Spatio-temporal expression profile of NGF and the two-receptor system, TrkA and p75NTR, in experimental autoimmune encephalomyelitis.

BACKGROUND: Nerve growth factor (NGF) and its receptors, tropomyosin receptor kinase A (TrkA) and pan-neurotrophin receptor p75 (p75NTR), are known to play bidirectional roles between the immune and nervous system. There are only few studies with inconclusive results concerning the expression pattern and role of NGF, TrkA, and p75NTR (NGF system) under the neuroinflammatory conditions in multiple sclerosis (MS) and its mouse model, the experimental autoimmune encephalomyelitis (EAE). The aim of this study is to investigate the temporal expression in different cell types of NGF system in the central nervous system (CNS) during the EAE course. METHODS: EAE was induced in C57BL/6 mice 6-8 weeks old. CNS tissue samples were collected on specific time points: day 10 (D10), days 20-22 (acute phase), and day 50 (chronic phase), compared to controls. Real-time PCR, Western Blot, histochemistry, and immunofluorescence were performed throughout the disease course for the detection of the spatio-temporal expression of the NGF system. RESULTS: Our findings suggest that both NGF and its receptors, TrkA and p75NTR, are upregulated during acute and chronic phase of the EAE model in the inflammatory lesions in the spinal cord. NGF and its receptors were co-localized with NeuN+ cells, GAP-43+ axons, GFAP+ cells, Arginase1+ cells, and Mac3+ cells. Furthermore, TrkA and p75NTR were sparsely detected on CNPase+ cells within the inflammatory lesion. Of high importance is our observation that despite EAE being a T-mediated disease, only NGF and p75NTR were shown to be expressed by B lymphocytes (B220+ cells) and no expression on T lymphocytes was noticed. CONCLUSION: Our results indicate that the components of the NGF system are subjected to differential regulation during the EAE disease course. The expression pattern of NGF, TrkA, and p75NTR is described in detail, suggesting possible functional roles in neuroprotection, neuroregeneration, and remyelination by direct and indirect effects on the components of the immune system.

Beneficial effects of environmental enrichment on behavior, stress reactivity and synaptophysin/BDNF expression in hippocampus following early life stress.

Exposure to environmental enrichment can beneficially influence the behavior and enhance synaptic plasticity. The aim of the present study was to investigate the mediated effects of environmental enrichment on postnatal stress-associated impact with regard to behavior, stress reactivity as well as synaptic plasticity changes in the dorsal hippocampus. Wistar rat pups were submitted to a 3 h maternal separation (MS) protocol during postnatal days 1-21, while another group was left undisturbed. On postnatal day 23, a subgroup from each rearing condition (maternal separation, no-maternal separation) was housed in enriched environmental conditions until postnatal day 65 (6 weeks duration). At approximately three months of age, adult rats underwent behavioral testing to evaluate anxiety (Elevated Plus Maze), locomotion (Open Field Test), spatial learning and memory (Morris Water Maze) as well as non-spatial recognition memory (Novel Object Recognition Test). After completion of behavioral testing, blood samples were taken for evaluation of stress-induced plasma corticosterone using an enzyme-linked immunosorbent assay (ELISA), while immunofluorescence was applied to evaluate hippocampal BDNF and synaptophysin expression in dorsal hippocampus. We found that environmental enrichment protected against the effects of maternal separation as indicated by the lower anxiety levels and the reversal of spatial memory deficits compared to animals housed in standard conditions. These changes were associated with increased BDNF and synaptophysin expression in the hippocampus. Regarding the neuroendocrine response to stress, while exposure to an acute stressor potentiated corticosterone increases in maternally-separated rats, environmental enrichment of these rats prevented this effect. The current study aimed at investigating the compensatory role of enriched environment against the negative outcomes of adverse experiences early in life concurrently on emotional and cognitive behaviors, HPA function and neuroplasticity markers.

Humoral response in experimental autoimmune encephalomyelitis targets neural precursor cells in the central nervous system of naive rodents.

BACKGROUND: Neural precursor cells (NPCs) located in the subventricular zone (SVZ), a well-defined NPC niche, play a crucial role in central nervous system (CNS) homeostasis. Moreover, NPCs are involved in the endogenous reparative process both in multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE). However, the possibility that NPCs may be vulnerable to immune-related components may not be ruled out. Therefore, we investigated the potential affinity of myelin oligodendrocyte glycoprotein (MOG)-induced humoral response(s) to NPCs. METHODS: MOG35-55-EAE was induced in C57BL/6 mice; blood-sampling was performed on days 17-21 (acute phase) along with a naive group and corresponding antisera (AS) were collected (EAE-AS, NAIVE-AS). The presence of anti-CNS autoantibodies was examined with western blotting. Furthermore, using the collected antisera and anti-MOG antibody (as positive control), immunohistochemistry and double immunofluorescence were implemented on normal neonatal, postnatal, and adult mouse brain sections. Targeted NPCs were identified with confocal microscopy. In vitro immunoreactivity assessment on NPCs challenged with autoantibodies was evaluated for apoptotic/autophagic activity. RESULTS: Western blotting verified the existence of autoantibodies in EAE mice and demonstrated bands corresponding to yet unidentified NPC surface epitopes. A dominant selective binding of EAE-AS in the subventricular zone in all age groups compared to NAIVE-AS (p < 0.001) was observed. Additionally, anti-BrdU+/EAE-AS+ colocalization was significantly higher than anti-BrdU+/anti-MOG+, a finding suggesting that the EAE humoral response colocalized with NPCs(BrdU+), cells that do not express MOG. Well-established NPC markers (Nestin, m-Musashi-1, Sox2, DCX, GFAP, NG2) were used to identify the distinct cell types which exhibited selective binding with EAE-AS. The findings verified that EAE-AS exerts cross-reactivity with NPCs which varies throughout the neonatal to adult stage, with a preference to cells of early developmental stages. Finally, increased expressions of Caspase 3 and Beclin 1 on NPCs were detected. CONCLUSION: We provide evidence for the first time that MOG35-55 EAE induces production of antibodies with affinity to SVZ of naive mice in three different age groups. These autoantibodies target lineage-specific NPCs as brain develops and have the potential to trigger apoptotic pathways. Thus, our findings provide indication that cross-talk between immunity and NPCs may lead to functional alteration of NPCs regarding their viability and potentially oligodendrogenesis and effective remyelination.

Long-term effects of enriched environment following neonatal hypoxia-ischemia on behavior, BDNF and synaptophysin levels in rat hippocampus: Effect of combined treatment with G-CSF.

Increasing evidence shows that exposure to an enriched environment (EE) is neuroprotective in adult and neonatal animal models of brain ischemia. However, the mechanisms underlying this effect remain unclear. The aim of the current study was to investigate whether post-weaning EE would be effective in preventing functional deficits and brain damage by affecting markers of synaptic plasticity in a neonatal rat model of hypoxia-ischemia (HI). We also examined the possibility that granulocyte-colony stimulating factor (G-CSF), a growth factor with known neuroprotective effects in a variety of experimental brain injury models, combined with EE stimulation could enhance the potential beneficial effect of EE. Seven-day-old Wistar rats of either sex were subjected to permanent ligation of the left common carotid artery followed by 60min of hypoxia (8% O2) and immediately after weaning (postnatal day 21) were housed in enriched conditions for 4weeks. A group of enriched-housed rats had been treated with G-CSF immediately after HI for 5 consecutive days (50µg/kg/day). Behavioral examination took place approximately at three months of age and included assessments of learning and memory (Morris water maze) as well as motor coordination (Rota-Rod). Infarct size and hippocampal area were estimated following behavioral assessment. Synaptic plasticity was evaluated based on BDNF and synaptophysin expression in the dorsal hippocampus. EE resulted in recovery of post-HI motor deficits and partial improvement of memory impairments which was not accompanied by reduced brain damage. Increased synaptophysin expression was observed in the contralateral to carotid ligation hemisphere. Hypoxia-ischemia alone or followed by enriched conditions did not affect BDNF expression which was increased only in enriched-housed normal rats. The combined therapy of G-CSF and EE further enhanced cognitive function compared to EE provided as monotherapy and prevented HI-induced brain damage by altering synaptic plasticity as reflected by increased synaptophysin expression. The above findings demonstrate that combination of neuroprotective treatments may result in increased protection and it might be a more effective strategy for the treatment of neonatal hypoxic-ischemic brain injury.

Nogo receptor complex expression dynamics in the inflammatory foci of central nervous system experimental autoimmune demyelination.

BACKGROUND: Nogo-A and its putative receptor NgR are considered to be among the inhibitors of axonal regeneration in the CNS. However, few studies so far have addressed the issue of local NgR complex multilateral localization within inflammation in an MS mouse model of autoimmune demyelination. METHODS: Chronic experimental autoimmune encephalomyelitis (EAE) was induced in C57BL/6 mice. Analyses were performed on acute (days 18-22) and chronic (day 50) time points and compared to controls. The temporal and spatial expression of the Nogo receptor complex (NgR and coreceptors) was studied at the spinal cord using epifluorescent and confocal microscopy or real-time PCR. Data are expressed as cells/mm2, as mean % ± SEM, or as arbitrary units of integrated density. RESULTS: Animals developed a moderate to severe EAE without mortality, followed by a progressive, chronic clinical course. NgR complex spatial expression varied during the main time points of EAE. NgR with coreceptors LINGO-1 and TROY was increased in the spinal cord in the acute phase whereas LINGO-1 and p75 signal seemed to be dominant in the chronic phase, respectively. NgR was detected on gray matter NeuN+ neurons of the spinal cord, within the white matter inflammatory foci (14.2 ± 4.3 % NgR+ inflammatory cells), and found to be colocalized with GAP-43+ axonal growth cones while no ß-TubIII+, SMI-32+, or APP+ axons were found as NgR+. Among the NgR+ inflammatory cells, 75.6 ± 9.0 % were microglial/macrophages (lectin+), 49.6 ± 14.2 % expressed CD68 (phagocytic ED1+ cells), and no cells were Mac-3+. Of these macrophages/monocytes, only Arginase-1+/NgR+ but not iNOS+/NgR+ were present in lesions both in acute and chronic phases. CONCLUSIONS: Our data describe in detail the expression of the Nogo receptor complex within the autoimmune inflammatory foci and suggest a possible immune action for NgR apart from the established inhibitory one on axonal growth. Its expression by inflammatory macrophages/monocytes could signify a possible role of these cells on axonal guidance and clearance of the lesioned area during inflammatory demyelination.

Maternal separation prior to neonatal hypoxia-ischemia: Impact on emotional aspects of behavior and markers of synaptic plasticity in hippocampus.

Exposure to early-life stress is associated with long-term alterations in brain and behavior, and may aggravate the outcome of neurological insults. This study aimed at investigating the possible interaction between maternal separation, a model of early stress, and subsequent neonatal hypoxia-ischemia on emotional behavior and markers of synaptic plasticity in hippocampus. Therefore, rat pups (N=60) were maternally separated for a prolonged (MS 180min) or a brief (MS 15min) period during the first six postnatal days, while a control group was left undisturbed. Hypoxia-ischemia was applied to a subgroup of each rearing condition on postnatal day 7. Emotional behavior was examined at three months of age and included assessments of anxiety (elevated plus maze), depression-like behavior (forced swimming) and spontaneous exploration (open field). Synaptic plasticity was evaluated based on BDNF and synaptophysin expression in CA3 and dentate gyrus hippocampal regions. We found that neonatal hypoxia-ischemia caused increased levels of anxiety, depression-like behavior and locomotor activity (ambulation). Higher anxiety levels were also seen in maternally separated rats (MS180min) compared to non-maternally separated rats, but prolonged maternal separation prior to HI did not potentiate the HI-associated effect. No differences among the three rearing conditions were found regarding depression-like behavior or ambulation. Immunohistochemical evaluation of synaptophysin revealed that both prolonged maternal separation (MS180min) and neonatal hypoxia-ischemia significantly reduced its expression in the CA3 and dentate gyrus. Decreases in synaptophysin expression in these areas were not exacerbated in rats that were maternally separated for a prolonged period prior to HI. Regarding BDNF expression, we found a significant decrease in immunoreactivity only in the hypoxic-ischemic rats that were subjected to the prolonged maternal separation paradigm. The above findings suggest that early-life stress prior to neonatal hypoxia-ischemia leads to significant alterations in synaptic plasticity of the dorsal hippocampus during adulthood, but does not exacerbate HI-related changes in emotional behavior.

Subcutaneous Transplantation of Neural Precursor Cells in Experimental Autoimmune Encephalomyelitis Reduces Chemotactic Signals in the Central Nervous System.

UNLABELLED: Neural precursor cell (NPC) transplantation has been proposed as a therapy for multiple sclerosis (MS) and other degenerative disorders of the central nervous system (CNS). NPCs are suggested to exert immune modulation when they are transplanted in the animal model of MS, experimental autoimmune encephalomyelitis (EAE). Herein, we explore whether the effect of NPC transplantation on the clinical course and the pathological features of EAE is combined with the modulation of chemokines levels expressed in the inflamed CNS. NPCs were isolated from brains of neonatal C57/Bl6 mice and were subcutaneously administered in female mice with myelin oligodendrocyte glycoprotein (MOG)-induced EAE. Clinical signs of the disease and transcript analysis of the CNS in the acute phase were performed. In addition, the presence of inflammatory components in the spinal cord was evaluated and ex vivo proliferation of lymphocytes was measured. NPC recipients exhibited ameliorated clinical outcome and less pronounced pathological features in their spinal cord. Downregulation of chemokine mRNA levels throughout the CNS was correlated with diminished Mac-3-, CD3-, and CD4-positive cells and reduced expression levels of antigen-presenting molecules in the spinal cord. Moreover, NPC transplantation resulted in lymphocyte-related, although not splenocyte-related, peripheral immunosuppression. We conclude that NPCs ameliorated EAE potentially by modulating the levels of chemokines expressed in the inflamed CNS, thus resulting in the impaired recruitment of immune cells. These findings further contribute to the better understanding of NPCs' immunomodulatory properties in neuroinflammatory disorders, and may lead to faster translation into potential clinical use. SIGNIFICANCE: Endogenous neural precursor cells of the central nervous system are able to migrate and differentiate toward mature cells to repair an injury. There is increasing evidence that autologous transplantation of these cells in experimental autoimmune encephalomyelitis, the animal model of multiple sclerosis, may have a beneficial effect on the disease process. Several mechanisms have been proposed-among them, the potentiation of endogenous precursor cell differentiation of the central nervous system and the modulation of demyelinating and neurodegenerative immune-mediated processes. This article provides evidence of interference in immune signaling within the central nervous system as a potential mechanism underlying the immunomodulatory properties of transplanted neural precursor cells.

Connexin43 and connexin47 alterations after neural precursor cells transplantation in experimental autoimmune encephalomyelitis.

Exogenous transplanted neural precursor cells (NPCs) exhibit miscellaneous immune-modulatory effects in models of autoimmune demyelination. However, the regional interactions of NPCs with the host brain tissue in remissive inflammatory events have not been adequately studied. In this study we used the chronic MOG-induced Experimental Autoimmune Encephalomyelitis (EAE) model in C57BL/six mice. Based on previous data, we focused on neuropathology at Day 50 post-induction (D50) and studied the expression of connexin43 (Cx43) and Cx47, two of the main glial gap junction (GJ) proteins, in relation to the intraventricular transplantation of GFP(+) NPCs and their integration with the host tissue. By D50, NPCs had migrated intraparenchymally and were found in the corpus callosum at the level of the lateral ventricles and hippocampus. The majority of GFP(+) cells differentiated with simple or ramified processes expressing mainly markers of mature GLIA (GFAP and NogoA) and significantly less of precursor glial cells. GFP(+) NPCs expressed connexins and formed GJs around the hippocampus more than lateral ventricles. The presence of NPCs did not alter the increase in Cx43 GJ plaques at D50 EAE, but prevented the reduction of oligodendrocytic Cx47, increased the number of oligodendrocytes, local Cx47 levels and Cx47 GJ plaques per cell. These findings suggest that transplanted NPCs may have multiple effects in demyelinating pathology, including differentiation and direct integration into the panglial syncytium, as well as amelioration of oligodendrocyte GJ loss, increasing the supply of potent myelinating cells to the demyelinated tissue.

Changes in thyroid hormone receptors after permanent cerebral ischemia in male rats.

Thyroid hormones (TH) and receptors (TRs) may play an important role in the pathophysiology of acute cerebral ischemia. In the present study, we sought to determine whether serum triodothyronine (T3)/thyroxine (T4) and brain TRs (TRα1, TRß1) might change after experimental stroke. Male adult Wistar rats were subjected to permanent middle cerebral artery occlusion (group P) and compared to sham-operated controls (group S). Animals were followed clinically for 14 days until brain collection for Western blot (WB) or neuropathological analysis of TRs in three different brain areas (infarcted tissue, E1; noninfarcted ipsilateral hemisphere, E2; and contralateral hemisphere, E3). Analysis of serum TH levels showed a reduction of T4 in group P (p = 0.002) at days 2 to 14, while half of the animals also displayed "low T3" values (p = 0.012) on day 14. This T4 reduction was inversely correlated to the clinical severity of stroke and the concomitant body weight loss (p < 0.005). WB analysis of TRα1 and TRß1 protein expression showed heterogenic responses at day 14: total and nuclear TRα1 were similar between the two groups, while total TRß1 decreased 7.5-fold within E1 (p ≤ 0.001) with a concomitant 1.8-fold increase of nuclear TRß1 in E2 area (p = 0.03); TRß1 expression did not differ in E3. Neuropathological analysis revealed that activated macrophages/microglia exclusively expressed nuclear TRα1 within the infarct core. Astrocytes mildly expressed nuclear TRα1 in and around the infarct, along with a prominent TRß nuclear signal restricted in the astrocytic scar. Neurons around the infarct expressed mainly TRα1 and, to a milder degree, TRß. Surprisingly enough, we detected for the first time a TRß expression in the paranodal region of Ranvier nodes, of unknown significance so far. Our data support that cerebral ischemia induces a low TH response, associated with significant and heterogenic changes in brain TR expression. These findings could imply an important role of TH signaling in cerebral ischemia.

Repeated immunization of mice with phosphorylated-tau peptides causes neuroinflammation.

The recent studies of others and of us showing robust efficacy of anti-tangle immunotherapy, directed against phosphorylated (phos)-tau protein, may pave the way to clinical trials of phos-tau immunotherapy in Alzheimer's-disease and other tauopathies. At this stage addressing the safety of the phos-tau-immunotherapy is highly needed, particularly since we have previously shown the neurotoxic potential of tau-immunotherapy, specifically of full-length unphosphorylated-tau vaccine under a CNS-proinflammatory milieu [induced by emulsification in complete-Freund's-adjuvant (CFA) and pertussis-toxin (PT)] in young wild-type (WT)-mice. The aim of our current study was to address safety aspects of the phos-tau-immunotherapy in both neurofibrillary-tangle (NFT)-mice as well as in WT-mice, under challenging conditions of repeated immunizations with phos-tau peptides under a CNS-proinflammatory milieu. NFT- and WT-mice were repeatedly immunized (7 injections in adult-, 4 in aged-mice) with phos-tau peptides emulsified in CFA-PT. A paralytic disease was evident in the phos-tau-immunized adult NFT-mice, developing progressively to 26.7% with the number of injections. Interestingly, the WT-mice were even more prone to develop neuroinflammation following phos-tau immunization, affecting 75% of the immunized mice. Aged mice were less prone to neuroinflammatory manifestations. Anti-phos-tau antibodies, detected in the serum of immunized mice, partially correlated with the neuroinflammation in WT-mice. This points that repeated phos-tau immunizations in the frame of a proinflammatory milieu may be encephalitogenic to tangle-mice, and more robustly to WT-mice, indicating that - under certain conditions - the safety of phos-tau immunotherapy is questionable.

Time course and spatial profile of Nogo-A expression in experimental autoimmune encephalomyelitis in C57BL/6 mice.

Inhibition of the myelin-associated neurite outgrowth inhibitor Nogo-A has been found to be beneficial in experimental autoimmune encephalomyelitis (EAE), but there are little data on its expression dynamics during the disease course. We analyzed Nogo-A mRNA and protein during the course of EAE in 27 C57BL/6 mice and in 8 controls. Histopathologic and molecular analyses were performed on Day 0 (naive), preclinical (Day 10), acute (Days 18-22) and chronic (Day 50) time points. In situ hybridization and real-time polymerase chain reaction analyses revealed reduced Nogo-A mRNA expression at preclinical (p < 0.0001) and acute phases (p < 0.0001), followed by upregulation during the chronic phase (p < 0.0001). Nogo-A mRNA was expressed in neurons and oligodendrocytes. By immunohistochemistry and Western blot, there was increased Nogo-A protein expression (p < 0.001) in the chronic phase. Moreover, spatial differences were observed within EAE lesions. The pattern of Nogo-A protein expression inversely correlated with axonal regeneration growth-associated protein 43-positive axons (60% of which were Nogo-A contact-free during the acute phase) and axonal injury (ß-amyloid precursor protein-positive axons). Cortical Nogo-66 receptor protein and mRNA levels increased during the chronic phase. The results indicate that Nogo-A and Nogo receptor are actively regulated in EAE lesions; this may indicate a specific time window for localized axonal regeneration in the acute phase of EAE.