Key Specifications Table
|Species Reactivity||Key Applications||Host||Format||Antibody Type|
|R||FC, IHC, IH(P), IP||M||Purified||Monoclonal Antibody|
|Presentation||The monoclonal is presented in phosphate buffered saline containing 10mM sodium azide.|
|Safety Information according to GHS|
|Storage and Shipping Information|
|Storage Conditions||For use within 1 month of purchase store at +4°C, for long term storage aliquot antibody into small volumes and store at -20°C.|
|Material Size||500 µg|
References | 35 Available | See All References
|Reference overview||Application||Pub Med ID|
|Interleukin-18 expression increases in response to neurovascular damage following soman-induced status epilepticus in rats. |
Johnson, EA; Guignet, MA; Dao, TL; Hamilton, TA; Kan, RK
Journal of inflammation (London, England) 12 43 2015
Status epilepticus (SE) can cause neuronal cell death and impaired behavioral function. Acute exposure to potent acetylcholinesterase inhibitors such as soman (GD) can cause prolonged SE activity, micro-hemorrhage and cell death in the hippocampus, thalamus and piriform cortex. Neuroinflammation is a prominent feature of brain injury with upregulation of multiple pro-inflammatory cytokines including those of the IL-1 family. The highly pleiotropic pro-inflammatory cytokine interleukin-18 (IL-18) belongs to the IL-1 family of cytokines and can propagate neuroinflammation by promoting immune cell infiltration, leukocyte and lymphocyte activation, and angiogenesis and helps facilitate the transition from the innate to the adaptive immune response. The purpose of this study is to characterize the regional and temporal expression of IL -18 and related factors in the brain following SE in a rat GD seizure model followed by localization of IL-18 to specific cell types.The protein levels of IL-18, vascular endothelial growth factor and interferon gamma was quantified in the lysates of injured brain regions up to 72 h following GD-induced SE onset using bead multiplex immunoassays. IL-18 was localized to various cell types using immunohistochemistry and transmission electron microscopy. In addition, macrophage appearance scoring and T-cell quantification was determined using immunohistochemistry. Micro-hemorrhages were identified using hematoxylin and eosin staining of brain sections.Significant increases in IL-18 occurred in the piriform cortex, hippocampus and thalamus following SE. IL-18 was primarily expressed by endothelial cells and astrocytes associated with the damaged neurovascular unit. The increase in IL-18 was not related to macrophage accumulation, neutrophil infiltration or T-cell appearance in the injured tissue.These data show that IL-18 is significantly upregulated following GD-induced SE and localized primarily to endothelial cells in damaged brain vasculature. IL-18 upregulation occurred following leukocyte/lymphocyte infiltration and in the absence of other IL-18-related cytokines, suggesting another function, potentially for angiogenesis related to GD-induced micro-hemorrhage formation. Further studies at more chronic time points may help to elucidate this function.
|Neuregulin-1 inhibits neuroinflammatory responses in a rat model of organophosphate-nerve agent-induced delayed neuronal injury. |
Li, Y; Lein, PJ; Ford, GD; Liu, C; Stovall, KC; White, TE; Bruun, DA; Tewolde, T; Gates, AS; Distel, TJ; Surles-Zeigler, MC; Ford, BD
Journal of neuroinflammation 12 64 2015
Neuregulin-1 (NRG-1) has been shown to act as a neuroprotectant in animal models of nerve agent intoxication and other acute brain injuries. We recently demonstrated that NRG-1 blocked delayed neuronal death in rats intoxicated with the organophosphate (OP) neurotoxin diisopropylflurophosphate (DFP). It has been proposed that inflammatory mediators are involved in the pathogenesis of OP neurotoxin-mediated brain damage.We examined the influence of NRG-1 on inflammatory responses in the rat brain following DFP intoxication. Microglial activation was determined by immunohistchemistry using anti-CD11b and anti-ED1 antibodies. Gene expression profiling was performed with brain tissues using Affymetrix gene arrays and analyzed using the Ingenuity Pathway Analysis software. Cytokine mRNA levels following DFP and NRG-1 treatment was validated by real-time reverse transcription polymerase chain reaction (RT-PCR).DFP administration resulted in microglial activation in multiple brain regions, and this response was suppressed by treatment with NRG-1. Using microarray gene expression profiling, we observed that DFP increased mRNA levels of approximately 1,300 genes in the hippocampus 24 h after administration. NRG-1 treatment suppressed by 50% or more a small fraction of DFP-induced genes, which were primarily associated with inflammatory responses. Real-time RT-PCR confirmed that the mRNAs for pro-inflammatory cytokines interleukin-1β (IL-1β) and interleukin-6 (IL-6) were significantly increased following DFP exposure and that NRG-1 significantly attenuated this transcriptional response. In contrast, tumor necrosis factor α (TNFα) transcript levels were unchanged in both DFP and DFP + NRG-1 treated brains relative to controls.Neuroprotection by NRG-1 against OP neurotoxicity is associated with the suppression of pro-inflammatory responses in brain microglia. These findings provide new insight regarding the molecular mechanisms involved in the neuroprotective role of NRG-1 in acute brain injuries.
|Involvement of phosphatase and tensin homolog deleted from chromosome 10 in rodent model of neuropathic pain. |
Huang, SY; Sung, CS; Chen, WF; Chen, CH; Feng, CW; Yang, SN; Hung, HC; Chen, NF; Lin, PR; Chen, SC; Wang, HM; Chu, TH; Tai, MH; Wen, ZH
Journal of neuroinflammation 12 59 2015
Many cancer research studies have extensively examined the phosphatase and tensin homolog deleted from chromosome 10 (PTEN) pathway. There are only few reports that suggest that PTEN might affect pain; however, there is still a lack of evidence to show the role of PTEN for modulating pain. Here, we report a role for PTEN in a rodent model of neuropathic pain.We found that chronic constriction injury (CCI) surgery in rats could elicit downregulation of spinal PTEN as well as upregulation of phosphorylated PTEN (phospho-PTEN) and phosphorylated mammalian target of rapamycin (phospho-mTOR). After examining such changes in endogenous PTEN in neuropathic rats, we explored the effects of modulating the spinal PTEN pathway on nociceptive behaviors. The normal rats exhibited mechanical allodynia after intrathecal (i.t.) injection of adenovirus-mediated PTEN antisense oligonucleotide (Ad-antisense PTEN). These data indicate the importance of downregulation of spinal PTEN for nociception. Moreover, upregulation of spinal PTEN by i.t. adenovirus-mediated PTEN (Ad-PTEN) significantly prevented CCI-induced development of nociceptive sensitization, thermal hyperalgesia, mechanical allodynia, cold allodynia, and weight-bearing deficits in neuropathic rats. Furthermore, upregulation of spinal PTEN by i.t. Ad-PTEN significantly attenuated CCI-induced microglia and astrocyte activation, upregulation of tumor necrosis factor-α (TNF-α) and phospho-mTOR, and downregulation of PTEN in neuropathic rats 14 days post injury.These findings demonstrate that PTEN plays a key, beneficial role in a rodent model of neuropathic pain.
|Suppression of spinal connexin 43 expression attenuates mechanical hypersensitivity in rats after an L5 spinal nerve injury. |
Xu, Q; Cheong, YK; He, SQ; Tiwari, V; Liu, J; Wang, Y; Raja, SN; Li, J; Guan, Y; Li, W
Neuroscience letters 566 194-9 2014
Activation of spinal astrocytes may contribute to neuropathic pain. Adjacent astrocytes can make direct communication through gap junctions formed by connexin 43 (Cx43) in the central nervous system. Yet, the role of spinal astroglial gap junctions in neuropathic pain is not fully understood. Since Cx43 is the connexin isoform expressed preferentially in astrocytes in the spinal cord, we used a small interfering RNA (siRNA) approach to examine whether suppression of spinal Cx43 expression inhibits mechanical hypersensitivity in rats after an L5 spinal nerve ligation (SNL). SNL rats were administered intrathecal Cx43 siRNA (3μg/15μl, twice/day) or an equal amount of mismatch siRNA (control) on days 14-17 post-SNL. Cx43 siRNA, but not mismatch siRNA, alleviated mechanical hypersensitivity in SNL rats. Furthermore, Western blot analysis showed that the pain inhibition induced by Cx43 siRNA correlated with downregulation of Cx43 expression, but not that of Cx36 (the neuronal gap junction protein) or glial fibrillary acidic protein (GFAP, a marker for reactive astrocytes) in the spinal cord of SNL rats. Western blot analysis and immunohistochemistry also showed that SNL increased GFAP expression, but decreased Cx43 expression, in spinal cord. Our results provide direct evidence that selective suppression of spinal Cx43 after nerve injury alleviates neuropathic mechanical hypersensitivity. These findings suggest that in the spinal cord, the enhanced function of astroglial gap junctions, especially those formed by Cx43, may be important to neuropathic pain in SNL rats.
|Flexibilide obtained from cultured soft coral has anti-neuroinflammatory and analgesic effects through the upregulation of spinal transforming growth factor-β1 in neuropathic rats. |
Chen, NF; Huang, SY; Lu, CH; Chen, CL; Feng, CW; Chen, CH; Hung, HC; Lin, YY; Sung, PJ; Sung, CS; Yang, SN; Wang, HM; Chang, YC; Sheu, JH; Chen, WF; Wen, ZH
Marine drugs 12 3792-817 2014
Chronic neuroinflammation plays an important role in the development and maintenance of neuropathic pain. The compound flexibilide, which can be obtained from cultured soft coral, possesses anti-inflammatory and analgesic effects in the rat carrageenan peripheral inflammation model. In the present study, we investigated the antinociceptive properties of flexibilide in the rat chronic constriction injury (CCI) model of neuropathic pain. First, we found that a single intrathecal (i.t.) administration of flexibilide significantly attenuated CCI-induced thermal hyperalgesia at 14 days after surgery. Second, i.t. administration of 10-μg flexibilide twice daily was able to prevent the development of thermal hyperalgesia and weight-bearing deficits in CCI rats. Third, i.t. flexibilide significantly inhibited CCI-induced activation of microglia and astrocytes, as well as the upregulated proinflammatory enzyme, inducible nitric oxide synthase, in the ipsilateral spinal dorsal horn. Furthermore, flexibilide attenuated the CCI-induced downregulation of spinal transforming growth factor-β1 (TGF-β1) at 14 days after surgery. Finally, i.t. SB431542, a selective inhibitor of TGF-β type I receptor, blocked the analgesic effects of flexibilide in CCI rats. Our results suggest that flexibilide may serve as a therapeutic agent for neuropathic pain. In addition, spinal TGF-β1 may be involved in the anti-neuroinflammatory and analgesic effects of flexibilide.
|Adenosine kinase, glutamine synthetase and EAAT2 as gene therapy targets for temporal lobe epilepsy. |
Young, D; Fong, DM; Lawlor, PA; Wu, A; Mouravlev, A; McRae, M; Glass, M; Dragunow, M; During, MJ
Gene therapy 21 1029-40 2014
Astrocytes are an attractive cell target for gene therapy, but the validation of new therapeutic candidates is needed. We determined whether adeno-associated viral (AAV) vector-mediated overexpression of glutamine synthetase (GS) or excitatory amino-acid transporter 2 (EAAT2), or expression of microRNA targeting adenosine kinase (miR-ADK) in hippocampal astrocytes in the rat brain could modulate susceptibility to kainate-induced seizures and neuronal cell loss. Transgene expression was found predominantly in astrocytes following direct injection of glial-targeting AAV9 vectors by 3 weeks postinjection. ADK expression in miR-ADK vector-injected rats was reduced by 94-96% and was associated with an ~50% reduction in the duration of kainate-induced seizures and greater protection of dentate hilar neurons but not CA3 neurons compared with miR-control vector-injected rats. In contrast, infusion of AAV-GS and EAAT2 vectors did not afford any protection against seizures or neuronal damage as the level of transcriptional activity of the glial fibrillary acidic promoter was too low to drive any significant increase in transgenic GS or EAAT2 relative to the high endogenous levels of these proteins. Our findings support ADK as a prime therapeutic target for gene therapy of temporal lobe epilepsy and suggest that alternative approaches including the use of stronger glial promoters are needed to increase transgenic GS and EAAT2 expression to levels that may be required to affect seizure induction and propagation.
|Runx1t1 (Runt-related transcription factor 1; translocated to, 1) epigenetically regulates the proliferation and nitric oxide production of microglia. |
Baby, N; Li, Y; Ling, EA; Lu, J; Dheen, ST
PloS one 9 e89326 2014
Microglia, the resident immune cells of the brain, undergo rapid proliferation and produce several proinflammatory molecules and nitric oxide (NO) when activated in neuropathological conditions. Runx1t1 (Runt-related transcription factor 1, translocated to 1) has been implicated in recruiting histone deacetylases (HDACs) for transcriptional repression, thereby regulating cell proliferation. In the present study, Runx1t1 expression was shown to localize in amoeboid microglial cells of the postnatal rat brain, being hardly detectable in ramified microglia of the adult brain. Moreover, a marked expression of Runx1t1was induced and translocated to nuclei in activated microglia in vitro and in vivo. In view of these findings, it was hypothesized that Runx1t1 regulates microglial functions during development and in neuropathological conditions.siRNA-mediated knockdown of Runx1t1 significantly decreased the expression level of cell cycle-related gene, cyclin-dependent kinase 4 (Cdk4) and proliferation index in activated BV2 microglia. It was also shown that HDAC inhibitor (HDACi) treatment mimics the effects of Runx1t1 knockdown on microglial proliferation, confirming that microglial proliferation is associated with Runx1t1 expression and HDACs activity. Further, Runx1t1 and HDACs were shown to promote neurotoxic effect of microglia by repressing expression of LAT2, L-aminoacid transporter-2 (cationic amino acid transporter, y+ system), which normally inhibits NO production. This was confirmed by chromatin immunoprecipitation (ChIP) assay, which revealed that Runx1t1 binds to the promoter region of LAT2 and this binding increased upon microglial activation. However, the enhanced binding of Runx1t1 to the LAT2 promoter could not repress the LAT2 expression when the BV2 microglia cells were treated with HDACi, indicating that Runx1t1 requires HDACs to transcriptionally repress the expression of LAT2.In conclusion, it is suggested that Runx1t1 controls proliferation and the neurotoxic effect of microglia by epigenetically regulating Cdk4 and LAT2 via its interaction with HDACs.
|Neurochemical properties of BDNF-containing neurons projecting to rostral ventromedial medulla in the ventrolateral periaqueductal gray. |
Yin, JB; Wu, HH; Dong, YL; Zhang, T; Wang, J; Zhang, Y; Wei, YY; Lu, YC; Wu, SX; Wang, W; Li, YQ
Frontiers in neural circuits 8 137 2014
The periaqueductal gray (PAG) modulates nociception via a descending pathway that relays in the rostral ventromedial medulla (RVM) and terminates in the spinal cord. Previous behavioral pharmacology and electrophysiological evidence suggests that brain-derived neurotrophic factor (BDNF) plays an important role in descending pain modulation, likely through the PAG-RVM pathway. However, detailed information is still lacking on the distribution of BDNF, activation of BDNF-containing neurons projecting to RVM in the condition of pain, and neurochemical properties of these neurons within the PAG. Through fluorescent in situ hybridization (FISH) and immunofluorescent staining, the homogenous distributions of BDNF mRNA and protein were observed in the four subregions of PAG. Both neurons and astrocytes expressed BDNF, but not microglia. By combining retrograde tracing methods and formalin pain model, there were more BDNF-containing neurons projecting to RVM being activated in the ventrolateral subregion of PAG (vlPAG) than other subregions of PAG. The neurochemical properties of BDNF-containing projection neurons in the vlPAG were investigated. BDNF-containing projection neurons expressed the autoreceptor TrkB in addition to serotonin (5-HT), neurotensin (NT), substance P (SP), calcitonin gene related peptide (CGRP), nitric oxide synthase (NOS), and parvalbumin (PV) but not tyrosine decarboxylase (TH). It is speculated that BDNF released from projection neurons in the vlPAG might participate in the descending pain modulation through enhancing the presynaptic release of other neuroactive substances (NSs) in the RVM.
|Toll-like receptor 4 mediates microglial activation and production of inflammatory mediators in neonatal rat brain following hypoxia: role of TLR4 in hypoxic microglia. |
Yao, L; Kan, EM; Lu, J; Hao, A; Dheen, ST; Kaur, C; Ling, EA
Journal of neuroinflammation 10 23 2013
Hypoxia induces microglial activation which causes damage to the developing brain. Microglia derived inflammatory mediators may contribute to this process. Toll-like receptor 4 (TLR4) has been reported to induce microglial activation and cytokines production in brain injuries; however, its role in hypoxic injury remains uncertain. We investigate here TLR4 expression and its roles in neuroinflammation in neonatal rats following hypoxic injury.One day old Wistar rats were subjected to hypoxia for 2 h. Primary cultured microglia and BV-2 cells were subjected to hypoxia for different durations. TLR4 expression in microglia was determined by RT-PCR, western blot and immunofluorescence staining. Small interfering RNA (siRNA) transfection and antibody neutralization were employed to downregulate TLR4 in BV-2 and primary culture. mRNA and protein expression of tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β) and inducible nitric oxide synthase (iNOS) was assessed. Reactive oxygen species (ROS), nitric oxide (NO) and NF-κB levels were determined by flow cytometry, colorimetric and ELISA assays respectively. Hypoxia-inducible factor-1 alpha (HIF-1α) mRNA and protein expression was quantified and where necessary, the protein expression was depleted by antibody neutralization. In vivo inhibition of TLR4 with CLI-095 injection was carried out followed by investigation of inflammatory mediators expression via double immunofluorescence staining.TLR4 immunofluorescence and protein expression in the corpus callosum and cerebellum in neonatal microglia were markedly enhanced post-hypoxia. In vitro, TLR4 protein expression was significantly increased in both primary microglia and BV-2 cells post-hypoxia. TLR4 neutralization in primary cultured microglia attenuated the hypoxia-induced expression of TNF-α, IL-1β and iNOS. siRNA knockdown of TLR4 reduced hypoxia-induced upregulation of TNF-α, IL-1β, iNOS, ROS and NO in BV-2 cells. TLR4 downregulation-mediated inhibition of inflammatory cytokines in primary microglia and BV-2 cells was accompanied by the suppression of NF-κB activation. Furthermore, HIF-1α antibody neutralization attenuated the increase of TLR4 expression in hypoxic BV-2 cells. TLR4 inhibition in vivo attenuated the immunoexpression of TNF-α, IL-1β and iNOS on microglia post-hypoxia.Activated microglia TLR4 expression mediated neuroinflammation via a NF-κB signaling pathway in response to hypoxia. Hence, microglia TLR4 presents as a potential therapeutic target for neonatal hypoxia brain injuries.
|The response of cerebral cortex to haemorrhagic damage: experimental evidence from a penetrating injury model. |
Purushothuman, S; Marotte, L; Stowe, S; Johnstone, DM; Stone, J
PloS one 8 e59740 2013
Understanding the response of the brain to haemorrhagic damage is important in haemorrhagic stroke and increasingly in the understanding the cerebral degeneration and dementia that follow head trauma and head-impact sports. In addition, there is growing evidence that haemorrhage from small cerebral vessels is important in the pathogenesis of age-related dementia (Alzheimer's disease). In a penetration injury model of rat cerebral cortex, we have examined the neuropathology induced by a needlestick injury, with emphasis on features prominent in the ageing and dementing human brain, particularly plaque-like depositions and the expression of related proteins. Needlestick lesions were made in neo- and hippocampal cortex in Sprague Dawley rats aged 3-5 months. Brains were examined after 1-30 d survival, for haemorrhage, for the expression of hyperphosphorylated tau, Aβ, amyloid precursor protein (APP), for gliosis and for neuronal death. Temporal cortex from humans diagnosed with Alzheimer's disease was examined with the same techniques. Needlestick injury induced long-lasting changes-haem deposition, cell death, plaque-like deposits and glial invasion-along the needle track. Around the track, the lesion induced more transient changes, particularly upregulation of Aβ, APP and hyperphosporylated tau in neurons and astrocytes. Reactions were similar in hippocampus and neocortex, except that neuronal death was more widespread in the hippocampus. In summary, experimental haemorrhagic injury to rat cerebral cortex induced both permanent and transient changes. The more permanent changes reproduced features of human senile plaques, including the formation of extracellular deposits in which haem and Aβ-related proteins co-localised, neuronal loss and gliosis. The transient changes, observed in tissue around the direct lesion, included the upregulation of Aβ, APP and hyperphosphorylated tau, not associated with cell death. The findings support the possibility that haemorrhagic damage to the brain can lead to plaque-like pathology.
|PPARγ activation blocks development and reduces established neuropathic pain in rats. |
Morgenweck, J; Griggs, RB; Donahue, RR; Zadina, JE; Taylor, BK
Neuropharmacology 70 236-46 2013
Peroxisome proliferator-activated receptor gamma (PPARγ) is emerging as a new pharmacotherapeutic target for chronic pain. When oral (3-30 mg/kg/day in chow for 7 wk) or twice-daily intraperitoneal (1-10 mg/kg/day for 2 wk) administration began before spared nerve injury (SNI), pioglitazone, a PPARγ agonist, dose-dependently prevented multiple behavioral signs of somatosensory hypersensitivity. The highest dose of intraperitoneal pioglitazone did not produce ataxia or reductions in transient mechanical and heat nociception, indicating that inhibitory effects on hypersensitivity were not secondary to adverse drug-induced behaviors or antinociception. Inhibitory effects on hypersensitivity persisted at least one week beyond cessation of pioglitazone administration, suggestive of long-lasting effects on gene expression. Blockade of PPARγ with GW9662, an irreversible and selective PPARγ antagonist, dose-dependently reduced the inhibitory effect of pioglitazone on hypersensitivity, indicating a PPARγ-dependent action. Remarkably, a single preemptive injection of pioglitazone 15 min before SNI attenuated hypersensitivity for at least 2 weeks; this was enhanced with a second injection delivered 12 h after SNI. Pioglitazone injections beginning after SNI also reduced hypersensitivity, albeit to a lesser degree than preemptive treatment. Intraperitoneal pioglitazone significantly reduced the nerve injury-induced up-regulation of cd11b, GFAP, and p-p38 in the dorsal horn, indicating a mechanism of action involving spinal microglia and/or astrocyte activation. Oral pioglitazone significantly reduced touch stimulus-evoked phospho-extracellular signal-related kinase (p-ERK) in lamina I-II, indicating a mechanism of action involving inhibition of central sensitization. We conclude that pioglitazone reduces spinal glial and stimulus-evoked p-ERK activation and that PPARγ activation blocks the development of and reduces established neuropathic pain.
|Gene expression patterns following unilateral traumatic brain injury reveals a local pro-inflammatory and remote anti-inflammatory response. |
White, TE; Ford, GD; Surles-Zeigler, MC; Gates, AS; Laplaca, MC; Ford, BD
BMC genomics 14 282 2013
Traumatic brain injury (TBI) results in irreversible damage at the site of impact and initiates cellular and molecular processes that lead to secondary neural injury in the surrounding tissue. We used microarray analysis to determine which genes, pathways and networks were significantly altered using a rat model of TBI. Adult rats received a unilateral controlled cortical impact (CCI) and were sacrificed 24 h post-injury. The ipsilateral hemi-brain tissue at the site of the injury, the corresponding contralateral hemi-brain tissue, and naïve (control) brain tissue were used for microarray analysis. Ingenuity Pathway Analysis (IPA) software was used to identify molecular pathways and networks that were associated with the altered gene expression in brain tissues following TBI.Inspection of the top fifteen biological functions in IPA associated with TBI in the ipsilateral tissues revealed that all had an inflammatory component. IPA analysis also indicated that inflammatory genes were altered on the contralateral side, but many of the genes were inversely expressed compared to the ipsilateral side. The contralateral gene expression pattern suggests a remote anti-inflammatory molecular response. We created a network of the inversely expressed common (i.e., same gene changed on both sides of the brain) inflammatory response (IR) genes and those IR genes included in pathways and networks identified by IPA that changed on only one side. We ranked the genes by the number of direct connections each had in the network, creating a gene interaction hierarchy (GIH). Two well characterized signaling pathways, toll-like receptor/NF-kappaB signaling and JAK/STAT signaling, were prominent in our GIH.Bioinformatic analysis of microarray data following TBI identified key molecular pathways and networks associated with neural injury following TBI. The GIH created here provides a starting point for investigating therapeutic targets in a ranked order that is somewhat different than what has been presented previously. In addition to being a vehicle for identifying potential targets for post-TBI therapeutic strategies, our findings can also provide a context for evaluating the potential of therapeutic agents currently in development.
|Transcriptome analysis of amoeboid and ramified microglia isolated from the corpus callosum of rat brain. |
Parakalan, R; Jiang, B; Nimmi, B; Janani, M; Jayapal, M; Lu, J; Tay, SS; Ling, EA; Dheen, ST
BMC neuroscience 13 64 2012
Microglia, the resident immune cells of the central nervous system (CNS), have two distinct phenotypes in the developing brain: amoeboid form, known to be amoeboid microglial cells (AMC) and ramified form, known to be ramified microglial cells (RMC). The AMC are characterized by being proliferative, phagocytic and migratory whereas the RMC are quiescent and exhibit a slow turnover rate. The AMC transform into RMC with advancing age, and this transformation is indicative of the gradual shift in the microglial functions. Both AMC and RMC respond to CNS inflammation, and they become hypertrophic when activated by trauma, infection or neurodegenerative stimuli. The molecular mechanisms and functional significance of morphological transformation of microglia during normal development and in disease conditions is not clear. It is hypothesized that AMC and RMC are functionally regulated by a specific set of genes encoding various signaling molecules and transcription factors.To address this, we carried out cDNA microarray analysis using lectin-labeled AMC and RMC isolated from frozen tissue sections of the corpus callosum of 5-day and 4-week old rat brain respectively, by laser capture microdissection. The global gene expression profiles of both microglial phenotypes were compared and the differentially expressed genes in AMC and RMC were clustered based on their functional annotations. This genome wide comparative analysis identified genes that are specific to AMC and RMC.The novel and specific molecules identified from the trancriptome explains the quiescent state functioning of microglia in its two distinct morphological states.
|Effects of translocator protein (18 kDa) ligands on microglial activation and neuronal death in the quinolinic-acid-injected rat striatum. |
Leaver, KR; Reynolds, A; Bodard, S; Guilloteau, D; Chalon, S; Kassiou, M
ACS chemical neuroscience 3 114-9 2012
There is evidence that excitotoxicity and prolonged microglial activation are involved in neuronal death in neurodegenerative disorders. Activated microglia express various molecules, including the translocator protein 18 kDa (TSPO; formerly known as the peripheral benzodiazepine receptor) on the outer mitochondrial membrane. The TSPO is a novel target for neuroprotective treatments which aim to reduce microglial activation. The effect of PK 11195 and three other TSPO ligands on the level of microglial activation and neuronal survival was evaluated in a quinolinic acid (QUIN) rat model of excitotoxic neurodegeneration. All three ligands were neuroprotective at a level comparable to PK 11195. All of the ligands decreased microglial activation following the injection of QUIN but had no effect on astrogliosis. Interestingly, we also observed neuroprotective effects from the vehicle, dimethyl sulfoxide (DMSO).
|Spatiotemporal pattern of neuronal injury induced by dFP in rats: a Model for delayed neuronal cell death following acute OP intoxication. |
Li Y, Lein PJ, Liu C, Bruun DA, Tewolde T, Ford G, Ford BD
Toxicology and applied pharmacology 253 261-9. Epub 2011 Apr 12. 2011
Organophosphate (OP) neurotoxins cause acute cholinergic toxicity and seizures resulting in delayed brain damage and persistent neurological symptoms. Testing novel strategies for protecting against delayed effects of acute OP intoxication has been hampered by the lack of appropriate animal models. In this study, we characterize the spatiotemporal pattern of cellular injury after acute intoxication with the OP diisopropylfluorophosphate (DFP). Adult male Sprague-Dawley rats received pyridostigmine (0.1 mg/kg, im) and atropine methylnitrate (20mg/kg, im) prior to DFP (9 mg/kg, ip) administration. All DFP-treated animals exhibited moderate to severe seizures within minutes after DFP injection but survived up to 72 h. AChE activity was significantly depressed in the cortex, hippocampus, subcortical brain tissue and cerebellum at 1h post-DFP injection and this inhibition persisted for up to 72 h. Analysis of neuronal injury by Fluoro-Jade B (FJB) labeling revealed delayed neuronal cell death in the hippocampus, cortex, amygdala and thalamus, but not the cerebellum, starting at 4h and persisting until 72 h after DFP treatment, although temporal profiles varied between brain regions. At 24h post-DFP injection, the pattern of FJB labeling corresponded to TUNEL staining in most brain regions, and FJB-positive cells displayed reduced NeuN immunoreactivity but were not immunopositive for astrocytic (GFAP), oligodendroglial (O4) or macrophage/microglial (ED1) markers, demonstrating that DFP causes a region-specific delayed neuronal injury mediated in part by apoptosis. These findings indicate the feasibility of this model for testing neuroprotective strategies, and provide insight regarding therapeutic windows for effective pharmacological intervention following acute OP intoxication.Copyright © 2011 Elsevier Inc. All rights reserved.
|Time-course of nigrostriatal neurodegeneration and neuroinflammation in the 6-hydroxydopamine-induced axonal and terminal lesion models of Parkinson's disease in the rat. |
S Walsh,D P Finn,E Dowd
Neuroscience 175 2011
The pathogenesis of Parkinson's disease is thought to involve a self-sustaining cycle of neuroinflammation and neurodegeneration. In order to develop novel anti-inflammatory therapies to break this cycle, it is crucial that the temporal relationship between neurodegeneration and neuroinflammation is characterised in pre-clinical models to maximise their predictive validity. Thus, this study aimed to investigate the progression of neuroinflammation relative to nigrostriatal neurodegeneration in the two most commonly-used rat models of Parkinson's disease. Male Sprague-Dawley rats were lesioned by terminal or axonal administration of 6-hydroxydopamine, and were sacrificed for quantitative immunohistochemistry (to assess nigrostriatal integrity (anti-tyrosine hydroxylase), microgliosis (anti-OX42) and astrocytosis (anti-GFAP)) at 6 h 24 h 72 h or 2 weeks post-lesion. Following terminal lesion, dopaminergic deafferentation of the striatum was evident from 6 h post-lesion and was accompanied by microglial and astroglial activation. Dopamine neuron loss from the substantia nigra did not occur until 2 weeks after terminal lesion, and this was preceded by microglial, but not astroglial, activation. Following axonal lesion, retraction of nigrostriatal terminals from the striatum was not observed until the 72 h time-point, and this was associated with a slight astrocytosis, but not microgliosis. Degeneration of dopaminergic neurons from the substantia nigra was also evident from 72 h after axonal lesion, and was accompanied by nigral microgliosis and astrocytosis by 2 weeks. This study highlights the temporal relationship between neurodegeneration and neuroinflammation in models of Parkinson's disease, and should facilitate use of these models in the development of anti-inflammatory therapies for the human condition.
|A novel rat forelimb model of neuropathic pain produced by partial injury of the median and ulnar nerves. |
Hanju Yi,Myung Ah Kim,Seung Keun Back,Jong Shin Eun,Heung Sik Na
European journal of pain (London, England) 15 2011
The vast majority of human peripheral nerve injuries occur in the upper limb, whereas the most animal studies have been conducted using the hindlimb models of neuropathic pain, involving damages of the sciatic or lumbar spinal nerve(s). We attempted to develop a rat forelimb model of peripheral neuropathy by partial injury of the median and ulnar nerves. The halves of each nerve were transected by microscissors at about 5mm proximal from the elbow joint and behavioral signs of neuropathic pain, such as mechanical and cold allodynia, and heat hyperalgesia, were monitored up to 126 days following nerve injury. Mechanical allodynia was assessed by measuring the forepaw withdrawal threshold to von Frey filaments, and cold allodynia was evaluated by measuring the time spent in lifting or licking the forepaw after applying acetone to it. Heat hyperalgesia was also monitored by investigating the forepaw withdrawal latencies using the Hargreaves' test. After the nerve injury, the experimental animals exhibited long-lasting clear neuropathic pain-like behaviors, such as reduced forepaw withdrawal threshold to von Frey filaments, the increased response duration of the forepaw to acetone application, and the decreased withdrawal latency to radiant heat stimulation. These behaviors were significantly alleviated by administration of gabapentin (5 or 50mg/kg, i.p.) in a dose-dependent manner. Therefore, these abnormal sensitivities are interpreted as the signs of neuropathic pain following injury of the median and ulnar nerves. Our rat forelimb model of neuropathic pain may be useful for studying human neuropathic pain and screening for valuable drug candidates.
|Expression of sphingosine kinase 1 in amoeboid microglial cells in the corpus callosum of postnatal rats. |
Lin, H; Baby, N; Lu, J; Kaur, C; Zhang, C; Xu, J; Ling, EA; Dheen, ST
Journal of neuroinflammation 8 13 2011
Sphingosine kinase 1 (SphK1), a key enzyme responsible for phosphorylating sphingosine into sphingosine-1-phosphate (S1P) has been shown to be expressed in monocytes and monocyte-derived peripheral macrophages. This study demonstrates SphK1 immunoexpression in amoeboid microglial cells (AMC), a nascent monocyte-derived brain macrophage in the corpus callosum of developing rat brain. SphK1 immunofluorescence expression, which appeared to be weak in AMC in normal brain, was markedly induced by lipopolysaccharide (LPS) or hypoxia treatment. Western blot analysis also showed increased expression level of SphK1 in the corpus callosum rich in AMC after LPS treatment. Detection of SphK1 mRNA and its upregulation after LPS treatment was confirmed in primary culture AMC by RT-PCR. Administration of N, N-dimethylsphingosine (DMS), a specific inhibitor of SphK1, effectively reduced upregulated SphK1 immunoexpression in AMC both in vivo and in vitro. This was corroborated by western blot which showed a decrease in SphK1 protein level of callosal tissue with DMS pretreatment. Remarkably, LPS-induced upregulation of the transcription factor NFκB was suppressed by DMS. We conclude that SphK1 expression in AMC may be linked to regulation of proinflammatory cytokines via an NFκB signaling pathway.Full Text Article
|Dexamethasone inhibits the Nox-dependent ROS production via suppression of MKP-1-dependent MAPK pathways in activated microglia. |
Huo, Y; Rangarajan, P; Ling, EA; Dheen, ST
BMC neuroscience 12 49 2011
Nox-2 (also known as gp91phox), a subunit component of NADPH oxidases, generates reactive oxygen species (ROS). Nox-dependent ROS generation and nitric oxide (NO) release by microglia have been implicated in a variety of diseases in the central nervous system. Dexamethasone (Dex) has been shown to suppress the ROS production, NO release and inflammatory reaction of activated microglial cells. However, the underlying mechanisms remain unclear.The present study showed that the increased ROS production and NO release in activated BV-2 microglial cells by LPS were associated with increased expression of Nox-2 and iNOS. Dex suppressed the upregulation of Nox-2 and iNOS, as well as the subsequent ROS production and NO synthesis in activated BV-2 cells. This inhibition caused by Dex appeared to be mediated by upregulation of MAPK phosphatase-1 (MKP-1), which antagonizes the activity of mitogen-activated protein kinases (MAPKs). Dex induced-suppression of Nox-2 and -upregulation of MKP-1 was also evident in the activated microglia from corpus callosum of postnatal rat brains. The overexpression of MKP-1 or inhibition of MAPKs (by specific inhibitors of JNK and p38 MAPKs), were found to downregulate the expression of Nox-2 and iNOS and thereby inhibit the synthesis of ROS and NO in activated BV-2 cells. Moreover, Dex was unable to suppress the LPS-induced synthesis of ROS and NO in BV-2 cells transfected with MKP-1 siRNA. On the other hand, knockdown of Nox-2 in BV-2 cells suppressed the LPS-induced ROS production and NO release.In conclusion, it is suggested that downregulation of Nox-2 and overexpression of MKP-1 that regulate ROS and NO may form the potential therapeutic strategy for the treatment of neuroinflammation in neurodegenerative diseases.Full Text Article
|Hypoxia induced amoeboid microglial cell activation in postnatal rat brain is mediated by ATP receptor P2X4. |
Li, F; Wang, L; Li, JW; Gong, M; He, L; Feng, R; Dai, Z; Li, SQ
BMC neuroscience 12 111 2011
Activation of amoeboid microglial cells (AMC) and its related inflammatory response have been linked to the periventricular white matter damage after hypoxia in neonatal brain. Hypoxia increases free ATP in the brain and then induces various effects through ATP receptors. The present study explored the possible mechanism in ATP induced AMC activation in hypoxia.We first examined the immunoexpression of P2X4, P2X7 and P2Y12 in the corpus callosum (CC) and subependyma associated with the lateral ventricles where both areas are rich in AMC. Among the three purinergic receptors, P2X4 was most intensely expressed. By double immunofluorescence, P2X4 was specifically localized in AMC (from P0 to P7) but the immunofluorescence in AMC was progressively diminished with advancing age (P14). It was further shown that P2X4 expression was noticeably enhanced in P0 day rats subjected to hypoxia and killed at 4, 24, 72 h and 7 d versus their matching controls by double labeling and western blotting analysis. P2X4 expression was most intense at 7 d whence the inflammatory response was drastic after hypoxia. We then studied the association of P2X4 with cytokine release in AMC after hypoxic exposure. In primary microglial cells exposed to hypoxia, IL-1β and TNF-α protein levels were up-regulated. Blockade of P2X4 receptor with 2', 3'-0-(2, 4, 6-Trinitrophenyl) adenosine 5'-triphosphate, a selective P2X1-7 blocker resulted in partial suppression of IL-1β (24% vs hypoxic group) and TNF-α expression (40% vs hypoxic group). However, pyridoxal phosphate-6-azo (benzene-2, 4-disulfonic acid) tetrasodium salt hydrate, a selective P2X1-3, 5-7 blocker did not exert any significant effect on the cytokine expression.It is concluded that P2X4 which is constitutively expressed by AMC in postnatal rats was enhanced in hypoxia. Hypoxia induced increase in IL-1β and TNF-α expression was reversed by 2', 3'-0-(2, 4, 6-Trinitrophenyl) adenosine 5'-triphosphate suggesting that P2X4 mediates ATP induced AMC activation and its production of proinflammatory cytokines.Full Text Article
|Increased expression of the chemokines CXCL1 and MIP-1α by resident brain cells precedes neutrophil infiltration in the brain following prolonged soman-induced status epilepticus in rats. |
Johnson, EA; Dao, TL; Guignet, MA; Geddes, CE; Koemeter-Cox, AI; Kan, RK
Journal of neuroinflammation 8 41 2011
Exposure to the nerve agent soman (GD) causes neuronal cell death and impaired behavioral function dependent on the induction of status epilepticus (SE). Little is known about the maturation of this pathological process, though neuroinflammation and infiltration of neutrophils are prominent features. The purpose of this study is to quantify the regional and temporal progression of early chemotactic signals, describe the cellular expression of these factors and the relationship between expression and neutrophil infiltration in damaged brain using a rat GD seizure model.Protein levels of 4 chemokines responsible for neutrophil infiltration and activation were quantified up to 72 hours in multiple brain regions (i.e. piriform cortex, hippocampus and thalamus) following SE onset using multiplex bead immunoassays. Chemokines with significantly increased protein levels were localized to resident brain cells (i.e. neurons, astrocytes, microglia and endothelial cells). Lastly, neutrophil infiltration into these brain regions was quantified and correlated to the expression of these chemokines.We observed significant concentration increases for CXCL1 and MIP-1α after seizure onset. CXCL1 expression originated from neurons and endothelial cells while MIP-1α was expressed by neurons and microglia. Lastly, the expression of these chemokines directly preceded and positively correlated with significant neutrophil infiltration in the brain. These data suggest that following GD-induced SE, a strong chemotactic response originating from various brain cells, recruits circulating neutrophils to the injured brain.A strong induction of neutrophil attractant chemokines occurs following GD-induced SE resulting in neutrophil influx into injured brain tissues. This process may play a key role in the progressive secondary brain pathology observed in this model though further study is warranted.
|Reduction of β-amyloid deposits by γ-secretase inhibitor is associated with the attenuation of secondary damage in the ipsilateral thalamus and sensory functional improvement after focal cortical infarction in hypertensive rats. |
Zhang, Y; Xing, S; Zhang, J; Li, J; Li, C; Pei, Z; Zeng, J
Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism 31 572-9 2011
Abnormal β-amyloid (Aβ) deposits in the thalamus have been reported after cerebral cortical infarction. In this study, we investigated the association of Aβ deposits, with the secondary thalamic damage after focal cortical infarction in rats. Thirty-six stroke-prone renovascular hypertensive rats were subjected to distal middle cerebral artery occlusion (MCAO) and then randomly divided into MCAO, vehicle, and N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT) groups and 12 sham-operated rats as control. The DAPT was administered orally at 72 hours after MCAO. Seven days after MCAO, sensory function, neuron loss, and glial activation and proliferation were evaluated using adhesive removal test, Nissl staining, and immunostaining, respectively. Thalamic Aβ accumulation was evaluated using immunostaining and enzyme-linked immunosorbent assay (ELISA). Compared with vehicle group, the ipsilateral thalamic Aβ, neuronal loss, glial activation and proliferation, and the mean time to remove the stimulus from right forepaw significantly decreased in DAPT group. The mean time to remove the stimulus from the right forepaw and thalamic Aβ burden were both negatively correlated with the number of thalamic neurons. These findings suggest that Aβ deposits are associated with the secondary thalamic damage. Reduction of thalamic Aβ by γ-secretase inhibitor may attenuate the secondary damage and improve sensory function after cerebral cortical infarction.
|Cuprizone-induced demyelination in CNP::GFP transgenic mice. |
Lucas Silvestroff,Sandra Bartucci,Eduardo Soto,Vittorio Gallo,Juana Pasquini,Paula Franco
The Journal of comparative neurology 518 2010
Cuprizone (bis-cyclohexanone oxaldihydrazone) was previously shown to induce demyelination in white matter enriched brain structures. In the present study we used the cuprizone demyelination model in transgenic mice expressing the enhanced green fluorescent protein (GFP) under the 2'-3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) promoter. The use of these particular transgenic mice allows easy detection of cells belonging to the entire oligodendroglial (OLG) lineage, ranging from OLG precursors to mature myelinating OLGs. We were able to evaluate the precise extent of oligodendroglial cell damage and recovery within the murine adult central nervous system (CNS) after inducing demyelination by acute cuprizone intoxication. A generalized loss of GFP+ cells was observed after cuprizone exposure and correlated with a decline in myelin basic protein (MBP) expression. OLGs were depleted in many brain areas that were previously thought to be unaffected by cuprizone treatment. Thus, in addition to the well-known cuprizone effects on the medial corpus callosum, we also found a loss of GFP+ cells in most brain structures, particularly in the caudatus putamen, cortex, anterior commissure, olfactory bulb, hippocampus, optic chiasm, brainstem, and cingulum. Loss of GFP+ cells was accompanied by extensive astrogliosis and microglial activation, although neurons were not affected. Interestingly, cuprizone-treated animals showed both activation of GFAP expression and a higher proliferation rate in subventricular zone cells. A week after cuprizone removal from the diet, GFP+ oligodendroglial cells began repopulating the damaged structures. GFP expression precedes that of MBP and allows OLG detection before myelin restoration.
|Neuroprotection and Functional Recovery Associated with Decreased Microglial Activation Following Selective Activation of mGluR2/3 Receptors in a Rodent Model of Parkinson's Disease. |
Chan, H; Paur, H; Vernon, AC; Zabarsky, V; Datla, KP; Croucher, MJ; Dexter, DT
Parkinson's disease 2010 190450. 2010
Clinical trials have demonstrated positive proof of efficacy of dual metabotropic glutamate receptor 2/3 (mGluR2/3) agonists in both anxiety and schizophrenia. Importantly, evidence suggests that these drugs may also be neuroprotective against glutamate excitotoxicity, implicated in the pathogenesis of Parkinson's disease (PD). However, whether this neuroprotection also translates into functional recovery is unclear. In the current study, we examined the neuroprotective efficacy of the dual mGluR2/3 agonist, 2R,4R-4-aminopyrrolidine-2,4-dicarboxylate (2R,4R-APDC), and whether this is accompanied by behavioral recovery in a rodent 6-hydroxydopamine (6-OHDA) model of PD. We now report that delayed post lesion treatment with 2R,4R-APDC (10 nmol), results in robust neuroprotection of the nigrostriatal system, which translated into functional recovery as measured by improved forelimb use asymmetry and reduced (+)-amphetamine-induced rotation compared to vehicle treated animals. Interestingly, these beneficial effects were associated with a decrease in microglial markers in the SNc, which may suggest an antiinflammatory action of this drug.Full Text Article
|The acute phase response and soman-induced status epilepticus: temporal, regional and cellular changes in rat brain cytokine concentrations. |
Johnson, EA; Kan, RK
Journal of neuroinflammation 7 40 2010
Neuroinflammation occurs following brain injury, including soman (GD) induced status epilepticus (SE), and may contribute to loss of neural tissue and declined behavioral function. However, little is known about this important pathological process following GD exposure. Limited transcriptional information on a small number of brain-expressed inflammatory mediators has been shown following GD-induced SE and even less information on protein upregulation has been elucidated. The purpose of this study is to further characterize the regional and temporal progression of the neuroinflammatory process following acute GD-induced SE.The protein levels of 10 cytokines was quantified using bead multiplex immunoassays in damaged brain regions (i.e., piriform cortex, hippocampus and thalamus) up to 72 hours following seizure onset. Those factors showing significant changes were then localized to neural cells using fluorescent IHC.A significant concentration increase was observed in all injured brain regions for four acute phase response (APR) induction cytokines: interleukin (IL)-1alpha, IL-1beta, IL-6, and tumor necrosis factor (TNF)-alpha. Increases in these APR cytokines corresponded both temporally and regionally to areas of known seizure damage and neuronal death. Neurotoxic cytokines IL-1alpha and IL-1beta were primarily expressed by activated microglia whereas the potentially neuroprotective cytokine IL-6 was expressed by neurons and hypertrophic astrocytes.Increases in neurotoxic cytokines likely play an active role in the progression of GD-induced SE neuropathology though the exact role that these and other cytokines play in this process require further study.
|Astrocytes and oligodendrocytes can be generated from NG2+ progenitors after acute brain injury: intracellular localization of oligodendrocyte transcription factor 2 is associated with their fate choice. |
Jing-Wei Zhao,Ruma Raha-Chowdhury,James W Fawcett,Colin Watts
The European journal of neuroscience 29 2009
Brain injury induces gliosis and scar formation; its principal cell types are mainly astrocytes and some oligodendrocytes. The origin of the astrocytes and oligodendrocytes in the scar remains unclear together with the underlying mechanism of their fate choice. We examined the response of oligodendrocyte transcription factor (Olig)2(+) glial progenitors to acute brain injury. Both focal cortical (mechanical or excitotoxic) and systemic (kainic acid-induced seizure or lipopolysaccharide-induced inflammation) injury caused cytoplasmic translocation of Olig2 (Olig2(TL)) exclusively in affected brain regions as early as 2 h after injury in two-thirds of Olig2(+) cells. Many of the proliferating Olig2(+) cells reacting to injury co-expressed chondroitin sulphate proteoglycan neuron/glia antigen 2 (NG2). Using 5-bromodeoxyuridine (BrdU) tracing protocols, proliferating Olig2(TL)GFAP(+)BrdU(+) cells were observed from 2 days post-lesion (dpl). Immature oligodendrocytes were also seen from 2 dpl and all of them retained Olig2 in the nucleus (Olig2(Nuc)). From 5 dpl Olig2(TL)NG2(+)GFAP(+) cells were observed in the wound and some of them were proliferative. From 5 dpl NG2(+)RIP(+) cells were also seen, all of which were Olig2(Nuc) and some of which were also BrdU(+). Our results suggest that, in response to brain injury, NG2(+) progenitors may generate a subpopulation of astrocytes in addition to oligodendrocytes and their fate choice was associated with Olig2(TL) or Olig2(Nuc). However, the NG2(+)GFAP(+) phenotype was only seen within a limited time window (5-8 dpl) when up to 20% of glial fibrillary acidic protein (GFAP) cells co-expressed NG2. We also observed Olig2(TL)GFAP(+) cells that appeared after injury and before the NG2(+)GFAP(+) phenotype. This suggests that not all astrocytes are derived from an NG2(+) population.
|Upregulation of Dpysl2 and Spna2 gene expression in the rat brain after ischemic stroke. |
Fransisca Indraswari,Peter T H Wong,Elgin Yap,Y K Ng,S Thameem Dheen
Neurochemistry international 55 2009
Ischemia activates the synthesis of potentially damaging and protective proteins in the central nervous system. Dihydropyrimidinase-like 2 (Dpysl2), a protein involved in neuronal differentiation and axonal guidance, and alpha-spectrin 2 (Spna2), a protein involved in maintaining neuronal membrane integrity, were found altered in various nervous system diseases. Modifications of Dpysl2 and Spna2 proteins have been reported in focal ischemic stroke, but their significance is not yet established. Therefore, this study was aimed to investigate the temporal expression of Dpysl2 and Spna2 genes in normal and stroke rat brain and to characterize stroke brains for cell areas, apoptosis, and microglia cells. The middle cerebral artery of rat brain was occluded and the brain tissue was sectioned for in situ hybridization of Dpysl2 and Spna2 genes, TUNEL, and OX-42 immunofluorescence staining. Dpysl2 and Spna2 mRNA expression was quantified by real-time RT-PCR. Characterization of stroke brain for apoptosis and microglia cells showed apoptotic cells and activated microglia, mainly in the infarct core of ipsilateral cortex and striatum of stroke brain. Significant upregulation of Dpysl2 and Spna2 mRNA expression in the penumbra region after stroke was observed predominantly in injured swollen cells in the cortex and striatum. Upregulation of Dpysl2 and Spna2 expression in hypertrophic cells in the penumbra regions of cortex and striatum of stroke brain indicates an early neuronal defense mechanism involving active neuronal repair, regeneration and development, as these genes are known to be involved in neurite outgrowth and plasticity.
|Erythropoietin reduces epileptogenic processes following status epilepticus. |
Kon Chu,Keun-Hwa Jung,Soon-Tae Lee,Jin-Hee Kim,Kyung-Muk Kang,Hyun-Kyung Kim,Jae-Sung Lim,Hee-Kwon Park,Manho Kim,Sang Kun Lee,Jae-Kyu Roh
Epilepsia 49 2008
Erythropoietin (EPO) has neuron and astroglial protective effects via reduction of tissue-injuring molecules such as reactive oxygen species, glutamate, inflammatory cytokines, and other damaging molecules. Although EPO may constitute an effective therapeutic modality in cases of epileptic insult, no study has been performed on the effects of exogenous EPO on the chronic seizure formation. In this study, we attempted to investigate if EPO could modulate the altered microenvironment in the epileptic rat brain.
|CXCR4 receptors in the dorsal medulla: implications for autonomic dysfunction. |
Hermann, GE; Van Meter, MJ; Rogers, RC
The European journal of neuroscience 27 855-64 2008
The chemokine receptor, CXCR4, plays an essential role in guiding neural development of the CNS. Its natural agonist, CXCL12 [or stromal cell-derived factor-1 (SDF-1)], normally is derived from stromal cells, but is also produced by damaged and virus-infected neurons and glia. Pathologically, this receptor is critical to the proliferation of the HIV virus and initiation of metastatic cell growth in the brain. Anorexia, nausea and failed autonomic regulation of gastrointestinal (GI) function cause morbidity and contribute to the mortality associated with these disease states. Our previous work on the peripheral cytokine, tumor necrosis factor-alpha, demonstrated that similar morbidity factors involving GI dysfunction are attributable to agonist action on neural circuit elements of the dorsal vagal complex (DVC) of the hindbrain. The DVC includes vagal afferent terminations in the solitary nucleus, neurons in the solitary nucleus (NST) and area postrema, and visceral efferent motor neurons in the dorsal motor nucleus (DMN) that are responsible for the neural regulation of digestive functions from the oral cavity to the transverse colon. Immunohistochemical techniques demonstrate a dense concentration of CXCR4 receptors on neurons throughout the DVC and the hypoglossal nucleus. CXCR4-immunoreactivity is also intense on microglia within the DVC, though not on the astrocytes. Physiological studies show that nanoinjection of SDF-1 into the DVC produces a significant reduction in gastric motility in parallel with an elevation in the numbers of cFOS-activated neurons in the NST and DMN. These results suggest that this chemokine receptor may contribute to autonomically mediated pathophysiological events associated with CNS metastasis and infection.
|HDAC inhibitor increases histone H3 acetylation and reduces microglia inflammatory response following traumatic brain injury in rats. |
Zhang, B; West, EJ; Van, KC; Gurkoff, GG; Zhou, J; Zhang, XM; Kozikowski, AP; Lyeth, BG
Brain research 1226 181-91 2008
Traumatic brain injury (TBI) produces a rapid and robust inflammatory response in the brain characterized in part by activation of microglia. A novel histone deacetylase (HDAC) inhibitor, 4-dimethylamino-N-[5-(2-mercaptoacetylamino)pentyl]benzamide (DMA-PB), was administered (0, 0.25, 2.5, 25 mg/kg) systemically immediately after lateral fluid percussion TBI in rats. Hippocampal CA2/3 tissue was processed for acetyl-histone H3 immunolocalization, OX-42 immunolocalization (for microglia), and Fluoro-Jade B histofluorescence (for degenerating neurons) at 24 h after injury. Vehicle-treated TBI rats exhibited a significant reduction in acetyl-histone H3 immunostaining in the ipsilateral CA2/3 hippocampus compared to the sham TBI group (pless than 0.05). The reduction in acetyl-histone H3 immunostaining was attenuated by each of the DMA-PB dosage treatment groups. Vehicle-treated TBI rats exhibited a high density of phagocytic microglia in the ipsilateral CA2/3 hippocampus compared to sham TBI in which none were observed. All doses of DMA-PB significantly reduced the density of phagocytic microglia (pless than 0.05). There was a trend for DMA-PB to reduce the number of degenerating neurons in the ipsilateral CA2/3 hippocampus (p=0.076). We conclude that the HDAC inhibitor DMA-PB is a potential novel therapeutic for inhibiting neuroinflammation associated with TBI.
|Phosphorylated FTY720 promotes astrocyte migration through sphingosine-1-phosphate receptors. |
Florian Mullershausen,Luis M Craveiro,Youngah Shin,Marta Cortes-Cros,Frederic Bassilana,Maribel Osinde,William L Wishart,Danilo Guerini,Michaela Thallmair,Martin E Schwab,Rajeev Sivasankaran,Klaus Seuwen,Kumlesh K Dev
Journal of neurochemistry 102 2007
Sphingosine-1-phosphate (S1P) receptors are widely expressed in the central nervous system where they are thought to regulate glia cell function. The phosphorylated version of fingolimod/FTY720 (FTY720P) is active on a broad spectrum of S1P receptors and the parent compound is currently in phase III clinical trials for the treatment of multiple sclerosis. Here, we aimed to identify which cell type(s) and S1P receptor(s) of the central nervous system are targeted by FTY720P. Using calcium imaging in mixed cultures from embryonic rat cortex we show that astrocytes are the major cell type responsive to FTY720P in this assay. In enriched astrocyte cultures, we detect expression of S1P1 and S1P3 receptors and demonstrate that FTY720P activates Gi protein-mediated signaling cascades. We also show that FTY720P as well as the S1P1-selective agonist SEW2871 stimulate astrocyte migration. The data indicate that FTY720P exerts its effects on astrocytes predominantly via the activation of S1P1 receptors, whereas S1P signals through both S1P1 and S1P3 receptors. We suggest that this distinct pharmacological profile of FTY720P, compared with S1P, could play a role in the therapeutic effects of FTY720 in multiple sclerosis.
|Expression of acute-phase response proteins in retinal Müller cells in diabetes |
Gerhardinger, Chiara, et al
Invest Ophthalmol Vis Sci, 46:349-57 (2005) 2005
|Immunohistochemical detection of T-cell subsets and other leukocytes in paraffin-embedded rat and mouse tissues with monoclonal antibodies. |
Whiteland, J L, et al.
J. Histochem. Cytochem., 43: 313-20 (1995) 1995
We describe a method for immunohistochemical localization of T-cells, CD4+ T-cells, CD8+ T-cells, B-cells, activated lymphocytes, major histocompatibility complex (MHC) class II antigens, macrophages, dendritic cells, and granulocytes in rat and mouse tissue fixed in periodate-lysine-paraformaldehyde (PLP) and embedded in paraffin. Rat and mouse spleen and eyes were fixed in PLP for 18-24 hr, rapidly dehydrated, infiltrated under vacuum with paraffin at 54 degrees C, sectioned, and stained with appropriate monoclonal antibodies (MAbs). Sections of PLP-fixed, paraffin-embedded spleen were compared with acetone-fixed frozen spleen sections with respect to morphology and staining quality. Nine of 10 MAbs to rat antigens and eight of nine MAbs to mouse antigens stained paraffin sections equally or more intensely than frozen sections. The two MAbs that showed weaker staining still gave good staining on paraffin sections. Paraffin-embedded rat and mouse eyes were easier to section serially than frozen eyes, showed superior morphology, and individually stained cells were readily identified. Therefore, a combination of PLP fixation and low-temperature paraffin embedding permits detection of the major types of immune cell in rat and mouse tissues while maintaining good morphology, particularly in diseased, damaged, or delicate tissues.
|Differential immunochemical markers reveal the normal distribution of brain macrophages and microglia in the developing rat brain |
Milligan C.E. et al.
J Comparative Neurol., 314:125-135 (1991) 1991
|Macrophage heterogeneity in the rat as delineated by two monoclonal antibodies MRC OX-41 and MRC OX-42, the latter recognizing complement receptor type 3 |
Robinson A.P. et al.
Immunol., 57:239-247 (1986) 1986
|Anti-Integrin alphaM [CD11b], clone OX-42 - Data Sheet|