (all analyzed by unpaired Student's t test).

In both naïve and injured aged rats, compared to young rats, tissue analysis revealed significant increases in 8-OHdG and Iba1, as well as inflammatory and NOX2 component gene expression. Further, injured aged rats showed greater lesion volume rostral and caudal to the injury epicenter. Finally, injured aged rats showed significantly reduced Basso-Beattie-Bresnahan (BBB) scores and stride length after SCI.

These results show that middle-aged rats demonstrate increased microglial activation, oxidative stress, and inflammatory gene expression, which may be related to elevated NOX2 expression, and contribute to worsened functional outcome following injury. These findings are essential to elucidating the mechanisms of age-related differences in response to SCI and developing age-appropriate therapeutics.