While the onset of mechanical hyperalgesia induced by endothelin-1 was delayed in female rats, compared to males, the duration was much longer. Given that the repeated test stimulus used to assess nociceptive threshold enhances hyperalgesia, a phenomenon we have referred to as stimulus induced enhancement of hyperalgesia, we also evaluated for sexual dimorphism in the impact of repeated application of the mechanical test stimulus on endothelin-1 hyperalgesia. In male and female rats, endothelin-1 induced hyperalgesia is already maximal at 30 min. At this time stimulus-induced enhancement of hyperalgesia, which is observed only in male rats, persisted for 3–4 hours. In contrast, in females, it develops only after a very long (15 day) delay, and is still present, without attenuation, at 45 days. Ovariectomy eliminated these differences between male and female rats. These findings suggest marked, ovarian-dependent sexual dimorphism in endothelin-1induced mechanical hyperalgesia and its enhancement by repeated mechanical stimulation.
Endothelin; Gender; Hyperalgesia; Rat; Sexual dimorphism; Stimulus; Enhancement
Iron-mediated free radical damage contributes to secondary damage after intracerebral hemorrhage (ICH). Iron is released from heme after hemoglobin breakdown and accumulates in the parenchyma over days and then persists in the brain for months (e.g., hemosiderin). This non-heme iron has been linked to cerebral edema and cell death. Deferoxamine, a ferric iron chelator, has been shown to mitigate iron-mediated damage, but results vary with less protection in the collagenase model of ICH. This study used rapid-scanning X-ray fluorescence (RS-XRF), a synchrotron-based imaging technique, to spatially map total iron and other elements (zinc, calcium and sulfur) at three survival times after collagenase-induced ICH in rats. Total iron was compared to levels of non-heme iron determined by a Ferrozine-based spectrophotometry assay in separate animals. Finally, using RS-XRF we measured iron levels in ICH rats treated with deferoxamine versus saline. The non-heme iron assay showed elevations in injured striatum at 3 days and 4 weeks post-ICH, but not at 1 day. RS-XRF also detected significantly increased iron levels at comparable times, especially notable in the peri-hematoma zone. Changes in other elements were observed in some animals, but these were inconsistent among animals. Deferoxamine diminished total parenchymal iron levels but did not attenuate neurological deficits or lesion volume at 7 days. In summary, ICH significantly increased non-heme and total iron levels. We evaluated the latter and found it to be significantly lowered by deferoxamine, but its failure to attenuate injury or functional impairment in this model raises concern about successful translation to patients.
PMID: 22226595 CAMSID: cams3545
Stroke; Hemorrhage; Iron; Synchrotron; X-ray fluorescence; Deferoxamine
We have previously shown the pathophysiological importance of the reactive nitrogen species peroxynitrite (PN) formed from the reaction of nitric oxide (•NO) and superoxide (O2
•−) radicals and its involvement in lipid peroxidation (LP) and protein nitration damage in brain tissue following traumatic brain injury (TBI). Nitric oxide is produced by at least three isoforms of the enzyme nitric oxide synthase (NOS) including: endothelial NOS (eNOS) in the CNS vasculature, neuronal NOS (nNOS), and inducible NOS (iNOS) in macrophages/microglia. In view of the requirement of ●NO synthesis for PN formation, we sought to address the time course of NOS expression (mRNA by real time quantitative PCR and protein by western blot) after TBI in comparison with the time course of PN-mediated protein nitration (3-nitrotyrosine, 3-NT) in ipsilateral cortex (CTX) and hippocampus (HIPP) between 3 hours and 1 week post-injury using a controlled cortical impact (CCI) mouse model of TBI in young adult CF-1 mice. Protein nitration showed a progressive posttraumatic increase that became significant in CTX at 24 hours and then peaked at 72 hours in both CTX and HIPP. During the increase in PN-derived 3-NT, there was no increase in either CTX or HIPP eNOS mRNA levels, whereas eNOS protein levels were significantly (p<0.05) increased at 48 and 72 hours in both brain regions. There was a significant decrease in HIPP, but not CTX nNOS mRNA; however, nNOS protein did not change except for a significant increase in CTX at 1 week. There was significantly increased CTX and HIPP iNOS mRNA levels at 24, 48, and 72 hours (p<.05) post-injury. In contrast, no change was seen in CTX or HIPP iNOS protein at any timepoint. Taken together, eNOS protein expression and iNOS mRNA appear to bear a coincident temporal relationship to the time course of PN-mediated protein nitrative damage after CCI-TBI suggesting that both constitutive and inducible NOS isoforms contribute ●NO for PN formation and 3-NT protein modification after TBI.
traumatic brain injury; peroxynitrite; oxidative damage; 3-nitrotyrosine; nitric oxide synthase
Maintenance of transmembrane ionic gradients and their restoration after cortical spreading depression (CSD) are energy dependent. We recently showed an inverse relationship between blood pressure and CSD duration that is independent of tissue oxygenation. Here, we tested the alternative hypothesis that glucose availability becomes rate-limiting for CSD recovery upon reduced blood pressure in anesthetized rats under full systemic physiological monitoring. Hypotension induced by controlled exsanguination significantly prolonged CSD durations, reduced propagation speeds, and diminished the blood flow response. Hyperglycemia failed to restore the prolonged CSD durations in hypotensive rats and did not significantly alter the propagation speed or the blood flow response. These data suggest that prolonged CSD durations during reduced cerebral perfusion pressure are independent of tissue energy status, and implicate alternative mechanisms of CSD recovery such as vascular clearance of extracellular K+.
Hyperglycemia; hypotension; spreading depression
Endothelial apoptosis plays a major role in the development of cerebral vascular spasm after subarachnoid hemorrhage (SAH). C/EBP homologous protein (CHOP) orchestrates apoptosis in a variety of cell types in response to endoplasmic reticulum (ER) stress, implicated in the brain injury after SAH. However, the role of CHOP in the mechanism of cerebral vasospasm (CVS) after SAH remains unexplored. The aim of this study was to evaluate the effect of CHOP silencing on endothelial apoptosis and CVS following subarachnoid hemorrhage in the rat. The study was conducted on 65 rats and employed endovascular perforation model of SAH. CHOP siRNAs were injected 24 hrs prior to the hemorrhage. At 72 hours after SAH brains with basilar arteries (BA) were collected from euthanized rats for laboratory investigations. Triple fluorescence stain revealed expression of CHOP in cerebral vascular endothelia after SAH. Marked reduction of CHOP protein and the reduction of its downstream signaling effectors, bim and caspase-3, were found in BA with Western blot analysis. CHOP silencing reduced number of apoptotic endothelial cells in BA, and increased BA diameter after SAH. The amelioration of CVS was associated with reduced neuronal injury in cerebral tissues. In conclusion, CHOP siRNA treatment can effectively combat apoptotic mechanisms of cerebral vasospasm set in motion by subarachnoid bleeding.
Apoptosis; bim; bcl-2; C/EBP homologous protein; caspase-3; cerebral vasospasm; siRNA; subarachnoid hemorrhage
Excitotoxicity (caused by over-activation of glutamate receptors) and inflammation both contribute to motor neuron (MN) damage in amyotrophic lateral sclerosis (ALS) and other diseases of the spinal cord. Microglial and astrocytic activation in these conditions results in release of inflammatory mediators, including the cytokine, tumor necrosis factor–alpha (TNF-α). TNF-α has complex effects on neurons, one of which is to trigger rapid membrane insertion of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) type glutamate receptors, and in some cases, specific insertion of GluA2 lacking, Ca2+ permeable AMPA receptors (Ca-perm AMPAr). In the present study, we use a histochemical stain based upon kainate stimulated uptake of cobalt ions (“Co2+ labeling”) to provide the first direct demonstration of the presence of substantial numbers of Ca-perm AMPAr in ventral horn MNs of adult rats under basal conditions. We further find that TNF-α exposure causes a rapid increase in the numbers of these receptors, via a phosphatidylinositol 3 kinase (PI3K) and protein kinase A (PKA) dependent mechanism. Finally, to assess the relevance of TNF-α to slow excitotoxic MN injury, we made use of organotypic spinal cord slice cultures. Co2+ labeling revealed that MNs in these cultures possess Ca-perm AMPAr. Addition of either a low level of TNF-α, or of the glutamate uptake blocker, trans-pyrrolidine-2,4-dicarboxylic acid (PDC) to the cultures for 48 h resulted in little MN injury. However, when combined, TNF-α+PDC caused considerable MN degeneration, which was blocked by the AMPA/kainate receptor blocker, 2,3-Dihydroxy-6-nitro-7-sulfamoylbenzo (F) quinoxaline (NBQX), or the Ca-perm AMPAr selective blocker, 1-naphthyl acetylspermine (NASPM). Thus, these data support the idea that prolonged TNF-α elevation, as may be induced by glial activation, acts in part by increasing the numbers of Ca-perm AMPAr on MNs to enhance injurious excitotoxic effects of deficient astrocytic glutamate transport.
tumor necrosis factor-alpha; amyotrophic lateral sclerosis; ALS; AMPA; Ca2+ permeable AMPA receptors; slice culture; motor neuron; protein kinase A; phosphatidylinositol 3 kinase
Brain-derived neurotrophic factor (BDNF) plays an essential role in sensory neuronal activation in response to visceral inflammation. Here we report that BDNF up-regulation in the primary afferent neurons in the dorsal root ganglia (DRG) in a rat model of colitis is mediated by the activation of endogenous extracellular signal-regulated protein kinases (ERK) 5 and by nerve growth factor (NGF) retrograde signaling. At 7 days of colitis, the expression level of BDNF is increased in conventional neuronal tracing dye Fast Blue labeled primary afferent neurons project to the distal colon. In these neurons, the phosphorylation (activation) level of ERK5 is also increased. In contrast, the level of phospho-ERK1/2 is not changed in the DRG during colitis. Prevention of the ERK5 activation in vivo with an intrathecal application of the MEK inhibitor PD98059 significantly attenuates the colitis-induced increases in BDNF expression in the DRG. Further studies show that BDNF up-regulation in the DRG is triggered by NGF retrograde signaling which also involves activation of the MEK/ERK pathways. Application of exogenous NGF exclusively to the compartment containing DRG nerve terminals in an ex vivo ganglia-nerve preparation has markedly increased the BDNF expression level in the DRG neuronal cell body that is placed in a different compartment; this BDNF elevation is attenuated by U0126, PD98059 and a specific ERK5 inhibitor BIX02188. These results demonstrate the mechanisms and pathways by which BDNF expression is elevated in primary sensory neurons following visceral inflammation that is mediated by increased activity of ERK5 and is likely to be triggered by the elevated NGF level in the inflamed viscera.
brain-derived neurotrophic factor; extracellular signal-regulated kinase; nerve growth factor; retrograde; primary afferent neuron
Astrocyte activation is a hallmark of the response to brain ischemia consisting of changes in gene expression and morphology. Heat shock protein 72 (Hsp72) protects from cerebral ischemia, and although several protective mechanisms have been investigated, effects on astrocyte activation have not been studied. To identify potential mechanisms of protection, microarray analysis was used to assess gene expression in the ischemic hemispheres of wild-type (WT) and Hsp72-overexpressing (Hsp72Tg) mice 24 hours after middle cerebral artery occlusion or sham surgery. After stroke both genotypes exhibited changes in genes related to apoptosis, inflammation, and stress, with more downregulated genes in Hsp72Tg and more inflammation-related genes increased in WT mice. Genes indicative of astrocyte activation were also upregulated in both genotypes. To measure the extent and time course of astrocyte activation after stroke, detailed histological and morphological analyses were performed in the cortical penumbra. We observed a marked and persistent increase in glial fibrillary acidic protein (GFAP) and a transient increase in vimentin. No change in overall astrocyte number was observed based on glutamine synthetase immunoreactivity. Hsp72Tg and WT mice were compared for density of astrocytes expressing activation markers and astrocytic morphology. In animals with comparable infarct size, overexpression of Hsp72 reduced the density of GFAP- and vimentin-expressing cells, and decreased astrocyte morphological complexity 72 h following stroke. However, by 30 days astrocyte activation was similar between genotypes. These data indicate that early modulation of astrocyte activation provides an additional novel mechanism associated with Hsp72 overexpression in the setting of ischemia.
Estrogen involvement in neuroprotection is now widely accepted, although the specific molecular and cellular mechanisms of estrogen action in neuroprotection remain unclear. This study examines estrogenic effects in a mixed population of cells in attempts to identify the contributing cells that result in estrogen-mediated neuroprotection. Utilizing primary mesencephalic neurons, we found expression of both estrogen receptor α (ERα) and estrogen receptor β (ERβ) with a predominance of ERα on both dopamine neurons and astrocytes. We also found that 17-β-estradiol protects dopamine neurons from injury induced by the complex I inhibitor, 1-methyl-4-phenyl pyridium (MPP+) in a time and ER dependent manner. At least 4 hr of estrogen pre-treatment was required to elicit protection, an effect that was blocked by the ER antagonist, ICI 182,780. Moreover, ERα mediated the protection afforded by estrogen since only the ERα agonist, HPTE but not the ERβ agonist, DPN protected against dopamine cell loss. Since glial cells were shown to express significant levels of ERα, we investigated a possible indirect mechanism of estrogen-mediated neuroprotection through glial cell interaction. Removal of glial cells from the cultures by application of the mitotic inhibitor, 5-Fluoro-2’-deoxyuridine significantly reduced the neuroprotective effects of estrogen. These data indicate that neuroprotection provided by estrogen against MPP+ toxicity is mediated by ERα and involves an interplay among at least two cell types.
estrogen; estrogen receptor; neuroprotection; MPP+; mesencephalon; dopamine neuron; astrocytes
Correct guidance of the migration of neural progenitor cells (NPCs) is essential for the development and repair of the central nervous system (CNS). Electric field (EF)-guided migration, electrotaxis, has been observed in many cell types. We report here that, in applied EFs of physiological magnitude, embryonic and adult NPCs show marked electrotaxis, which is dependent on the PI3K/Akt pathway. The electrotaxis was also evidenced by ex vivo investigation that transplanted NPCs migrated directionally towards cathode in organotypic spinal cord slice model when treated with EFs. Genetic disruption or pharmacological inhibition of phosphoinositide 3-kinase (PI3K) impaired electrotaxis, whereas EF exposure increased Akt phosphorylation in a growth factor-dependent manner and increased phosphatidylinositol-3,4,5-trisphosphate (PIP3) levels. EF treatments also induced asymmetric redistribution of PIP3, growth factor receptors, and actin cytoskeleton. Electrotaxis in both embryonic and adult NPCs requires epidermal growth factor (EGF) and fibroblast growth factor (FGF). Our results demonstrate the importance of the PI3K/Akt pathway in directed migration of NPCs driven by EFs and growth factors and highlight the potential of EFs to enhance the guidance of various NPC populations in CNS repair therapies.
Neural progenitor cells; Electric field; Cell migration; PI3K; Akt; Growth factors
In this study, a modified infusion procedure and a novel infusion device designed for use in humans (Clinical Device B) were evaluated for delivery of recombinant adeno-associated virus (AAV2) to brain. The device is composed of 1.2 m of fused silica inserted through a 24.6-cm surgical steel cannula designed to fit a standard Leksell® clinical stereotaxic frame and micro-infusion syringe pump. AAV2 encoding the human aromatic L-amino acid decarboxylase gene (AAV-hAADC-2) was infused into the putamen of 4 normal rhesus monkeys as a supportive study for a clinical trial in Parkinson&apos ;s disease (PD) patients. Two infusion protocols were tested: a ramped procedure (slow stepwise increases in rate from 0.2 μL/min to 1μL/min), thought to be essential for convection-enhanced delivery (CED), and a non-ramped infusion at a constant rate of 1 μL/min. The primary endpoints were safety evaluation of the infusion procedures and assessment of transgene expression at 5.5 weeks post-infusion. Clinical observations after vector infusions revealed no behavioral abnormalities during the study period. No differences in gross pathology with either the ramped or non-ramped infusion procedure were observed. Histopathology of the putamen was comparable with both procedures, and revealed only minimal localized inflammatory tissue reaction along the needle track in response to cannula placement and vector infusion. AADC immunohistochemistry demonstrated that vector was distributed throughout the putamen, with no significant difference in volume of immunostaining with either infusion procedure. Serum antibody levels against AAV2 vector exhibited a minor increase after infusion. These results validate the clinical utility of this new infusion device and non-ramped infusion conditions for intraputamenal gene therapy, and have the potential to impact a number of human diseases in which delivery of therapeutics to brain is indicated.
Adeno-associated virus; Aromatic L-amino acid decarboxylase; Parkinson's disease; Brain; Infusion device; Putamen; Convection-enhanced delivery; AAV-hAADC-2
Rats exposed to the odor of a predator or to the elevated plus maze express fear behaviors without a prior exposure to either stimulus. The expression of innate fear provides for an ideal model of anxiety which can aid in the elucidation of brain circuits involved in anxiety-related behaviors. The current experiments compared activation of neuropeptide-containing neuronal populations in the amygdala of rats exposed to either the elevated plus maze (EPM; 5 minutes) versus home cage controls, or predator ferret odor versus butyric acid, or no odor (30 minutes). Sections of the brains were prepared for dual-labeled immunohistochemistry and counts of c-Fos co-localized with somatostatin (SOM) or neuropeptide Y (NPY) were made in the basolateral (BLA), central (CEA), medial (MEA) nucleus of the amygdala. Ferret odor and butyric acid exposure significantly decreased the percentage of SOM–positive neurons also immunoreactive for c-Fos in the anterior BLA compared to controls, whereas EPM exposure yielded a significant increase in the activation of SOM-positive neurons versus home cage controls. In the CEA, ferret odor and butyric exposure significantly decreased the percentage of SOM-positive neurons also immunoreactive for c-Fos compared to no-odor controls whereas EPM exposure yielded no change versus controls. In the MEA, both ferret odor exposure and EPM exposure resulted in increased SOM co-localized with c-Fos compared to control groups whereas NPY co-localized with c-Fos occurred following ferret odor exposure, but not EPM exposure. These results indicate that phenotypically distinct neuronal populations of the amygdala are differentially activated following exposure to different anxiogenic stimuli. These studies further elucidate the fundamental neurocircuitry of anxiety and could possibly explain the differential behavioral effects of predator versus novelty-induced stress.
Recovery of respiratory activity in an upper cervical hemisection model (C2H) of spinal cord injury (SCI) can be induced by systemic theophylline administration 24–48 h after injury. The objectives in the present study are (1) to identify pro-inflammatory and neurotrophic factors expressed after C2H and (2) molecular signals involved in functional recovery. Four groups of adult female rats classified as (i) sham (SH) controls, (ii) subjected to a left C2 hemisection (C2H) only, (iii) C2H rats administered theophylline for 3 consecutive days 2 days after C2H (C2H-T Day 5) and (iv) C2H rats treated with theophylline for 3 consecutive days 2 days after C2H and then weaned for 12 days (C2H-T Day 17) prior to assessment of respiratory function and molecular analysis were employed. Corresponding Sham controls, C2H untreated (vehicle only controls) and C2H treated (theophylline) rats were sacrificed, C3-C6 spinal cord segments quickly dissected and left (ipsilateral) hemi spinal cord and right (contralateral) hemi spinal cord were separately harvested 2 days post surgery. SHAM operated and C2H untreated-controls corresponding to C2H-T Day 5 and C2H-T Day 17 rats, respectively, were prepared similarly. Messenger RNA levels for pro-inflammatory genes (TXNIP, IL-1β, TNF-α and iNOS) and neurotrophic and survival factors (BDNF, GDNF, and Bcl2) were analyzed by real time quantitative PCR. Gene expression pattern was unaltered in SH rats. TXNIP, iNOS, BDNF, GDNF and Bcl2 mRNA levels were significantly increased in the ipsilateral hemi spinal cord in C2H rats. BDNF, GDNF and Bcl2 levels remained elevated in the ipsilateral hemi spinal cord in C2H-T Day 5 rats. In this same group, there was further enhancement in TXNIP and IL-1β while iNOS returned to basal levels. Theophylline increased DNA binding activity of transcription factors - cyclic AMP responsive element (CRE) binding protein (CREB) and pro-inflammatory NF-κB. Messenger RNA levels for all genes returned to basal levels in C2H-T Day 17 rats. However, BDNF mRNA levels remained significantly elevated after weaning from the drug. Our results suggest that enhanced resolution of early inflammatory processes and expression of pro-survival factors may underlie theophylline-induced respiratory recovery. The results identify potential targets for gene and drug therapies.
Spinal cord injury; Theophylline; Respiratory Recovery; Neurotrophic factor; Transcription factor; Inflammation Resolution
Circadian rhythm disorders constitute a group of phenotypes that usually present as altered sleep-wake schedules. Until a human genetics approach was applied to investigate these traits, the genetic components regulating human circadian rhythm and sleep behaviors remained mysterious. Steady advances in the last decade have dramatically improved our understanding of the genes involved in circadian rhythmicity and sleep regulation. Finding these genes presents new opportunities to use a wide range of approaches, including in vitro molecular studies and in vivo animal modeling, to elevate our understanding of how sleep and circadian rhythms are regulated and maintained. Ultimately, this knowledge will reveal how circadian and sleep disruption contribute to various ailments and shed light on how best to maintain and recover good health.
Circadian rhythm; Circadian rhythm sleep disorders; Advanced sleep phase; Delayed sleep phase; Advanced sleep phase disorder
The loss or injury of neurons associated with oxidative and nitrosative redox stress plays an important role in the onset of various neurodegenerative diseases. Specifically, nitric oxide (NO), can affect neuronal survival through a process called S-nitrosylation, by which the NO group undergoes a redox reaction with specific protein thiols. This in turn can lead to the accumulation of misfolded proteins, which generally form aggregates in Alzheimer’s, Parkinson’s, and other neurodegenerative diseases. Evidence suggests that S-nitrosylation can also impair mitochondrial function and lead to excessive fission of mitochondria and consequent bioenergetic compromise via effects on the activity of the fission protein dynamin-related protein 1 (Drp1). This insult leads to synaptic dysfunction and loss. Additionally, high levels of NO can S-nitrosylate a number of aberrant targets involved in neuronal survival pathways, including the antiapoptotic protein XIAP, inhibiting its ability to prevent apoptosis.
Huntington’s disease (HD) is a genetic brain disease characterized by loss of capacity in movement control, cognition, and emotional regulation over a period of about 30 years. Since it is well established that clinical impairments and brain atrophy can be detected decades prior to receiving a clinical diagnosis, functional neuroimaging efforts have gained momentum in HD research. In most brain disorders, there is accumulating evidence that the clinical manifestations of disease do not simply depend on the extent of tissue loss, but represent a complex balance among neuronal dysfunction, tissue repair, and circuitry reorganization. Based upon this premise, functional neuroimaging modalities may be more sensitive to the earliest changes in HD than are structural imaging approaches. For this review, PET and fMRI studies conducted in HD samples were summarized. Strengths and limitations of the utilization of functional imaging in HD are discussed and recommendations are offered to facilitate future research endeavors.
Huntington’s disease; Functional imaging; PET; fMRI
The central nervous system (CNS) is a remarkably complex organ system, requiring an equally complex network of molecular pathways controlling the multitude of diverse, cellular activities. Gene expression is a critical node at which regulatory control of molecular networks is implemented. As such, elucidating the various mechanisms employed in the physiological regulation of gene expression in the CNS is important both for establishing a reference for comparison to the diseased state and for expanding the set of validated drug targets available for disease intervention. MicroRNAs (miRNAs) are an abundant class of small RNA that mediates potent inhibitory effects on global gene expression. Recent advances have been made in methods employed to study the contribution of these miRNAs to gene expression. Here we review these latest advances and present a methodological workflow from the perspective of an investigator studying the physiological regulation of a gene of interest. We discuss methods for identifying putative miRNA target sites in a transcript of interest, strategies for validating predicted target sites, assays for detecting miRNA expression, and approaches for disrupting endogenous miRNA function. We consider both advantages and limitations, highlighting certain caveats that inform the suitability of a given method for a specific application. Through careful implementation of the appropriate methodologies discussed herein, we are optimistic that important discoveries related to miRNA participation in CNS physiology and dysfunction are on the horizon.
Alzheimer; Predictors; RIP-Chip; HITS-CLIP; PAR-CLIP; Non-coding RNA; Method; Post-transcriptional
Müllerian Inhibiting Substance (MIS, Anti-Müllerian hormone) is a gonadal hormone that contributes to the subtle sex-biases in the nervous system. Mature neurons of both sexes also produce MIS, suggesting that MIS may be a paracrine regulator of adult neural networks. We report here that murine hypoglossal motor neurons produce MIS and its receptors, MISRII and bone morphogenetic protein receptor 1A (BMPR1A, ALK3), but differentially transport them, with only MIS being detectable in axons. The production of MIS and its receptors were rapidly down regulated after axonal damage, which is a characteristic of genes involved in mature neuronal function. MIS is a survival factor for embryonic spinal motor neurons, but the rate of cell loss after hypoglossal nerve avulsion was normal in Mis−/− mice and was not attenuated by intraventricular administration of MIS. These observations suggest that MIS may be involved in anterograde rather than autocrine or retrograde regulation of neurons.
Axonal transport; Avulsion; MISRII; ALK3
Newborn feeding, maternal, bonding, growth and wellbeing depend upon intact odor recognition in the early postnatal period. Antenatal stress may affect postnatal odor recognition. We investigated the exact role of a neurotransmitter, nitric oxide (NO), in newborn olfactory function. We hypothesized that olfactory neuron activity depended on NO generated by neuronal NO synthase (NOS). Utilizing in vivo functional manganese enhanced MRI (MEMRI) in a rabbit model of cerebral palsy we had shown previously that in utero hypoxia ischemia (H-I) at E22 (70% gestation) resulted in impaired postnatal response to odorants and poor feeding. With the same antenatal insult, we manipulated NO levels in the olfactory neuron in postnatal day 1 (P1) kits by administration of intranasal NO donors or a highly selective nNOS inhibitor. Olfactory function was quantitatively measured by the response to amyl acetate stimulation by MEMRI. The relevance of nNOS to normal olfactory development was confirmed by the increase of nNOS gene expression from fetal ages to P1 in olfactory epithelium and bulbs. In control kits, nNOS inhibition decreased NO production in the olfactory system and increased MEMRI slope enhancement. In H-I kits the MEMRI slope did not increase, implicating modification of endogenous NO-mediated olfactory function by the antenatal insult. NO donors as a source of exogenous NO did not significantly change function in either group. In conclusion, olfactory epithelium nNOS in newborn rabbits probably modulates olfactory signal transduction. Antenatal H-I injury remote from delivery may affect early functional development of the olfactory system by decreasing NO-dependent signal transduction.
nitric oxide; nNOS; olfactory development; MEMRI; olfactory injury
Parkinson’s disease (PD) is characterized by widespread alpha-synuclein pathology and neuronal loss, primarily of the nigrostriatal dopaminergic neurons. Inflammation has been implicated in PD, and alpha-synuclein can initiate microglial activation; however, the kinetics and distribution of inflammatory responses to alpha-synuclein overexpression in vivo are not well understood. We have examined the regional and temporal pattern of microglial activation and pro-inflammatory cytokine production in mice over-expressing wild-type human alpha-synuclein driven by the Thy1-promoter (Thy1-aSyn mice). Increased number of activated microglia, and increased levels of TNF-α mRNA and protein were first detected in the striatum (1 month of age) and later in the substantia nigra (5–6 months), but not cerebral cortex or cerebellum; in contrast, IL-1β and TGFβ remained unchanged in striatum and substantia nigra at all ages examined. Microglial activation persisted up to 14 months of age in these regions and only minimal increases were observed in other regions at this later age. Increased concentrations of serum TNF-α were observed at 5–6 months, but not 1 month of age. The expression of toll-like receptors (TLR) 1, TLR 4 and TLR 8, which are possible mediators of microglial activation, was increased at 5–6 months in the substantia nigra but not in the cerebral cortex, and TLR 2 was increased in the substantia nigra at 14 months of age. With the exception of a slight increase in the striatum of 14 months old Thy1-aSyn mice, MHCII staining was not detected in the regions and ages examined. Similarly, peripheral CD4 and CD8-postive T cells were increased in the blood but only at 22 months of age, suggesting later involvement of the adaptive immune response. These data indicate that, despite the presence of high levels of alpha-synuclein in other brain regions, alpha-synuclein overexpression caused a selective early inflammatory response in regions containing the axon terminals and cell bodies of the nigrostriatal pathway. Our results suggest that specific factors, possibly involving a regionally and temporally selective increase in TLRs, mediate alpha-synuclein-induced inflammatory responses in the SN, and may play a role in the selective vulnerability of nigrostriatal dopaminergic neurons in PD.
Parkinson’s disease; alpha-synuclein; overexpression; transgenic; neurodegeneration; microglia; cytokines; inflammation
Nigrostriatal reserve refers to the threshold of neuronal injury to dopaminergic cell bodies and their terminal fields required to produce parkinsonian motor deficits. Inferential studies have estimated striatal dopamine reserve to be at least 70%. Knowledge of this threshold is critical for planning interventions to prevent symptom onset or reverse nigrostriatal injury sufficient to restore function in people with Parkinson disease. In this study, we determine the nigrostriatal reserve in a non-human primate model that mimics the motor manifestations of Parkinson disease.
Fifteen macaque monkeys received unilateral randomized doses of the selective dopaminergic neuronal toxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. We compared blinded validated ratings of parkinsonism to in vitro measures of striatal dopamine and unbiased stereologic counts of nigral neurons after tyrosine hydroxylase immunostaining.
The percent of residual cell counts in lesioned nigra correlated linearly with the parkinsonism score at 2 months (r = −0.87, p <0.0001). The parkinsonism score at 2 months correlated linearly with the percent residual striatal dopamine (r = −0.77, p = 0.016) followed by a flooring effect once nigral cell loss exceeded 50%. A reduction of about 14 to 23% of nigral neuron counts or 14 to 37% of striatal dopamine was sufficient to induce mild parkinsonism.
The nigral cell body and terminal field injury needed to produce parkinsonian motor manifestations may be much less than previously thought.
Parkinsonism; MPTP; substantia nigra; dopamine
Globoid Cell Leukodystrohpy (GLD, Krabbe Disease) is a lysosomal storage disease, resulting from the genetic deficiency of Galactosylceramidase (GALC). This disease is marked by accumulation of the cytotoxic lipid psychosine (Psy). Psychosine is known to induce oxidative stress in cultured cells, and this stress can be ameliorated through co-treatment with the antioxidant N-acetyl cysteine (NAC). Oxidative stress has also been observed in vivo in the mouse model of GLD, the Twitcher mouse (Twi). We hypothesized that treating oxidative stress with NAC; either alone or in combination with bone marrow transplant (BMT) would improve the course of disease. All breeding cages were maintained on water containing NAC. Once born, the pups received IP boluses of NAC three times per week, and were maintained on NAC-containing water. A separate cohort of animals received the same regimen of NAC in addition to a BMT on post-natal day 2–3. Although NAC lowers the level of oxidized proteins in the brains of Twi mice, and dramatically improves immunohistochemical markers of disease, neither treatment results in any clinical improvements in the Twi mouse. Our data suggest that oxidative stress may be sufficiently down-stream in the pathogenic cascade initiated by Psy accumulation as to be difficult or impossible to treat with standard pharmacologic agents. It is possible that NAC may synergize with other therapies or combinations of therapies. A better understanding of the initiating effects of Psy toxicity and oxidative damage may uncover treatable therapeutic targets.
Krabbe Disease; N-acetyl cysteine; bone marrow transplant; Twitcher; Lysosomal storage disease; oxidative stress
Brief seizures (epileptic/seizure preconditioning) are capable of activating endogenous protective pathways in the brain which can temporarily generate a damage-refractory state against subsequent and otherwise harmful episodes of prolonged seizures (tolerance). Altered expression of microRNAs, a class of non-coding RNAs that function post-transcriptionally to regulate mRNA translation has recently been implicated in the molecular mechanism of epileptic tolerance. Here we characterized the effect of seizure preconditioning induced by low-dose systemic kainic acid on microRNA expression in the hippocampus of mice. Seizure preconditioning resulted in up-regulation of 25 mature microRNAs in the CA3 subfield of the mouse hippocampus, with the highest levels detected for miR-184. This finding was supported by real-time PCR and in situ hybridization showing increased neuronal miR-184 levels and a reduction in protein levels of a miR-184 target. Inhibiting miR-184 expression in vivo resulted in the emergence of neuronal death after preconditioning seizures and increased seizure-induced neuronal death following status epilepticus in previously preconditioned animals, without altered electrographic seizure durations. The present study suggests miRNA up-regulation after preconditioning may contribute to development of epileptic tolerance and identifies miR-184 as a novel contributor to neuronal survival following both mild and severe seizures.
Dicer; Epigenetics; Hippocampus; Status epilepticus; Temporal lobe Epilepsy
Myotonia congenita (MC) is caused by loss-of-function mutations of the muscle ClC-1 chloride channel. Clinical manifestations include the variable association of myotonia and transitory weakness. We recently described a cohort of recessive MC patients showing, at a low rate repetitive nerves stimulation protocol, different values of compound muscle action potential (CMAP) transitory depression, which is considered the neurophysiologic counterpart of transitory weakness. From among this cohort, we studied the chloride currents generated by G190S (associated with pronounced transitory depression), F167L (little or no transitory depression), and A531V (variable transitory depression) hClC-1 mutants in transfected HEK293 cells using patch-clamp. While F167L had no effect on chloride currents, G190S dramatically shifts the voltage dependence of channel activation and A531V reduces channel expression. Such variability in molecular mechanisms observed in the hClC-1 mutants may help to explain the different clinical and neurophysiologic manifestations of each ClCN1 mutation. In addition we examined five different mutations found in compound heterozygosis with F167L, including the novel P558S, and we identified additional molecular defects. Finally, the G190S mutation appeared to impair acetazolamide effects on chloride currents in vitro.
•Myotonia congenita is a muscle disorder due to mutations in ClC-1 chloride channel.•Eight ClC-1 channel mutants were studied using patch-clamp technique.•Mutations induce a variety of molecular defects in ClC-1 channel function.•We discuss the relationship between genotype and clinical phenotype.
Acetazolamide; Chloride channel mutation; ClC-1 chloride channel; Genotype–phenotype relationship; Myotonia congenita; Non-dystrophic myotonia; Patch-clamp; Transitory weakness