CaV α2δ Autoimmune Encephalitis: A Novel Antibody and its Characteristics.

OBJECTIVE: Discovery of a novel antibody would enable diagnosis and early treatment of autoimmune encephalitis. The aim was to discover a novel antibody targeting a synaptic receptor and characterize the pathogenic mechanism. METHOD: We screened for unknown antibodies in serum and cerebrospinal fluid samples from autoimmune encephalitis patients. Samples with reactivity to rat brain sections and no reactivity to conventional antibody tests underwent further processing for antibody discovery, using immunoprecipitation to primary neuronal cells, mass-spectrometry analysis, an antigen-binding assay on an antigen-overexpressing cell line, and an electrophysiological assay with cultured hippocampal neurons. RESULTS: Two patients had a novel antibody against CaV α2δ (voltage-gated calcium channel alpha-2/delta subunit). The patient samples stained neuropils of the hippocampus, basal ganglia, and cortex in rat brain sections and bound to a CaV α2δ-overexpressing cell line. Knockdown of CaV α2δ expression in cultured neurons turned off the immunoreactivity of the antibody from the patients to the neurons. The patients were associated with preceding meningitis or neuroendocrine carcinoma and responded to immunotherapy. In cultured neurons, the antibody reduced neurotransmitter release from presynaptic nerve terminals by interfering with tight coupling of calcium channels and exocytosis. INTERPRETATION: Here, we discovered a novel autoimmune encephalitis associated with anti-CaV α2δ antibody. Further analysis of the antibody in autoimmune encephalitis might promote early diagnosis and treatment. ANN NEUROL 2021;89:740-752.

Dysregulation of long non-coding RNAs in mouse models of localization-related epilepsy.

Genome-wide profiling has revealed that eukaryotic genomes are transcribed into numerous non-coding RNAs. In particular, long non-coding RNAs (lncRNAs) have been implicated in various human diseases due to their biochemical and functional diversity. Epileptic disorders have been characterized by dysregulation of epigenetic regulatory mechanisms, and recent studies have identified several lncRNAs involved in neural development and network function. However, comprehensive profiling of lncRNAs implicated in chronic epilepsy has been lacking. In this study, microarray analysis was performed to obtain the expression profile of lncRNAs dysregulated in pilocarpine and kainate models, two models of temporal lobe epilepsy commonly used for studying epileptic mechanisms. Total of 4622 lncRNAs were analyzed: 384 lncRNAs were significantly dysregulated in pilocarpine model, and 279 lncRNAs were significantly dysregulated in kainate model compared with control mice (≥3.0-fold, p < 0.05). Among these, 54 and 14 lncRNAs, respectively, had adjacent protein-coding genes whose expressions were also significantly dysregulated (≥2.0-fold, p < 0.05). Majority of these pairs of lncRNAs and adjacent genes shared the same direction of dysregulation. For the selected adjacent gene-lncRNA pairs, significant Gene Ontology terms were embryonic appendage morphogenesis and neuron differentiation. This was the first study to comprehensively identify dysregulated lncRNAs in two different models of chronic epilepsy and will likely provide a novel insight into developing lncRNA therapeutics.

Enhanced performance on polymeric light-emitting diode with non-metallic ion in the water soluble non-conjugated polymer.

We have fabricated a polymer light-emitting diode (PLED) from the conventional blue-emitting polymer, polyfluorene (PFO), by constructing a multilayer structure with non-metal ion containing water soluble non-conjugated polymer, polyurethane with F- ion (PU:F-), on the top of the PFO. The device with PU:F- layer shows a maximum luminance of 5294 cd/m2 at an applied voltage of 10 V while the one without PU:F- layer shows only 4439 cd/m2 at the same applied voltage. We propose the improvement of device performance with PU:F- layer was due to not only an effective hole blocking at the polymer-polymer interface but also increase of electric field strength with anode after electro-stactic repulsion between electrons from the cathod and anions from the water soluble polymer layer. We will discuss the effect of multilayer polymer structure in PLED in terms of current/voltage characteristics, luminance, and quantum efficiency related with the applied bias.

Therapeutic Target MicroRNA Identification Based on Circular RNA Expression Signature After Intracerebral Hemorrhage.

We investigated circular RNA (circRNA) expression pattern from a rat intracerebral hemorrhage (ICH) model and tested therapeutic strategy. Hemorrhagic stroke was induced by stereotactic collagenase injection. Brain was harvested at 1, 3, and 7 days after ICH induction to study circRNA expression. Significantly altered circRNAs from microarray were examined by quantitative real-time polymerase chain reaction. Predicted target microRNA and nearby messenger RNA levels of significantly altered circRNAs were validated from previously published database. Therapeutic strategy based on potential target microRNA of significantly depressed circRNA was examined using in vitro and in vivo hemorrhagic model. Both significantly elevated/downregulated circRNA increased as time passed after ICH: 9, 159, and 704 circRNAs were significantly elevated, whereas 19, 276, and 656 circRNAs were significantly depressed at 1, 3 and 7 days after ICH induction, respectively, out of 13,298 studied circRNAs. The most elevated circRNAs were rno_circRNA_002714 and rno_circRNA_002715, which are located closely each other in chromosome 10, within exon sequence of glial fibrillary acidic protein. The most significantly downregulated circRNA was rno_circRNA_016465, which has several complementary sequences for miR-466b. The most commonly predicted microRNA response element of significantly depressed circRNAs was miR-466b. The antagonistic sequence against miR-466b significantly decreased neuronal cell death and improved neurological recovery in a hemorrhagic stroke model by upregulating insulin like growth factor receptors 1 and 2. This study illustrated dynamic circRNA expression pattern in a hemorrhagic stroke model, which correlated with microRNA and messenger RNA expression, suggesting the regulatory role of RNA dynamics in ICH.

Gut microbiome diversity in a febrile seizure mouse model.

Purpose: Febrile seizures at a young age can provoke late-onset temporal lobe epilepsy. Since recent evidence has suggested that the gut microbiome affects central nervous system pathology across the blood-brain barrier, we hypothesized that febrile seizures alter the composition of the gut microbiome to provoke epilepsy. Methods: Third-generation C57BL/6 mice were separated into two groups (n = 5 each), and hot air was applied to only one group to cause febrile seizures. After two weeks of heat challenge, the fecal pellets acquired from each group were analyzed. Results: The gut microbiota of fecal pellets from each group revealed five taxa at the genus level and eight taxa at the species level that were significantly different in proportion between the groups. Conclusion: Although there was no significant difference in the overall diversity of the gut microbiota between the two groups, the identified heterogeneity may imply the pathognomonic causative relevance of febrile seizures and the development of epilepsy.

Neuroprotective effect of a cell-free extract derived from human adipose stem cells in experimental stroke models.

A recent study suggested that a cell-free extract of human adipose stem cells (hASCs-E) has beneficial effects on neurological diseases by modulating the host environment. Here, we investigated the effects of hASCs-E in several experimental models of stroke in vitro (oxygen and glucose deprivation, OGD) and in vivo (transient or permanent focal cerebral ischemia and intracerebral hemorrhage, ICH). Ischemia was induced in vitro in Neuro2A cells, and the hASCs-E was applied 24h before the OGD or concurrently. Focal cerebral ischemia was induced by unilateral intraluminal thread occlusion of the middle cerebral artery (MCA) in rats for 90min or permanently, or by unilateral MCA microsurgical direct electrocoagulation in mice. The ICH model was induced with an intracerebral injection of collagenase in rats. The hASCs-E was intraperitoneally administered 1h after the stroke insults. Treatment of the hASCs-E led to a substantially high viability in the lactate dehydrogenase and WST-1 assays in the in vitro ischemic model. The cerebral ischemic and ICH model treated with hASCs-E showed decreased ischemic volume and reduced brain water content and hemorrhage volume. The ICH model treated with hASCs-E exhibited better performance on the modified limb placing test. The expression of many genes related to inflammation, immune response, and cell-death was changed substantially in the ischemic rats or neuronal cells treated with the hASCs-E. These results reveal a neuroprotective role of hASCs-E in animal models of stroke, and suggest the feasible application of stem cell-based, noninvasive therapy for treating stroke.

miR-206 regulates brain-derived neurotrophic factor in Alzheimer disease model.

OBJECTIVE: Alzheimer disease (AD) brains are deficient in brain-derived neurotrophic factor (BDNF), which regulates synaptic plasticity and memory. MicroRNAs (miRNAs) are ∼22-nucleotide small noncoding RNAs that control a variety of physiological and disease processes. Here, we show that miR-206 regulates BDNF and memory function in AD mice. METHODS: Expression of miRNAs was analyzed in Tg2576 AD transgenic mice and human AD brain samples. Regulation of BDNF by a selected miRNA was validated by in silico prediction, target gene luciferase assay, and dendritic spine responses in neurons. AM206, a neutralizing inhibitor of miR-206 (antagomir), was injected into the third ventricle of Tg2576 mice, after which memory function, synaptogenesis, neurogenesis, and target gene expression were assessed. For noninvasive delivery, antagomirs were administered intranasally. RESULTS: The brains of Tg2576 mice and the temporal cortex of human AD brains had increased levels of miR-206. This miRNA targeted BDNF transcripts, and AM206 prevented the detrimental effects of amyloid-ß42 on BDNF and dendritic spine degeneration in Tg2576 neurons. Injection of AM206 into the cerebral ventricles of AD mice increased the brain levels of BDNF and improved their memory function. In parallel, AM206 enhanced the hippocampal synaptic density and neurogenesis. Furthermore, intranasally administered AM206 also reached the brain and increased BDNF levels and memory function in AD mice. INTERPRETATION: Our findings demonstrate a novel miRNA-dependent regulation of BDNF in AD and suggest possible therapeutic approaches, such as noninvasive intranasal delivery of AM206.

Modified polyvinyl alcohol layer with hydrophobic surface for the passivation of pentacene thin-film transistor.

We modified the surface of a polyvinyl alcohol (PVA) layer by self assembly monolayer technique using a fluorine substituted silane compound (1H,1H,2H,2H-perfluorooctyl-trichlorosilane: FTS) to protect a pentacene thin-film transistor (TFT) from O2 and H2O. Surface modified PVA showed very low surface energy with water contact angle of 106.2 degrees. Surface treatment of PVA layer on pentacene TFT device was done in toluene solvent and we did not observe any damage to the PVA layer or pentacene TFT devices during surface modification process. Pentacene TFT with surface modified PVA passivation layer exhibited very stable TFT operation with almost no field effect mobility drop or threshold voltage shift up to 400 hrs. The performance of unpassivated OTFTs exponentially degraded and almost failed in 290 hrs. We propose that modified PVA layer can be used as a good passivation layer for oxygen and water in OTFT.

Effect of Fluence and Multi-Pass on Groove Morphology and Process Efficiency of Laser Structuring for 3D Electrodes of Lithium-Ion Batteries.

Lithium-ion batteries (LIBs) are widely used as energy storage systems. With the growing interest in electric vehicles, battery performance related to traveling distance has become more important. Therefore, there are various studies going on to achieve high-power and high-energy batteries. Laser structuring of electrodes involves a groove being produced on electrodes by a laser. This technique was used to show that battery performance can be enhanced due to improving Li-ion diffusion. However, there is a lack of studies about the morphological variation of grooves and process efficiency in laser parameters in the laser structuring of electrodes. In this study, the LiFePO4 cathode is structured by a nanosecond laser to analyze the morphological variation of grooves and process efficiency depending on laser fluence and the number of passes. First, the various morphologies of grooves are formed by a combination of fluences and the number of passes. At a fluence of 0.86 J/cm2 and three passes, the maximum aspect ratio of 1.58 is achieved and the surface area of structured electrodes is greater than that of unstructured electrodes. Secondly, three ablation phenomena observed after laser structuring are classified according to laser parameters through SEM images and EDX analysis. Finally, we analyze the amount of active material removal and process efficiency during laser structuring. In conclusion, applying low fluence and multi-pass is assumed to be advantageous for laser structuring of electrodes.

Proteins related to ictogenesis and seizure clustering in chronic epilepsy.

Seizure clustering is a common phenomenon in epilepsy. Protein expression profiles during a seizure cluster might reflect the pathomechanism underlying ictogenesis. We performed proteomic analyses to identify proteins with a specific temporal expression pattern in cluster phases and to demonstrate their potential pathomechanistic role. Pilocarpine epilepsy model mice with confirmed cluster pattern of spontaneous recurrent seizures by long-term video-electroencpehalography were sacrificed at the onset, peak, or end of a seizure cluster or in the seizure-free period. Proteomic analysis was performed in the hippocampus and the cortex. Differentially expressed proteins (DEPs) were identified and classified according to their temporal expression pattern. Among the five hippocampal (HC)-DEP classes, HC-class 1 (66 DEPs) represented disrupted cell homeostasis due to clustered seizures, HC-class 2 (63 DEPs) cluster-onset downregulated processes, HC-class 3 (42 DEPs) cluster-onset upregulated processes, and HC-class 4 (103 DEPs) consequences of clustered seizures. Especially, DEPs in HC-class 3 were hippocampus-specific and involved in axonogenesis, synaptic vesicle assembly, and neuronal projection, indicating their pathomechanistic roles in ictogenesis. Key proteins in HC-class 3 were highly interconnected and abundantly involved in those biological processes. This study described the seizure cluster-associated spatiotemporal regulation of protein expression. HC-class 3 provides insights regarding ictogenesis-related processes.

Effects of long term nitrite therapy on functional recovery in experimental ischemia model.

Our data have shown that nitrite therapy can rescue the ischemic brain when injected <3h after cerebral ischemic-reperfusion (I/R) injury and its effects can be prolonged to 4.5h in combination with memantine. We investigated whether or not long-term nitrite therapy is beneficial in ischemic brains. Sodium nitrite (1-100 µg/kg ip) or saline were administered to rats subjected to focal I/R injury for 7 days beginning 24h after I/R. Behavioral tests for 5 weeks revealed better functional recovery in the high-dose nitrite group than the control group. Other nitrite groups with relatively low doses showed no functional benefits. Hemispheric atrophy was attenuated by approximately 30% in the high-dose nitrite group. High-dose nitrite therapy also reduced inflammatory cytokine levels and caspase activity in the subacute period, and increased BrdU(+)MAP2(+) and BrdU(+)laminin(+) cells, and vascular density in the 5-week ischemic brain. Long-term nitrite therapy, when initiated 24h after I/R, corrected the subacute hostile environment, induced tissue and vascular regeneration, and improved functional recovery. Early and subsequent long term nitrite therapy may be effective in the management for ischemic stroke patients.

Male-like sexual behavior of female mouse lacking fucose mutarotase.

BACKGROUND: Mutarotases are recently characterized family of enzymes that are involved in the anomeric conversions of monosaccharides. The mammalian fucose mutarotase (FucM) was reported in cultured cells to facilitate fucose utilization and incorporation into protein by glycosylation. However, the role of this enzyme in animal has not been elucidated. RESULTS: We generated a mutant mouse specifically lacking the fucose mutarotase (FucM) gene. The FucM knockout mice displayed an abnormal sexual receptivity with a drastic reduction in lordosis score, although the animals were fertile due to a rare and forced intromission by a typical male. We examined the anteroventral periventricular nucleus (AVPv) of the preoptic region in brain and found that the mutant females showed a reduction in tyrosine hydoxylase positive neurons compared to that of a normal female. Furthermore, the mutant females exhibited a masculine behavior, such as mounting to a normal female partner as well as showing a preference to female urine. We found a reduction of fucosylated serum alpha-fetoprotein (AFP) in a mutant embryo relative to that of a wild-type embryo. CONCLUSIONS: The observation that FucM-/- female mouse exhibits a phenotypic similarity to a wild-type male in terms of its sexual behavior appears to be due to the neurodevelopmental changes in preoptic area of mutant brain resembling a wild-type male. Since the previous studies indicate that AFP plays a role in titrating estradiol that are required to consolidate sexual preference of female mice, we speculate that the reduced level of AFP in FucM-/- mouse, presumably resulting from the reduced fucosylation, is responsible for the male-like sexual behavior observed in the FucM knock-out mouse.

Altered long noncoding RNA profile after intracerebral hemorrhage.

OBJECTIVE: We investigated the expression pattern of long noncoding RNAs (lncRNA) and messenger RNAs (mRNA) from two different intracerebral hemorrhage (ICH) rat models, and performed gene ontology and gene/protein interaction analyses. METHODS: We harvested hemorrhagic brain 1, 3, and 7 days after ICH induction by stereotactic collagenase injection. We performed microarray analyses with Agilent array platform to compare the expression of lncRNA and mRNAs from hemorrhagic and normal brains. The RNA expression patterns were also examined from the autologous blood injection ICH model at days 1 and 3, and significantly altered lncRNAs from two ICH models were validated by quantitative reverse transcriptase-polymerase chain reaction. Gene ontology analysis and pathway analysis were performed with differentially expressed mRNAs after ICH. Gene and protein interaction analysis was performed to elucidate the functional role of upregulated lncRNA in neuronal damage. RESULTS: Among the 13,661 lncRNAs studied, 83, 289, and 401 lncRNAs were significantly elevated after 1, 3, and 7 days after collagenase-induced ICH, respectively. NR_027324, or H19, was the most upregulated lncRNA after 1 day from the two ICH models and its elevation persisted until the 7th day. Gene ontology analysis revealed that immune-related biological processes such as immune response, immune system process, and defense response were upregulated from both ICH models. Gene and protein interaction study demonstrated that NR_027324 was closely related to the type I interferon signaling pathway. INTERPRETATION: This study illustrates the dynamic expression pattern of the lncRNA profile following ICH, and that H19 is the most consistently upregulated lncRNA after ICH.

Possible epigenetic regulatory effect of dysregulated circular RNAs in Alzheimer's disease model.

As circular RNAs (circRNAs) regulates the effect of micro RNAs (miRNAs), circRNA-miRNA-mRNA network might be implicated in various disease pathogenesis. Therefore, we evaluated the dysregulated circRNAs in the Tg2576 mouse Alzheimer's disease (AD) model, their possible regulatory effects on downstream target mRNAs, and their pathomechanistic role during the disease progression. The microarray-based circRNA expression analysis at seven- and twelve-months of ages (7 M and 12 M) returned 101 dysregulated circRNAs at 7 M (55 up-regulated and 46 down-regulated) and twelve dysregulated circRNAs at 12 M (five up-regulated and seven down-regulated). For each dysregulated circRNA, potential target miRNAs and their downstream target mRNAs were searched. Dysregulation of circRNAs was associated with increased frequency of relevant dysregulation of their downstream target mRNAs. Those differentially expressed circRNA-miRNA-mRNA regulatory network included 2,275 networks (876 for up-regulated circRNAs and 1,399 for down-regulated circRNAs) at 7 M and 38 networks (25 for up-regulated circRNAs and 13 for down-regulated circRNAs) at 12 M. Gene ontology (GO) and pathway analyses demonstrated that the dysregulated mRNAs in those networks represent the AD pathomechanism at each disease stage. We concluded that the dysregulated circRNAs might involve in the AD pathogenesis by modulating disease relevant mRNAs via circRNA-miRNA-mRNA regulatory networks.

Possible epigenetic regulatory effect of dysregulated circular RNAs in epilepsy.

Circular RNAs (circRNAs) involve in the epigenetic regulation and its major mechanism is the sequestration of the target micro RNAs (miRNAs). We hypothesized that circRNAs might be related with the pathophysiology of chronic epilepsy and evaluated the altered circRNA expressions and their possible regulatory effects on their target miRNAs and mRNAs in a mouse epilepsy model. The circRNA expression profile in the hippocampus of the pilocarpine mice was analyzed and compared with control. The correlation between the expression of miRNA binding sites (miRNA response elements, MRE) in the dysregulated circRNAs and the expression of their target miRNAs was evaluated. As miRNAs also inhibit their target mRNAs, circRNA-miRNA-mRNA regulatory network, comprised of dysregulated RNAs that targets one another were searched. For the identified networks, bioinformatics analyses were performed. As the result, Forty-three circRNAs were dysregulated in the hippocampus (up-regulated, 26; down-regulated, 17). The change in the expression of MRE in those circRNAs negatively correlated with the change in the relevant target miRNA expression (r = -0.461, P<0.001), supporting that circRNAs inhibit their target miRNA. 333 dysregulated circRNA-miRNA-mRNA networks were identified. Gene ontology and pathway analyses demonstrated that the up-regulated mRNAs in those networks were closely related to the major processes in epilepsy. Among them, STRING analysis identified 37 key mRNAs with abundant (≥4) interactions with other dysregulated target mRNAs. The dysregulation of the circRNAs which had multiple interactions with key mRNAs were validated by PCR. We concluded that dysregulated circRNAs might have a pathophysiologic role in chronic epilepsy by regulating multiple disease relevant mRNAs via circRNA-miRNA-mRNA interactions.

Maternal immune activation alters brain microRNA expression in mouse offspring.

OBJECTIVE: Maternal immune activation (MIA) is associated with an increased risk of autism spectrum disorder (ASD) in offspring. Herein, we investigate the altered expression of microRNAs (miRNA), and that of their target genes, in the brains of MIA mouse offspring. METHODS: To generate MIA model mice, pregnant mice were injected with polyriboinosinic:polyribocytidylic acid on embryonic day 12.5. We performed miRNA microarray and mRNA sequencing in order to determine the differential expression of miRNA and mRNA between MIA mice and controls, at 3 weeks of age. We further identified predicted target genes of dysregulated miRNAs, and miRNA-target interactions, based on the inverse correlation of their expression levels. RESULTS: Mice prenatally subjected to MIA exhibited behavioral abnormalities typical of ASD, such as a lack of preference for social novelty and reduced prepulse inhibition. We found 29 differentially expressed miRNAs (8 upregulated and 21 downregulated) and 758 differentially expressed mRNAs (542 upregulated and 216 downregulated) in MIA offspring compared to controls. Based on expression levels of the predicted target genes, 18 downregulated miRNAs (340 target genes) and three upregulated miRNAs (60 target genes) were found to be significantly enriched among the differentially expressed genes. miRNA and target gene interactions were most significant between mmu-miR-466i-3p and Hfm1 (ATP-dependent DNA helicase homolog), and between mmu-miR-877-3p and Aqp6 (aquaporin 6). INTERPRETATION: Our results provide novel information regarding miRNA expression changes and their putative targets in the early postnatal period of brain development. Further studies will be needed to evaluate potential pathogenic roles of the dysregulated miRNAs.

Dysregulated long non-coding RNAs in the temporal lobe epilepsy mouse model.

PURPOSE: To perform comprehensive profiling of long non-coding RNAs (LncRNAs) in temporal lobe epilepsy. METHODS: We performed extensive profiling of LncRNAs and mRNAs in the mouse pilocarpine model in specific brain regions, the hippocampus and cortex, and compared the results to those of the control mouse. Differentially expressed LncRNAs and mRNAs were identified with a microarray analysis (Arraystar Mouse LncRNA Expression Microarray V3.0). Then, gene ontology (GO) and pathway analysis were performed to investigate the potential roles of the differentially expressed mRNAs in the pilocarpine model. Protein-protein interactions transcribed by dysregulated mRNAs with/without co-dysregulated LncRNAs were analyzed using STRING v10 (http://string-db.org/). RESULTS: A total of 22 and 83 LncRNAs were up- and down-regulated (≥2.0-fold, all P < .05), respectively, in the hippocampus of the epilepsy model, while 46 and 659 LncRNAs were up- and down-regulated, respectively, in the cortex of the epilepsy model. GO and pathway analysis revealed that the dysregulated mRNAs were closely associated with a process already known to be involved in epileptogenesis: acute inflammation, calcium ion regulation, extracellular matrix remodeling, and neuronal differentiation. Among the LncRNAs, we identified 10 LncRNAs commonly dysregulated with corresponding mRNAs in the cortex. The STRING analysis showed that the dysregulated mRNAs were interconnected around two centers: the mTOR pathway-related genes and REST pathway-related genes. CONCLUSION: LncRNAs were dysregulated in the pilocarpine mouse model according to the brain regions of the hippocampus and cortex. The dysregulated LncRNAs with co-dysregulated mRNAs might be possible therapeutic targets for the epigenetic regulation of chronic epilepsy.

Pharmacological Induction of Ischemic Tolerance by Glutamate Transporter-1 (EAAT2) Upregulation.

BACKGROUND AND PURPOSE: Astrocytic glutamate transporter protein, GLT-1 (EAAT2), recovers extracellular glutamate and ensures that neurons are protected from excess stimulation. Recently, beta-lactam antibiotics, like ceftriaxone (CTX), were reported to induce the upregulation of GLT-1. Here, we investigated ischemic tolerance induction by CTX in an experimental model of focal cerebral ischemia. METHODS: CTX (200 mg/kg per day, IP) was administered for 5 consecutive days before transient focal ischemia, which was induced by intraluminal thread occlusion of the middle cerebral artery for 90 minutes or permanently. RESULTS: Repeated CTX injections enhanced GLT-1 mRNA and protein expressions after 3 and 5 days of treatment, respectively. CTX-pretreated animals showed a reduction in infarct volume by 58% (reperfusion) and 39% (permanent), compared with the vehicle-pretreated animals at 24 hours postischemia (P<0.01). Lower doses of CTX (20 mg/kg per day and 100 mg/kg per day) reduced infarct volumes to a lesser degree. The injection of GLT-1 inhibitor (dihydrokainate) at 30 minutes before ischemia ameliorated the effect of CTX pretreatment. However, CTX administration at 30 minutes after ischemia produced no significant reduction in infarct volume. CTX reduced the levels of proinflammatory cytokines (tumor necrosis factor-alpha, FasL), matrix metalloproteinase (MMP)-9, and activated caspase-9 (P<0.01). In addition, CTX-pretreated animals showed better functional recovery at day 1 to week 5 after ischemia (P<0.05). CONCLUSIONS: This study presents evidence that CTX induces ischemic tolerance in focal cerebral ischemia and that this is mediated by GLT-1 upregulation.

Proteasomal inhibition in intracerebral hemorrhage: neuroprotective and anti-inflammatory effects of bortezomib.

Inflammation is an important pathophysiologic mechanism of injury induced by intracerebral hemorrhage (ICH). The ubiquitin-proteasome system (UPS) regulates the inflammatory responses via the up-regulation of several pro-inflammatory molecules. In this study, we determined that a potent proteasome inhibitor, bortezomib, exerted therapeutic effects in experimental model of ICH. Either bortezomib (0.05, 0.2, 0.5, 1mg/kg) or vehicle was intravenously administered 2h after ICH induction. The high doses of bortezomib caused high mortality rates. Bortezomib at 0.2 mg/kg reduced the early hematoma growth and alleviated hematoma volume and brain edema at 3 days after ICH, compared with the ICH-vehicle group. The numbers of myeloperoxidase(+) neutrophils, Ox42(+) microglia, and TUNEL(+) cells in the perihematomal regions were decreased by bortezomib. Bortezomib induced significant decrements of mRNA expression of TNF-alpha and IL-6. The production of iNOS and COX2 was also reduced significantly by bortezomib. We concluded that the early treatment with bortezomib induced a reduction in the early hematoma growth and mitigated the development of brain edema, coupled with a marked inhibitory effect on inflammation in ICH.

Pharmacological induction of heat shock protein exerts neuroprotective effects in experimental intracerebral hemorrhage.

Heat shock proteins (HSPs) are reported to reduce inflammation and apoptosis in a variety of brain insults. Geranylgeranylacetone (GGA), developed as an antiulcer in Japan, has been known to induce HSP70 and to exert cytoprotective effects. In this study, we investigated whether GGA, as a specific HSP inducer, exerts therapeutic effects in experimentally induced intracerebral hemorrhage (ICH). ICH was induced with male Sprague-Dawley rats via the collagenase infusion. GGA (800 mg/kg) was administered via oral tube according to various schedules of treatment. The treatment with GGA, beginning before the induction of ICH and continuing until day 3, showed the reduction of brain water content and the increased level of HSP70 protein, as compared to the treatment with vehicle, although GGA started after the induction of ICH or administered as a single dose before ICH failed to up-regulate HSP70 and to reduce brain edema. The rats treated with GGA exhibited better functional recovery than those treated with vehicle. In the pre- and post- treatment group, inflammatory cells and cell death in the perihematomal regions were found to have been decreased. The treatment of GGA inhibited the mRNA expression of MMP-9, uPA, IL-6 and MIP-1, with concomitant increment of eNOS and phosphorylated STAT3 and Akt after ICH. We demonstrated that GGA induced a reduction in the brain edema along with marked inhibitory effects on inflammation and cell death after ICH.