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More than a quarter of veterans of the 1990–1991 Persian Gulf War suffer from Gulf War Illness (GWI), a chronic, multi-symptom illness that commonly includes musculoskeletal pain. Exposure to a range of toxic chemicals, including sarin nerve agent, are a suspected root cause of GWI. Moreover, such chemical exposures induce a neuroinflammatory response in rodents, which has been linked to several GWI symptoms in rodents and veterans with GWI. To date, a neuroinflammatory basis for pain associated with GWI has not been investigated. Here, we evaluated development of nociceptive hypersensitivity in a model of GWI. Male Sprague Dawley rats were treated with corticosterone in the drinking water for 7 days, to mimic high physiological stress, followed by a single injection of the sarin nerve agent surrogate, diisopropyl fluorophosphate. These exposures alone were insufficient to induce allodynia. However, an additional sub-threshold challenge (a single intramuscular injection of pH 4 saline) induced long-lasting, bilateral allodynia. Such allodynia was associated with elevation of markers for activated microglia/macrophages (CD11b) and astrocytes/satellite glia (GFAP) in the lumbar dorsal spinal cord and dorsal root ganglia (DRG). Additionally, Toll-like receptor 4 (TLR4) mRNA was elevated in the lumbar dorsal spinal cord, while IL-1β and IL-6 were elevated in the lumbar dorsal spinal cord, DRG, and gastrocnemius muscle. Demonstrating a casual role for such neuroinflammatory signaling, allodynia was reversed by treatment with either minocycline, the TLR4 inhibitor (+)-naltrexone, or IL-10 plasmid DNA. Together, these results point to a role for neuroinflammation in male rats in the model of musculoskeletal pain related to GWI. Therapies that alleviate persistent immune dysregulation may be a strategy to treat pain and other symptoms of GWI.

Aims Growing evidence suggests that Gulf War Illness (GWI) is the result of underlying neuroimmune dysfunction. For example, previously we found that several GWI-relevant organophosphate acetylcholinesterase inhibitors produce heightened neuroinflammatory responses following subchronic exposure to stress hormone as a mimic of high physiological stress. The goal of the current study was to evaluate the potential for the β-adrenergic receptor inhibitor and anti-inflammatory drug, propranolol, to treat neuroinflammation in a novel long-term mouse model of GWI. Main methods Adult male C57BL/6J mice received a subchronic exposure to corticosterone (CORT) at levels mimicking high physiological stress followed by exposure to the sarin surrogate, diisopropyl fluorophosphate (DFP). These mice were then re-exposed to CORT every other week for a total of five weeks, followed by a systemic immune challenge with lipopolysaccharide (LPS). Animals receiving the propranolol treatment were given a single dose (20 mg/kg, i.p.) either four or 11 days prior to the LPS challenge. The potential anti-neuroinflammatory effects of propranolol were interrogated by analysis of cytokine mRNA expression. Key findings We found that our long-term GWI model produces a primed neuroinflammatory response to subsequent immune challenge that is dependent upon GWI-relevant organophosphate exposure. Propranolol treatment abrogated the elaboration of inflammatory cytokine mRNA expression in the brain instigated in our model, having no treatment effects in non-DFP exposed groups. Significance Our results indicate that propranolol may be a promising therapy for GWI with the potential to treat the underlying neuroinflammation associated with the illness.

Published: 9 August 2021 GulfWar Illness (GWI) is a persistent chronic neuroinflammatory illness exacerbated by external stressors and characterized by fatigue, musculoskeletal pain, cognitive, and neurological problems linked to underlying immunological dysfunction for which there is no known treatment. As the immune system and the brain communicate through several signaling pathways, including the hypothalamic–pituitary–adrenal (HPA) axis, it underlies many of the behavioral and physiological responses to stressors via blood-borne mediators, such as cytokines, chemokines, and hormones. Signaling by these molecules is mediated by the semipermeable blood–brain barrier (BBB) made up of a monocellular layer forming an integral part of the neuroimmune axis. BBB permeability can be altered and even diminished by both external factors (e.g., chemical agents) and internal conditions (e.g., acute or chronic stress, or cross-signaling from the hypothalamic–pituitary–gonadal (HPG) axis). Such a complex network of regulatory interactions that possess feed-forward and feedback connections can have multiple response dynamics that may include several stable homeostatic states beyond normal health. Here we compare immune and hormone measures in the blood of human clinical samples and mouse models of Gulf War Illness (GWI) subtyped by exposure to traumatic stress for subtyping this complex illness. We do this via constructing a detailed logic model of HPA–HPG–Immune regulatory behavior that also considers signaling pathways across the BBB to neuronal–glial interactions within the brain. We apply conditional interactions to model the effects of changes in BBB permeability. Several stable states are identified in the system beyond typical health. Following alignment of the human and mouse blood profiles in the context of the model, mouse brain sample measures were used to infer the neuroinflammatory state in human GWI and perform treatment simulations using a genetic algorithm to optimize the Monte Carlo simulations of the putative treatment strategies aimed at returning the ill system back to health. We identify several ideal multi-intervention strategies and potential drug candidates that may be used to treat chronic neuroinflammation in GWI. This research was funded by the U.S. Department of Defense—Congressionally Directed Medical Research Programs—GulfWar Illness Research Program, grant numbers W81XWH-16-1-0632 (Craddock PI), and W81XWH-16-1-0552 (Craddock PI), W81XWH-13-2-0085 (Morris PI). Intramural funds from the Centers for Disease Control—National Institute for Occupational Safety and Health (O’Callaghan PI). The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Paraquat (PQ) is an herbicide used in many countries, including the United States. It is also implicated as a risk factor for sporadic Parkinson’s disease, especially in those living in agricultural areas and drinking well water. Studies linking PQ to sporadic Parkinson’s disease are not consistent however and there appears to be interindividual differential susceptibility. One likely reason is genetically based differential susceptibility to paraquat neurotoxicity in subpopulations. To address this issue, we tested the effects of paraquat in a genetic reference population of mice (the BXD recombinant inbred strain family). In our earlier work, we showed that in genetically susceptible mice, paraquat increases iron in the ventral midbrain, the area containing the substantia nigra. Our hypothesis is that genetic variability contributes to diverse PQ-related susceptibility and iron concentration. To test this hypothesis, we treated male mice from 28 to 39 BXD strains plus the parental strains with 1 of 3 doses of paraquat, 1, 5, and 10 mg/kg 3 times on a weekly basis. At the end of the treatment period, we analyzed the ventral midbrain for concentrations of iron, copper, and zinc, also we measured the concentration of paraquat in cerebellum, and proinflammatory cytokines in serum and cerebellum. The effect on paraquat-treated mice with 5 mg/kg and principal component analysis of iron showed suggestive quantitative trait loci on chromosome 5. Overall, our results suggest that gene Prkag2 and related networks may serve as potential targets against paraquat toxicity and demonstrate the utility of genetically diverse mouse models for the study of complex human toxicity.

Gulf War Illness (GWI) is a chronic multi-symptom disorder experienced by as many as a third of the veterans of the 1991 Gulf War; the constellation of “sickness behavior” symptoms observed in ill veterans is suggestive of a neuroimmune involvement. Various chemical exposures and conditions in theater have been implicated in the etiology of the illness. Previously, we found that GW-related organophosphates (OPs), such as the sarin surrogate, DFP, and chlorpyrifos, cause neuroinflammation. The combination of these exposures with exogenous corticosterone (CORT), mimicking high physiological stress, exacerbates the observed neuroinflammation. The potential relationship between the effects of OPs and CORT on the brain versus inflammation in the periphery has not been explored. Here, using our established GWI mouse model, we investigated the effects of CORT and DFP exposure, with or without a chronic application of pyridostigmine bromide (PB) and N,N-diethyl-meta-toluamide (DEET), on cytokines in the liver and serum. While CORT primed DFP-induced neuroinflammation, this effect was largely absent in the periphery. Moreover, the changes found in the peripheral tissues do not correlate with the previously reported neuroinflammation. These results not only support GWI as a neuroimmune disorder, but also highlight the separation between central and peripheral effects of these exposures.

Many veterans of the 1991 Persian Gulf War (GW) returned with a chronic multisymptom illness that has been termed Gulf War Illness (GWI). Previous GWI studies have suggested that exposure to acetylcholinesterase inhibitors (AChEIs) in theater, such as sarin and/or pesticides, may have contributed to the symptomatology of GWI. Additionally, concomitant high physiological stress experienced during the war may have contributed to the initiation of the GWI phenotype. Although inhibition of AChE leading to accumulation of acetylcholine (ACh) will activate the cholinergic anti-inflammatory pathway, the signature symptomatology of GWI has been shown to be associated with neuroinflammation. To investigate the relationship between ACh and neuroinflammation in discrete brain regions, we used our previously established mouse model of GWI, which combines an exposure to a high physiological stress mimic, corticosterone (CORT), with GW-relevant AChEIs. The AChEIs used in this study were diisopropyl fluorophosphate (DFP), chlorpyrifos oxon (CPO), and physostigmine (PHY). After AChEI exposure, ACh concentrations for cortex (CTX), hippocampus (HIP), and striatum (STR) were determined using hydrophilic interaction liquid chromatography with ultraperformance liquid chromatography-tandem-mass spectrometry (MS/MS). CORT pretreatment ameliorated the DFP-induced ACh increase in HIP and STR, but not CTX. CORT pretreatment did not significantly alter ACh levels for CPO and PHY. Further analysis of STR neuroinflammatory biomarkers revealed an exacerbated CORT + AChEI response, which does not correspond to measured brain ACh. By utilizing this new analytical method for discrete brain region analysis of ACh, this work suggests the exacerbated neuroinflammatory effects in our mouse model of GWI are not driven by the accumulation of brain region-specific ACh.

Background Gulf War illness (GWI) is an archetypal, medically unexplained, chronic condition characterised by persistent sickness behaviour and neuroimmune and neuroinflammatory components. An estimated 25–32% of the over 900,000 veterans of the 1991 Gulf War fulfil the requirements of a GWI diagnosis. It has been hypothesised that the high physical and psychological stress of combat may have increased vulnerability to irreversible acetylcholinesterase (AChE) inhibitors leading to a priming of the neuroimmune system. A number of studies have linked high levels of psychophysiological stress and toxicant exposures to epigenetic modifications that regulate gene expression. Recent research in a mouse model of GWI has shown that pre-exposure with the stress hormone corticosterone (CORT) causes an increase in expression of specific chemokines and cytokines in response to diisopropyl fluorophosphate (DFP), a sarin surrogate and irreversible AChE inhibitor. Methods C57BL/6J mice were exposed to CORT for 4 days, and exposed to DFP on day 5, before sacrifice 6 h later. The transcriptome was examined using RNA-seq, and the epigenome was examined using reduced representation bisulfite sequencing and H3K27ac ChIP-seq. Results We show transcriptional, histone modification (H3K27ac) and DNA methylation changes in genes related to the immune and neuronal system, potentially relevant to neuroinflammatory and cognitive symptoms of GWI. Further evidence suggests altered proportions of myelinating oligodendrocytes in the frontal cortex, perhaps connected to white matter deficits seen in GWI sufferers. Conclusions Our findings may reflect the early changes which occurred in GWI veterans, and we observe alterations in several pathways altered in GWI sufferers. These close links to changes seen in veterans with GWI indicates that this model reflects the environmental exposures related to GWI and may provide a model for biomarker development and testing future treatments. Electronic supplementary material The online version of this article (10.1186/s12974-018-1113-9) contains supplementary material, which is available to authorized users.

Neurotoxicity has been linked with exposure to a number of common drugs and chemicals, yet efficient, accurate, and minimally invasive methods to detect it are lacking. Fluid-based biomarkers such as those found in serum, plasma, urine, and cerebrospinal fluid have great potential due to the relative ease of sampling but at present, data on their expression and translation are lacking or inconsistent. In this pilot study using a trimethyl tin rat model of central nervous system toxicity, we have applied state-of-the-art assessment techniques to identify potential individual biomarkers and patterns of biomarkers in serum, plasma, urine or cerebral spinal fluid that may be indicative of nerve cell damage and degeneration. Overall changes in metabolites and microRNAs were observed in biological fluids that were associated with neurotoxic damage induced by trimethyl tin. Behavioral changes and magnetic resonance imaging T2 relaxation and ventricle volume changes served to identify animals that responded to the adverse effects of trimethyl tin. Impact statement These data will help design follow-on studies with other known neurotoxicants to be used to assess the broad applicability of the present findings. Together this approach represents an effort to begin to develop and qualify a set of translational biochemical markers of neurotoxicity that will be readily accessible in humans. Such biomarkers could prove invaluable for drug development research ranging from preclinical studies to clinical trials and may prove to assist with monitoring of the severity and life cycle of brain lesions.

Gulf War Illness (GWI) is a chronic multi‐symptom disorder affecting veterans of the 1991 Gulf War. Among the symptoms of GWI are those associated with sickness behavior, observations suggestive of underlying neuroinflammation. We have shown that exposure of mice to the stress hormone, corticosterone (CORT), and to diisopropyl fluorophosphate (DFP), as a nerve agent mimic, results in marked neuroinflammation, findings consistent with a stress/neuroimmune basis of GWI. Here, we examined the contribution of irreversible and reversible acetylcholinesterase (AChE) inhibitors to neuroinflammation in our mouse model of GWI. Male C57BL/6J mice received 4 days of CORT (400 mg/L) in the drinking water followed by a single dose of chlorpyrifos oxon (CPO; 8 mg/kg, i.p.), DFP (4 mg/kg, i.p.), pyridostigmine bromide (PB; 3 mg/kg, i.p.), or physostigmine (PHY; 0.5 mg/kg, i.p.). CPO and DFP alone caused cortical and hippocampal neuroinflammation assessed by qPCR of tumor necrosis factor‐alpha, IL‐6, C–C chemokine ligand 2, IL‐1β, leukemia inhibitory factor and oncostatin M; CORT pretreatment markedly augmented these effects. Additionally, CORT exposure prior to DFP or CPO enhanced activation of the neuroinflammation signal transducer, signal transducer and activator of transcription 3 (STAT3). In contrast, PHY or PB alone or with CORT pretreatment did not produce neuroinflammation or STAT3 activation. While all of the CNS‐acting AChE inhibitors (DFP, CPO, and PHY) decreased brain AChE activity, CORT pretreatment abrogated these effects for the irreversible inhibitors. Taken together, these findings suggest that irreversible AChE inhibitor‐induced neuroinflammation and particularly its exacerbation by CORT, result from non‐cholinergic effects of these compounds, pointing potentially to organophosphorylation of other neuroimmune targets.

The rise in popularity of substituted methcathinones (aka “bath salts”) has increased the focus on their neurotoxic effects. Two commonly abused methcathinones, 3,4-methylenedioxymethcathinone (methylone, MDMC) and 3,4-methylenedioxypyrovalerone (MDPV), are often concomitantly ingested with the illicit drug 3,4-methylenedioxymethamphetamine (MDMA). To examine potential neurotoxic effects of these drug combinations, C57BL/6J mice were administered 4 i.p. injection of the drugs, at 2 h intervals, either singularly: MDMA 15 or 30 mg/kg, methylone 20 mg/kg, MDPV 1 mg/kg; or in combination: methylone/MDMA 20/15 mg/kg, MDPV/MDMA 1/15 mg/kg. Drug effects on thermoregulation were characterized and striatal tissue analyzed after 2 or 7 days for dopamine (DA) and tyrosine hydroxylase (TH) levels, as well as glial fibrillary acidic protein (GFAP) expression. Two days following drug administration, DA and TH were decreased only in the MDMA 30 mg/kg group, whereas GFAP expression was dose-dependently increased by MDMA alone. While the combination of the methcathinones with the lower MDMA dose did not affect DA or TH levels, both blocked the MDMA-induced increase in GFAP expression. Seven days following drug administration, there were no significant differences in DA, TH, or GFAP for any treatment group, indicating that changes in DA, TH, and GFAP were transient. Five of the six drug groups exhibited acute hypothermia followed by gradually increasing temperatures. Animals treated with MDPV did not exhibit these biphasic temperature changes, and resembled the saline group. These results indicate that specific effects of both methylone and MDPV on DA depletion or astrocyte activation in the striatum are not additive with effects of MDMA, but block astrogliosis caused by MDMA alone. Additionally, MDPV modulates thermoregulation through a different mechanism than methylone or MDMA.

Gulf War Illness (GWI) is a multi‐symptom disorder with features characteristic of persistent sickness behavior. Among conditions encountered in the Gulf War (GW) theater were physiological stressors (e.g., heat/cold/physical activity/sleep deprivation), prophylactic treatment with the reversible AChE inhibitor, pyridostigmine bromide (PB), the insect repellent, N,N‐diethyl‐meta‐toluamide (DEET), and potentially the nerve agent, sarin. Prior exposure to the anti‐inflammatory glucocorticoid, corticosterone (CORT), at levels associated with high physiological stress, can paradoxically prime the CNS to produce a robust proinflammatory response to neurotoxicants and systemic inflammation; such neuroinflammatory effects can be associated with sickness behavior. Here, we examined whether CORT primed the CNS to mount neuroinflammatory responses to GW exposures as a potential model of GWI. Male C57BL/6 mice were treated with chronic (14 days) PB/ DEET, subchronic (7–14 days) CORT, and acute exposure (day 15) to diisopropyl fluorophosphate (DFP), a sarin surrogate and irreversible AChE inhibitor. DFP alone caused marked brain‐wide neuroinflammation assessed by qPCR of tumor necrosis factor‐α, IL6, chemokine (C‐C motif) ligand 2, IL‐1β, leukemia inhibitory factor, and oncostatin M. Pre‐treatment with high physiological levels of CORT greatly augmented (up to 300‐fold) the neuroinflammatory responses to DFP. Anti‐inflammatory pre‐treatment with minocycline suppressed many proinflammatory responses to CORT+DFP. Our findings are suggestive of a possible critical, yet unrecognized interaction between the stressor/environment of the GW theater and agent exposure(s) unique to this war. Such exposures may in fact prime the CNS to amplify future neuroinflammatory responses to pathogens, injury, or toxicity. Such occurrences could potentially result in the prolonged episodes of sickness behavior observed in GWI. Gulf War (GW) veterans were exposed to stressors, prophylactic medicines and, potentially, nerve agents in theater. Subsequent development of GW Illness, a persistent multi‐symptom disorder with features characteristic of sickness behavior, may be caused by priming of the CNS resulting in exaggerated neuroinflammatory responses to pathogens/insults. Nerve agent, diisopropyl fluorophosphate (DFP), produced a neuroinflammatory response that was exacerbated by pre‐treatment with levels of corticosterone simulating heightened stressor conditions. While prophylactic treatments reduced DFP‐induced neuroinflammation, this effect was negated when those treatments were combined with corticosterone.

Veterans of the 1991 Gulf War were potentially exposed to a variety of toxic chemicals, including sarin nerve agent and pesticides, which have been suspected to be involved in the development of Gulf War Illness (GWI). Several of these exposures cause a neuroinflammatory response in mice, which may serve as a basis for the sickness behavior-like symptoms seen in veterans with GWI. Furthermore, conditions mimicking the physiological stress experienced during the war can exacerbate this effect. While neuroinflammation has been observed post-exposure using animal models, it remains a challenge to evaluate neuroinflammation and its associated cellular and molecular changes in vivo in veterans with GWI. Here, we evaluated neuroimmune-associated alterations in intact brains, applying our existing GWI mouse model to rats, by exposing them to 4 days of corticosterone (CORT; 200 mg/L in the drinking water), to mimic high physiological stress, followed by a single injection of the sarin nerve agent surrogate, diisopropyl fluorophosphate (DFP; 1.5 mg/kg, i.p.). Then, we evaluated the neuroinflammatory responses using qPCR of cytokine mRNA and also examined brain structure with a novel high-order diffusion MRI. We found a CORT-enhancement of DFP-induced neuroinflammation, extending our mouse GWI model to the rat. High order diffusion MRI revealed different patterns among the different treatment groups. Particularly, while the CORT + DFP rats had more restricted spatial patterns in the hippocampus and the hypothalamus, the highest and most wide-spread differences were shown in DFP-treated rats compared to the controls in the thalamus, the amygdala, the piriform cortex and the ventral tegmental area. The association of these diffusion changes with neuroinflammatory cytokine expression indicates the potential for GW-relevant exposures to result in connectivity changes in the brain. By transferring this high order diffusion MRI into in vivo imaging in veterans with GWI, we can achieve further insights on the trajectories of the neuroimmune response over time and its impacts on behavior and potential neurological damage.

Systemic exposure to the inflammagen and bacterial endotoxin lipopolysaccharide (LPS) has been widely used to evaluate inflammation and sickness behavior. While many inflammatory conditions occur in the periphery, it is well established that peripheral inflammation can affect the brain. Neuroinflammation, the elaboration of proinflammatory mediators in the CNS, commonly is associated with behavioral symptoms (e.g., lethargy, anhedonia, anorexia, depression, etc.) termed sickness behavior. Stressors have been shown to interact with and alter neuroinflammatory responses and associated behaviors. Here, we examined the effects of the stress hormone, corticosterone (CORT), as a stressor mimic, on neuroinflammation induced with a single injection (2mg/kg, s.c.) or inhalation exposure (7.5 μg/m3) of LPS or polyinosinic:polycytidylic acid (PIC; 12mg/kg, i.p.) in adult male C57BL/6J mice. CORT was given in the drinking water (200 mg/L) for 1 week or every other week for 90 days followed by LPS. Proinflammatory cytokine expression (TNFα, IL-6, CCL2, IL-1β, LIF, and OSM) was measured by qPCR. The activation of the neuroinflammation downstream signaling activator, STAT3, was assessed by immunoblot of pSTAT3Tyr705. The presence of astrogliosis was assessed by immunoassay of GFAP. Acute exposure to LPS caused brain-wide neuroinflammation without producing astrogliosis; exposure to CORT for 1 week caused marked exacerbation of the LPS-induced neuroinflammation. This neuroinflammatory "priming" by CORT was so pronounced that sub-neuroinflammatory exposures by inhalation instigated neuroinflammation when paired with prior CORT exposure. This effect also was extended to another common inflammagen, PIC (a viral mimic). Furthermore, a single week of CORT exposure maintained the potential for priming for 30 days, while intermittent exposure to CORT for up to 90 days synergistically primed the LPS-induced neuroinflammatory response. These findings highlight the possibility for an isolated inflammatory event to be exacerbated by a temporally distant stressful stimulus and demonstrates the potential for recurrent stress to greatly aggravate chronic inflammatory disorders.

Our previous studies have raised the possibility that altered blood glucose levels may influence and/or be predictive of methamphetamine (METH) neurotoxicity. This study evaluated the effects of exogenous glucose and corticosterone (CORT) pretreatment alone or in combination with METH on blood glucose levels and the neural and vascular toxicity produced. METH exposure consisted of four sequential injections of 5, 7.5, 10, and 10 mg/kg (2h between injections) D-METH. The three groups given METH in combination with saline, glucose (METH+Glucose), or CORT (METH+CORT) had significantly higher glucose levels compared to the corresponding treatment groups without METH except at 3 h after the last injection. At this last time point, the METH and METH+Glucose groups had lower levels than the non-METH groups, while the METH+CORT group did not. CORT alone or glucose alone did not significantly increase blood glucose. Mortality rates for the METH+CORT (40%) and METH+Glucose (44%) groups were substantially higher than the METH (< 10%) group. Additionally, METH+CORT significantly increased neurodegeneration above all other treatments (≈ 2.5-fold in the parietal cortex). Thus, maintaining elevated levels of glucose during METH exposure increases lethality and may exacerbate neurodegeneration. Neuroinflammation, specifically microglial activation, was associated with degenerating neurons in the parietal cortex and thalamus after METH exposure. The activated microglia in the parietal cortex were surrounding vasculature in most cases and the extent of microglial activation was exacerbated by CORT pretreatment. Our findings implicate elevated blood levels of glucose and hyperthermia in METH-induced neurotoxicity, neurovascular damage, and lethality, and that acute elevation of CORT exacerbates both neurotoxicity and neuroinflammation. This article is protected by copyright. All rights reserved.

Sprague–Dawley rats were exposed via inhalation to vapor condensates of either gasoline or gasoline combined with various fuel oxygenates to assess potential neurotoxicity of evaporative emissions. Test articles included vapor condensates prepared from “baseline gasoline” (BGVC), or gasoline combined with methyl tertiary butyl ether (G/MTBE), ethyl t-butyl ether (G/ETBE), t-amyl methyl ether (G/TAME), diisopropyl ether (G/DIPE), ethanol (G/EtOH), or t-butyl alcohol (G/TBA). Target concentrations were 0, 2000, 10,000 or 20,000mg/mg3 and exposures were for 6h/day, 5days/week for 13weeks. The functional observation battery (FOB) with the addition of motor activity (MA) testing, hematoxylin and eosin staining of brain tissue sections, and brain regional analysis of glial fibrillary acidic protein (GFAP) were used to assess behavioral changes, traditional neuropathology and astrogliosis, respectively. FOB and MA data for all agents, except G/TBA, were negative. G/TBA behavioral effects resolved during recovery. Neuropathology was negative for all groups. Analyses of GFAP revealed increases in multiplebrain regions largely limited to males of the G/EtOH group, findings indicative of minor gliosis, most significantly in the cerebellum. Small changes (both increases and decreases) in GFAP were observed for other test agents but effects were not consistent across sex, brain region or exposure concentration.

Attention Deficit Hyperactivity Disorder (ADHD) is estimated to affect 4-5% of the adult human population (Kessler et al., 2006; Willcutt, 2012). Often prescribed to attenuate ADHD symptoms (Nair and Moss, 2009), methylphenidate hydrochloride (MPH) can have substantial positive effects. However, there is a paucity of literature regarding its use during pregnancy. Thus, adult women with ADHD face a difficult decision when contemplating pregnancy. In this study, pregnant Sprague-Dawley rats were orally treated a total of 0 (water), 6 (low), 18 (medium), or 42 (high) mg MPH/kg body weight/day (divided into three doses) on gestational days 6-21 (i.e., the low dose received 2 mg MPH/kg body weight 3×/day). Offspring were orally treated with the same daily dose as their dam (divided into two doses) on postnatal days (PNDs) 1-21. One offspring/sex/litter was sacrificed at PND 22 or PND 104 (n=6-7/age/sex/treatment group) and the striatum was quickly dissected and frozen. High Performance Liquid Chromatography (HPLC) coupled to a Photo Diode Array detector (PDA) was used to analyze monoamine content in the striatum of one side while a sandwich ELISA was used to analyze tyrosine hydroxylase (TH) from the other side. Age significantly affected monoamine and metabolite content as well as turnover ratios (i.e., DA, DOPAC, HVA, DOPAC/DA, HVA/DA, 5-HT and 5-HIAA); however, there were no significant effects of sex. Adult rats of the low MPH group had higher DA levels than control adults (p

Background Brain microglial activations and damage responses are most commonly associated with neurodegeneration or systemic innate immune system activation. Here, we used histological methods to focus on microglial responses that are directed towards brain vasculature, previously undescribed, after a neurotoxic exposure to methamphetamine. Methods Male rats were given doses of methamphetamine that produce pronounced hyperthermia, hypertension, and toxicity. Identification of microglia and microglia-like cells (pericytes and possibly perivascular cells) was done using immunoreactivity to allograft inflammatory factor 1 (Aif1 a.k.a Iba1) and alpha M integrin (Itgam a.k.a. Cd11b) while vasculature endothelium was identified using rat endothelial cell antigen 1 (RECA-1). Regions of neuronal, axonal, and nerve terminal degeneration were determined using Fluoro-Jade C. Results Dual labeling of vasculature (RECA-1) and microglia (Iba1) showed a strong association of hypertrophied cells surrounding and juxtaposed to vasculature in the septum, medial dorsal hippocampus, piriform cortex, and thalamus. The Iba1 labeling was more pronounced in the cell body while Cd11b more so in the processes of activated microglia. These regions have been previously identified to have vascular leakage after neurotoxic methamphetamine exposure. Dual labeling with Fluoro-Jade C and Iba1 indicated that there was minimal or no evidence of neuronal damage in the septum and hippocampus where many hypertrophied Iba1-labeled cells were found to be associated with vasculature. Although microglial activation around the prominent neurodegeneration was found in the thalamus, there were also many examples of activated microglia associated with vasculature. Conclusions The data implicate microglia, and possibly related cell types, in playing a major role in responding to methamphetamine-induced vascular damage, and possibly repair, in the absence of neurodegeneration. Identifying brain regions with hypertrophied/activated microglial-like cells associated with vasculature has the potential for identifying regions of more subtle examples of vascular damage and BBB compromise. Electronic supplementary material The online version of this article (doi:10.1186/s12974-016-0526-6) contains supplementary material, which is available to authorized users.

Damage to the CNS results in a complex series of molecular and cellular changes involving the affected targets and the ensuing glial reaction. The initial gene expression events that underlie these cellular responses may serve as early biomarkers of neurotoxicity. Here, we examined gene expression profiles during the initial phase of hippocampal damage resulting from systemic exposure of rats to the organometallic neurotoxicant, trimethyltin (TMT, 8.0 mg/kg, i.p.). Using TMT as a neurodegeneration tool confers several advantages for evaluating molecular events associated with neural damage: 1) regional and cellular targets and time course of damage are known, 2) the blood-brain barrier is not compromised, which limits the contribution of blood-borne factors, e.g. immune, to neural injury responses and 3) known protein and mRNA signatures of TMT-induced neurotoxicity can be used as positive controls to validate novel expression events associated with exposure to this neurotoxicant. Using Affymetrix Gene Chip® to assess gene expression after TMT, combined with Ingenuity Pathway Analysis®, we observed changes consistent for genes known to be affected in hippocampus, while corresponding changes were not detected in cerebellum, a non-target region. In agreement with previous observations, limited changes in expression of inflammation-related genes were observed. Correlated expression profiles were found after exposure to TMT, including changes in gene ontologies associated with neurological disease, cellular assembly and maintenance, as well as signaling pathways associated with cellular stress, energy metabolism and glial activation. Selected gene changes were confirmed from each category by q-RT-PCR and immunoblot analysis. The canonical relationships identified implicate molecular pathways and processes relevant to detection of early stages of hippocampal damage in the TMT model. These observations provide new insight into early events associated with neuronal degeneration and associated glial activation that may serve as the basis for discovery and development of biomarkers of neurotoxicity.

Serious questions have been raised by occupational health investigators regarding a possible causal association between neurological effects in welders and the presence of manganese (Mn) in welding fume. Male Sprague-Dawley rats were exposed by inhalation to 40 mg/m(3) of gas metal arc-mild steel (MS) welding fume for 3 h/day for 10 days. Generated fume was collected in the animal chamber during exposure, and particle size, composition, and morphology were characterized. At 1 day after the last exposure, metal deposition in different organ systems and neurological responses in dopaminergic brain regions were assessed in exposed animals. The welding particles were composed primarily of a complex of iron (Fe) and Mn and were arranged as chain-like aggregates with a significant number of particles in the nanometer size range. Mn was observed to translocate from the lungs to the kidney and specific brain regions (olfactory bulb, cortex, and cerebellum) after MS fume inhalation. In terms of neurological responses, short-term MS fume inhalation induced significant elevations in divalent metal ion transporter 1 (Dmt1) expression in striatum and midbrain and significant increases in expression of proinflammatory chemokines (Ccl2, Cxcl2) and cytokines (IL1beta, TNFalpha) in striatum. In addition, mRNA and protein expression of glial fibrillary acidic protein (GFAP) was significantly increased in striatum after MS fume exposure. However, the 10-day MS welding fume inhalation did not cause any changes in dopamine and its metabolites or GABA in dopaminergic brain regions nor did it produce overt neural cell damage as assessed by histopathology. In summary, short-term MS welding fume exposure led to translocation of Mn to specific brain regions and induced subtle changes in cell markers of neuroinflammatory and astrogliosis. The neurofunctional significance of these findings currently is being investigated in longer, more chronic welding fume exposure studies.

Continuing our previous work in which we showed wide-ranging strain differences in MPTP neurotoxicity in male mice among ten BXD recombinant inbred strains, we replicated our work in females from nine of the same strains. Mice received a single s.c. injection of 12.5 mg/kg MPTP or saline. Forty-eight hours later the striatum was dissected for neurochemical analysis. Striatal dopamine (DA) and its metabolites, DOPAC and HVA, striatal serotonin (5-HT) and its metabolite, 5-HIAA, were analyzed using HPLC. Tyrosine hydroxylase (TH) and glial fibrillary acidic protein (GFAP), an astrocytic protein that increases during the astroglial response to neural injury, were measured using ELISA. There were wide genetic variations in the DA, DOPAC, HVA, TH and GFAP responses to MPTP. We also performed principal component analysis (PCA) on the difference values, saline minus MPTP, for DA, DOPAC, HVA and TH and mapped the dominant principal component to a suggestive QTL on chromosome 1 at the same location that we observed previously for males. Moreover, there were significant correlations between the sexes for the effect of MPTP on DA, HVA, and TH. Our findings suggest that the systems genetic approach as utilized here can help researchers understand the role of sex in individual differences. The same approach can pave the way to understand and pinpoint the genetic bases for individual differences in pathology attributable to toxicants. Such systems genetics approach has broad implications for elucidating gene-environment contributions to neurodegenerative diseases.

Expression of the chemokine (C-C motif) ligand 2 (CCL2), also known as, monocyte chemoattractant protein (MCP)-1, increases in response to disease-, trauma-, or toxicant-induced damage to the central nervous system (CNS). In the periphery, endogenous and exogenous glucocorticoids are known to suppress CCL2 expression associated with inflammatory conditions. However, such actions of glucocorticoids on CCL2 expression in the CNS remain unknown. Here, we explored the effects of the glucocorticoid, corticosterone (CORT), on the expression of CCL2 and its receptors, CCR2 and CCR5, in the hippocampal formation using intact, adrenalectomized (ADX) and trimethyltin (TMT)-treated rats. An immunosuppressive regimen of CORT did not alter the mRNA expression of CCL2 or its receptors in the hippocampus. ADX, however, markedly increased the expression of CCL2 and CCR2 mRNAs in the hippocampus, while CORT replacement reversed the effects of ADX on CCL2 gene expression. Hippocampal damage resulting from systemic administration of the organometallic neurotoxicant, TMT, was associated with microglial activation, as evidenced by enhanced expression of microglial markers integrin alphaM (CD11b) and F4/80, as well as, microglia-associated factors, CCL2 and IL-1alpha. An immunosuppressive dose of CORT, suppressed TMT-induced expression of CCL2. Given the association of CCL2 with microglial activation, it appears that CORT may play a role in regulating microglial activation. However, CORT treatment did not alter TMT-mediated neuronal damage and astrogliosis. Such observations suggest that injury-related expression of microglia-associated chemokines and cytokines may subserve a role unrelated to neuronal damage. In summary, our data indicate that in the CNS, CCL2 gene expression is under negative regulation by glucocorticoids.

A sample preparation method that combines a modified target plate with a nanoscale reversedphase column (nanocolumn) was developed for detection of neuropeptides by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). A gold-coated MALDI plate was modified with an octadecanethiol (ODT) self-assembled monolayer to create a hydrophobic surface that could concentrate peptide samples into a ∼200‐500-µm diameter spot. The spot sizes generated were comparable to those obtained for a substrate patterned with 200-µm hydrophilic spots on a hydrophobic substrate. The sample spots on the ODT-coated plate were 100-fold smaller than those formed on an unmodified gold plate with a 1-µl sample and generated 10 to 50 times higher mass sensitivity for peptide standards by MALDI-TOF MS. When the sample was deposited on an ODT-modified plate from a nanocolumn, thedetectionlimitforpeptideswasaslowas20 pMfor5-µlsamplescorresponding to80 amol deposited. This technique was used to analyze extracts of microwave-fixed tissue from rat brain striatum. Ninety-eight putative peptides were detected including several that had masses matching neuropeptides expected in this brain region such as substance P, rimorphin, and neurotensin. Twenty-three peptides had masses that matched peaks detected by capillary liquid chromatography with electrospray ionization MS. 1,2 Copyright  2005 John Wiley & Sons, Ltd.

1-Methyl-4-(2'-aminophenyl)-1,2,3,6-tetrahydropyridine (2'-NH2-MPTP) causes long-term loss of forebrain serotonin (5-HT) and norepinephrine (NE) and consequently, is unlike 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and its other 2'-analogs that primarily deplete striatal dopamine (DA). In the present investigation into the acute effects of 2'-NH2-MPTP in mice, profound decreases in cortical and hippocampal 5-HT and NE to 10 to 40% of control were observed as early as 30 min post-treatment and lasted throughout the ensuing 21 days. Striatal DA was decreased to 60 to 80% of control during the first 48 h but returned to normal by 72 h. Reactive gliosis, which occurs in response to neurodegeneration was not evident by immunocytochemistry but was detected by enzyme-linked immunosorbent assay, where glial fibrillary acidic protein (GFAP) was increased to 130% of control in cortex, hippocampus, and brain stem 48 to 72 h post-treatment. To explore the possibility that 5-HT modulates the astrocytic response to injury, 2'-NH2-MPTP was used to damage 5-HT axons 2 weeks before administration of the potent DA neurotoxin 1-methyl-4-(2'-methylphenyl)-1,2,3,6-tetrahydropyridine (2'-CH3-MPTP). Despite a 90% decrement in striatal DA in 2'-NH2-MPTP/2'-CH3-MPTP-treated mice, increases in GFAP were attenuated compared to mice treated with 2'-CH3-MPTP alone. Thus, 2'-NH2-MPTP causes severe and immediate decrements in 5-HT and NE in frontal cortex and hippocampus, yet induces a modest GFAP response compared with other MPTP analogs that have their primary effect on DA. These results demonstrate the importance of obtaining quantitative assessments of GFAP to detect astroglial responses associated with selective damage to neurotransmitter systems with low-density innervation and suggest that serotonin may facilitate the astrocytic response to striatal injury.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is a pro-neurotoxicant that must be metabolized to 1-methyl-4-phenylpyridinium (MPP + ) and taken up into striatal dopaminergic neurons to produce neurodegeneration. Recently, we showed wide genetic variability in MPTP-associated neuronal damage in a panel of recombinant inbred mouse strains. Here we examined the amount of MPP + produced in the striatum in the same strains of inbred BXD mice. This allowed us to determine if the differences in the dopaminergic neurotoxicity and associated astrogliosis among the BXD mouse strains were due to differential metabolism of MPTP to MPP + . Using the same BXD mouse strains examined previously (Jones et al., 2013) we found that the extent of the striatal damage produced following MPTP treatment is not correlated quantitatively with the production of MPP + in the striatum. Our findings also extend those of others regarding strain differences in MPTP-induced dopaminergic neurotoxicity. Importantly, our finding suggests that additional factors influence the neurodegenerative response other than the presence and amount of the toxicant at the target site.

A long-term induction of Fos-related antigens has been shown in neurons after brain injury, suggesting that Fos-related antigens are involved in enhancing the transcription of genes related to the process of regeneration and repair. In the present study, we report that levels of Fos-related antigen-2 are elevated in several models of chemically induced brain injury. Trimethyltin, which causes degeneration of neurons primarily in the hippocampus and other limbic regions, results in a five-fold induction of Fos-related antigen-2 immunoreactivity in neurons in the pyramidal and dentate layers of the hippocampus starting at seven days post-treatment and persisting for 60 days. Methamphetamine and methylenedioxymethamphetamine, agents which cause degeneration of dopaminergic nerve terminals in the striatum of the mouse, cause an increase in Fos-related antigen-2 immunoreactivity which begins at three days post-treatment and returns to basal levels by days 5 and 15, respectively. Treatment with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine elevated levels of Fos-related antigen-2 in the mouse striatum at three days post-treatment. This abbreviated time-course of Fos-related antigen-2 induction is consistent with less severe insult (terminal damage) relative to trimethyltin (cell death), but induction occurs during the period of regeneration and repair in both models. Dexfenfluramine, a non-neurotoxic amphetamine, does not induce Fos-related antigen-2 expression. Decreasing core temperature of the mouse, which blocks amphetamine-induced neurotoxicity, also blocks Fos-related antigen-2 induction. In summary, Fos-related antigen-2 is induced in models of both cell death and terminal degeneration, suggesting that this transcription factor may serve as a universal signal transduction molecule involved in the regulation of genes related to regeneration and repair in the CNS.

A number of substituted amphetamines, including methamphetamine (METH) are considered dopaminergic neurotoxicants. METH causes long-term depletions of striatal dopamine (DA) and its metabolites (DOPAC and HVA) that are accompanied by other changes indicative of nerve terminal degeneration. These include argyrophilia as detected by silver degeneration stains and an elevation in glial fibrillary acidic protein (GFAP), a marker of reactive gliosis in response to injury, as well as a long-term decrease in tyrosine hydroxylase (TH) protein levels. The susceptibility to the dopaminergic neurotoxicity of METH and the other amphetamines can be affected by a number of factors including age, gender, stress, and environment. Many of these susceptibility factors have been extensively investigated in the rat but less so in the mouse. As the availability of genetically altered mice continues to expand, this species is increasingly selected for study. Thus, in previous work we determined that stress, gender, and the environment can significantly impact the neurotoxicity of the amphetamines. Here we determined how age affects the striatal DA depletion and GFAP elevation induced by d-METH in C57BL/6 mice. Age was a significant determinant of the ability of a known neurotoxic regimen of d-METH (10 mg/kg x 4) to produce striatal DA depletion with one-month-old C57BL16 mice displaying minimal and nonpersistent depletion of DA or its metabolites while mice 12 months of age displayed large and persistent depletions of DA (87%), DOPAC (71%), and HVA (94%). Large elevations in striatal GFAP were induced in mice 2-23 months of age by d-METH, with lower dosages of d-METH being effective in the older mice. In contrast, the usual neurotoxic regimen of d-METH was minimally effective in inducing GFAP elevations (49% over control) in one-month-old mice, despite elevations in body temperature equivalent to those observed in older mice. Although increasing the dosage of d-METH (20 to 80 mg/kg) did increase the GFAP response (100% over control), it was still well below that usually exhibited at the usual neurotoxic dosage (300-400% over control) in fully mature mice. These data suggest maturity of striatal dopamine systems may be an essential element in the striatal damage induced by the neurotoxic amphetamines.

AP-1 transcription factors have been shown to be induced in the brain after ischemic injury. However, their roles in neuronal survival or death have yet to be defined. Here, we report the discovery of elevated nuclear levels of fos-related antigen-2 (FRA-2) in the nuclei of hippocampal neurons seven days after middle cerebral artery occlusion (MCAO). Expression of FRA-2 and AP-1 DNA binding activity is elevated in hippocampi ipsilateral as well as contralateral to MCAO. Using Fluoro-Jade staining as a marker of neurodegeneration, FRA-2 was not found to be expressed in degenerating neurons. Thus, FRA-2 is expressed in neurons that survive ischemic insult suggesting a role for this transcription factor in neuronal adaptation to the post-injury state.

We compared the effects of treatment with high doses of fluoxetine, sibutramine, sertraline, and dexfenfluramine for 4 days on brain serotonergic nerve terminals in rats. Methylenedioxymethamphetamine (MDMA) and 5,7-dihydroxytryptamine (5,7-DHT) were used as positive controls because both compounds deplete brain serotonin. Food intake and body weight changes were also monitored and yoked, pair-fed animals were used to control for possible changes in morphology due to nutritional deficits. Fluoxetine, sibutramine, sertraline and dexfenfluramine all produced a significant reduction in body weight. Fluoxetine, sibutramine and sertraline treatment resulted in no depletion of brain serotonin but produced morphological abnormalities in the serotonergic immunoreactive nerve network. In contrast, dexfenfluramine and MDMA depleted brain serotonin and produced morphological changes in the serotonin nerve network. These results indicate that even though fluoxetine, sibutramine and sertraline do not deplete brain serotonin, they do produce morphological changes in several brain regions (as identified by serotonin immunohistochemistry). Dexfenfluramine and MDMA, on the other hand, markedly deplete brain serotonin and also produce morphological changes. Collectively, these results lend support to the concept that all compounds acting on brain serotonin systems, whether capable of producing serotonin depletion or not, could produce similar effects on the morphology of cerebral serotonin systems.

Dopaminergic neurons exert a major modulatory effect on the forebrain. Dopamine and adenosine 3′,5′-monophosphate–regulated phosphoprotein (32 kilodaltons) (DARPP-32), which is enriched in all neurons that receive a dopaminergic input, is converted in response to dopamine into a potent protein phosphatase inhibitor. Mice generated to contain a targeted disruption of the DARPP-32 gene showed profound deficits in their molecular, electrophysiological, and behavioral responses to dopamine, drugs of abuse, and antipsychotic medication. The results show that DARPP-32 plays a central role in regulating the efficacy of dopaminergic neurotransmission.

Methamphetamine (METH) exposure results in dopaminergic neurotoxicity in striatal regions of the brain, an effect that has been linked to an increased risk of Parkinson's disease. Various aspects of neuroinflammation, including astrogliosis, are believed to be contributory factors in METH neurotoxicity. METH interacts with sigma receptors at physiologically relevant concentrations and treatment with sigma receptor antagonists has been shown to mitigate METH-induced neurotoxicity in rodent models. Whether these compounds alter the responses of glial cells within the central nervous system to METH however has yet to be determined. Therefore, the purpose of the current study was to determine whether the sigma receptor antagonist, SN79, mitigates METH-induced striatal reactive astrogliosis. Male, Swiss Webster mice treated with a neurotoxic regimen of METH exhibited time-dependent increases in striatal gfap mRNA and concomitant increases in GFAP protein, indicative of astrogliosis. This is the first report that similar to other neurotoxicants that induce astrogliosis through the activation of JAK2/STAT3 signaling by stimulating gp-130-linked cytokine signaling resulting from neuroinflammation, METH treatment also increases astrocytic oncostatin m receptor (OSMR) expression and the phosphorylation of STAT3 (Tyr-705) in vivo. Pretreatment with SN79 blocked METH-induced increases in OSMR, STAT3 phosphorylation and astrocyte activation within the striatum. Additionally, METH treatment resulted in striatal cellular degeneration as measured by Fluoro-Jade B, an effect that was mitigated by SN79. The current study provides evidence that sigma receptor antagonists attenuate METH-induced astrocyte activation through a pathway believed to be shared by various neurotoxicants.

We analyzed genetic variation in severity of neuronal damage using the known dopaminergic neurotoxicant, MPTP, as a prototypical chemical denervation agent. Male mice from ten members of the BXD family of recombinant inbred strains received 12.5 mg/kg MPTP s.c. (vs. saline) and 48 h later brains were taken for multiple related biochemical analyses. Striatal dopamine (DA) and its metabolites, DOPAC and HVA, and serotonin and its metabolite, 5-HIAAA, were analyzed by HPLC. DA turnover was assessed using DOPAC/DA and HVA/DA ratios. Striatal tyrosine hydroxylase (TH), glial fibrilary acidic protein (GFAP), and iron content in ventral midbrain were quantified. All dopamine measures, as well as TH and GFAP, demonstrated wide, genotype-dependent differences in response to MPTP. Serotonin was largely unaffected. Principal components analysis (PC) on difference values, saline minus MPTP, for DA, DOPAC, HVA, and TH, yielded a dominant principal component. The PC trait residuals for each genotype were compared against complementary expression data for striatum of the same strains. Three transcripts representing Mtap2, Lancl 1, and Kansl1l were highly correlated with the PC, as was the difference score, MPTP minus saline for GFAP. This systems approach to the study of environmental neurotoxicants holds promise to define individual genetic differences that contribute to variability in susceptibility to risk factors for diseases such as Parkinson's disease.

Acute administration of high doses of ibogaine (IBG) to the male rat results in degeneration of Purkinje cells and reactive gliosis in the cerebellar vermis. We examined whether acute and chronic administration of IBG to male and female rats results in gliosis as determined by quantification of the astroglial intermediate filament protein, glial fibrillary acidic protein (GFAP). After acute administration of IBG, rats of both sexes showed dose-related increases in GFAP that were not confined to the cerebellar vermis. After chronic administration of IBG, female, but not male rats, showed large (as much as 200% of control), dose-related increases in GFAP in hippocampus, olfactory bulbs, brain stem and striatum, but not cerebellum. In hippocampus, the cytoskeletal proteins, neurofilament 68 (NF-68) and beta-tubulin were increased in females treated chronically with IBG, findings consistent with a damage-induced sprouting response. Together, the data indicate that IBG damages areas of the brain outside the cerebellum and that the sites damaged are dependent on sex and dosage regimen.

Glial fibrillary acidic protein (GFAP) immunostaining is the most commonly used method to examine the distribution of astrocytes and the hypertrophy of astrocytes in response to neural degeneration or injury. More recently, a variety of biochemical assays for GFAP have been developed. Both qualitative immunocytochemical evaluations of GFAP and quantitative biochemical measurements of GFAP have been used to examine the regional distribution of GFAP within the central nervous system (CNS). The former method has largely been based on aldehyde-fixed tissue, while the latter approach has been based on the use of fresh tissue extracts or homogenates. In the present study, we used ethanol as a fixative to permit both immunocytochemical and biochemical procedure to be carried out on brain tissue from a single animal. Normal adult rats and mice were perfused with a 70% ethanol/saline solution, and each brain was hemisectioned. The concentration of GFAP was measured in regions of 1 hemisection, using an enzyme-linked immunosorbent assay (ELISA), while the other hemisection was used for GFAP immunostaining. Regional differences occurred in the brains of both species, with the highest concentration of GFAP found in the brainstem, and the lowest concentrations found in the striatum and cortex. The specific patterns of GFAP immunoreactivity corresponded to regional concentrations in most brain area of both species. These data show that it is possible to assay GFAP concentrations in tissue prepared for immunocytochemical analysis, providing both qualitative and quantitative information from one set of tissue.

Transgenic mice, expressing the gene for bovine growth hormone (bGH), exhibit increased body size, reduced reproductive capacity, and high basal levels of several hormones including corticosterone. Their shortened life span may be indicative of accelerated aging. As prominent astrogliosis of the CNS accompanies aging in rodents, bGH transgenic mice were examined for astrogliosis, as quantified by an ELISA for the astrocyte-localized protein, glial fibrillary acidic protein (GFAP). Transgenic mice were produced by mating C57BL/6 x C3H F1 hybrid females with male descendants of animals produced by microinjection of fertilized eggs with phosphoenolpyruvate carboxykinase (PEPCK)/bGH-hybrid gene. Transgenic mice (approximately 3.5 and approximately 12 months of age) weighed significantly more than same age or older (approximately 20 month) controls. Most of their internal organs, including the heart, kidneys, adrenals, liver, and spleen, were also heavier. In contrast, the thymus was heavier only in the younger transgenic mice. Serum corticosterone was highest in the older transgenic mice. A small but significant increase in whole brain, cortex, and cerebellar weight, relative to controls and the older transgenic mice, was found in the younger transgenic mice. Control mice exhibited large, significant age-related increases in GFAP. Increases of 35, 70, 68, 89, 79, and 95% for cortex, cerebellum, striatum, hippocampus, midbrain, and brain stem, respectively, were found when comparing the oldest (approximately 20 months) control mice to the youngest (approximately 3.5 months). In contrast, in the olfactory bulbs and the hypothalamus there were no age-related changes in the levels of GFAP in control mice. Transgenic mice (approximately 3.5 months) had significantly elevated GFAP levels relative to the same-age controls in all brain areas examined. In some brain areas, the GFAP levels found in the younger transgenic mice were equivalent to those found in the oldest controls. No differences between controls and transgenics were found in tyrosine hydroxylase protein levels of striatum or hypothalamus. The elevated GFAP levels of transgenic mice may reflect increased neural damage due to accelerated aging processes or damage associated with high circulating levels of bGH or corticosterone. Alternatively, the increased expression of GFAP in the transgenic mice may reflect altered regulation of GFAP rather than an increase signaled by neural damage.

Up-regulation of proinflammatory cytokines and chemokines in brain (“neuroinflammation”) accompanies neurological disease and neurotoxicity. Previously, we documented a striatal neuroinflammatory response to acute administration of a neurotoxic dose of methamphetamine (METH), i.e. one associated with evidence of dopaminergic terminal damage and activation of microglia and astroglia. When we used minocycline to suppress METH-induced neuroinflammation, indices of dopaminergic neurotoxicity were not affected, but suppression of neuroinflammation was incomplete. Here, we administered the classic anti-inflammatory glucocorticoid, corticosterone (CORT), in an attempt to completely suppress METH-related neuroinflammation. METH alone caused large increases in striatal proinflammatory cytokine/chemokine mRNA and subsequent astrocytic hypertrophy, microglial activation, and dopaminergic nerve terminal damage. Pre-treatment of mice with acute CORT failed to prevent neuroinflammatory responses to METH. Surprisingly, when mice were pre-treated with chronic CORT in the drinking water, an enhanced striatal neuroinflammatory response to METH was observed, an effect that was accompanied by enhanced METH-induced astrogliosis and dopaminergic neurotoxicity. Chronic CORT pre-treatment also sensitized frontal cortex and hippocampus to mount a neuroinflammatory response to METH. Because the levels of chronic CORT used are associated with high physiological stress, our data suggest that chronic CORT therapy or sustained physiological stress may sensitize the neuroinflammatory and neurotoxicity responses to METH.

Although organochlorines have been reported more frequently in Parkinson's disease (PD) brains than in controls, the association with brain Lewy pathology is unknown. Honolulu-Asia Aging Study (HAAS) participants, exposed to organochlorines from a variety of sources during midlife, represent a population well suited to determining the relationship of brain organochlorines with Lewy pathology in decedents from the longitudinal HAAS. The study design included the measurement of 21 organochlorine levels in frozen occipital lobe samples from HAAS decedents. Alpha-synuclein immunostaining performed on 225 brains was used to identify Lewy bodies and Lewy neurites. With the potential for spurious associations to appear between Lewy pathology and 17 organochlorine compounds found in at least 1 brain, initial assessments identified heptachlor epoxide isomer b, methoxychlor, and benzene hexachloride b as being most important. The prevalence of Lewy pathology was 75% (6 of 8) among brains with any 2 of the 3 compounds, 48.8% (79 of 162) among those with 1, and 32.7% (18 of 55) for those with neither (P = .007 test for trend). Although findings persisted after removing cases with PD and dementia with Lewy bodies and after adjustment for age at death, body mass index, pack-years of cigarette smoking, and coffee intake (P = .013), the results were insignificant when correcting for multiple testing. Although consistent with earlier accounts of an association between organochlorines and clinical PD, associations with Lewy pathology warrant further study.

Developmental Neurotoxicity: Evaluation of Testing Procedures with Methylazoxymethanol and Methylmercury. Goldey, E. S., O'Callaghan, J. P., Stanton, M. E., Barone, S., Jr., and Crofton, K. M. (1994). Fundam. Appl. Toxicol. 23, 447-464. Testing procedures for identification of potential developmental neurotoxicants were evaluated using two prototypical developmental neurotoxicants, methylazoxymethanol (MAM) and methylmercury (MeHg). Evaluation of offspring of Long-Evans rats incorporated assessments of developmental toxicity, neurochemistry, histology, and behavior, with most testing being completed near weaning. A number of endpoints in the testing strategy were sensitive to the effects of prenatal exposure to MAM [30 mg/kg on Gestation Day (GD) 15]; (1) MAM caused reduced neonatal body weights but did not effect viability or postnatal survivorship; (2) measurement of total and regional brain weight and histological analysis showed that a number of regions, the cortex and hippocampus in particular, were affected by MAM exposure; (3) an assay for glial fibrillary acidic protein (GFAP) showed that the concentration of this protein was significantly increased in the cortex and hippocampus of treated offspring; (4) a T-maze delayed-alternation procedure indicated that MAM-treated pups were slower in the acquisition phase of the task relative to control pups; (5) motor activity testing revealed hyperactivity in treated offspring that persisted into adulthood; and (6) acoustic startle procedures revealed reduced startle amplitudes in preweanlings. Few endpoints were significantly affected by prenatal MeHg exposure (1, 2, or 4 mg/kg on GD 6-15). High fetal and neonatal mortality and lower neonatal body weights were detected at the highest dose of MeHg. Although minimal effects of MeHg may reflect a relative insensitivity of the test species and/or the test methods, the combined results from both chemicals suggest that some procedures not currently required in the developmental neurotoxicity guideline may be useful in hazard identification, and further evaluation with other chemicals, species, strains, and/or exposure paradigms may be warranted.

The neurological effects of organophosphate (OP) pesticides, commonly used on foods and in households, are an important public health concern. Furthermore, subclinical exposure to combinations of organophosphates is implicated in Gulf War illness. Here, we characterized the effects of the broadly used insecticide chlorpyrifos (CPF) on dopamine and glutamatergic neurotransmission effectors in corticostriatal motor/reward circuitry. CPF potentiated protein kinase A (PKA)-dependent phosphorylation of the striatal protein dopamine- and cAMP-regulated phosphoprotein of M(r) 32 kDa (DARPP-32) and the glutamate receptor 1 (GluR1) subunit of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors in mouse brain slices. It also increased GluR1 phosphorylation by PKA when administered systemically. This correlated with enhanced glutamate release from cortical projections in rat striatum. Similar effects were induced by the sarin congener, diisopropyl fluorophosphate, alone or in combination with the putative neuroprotectant, pyridostigmine bromide and the pesticide N,N-diethyl-meta-toluamide (DEET). This combination, meant to mimic the neurotoxicant exposure encountered by veterans of the 1991 Persian Gulf War, also induced hyperphosphorylation of the neurofibrillary tangle-associated protein tau. Diisopropyl fluorophosphate and pyrodostigmine bromide, alone or in combination, also increased the aberrant activity of the protein kinase, Cdk5, as indicated by conversion of its activating cofactor p35 to p25. Thus, consistent with recent findings in humans and animals, organophosphate exposure causes dysregulation in the motor/reward circuitry and invokes mechanisms associated with neurological disorders and neurodegeneration.

The effects of repeated treatment with the phosphodiesterase-4 (PDE4) inhibitors rolipram, piclamilast, and 4-(2-(3-(cyclopentyloxy)-4-methoxyphenyl)-2-phenylethyl)pyridine (CDP840), which differ in their interactions with high- and low-affinity binding conformers of the enzyme, were contrasted to those of acute treatment on cAMP signaling, hippocampal cell proliferation, and immobility in the forced-swim test in rats. Repeated treatment with rolipram (1 and 3 mg/kg), piclamilast (0.3 and 1 mg/kg), or CDP840 (10 and 30 mg/kg) for 16 days increased cAMP and phosphorylation of cAMP response element binding protein (pCREB) in hippocampus and prefrontal cortex. In addition, repeated treatment with the PDE4 inhibitors increased proliferation and survival of newborn cells in the hippocampus and produced antidepressant-like effects on behavior, as evidenced by decreased immobility in the forced-swim test. Acute treatment with rolipram (3 mg/kg), piclamilast (1 mg/kg), or CDP840 (30 mg/kg) induced transient increases in cAMP and pCREB in hippocampus and prefrontal cortex, but the dose and time dependence of these effects did not parallel the behavioral effects. Compared with rolipram and piclamilast, repeated treatment with CDP840 exerted lesser effects on neural and behavioral measures, probably because of its weak interaction with the high-affinity binding conformer of PDE4. This suggests the relative importance of the high-affinity binding conformer in the mediation of the long-term effects of PDE4 inhibition on cAMP/pCREB signaling, hippocampal cell proliferation, and antidepressant-like effects on behavior.

The astrocyte response to central nervous system injury induced by neonatal alcohol exposure was evaluated using radioimmunoassay and immunocytochemistry of glial fibrillary acidic protein (GFAP). Rat pups were exposed to alcohol on postnatal days 4 through 9 via artificial rearing. Alcohol solutions were administered as one of the following treatments: 10.2% (v/v) in two feedings (4.5 g/kg/day), 5.1% (v/v) in four feedings (4.5 g/kg/day), or 2.5% (v/v) in 12 feedings (6.6 g/kg/day), producing mean blood alcohol concentrations (BACs) of approximately 300, 180, and 50 mg/dl, respectively. Littermates were included as gastrostomy controls (GC) and suckle controls (SC). On postnatal day 10, GFAP concentration increased as a function of BAC, and the 10.2% alcohol treatment significantly and dramatically increased GFAP in the cortex (325% of SC). GFAP immunocytochemistry revealed frequent loci of heavily labeled reactive astrocytes surrounding small cortical blood vessels in the 10.2% group. In addition, a generalized increase in GFAP immunoreactivity was present in the deep layers of the cortex in all alcohol groups, marked by astrocytic fibrillary hypertrophy and increased density. Three-dimensional counts in layer V of parietal cortex using confocal microscopy indicated that the density of GFAP-labeled astrocytes of the 10.2% group was twice that of controls. The layer V gliosis was observable even at low BACs, while gliosis around the vasculature occurred only with high BACs. By postnatal day 15, the astroglial effects were no longer evident. These transient astroglial reactions likely constitute an important aspect of cortical pathophysiology resulting from binge alcohol exposure during the brain growth spurt of the third trimester equivalent.

Objective. This preliminary study assessed possible relationships between plasma and/or cerebrospinal fluid (CSF) concentrations of the pleiotropic cytokine, interleukin (IL)-6, the anti-inflammatory cytokine, IL-10, and levels of pain reported by patients receiving intrathecal (i.t.) opioids. Design. A prospective study quantifying IL-6 and IL-10 concentrations using enzyme-linked immunoassays in samples of plasma and CSF as well as assessment of pain scores in patients receiving intrathecal opioids for management of chronic noncancer pain. Setting. Outpatient pain clinics. Patients. Patients with chronic pain receiving intrathecal morphine or hydromorphone alone or in combination with local anesthetics. Interventions. Two groups of patients were studied. The first group (n = 50) had been receiving long-term i.t. opioids by chronically implanted pump for ∼5 years; paired samples of plasma and CSF were collected at the time of i.t. pump refill. For the second patient group (n = 10), possible temporal changes in the plasma and/or CSF concentrations of IL-6 and IL-10 were investigated for 3 months after initiation of i.t. opioid infusions. Results. For patients receiving long-term i.t. opioid infusions, there were significant inverse correlations ( P ≤ 0.05) between pain intensity and the plasma (but not CSF) IL-10 and IL-6 concentrations. Despite the considerable inter-patient variability in the CSF concentrations of IL-6 in the long-term cohort, the mean CSF IL-6 concentration was approximately fivefold higher in patients receiving long-term i.t. opioids relative to those receiving i.t. opioids for only 3 months. Conclusions. The significant inverse correlations observed between pain intensity and the plasma IL-6 and IL-10 concentrations in patients receiving longterm i.t. opioids for chronic pain management, suggests that these cytokines are worthy of further investigation as possible biomarkers of persistent pain.

Kindling is a process in which episodic electrical stimulation permanently increases seizure susceptibility. One mechanism to account for a change in seizure susceptibility is some alteration in signal transduction, possibly at the level of second messenger systems. In this study, male Long-Evans rats were kindled in the amygdala, and Ca 2+ /calmodulin (Ca 2+ /CaM)-dependent protein phosphorylation was assessed at the site of the primary kindled focus using one- and two-dimensional gel electrophoresis. In vitro phosphorylation of membrane and cytosol fractions in the presence or absence of Ca 2+ /CaM did not differentiate kindled from nonkindled amygdaloid tissue. These results suggest that changes in Ca 2+ /CaM-dependent phosphorylation are not related to the mechanism(s) underlying the establishment of an amygdaloid kindled focus.

3,4-Methylenedioxymethamphetamine (MDMA), a substituted amphetamine analogue which stimulates serotonin release in the CNS, has been shown to induce near lethal elevations in core temperature in the rat. To characterize the effects of MDMA on temperature regulation, we measured metabolic rate (MR), evaporative water loss (EWL), motor activity (MA), and colonic temperature (Tc) in male, Long-Evans rats at 60 min following 30 mg/kg (SC) MDMA or saline at ambient temperatures (Ta) of 10, 20, and 30°C. MDMA caused an elevation in MR at Ta's of 20 and 30°C but had no effect at 10°C. At a Ta of 30°C, MR of the MDMA group was double that of the saline group. EWL was elevated by MDMA, an effect which was potentiated with increasing Ta. MDMA also elicited an increase in MA at all three Ta's. MDMA led to a 3.2°C increase in Tc at 30°C, no change in Tc at 20°C, and a 2.0°C decrease in Tc at 10°C. A second study found that treatment with 20 mg/kg MDMA failed to elicit an increase in blood flow to the tail in spite of a hyperthermic core temperature of 41.4°C. Preliminary studies using radiotelemetry methodology suggested that MDMA lethality is preceded by precipitous elevations in heart rate and core temperature. The data suggest that, at relatively warm Ta's, MDMA-induced stimulation of serotonergic pathway causes an elevation in MR and peripheral vasoconstriction, thus producing life-threatening elevations in Tc. The increase in EWL following MDMA partially attenuates the hyperthermia at warm Ta's, but leads to hypothermia in the rat maintained at a cold Ta of 10°C.

The aim of this randomized double-blind, placebo-controlled, parallel-group study was to evaluate the efficacy, safety, and tolerability of pregabalin in combination with oxycodone or placebo, in patients with either postherpetic neuralgia (PHN) or painful diabetic neuropathy (PDN). After a 7-day washout period, 62 patients were randomized to receive either oxycodone mixture 10 mg/day or placebo mixture for 1 week. Patients were then started on open-label pregabalin (75, 150, 300 and 600 mg/day) according to a forced titration dosing regimen, while continuing the same dosage of oxycodone or placebo for 4 weeks. The primary efficacy measure was a decrease in the pain-intensity score of at least 2cm and a pain score4cm measured using a 10-cm visual analogue scale (VAS) following pregabalin dosage escalation and treatment for 4 weeks. Secondary efficacy measures included sleep interference and the Neuropathic Pain Scale. There were similar levels of overall efficacy between pregabalin/oxycodone and pregabalin/placebo groups in relieving PHN and PDN related pain.Peripheral neuropathic pain presents commonly in clinical practice, and 2 of its most prevalent types are PHN and PDN. Currently available treatments provide some degree of pain relief in approximately 40-60% of patients, leaving the remainder with unremitting pain. Although this study supports the effectiveness of pregabalin in the treatment of PHN or PDN, it also shows that the addition of a low dose of oxycodone at 10mg/day does not enhance the pain-relieving effects of pregabalin.

Phosphodiesterase (PDE) is a critical regulator of cAMP/protein kinase A (PKA) signaling in cells. Multiple PDEs with different substrate specificities and subcellular localization are expressed in neurons. Dopamine plays a central role in the regulation of motor and cognitive functions. The effect of dopamine is largely mediated through the cAMP/PKA signaling cascade, and therefore controlled by PDE activity. We usedin vitroandin vivobiochemical techniques to dissect the roles of PDE4 and PDE10A in dopaminergic neurotransmission in mouse striatum by monitoring the ability of selective PDE inhibitors to regulate phosphorylation of presynaptic [e.g., tyrosine hydroxylase (TH)] and postsynaptic [e.g., dopamine- and cAMP-regulated phosphoprotein of Mr32 kDa (DARPP-32)] PKA substrates. The PDE4 inhibitor, rolipram, induced a large increase in TH Ser40 phosphorylation at dopaminergic terminals that was associated with a commensurate increase in dopamine synthesis and turnover in striatumin vivo. Rolipram induced a small increase in DARPP-32 Thr34 phosphorylation preferentially in striatopallidal neurons by activating adenosine A2Areceptor signaling in striatum. In contrast, the PDE10A inhibitor, papaverine, had no effect on TH phosphorylation or dopamine turnover, but instead robustly increased DARPP-32 Thr34 and GluR1 Ser845 phosphorylation in striatal neurons. Inhibition of PDE10A by papaverine activated cAMP/PKA signaling in both striatonigral and striatopallidal neurons, resulting in potentiation of dopamine D1receptor signaling and inhibition of dopamine D2receptor signaling. These biochemical results are supported by immunohistochemical data demonstrating differential localization of PDE10A and PDE4 in striatum. These data underscore the importance of individual brain-enriched cyclic-nucleotide PDE isoforms as therapeutic targets for neuropsychiatric and neurodegenerative disorders affecting dopamine neurotransmission.

Developing accessible biomarkers of neurotoxic effects which are readily applicable to human populations poses a challenge for neurotoxicology. In the past, the neurotoxic organometal trimethyltin (TMT) has been used as a denervation tool to validate the enhanced expression of GFAP as a biomarker of astrogliosis and neurotoxicity resulting from chemical exposures. In the present study, TMT was used to assess the detection of serum autoantibodies as biomarkers of neurotoxicity. Previous studies in both human and animals have demonstrated the presence of serum autoantibodies to neurotypic [e.g., neurofilament triplet (NF)] and gliotypic proteins [myelin basic protein (MBP) and glial fibrillary acidic protein (GFAP)] as a peripheral marker of neurodegeneration that may be applicable to humans and experimental studies. Male Long-Evans rats (45 days of age) were administered either TMT (8 mg/kg; s) or an equal volume of sterile 0.9% saline. At 1, 2, and 3 weeks post-administration, serum was collected, and rats were sacrificed for the collection of brains. Serum autoantibodies (both IgM and IgG isotypes) to NF68, NF160, NF200, MBP, and GFAP were assayed using an ELISA. Saline only rats did not have detectable levels of autoantibodies. Only sera from TMT-exposed rats had detectable titers of autoantibodies to NFs with IgG predominating starting week 2. Anti-NF68 titers were highest compared to NF160, or NF200. Autoantibodies to MBP and GFAP also were detected; however, there was no significant increase in their titers until week 3. Hippocampal GFAP, detected at these time points, was significantly (p

Compulsive drug abuse has been conceptualized as a behavioral state where behavioral stimuli override normal decision making. Clinical studies of methamphetamine users have detailed decision making changes and imaging studies have found altered metabolism and activation in the parietal cortex. To examine the molecular effects of amphetamine (AMPH) on the parietal cortex, gene expression responses to amphetamine challenge (7.5 mg/kg) were examined in the parietal cortex of rats pretreated for nine days with either saline, non-neurotoxic amphetamine, or neurotoxic AMPH dosing regimens. The neurotoxic AMPH exposure [three doses of 7.5 mg/kg/day AMPH (6 h between doses), for nine days] produced histological signs of neurotoxicity in the parietal cortex while a non-neurotoxic dosing regimen (2.0 mg/kg/day x 3) did not. Neurotoxic AMPH pretreatment resulted in significantly diminished AMPH challenge-induced mRNA increases of activity-regulated cytoskeletal protein (ARC), nerve growth-factor inducible protein A (NGFI-A), and nerve growth-factor inducible protein B (NGFI-B) in the parietal cortex while neither saline pretreatment nor non-neurotoxic AMPH pretreatment did. This effect was specific to these genes as tissue plasminogen activator (t-PA), neuropeptide Y (NPY) and c-jun expression in response to AMPH challenge was unaltered or enhanced by amphetamine pretreatments. In the striatum, there were no differences between saline, neurotoxic AMPH, and non-neurotoxic AMPH pretreatments on ARC, NGFI-A or NGFI-B expression elicited by the AMPH challenge. These data indicate that the responsiveness of synaptic plasticity-related genes is sensitive to disruption specifically in the parietal cortex by threshold neurotoxic AMPH exposures.

Capillary liquid chromatography (CLC) coupled off-line with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and TOF/TOF-MS were explored for identification and quantification of neuropeptides in microwave-fixed rat brain tissue. Sample was separated by gradient elution on 50-mum-inner diameter reversed-phase columns at 180 nL/min. Effluent was mixed with matrix solution and transferred to a MALDI target plate by pulsed electric field deposition, yielding sample spots with 200-300-mum diameter. Mass detection limits as low as 2 amol, corresponding to 1 pM concentration, were achieved for neuropeptides. CLC-MALDI-TOF-MS analysis of microwave-fixed rat striatum tissue yielded detection of over 400 distinctive peaks. CLC-MALDI-TOF/TOF-MS allowed identification of 10 peptides including 3 novel peptides. Quantification was evaluated using substance P as analyte and 15N3-labeled substance P as an internal standard. Quantification of substance P revealed approximately 6.8-fold higher levels than previously reported in the rat striatum. This increase is attributed to use of microwave fixation, which prevented degradation of the peptide, aggressive extraction procedures, and accounting for oxidation of substance P in the analysis. These results demonstrate that CLC-MALDI-TOF-MS is a versatile tool for neuropeptide analysis in brain tissue by allowing for detection, identification, and quantification.

Enhanced expression of tumor necrosis factor (TNF) -alpha, is associated with the neuropathological effects underlying disease-, trauma- and chemically induced neurodegeneration. Previously, we have shown that deficiency of TNF receptors protects against MPTP-induced striatal dopaminergic neurotoxicity, findings suggestive of a role for TNF-alpha in neurodegeneration. Here, we demonstrate that deficiency of TNF receptors suppresses microglial activation and alters the susceptibility of brain regions to MPTP. MPTP-induced expression of microglia-derived factors, TNF-alpha, MCP-1, and IL-1alpha, preceded the degeneration of striatal dopaminergic nerve terminals and astrogliosis, as assessed by loss of striatal dopamine and TH, and an increase in striatal GFAP. Pharmacological neuroprotection with the dopamine reuptake inhibitor, nomifensine, abolished striatal dopaminergic neurotoxicity and associated microglial activation. Similarly, in mice lacking TNF receptors, microglial activation was suppressed, findings consistent with a role for TNF-alpha in striatal MPTP neurotoxicity. In the hippocampus, however, TNF receptor-deficient mice showed exacerbated neuronal damage after MPTP, as evidenced by Fluoro Jade-B staining (to identify degenerating neurons) and decreased microtubule-associated protein-2 (MAP-2) immunoreactivity. These effects were not accompanied by microglial activation, but were associated with increased oxidative stress (nitrosylation of tyrosine residues). These findings suggest that TNF-alpha exerts a neurotrophic/neuroprotective effect in hippocampus. The marked differences we observed in the regional density, distribution and/or activity of microglia and microglia-derived factors may influence the region-specific role for this cell type. Taken together, our results are indicative of a region-specific and dual role for TNF-alpha in the brain: a promoter of neurodegeneration in striatum and a protector against neurodegeneration in hippocampus.

Activated microglia are implicated in the pathogenesis of disease-, trauma- and toxicant-induced damage to the CNS, and strategies to modulate microglial activation are gaining impetus. A novel action of the tetracycline derivative minocycline is the ability to inhibit inflammation and free radical formation, factors that influence microglial activation. Minocycline is therefore being tested as a neuroprotective agent to alleviate CNS damage, although findings so far have yielded mixed results. Here, we showed that administration of a single low dose of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) or methamphetamine (METH), a paradigm that causes selective degeneration of striatal dopaminergic nerve terminals without affecting the cell body in substantia nigra, increased the expression of mRNAs encoding microglia-associated factors F4/80, interleukin (IL)-1alpha, IL-6, monocyte chemoattractant protein-1 (MCP-1, CCL2) and tumor necrosis factor (TNF)-alpha. Minocycline treatment attenuated MPTP- or METH-mediated microglial activation, but failed to afford neuroprotection. Lack of neuroprotection was shown to be due to the inability of minocycline to abolish the induction of TNF-alpha and its receptors, thereby failing to modulate TNF signaling. Thus, TNF-alpha appeared to be an obligatory component of dopaminergic neurotoxicity. To address this possibility, we examined the effects of MPTP or METH in mice lacking genes encoding IL-6, CCL2 or TNF receptor (TNFR)1/2. Deficiency of either IL-6 or CCL2 did not alter MPTP neurotoxicity. However, deficiency of both TNFRs protected against the dopaminergic neurotoxicity of MPTP. Taken together, our findings suggest that attenuation of microglial activation is insufficient to modulate neurotoxicity as transient activation of microglia may suffice to initiate neurodegeneration. These findings support the hypothesis that TNF-alpha may play a role in the selective vulnerability of the nigrostriatal pathway associated with dopaminergic neurotoxicity and perhaps Parkinson's disease.