Intracerebral Hemorrhage Induction Research Articles

Mature Adult Mice With Exercise-Preconditioning Show Better Recovery After Intracerebral Hemorrhage.

Physical exercise offers therapeutic potentials for several central nervous system disorders, including stroke and cardiovascular diseases. However, it is still mostly unknown whether and how exercise preconditioning affects the prognosis of intracerebral hemorrhage (ICH). In this study, we examined the effects of preconditioning on ICH pathology in mature adult mice using treadmill exercise. Male C57BL/6J (25-week old) mice were subjected to 6 weeks of treadmill exercise followed by ICH induction. Outcome measurements included various neurological function tests at multiple time points and the assessment of lesion volume at 8 days after ICH induction. In addition, plasma soluble factors and phagocytotic microglial numbers in the peri-lesion area were also measured to determine the mechanisms underlying the effects of exercise preconditioning. The 6-week treadmill exercise preconditioning promoted recovery from ICH-induced neurological deficits in mice. In addition, mice with exercise preconditioning showed smaller lesion volumes and increased numbers of phagocytotic microglia. Furthermore, the levels of several soluble factors, including endostatin, IGFBP (insulin-like growth factor-binding protein)-2 and -3, MMP (matrix metallopeptidase)-9, osteopontin, and pentraxin-3, were increased in the plasma samples from ICH mice with exercise preconditioning compared with ICH mice without exercise. These results suggest that mice with exercise preconditioning may suffer less severe injury from hemorrhagic stroke, and therefore, a habit of physical exercise may improve brain health even in middle adulthood.

MiR-181a Mediates Inflammatory Gene Expression After Intracerebral Hemorrhage: An Integrated Analysis of miRNA-seq and mRNA-seq in a Swine ICH Model.

Intracerebral hemorrhage (ICH) is a severe neurological disorder with no proven treatment. Inflammation after ICH contributes to clinical outcomes, but the relevant molecular mechanisms remain poorly understood. In studies of peripheral leukocyte counts and mRNA-sequencing (mRNA-seq), our group previously reported that monocytes and Interleukin-8 (IL-8) were important contributors to post-ICH inflammation. microRNA (miRNA) are powerful regulators of gene expression and promising therapeutic targets. We now report findings from an integrated analysis of miRNA-seq and mRNA-seq in peripheral blood mononuclear cells (PBMCs) from a swine ICH model. In 10 pigs, one PBMC sample was collected immediately prior to ICH induction and a second 6 h later; miRNA-seq and mRNA-seq were completed for each sample. An aggregate score calculation determined which miRNA regulated the differentially expressed mRNA. Networks of molecular interactions were generated for the combined miRNA/target mRNA. A total of 227 miRNA were identified, and 46 were differentially expressed after ICH (FDR<0.05). The anti-inflammatory miR-181a was decreased post-ICH, and it was the most highly connected miRNA in the miRNA/mRNA bioinformatic network analysis. miR-181a has interconnected pathophysiology with IL-8 and monocytes; in prior studies, we found that IL-8 and monocytes contributed to post-ICH inflammation and ICH clinical outcome, respectively. miR-181a was a significant mediator of post-ICH inflammation and is promising for further study, including as a potential therapeutic target. This investigation also demonstrated feasible methodology for miRNA-seq/mRNA-seq analysis in swine that is innovative, and with unique challenges, compared with transcriptomics research in more established species.

Electroacupuncture Enhance Therapeutic Efficacy of Mesenchymal Stem Cells Transplantation in Rats With Intracerebral Hemorrhage.

Previous studies have showed the beneficial effects of mesenchymal stem cells (MSCs) on experimental intracerebral hemorrhage (ICH) animal. Enhancement of the treatment efficacy of MSCs in ICH is essential, considering the diseases association with high rates of disability and mortality. Some auxiliary methods to enhance the beneficial efficacy of MSCs have been introduced. However, the effect of electroacupuncture (EA) on the therapeutic efficacy of MSCs transplantation in hemorrhagic stroke and its potential mechanism is not explored. ICH rat models were established using collagenase and heparin. 48h after ICH induction, the rats were randomly divided into model control (MC), MSCs transplantation (MSCs), EA stimulation (EA) and MSCs transplantation combined with EA stimulation (MSCs + EA) groups. We used mNSS test and gait analysis to assess neurological function of rats, and PET/CT to evaluate the volume of hemorrhage focus and level of glucose uptake. The concentrations of MDA, SOD, NSE, S100B and MBP in serum or plasma were examined with ELISA. Neural differentiation of MSCs, and the expressions of Bcl-2, Bax, Arg-1 and iNOS proteins around hematoma were detected by immunofluorescence and immunohistochemistry staining respectively. Western blot was carried out to analyze the expression levels of COX4, OGDH, PDH-E1α, Bcl-2 and Bax proteins. TUNEL staining was used to estimate cell apoptosis and transmission electron microscopy (TEM) was used to observe the ultrastructure and number of mitochondria. Our data showed that EA promoted neuron-like differentiation of transplanted MSCs and the expressions of BDNF and NGF proteins in ICH rats. The score of mNSS and the gait analysis showed that the recovery of the neurological function in the MSCs + EA group was better than that in the MSCs and EA groups. EA improved the structure of brain tissue, and alleviated brain injury further after MSCs transplantation in ICH rats. When compared with the MSCs and EA groups, the level of glucose uptake and numbers of mitochondria and Arg-1 positive cells in MSCs + EA group increased significantly, but the numbers of apoptotic cells and iNOS positive cells and volume of hemorrhage focus reduced. The expressional levels of COX4, OGDH, PDH-E1α and Bcl-2 proteins increased, while the expressional level of Bax protein decreased compared with those in the MSCs and EA groups. Our results reveal that EA improve therapeutic efficacy of MSCs transplantation in ICH rats.

Early Growth Response Protein 1 Knockdown Alleviates the Cerebral Injury in Rats with Intracerebral Hemorrhage via STAT3/NF-κB Pathway by Reducing RXRα Acetylation Level

Brain EGR1 (early growth response protein 1) overexpression aggravates focal ischemic brain injury, but its role in intracerebral hemorrhage (ICH) induced cerebral injury remains obscure. In this study, a rat ICH model was established by injecting type VII collagenase into the brain, and EGR1 knockdown reversed the increase of hematoma area, neurological function score, brain water content, blood–brain barrier (BBB) permeability, inflammation, p300 and retinoid a X receptor-α (RXRα) protein levels, as well as RXRα acetylation level induced by ICH. EGR1 expression was up-regulated in primary brain microvascular endothelial cells (BMECs), neurons, and astrocytes after ICH induction, and the up-regulation was most significant in BMECs. We also found that EGR1 promoted RXRα acetylation level by regulating p300 in BMECs. Silencing EGR1 rescued the upregulation of cell inflammation and the reduction of cell viability and TEER (transendothelial electric resistance) caused by OGD (oxygen glucose deprivation) plus hemin via p300-mediated RXRα acetylation. Furthermore, the STAT3/NF-κB pathway was activated after treatment with OGD plus hemin, which was suppressed by silencing EGR1. Treatment with Stattic (an inhibitor of STAT3) restrained the effect of OGD plus hemin on NF-κB pathway activity, inflammation, cell viability and TEER. In conclusion, EGR1 increased RXRα acetylation level by regulating p300, thereby aggravating brain damage in ICH rat model and dysfunction in BMECs, Through the STAT3/NF-κB pathway.

Albumin Reduces Oxidative Stress and Neuronal Apoptosis via the ERK/Nrf2/HO-1 Pathway after Intracerebral Hemorrhage in Rats.

Background Albumin has been regarded as a potent antioxidant with free radical scavenging activities. Oxidative stress and neuronal apoptosis are responsible for its highly damaging effects on brain injury after intracerebral hemorrhage (ICH). Here, the present study investigated the neuroprotective effect of albumin against early brain injury after ICH and the potential underlying mechanisms. Methods Adult male Sprague-Dawley rats were subjected to intrastriatal injection of autologous blood to induce ICH. Human serum albumin was given by intravenous injection 1 h after ICH. U0126, an inhibitor of extracellular signal-regulated kinase (ERK1/2), and ML385, an inhibitor of nuclear factor-E2-related factor 2 (Nrf2), were intraperitoneally administered 1 h before ICH induction. Short- and long-term neurobehavioral tests, western blotting, immunofluorescence staining, oxidative stress evaluations, and apoptosis measurements were performed. Results Endogenous expression of albumin (peaked at 5 days) and heme oxygenase 1 (HO-1, peaked at 24 h) was increased after ICH compared with the sham group. Albumin and HO-1 were colocalized with neurons. Compared with vehicle, albumin treatment significantly improved short- and long-term neurobehavioral deficits and reduced oxidative stress and neuronal death at 72 h after ICH. Moreover, albumin treatment significantly promoted the phosphorylation of ERK1/2; increased the expression of Nrf2, HO-1, and Bcl-2; and downregulated the expression of Romo1 and Bax. U0126 and ML385 abolished the treatment effects of albumin on behavior and protein levels after ICH. Conclusions Albumin attenuated oxidative stress-related neuronal death may in part via the ERK/Nrf2/HO-1 signaling pathway after ICH in rats. Our study suggests that albumin may be a novel therapeutic method to ameliorate brain injury after ICH.

Entinostat improves acute neurological outcomes and attenuates hematoma volume after Intracerebral Hemorrhage

Intracerebral hemorrhage (ICH) or hemorrhagic stroke is a major public health problem with no effective treatment. Given the emerging role of epigenetic mechanisms in the pathophysiology of ICH, we tested the hypothesis that a class 1 histone deacetylase inhibitor (HDACi), Entinostat, attenuates neurodegeneration and improves neurobehavioral outcomes after ICH. To address this, we employed a preclinical mouse model of ICH and Entinostat was administered intraperitoneally one-hour post induction of ICH. Entinostat treatment significantly reduced the number of degenerating neurons and TUNEL-positive cells after ICH in comparison to vehicle-treated controls. Moreover, Entinostat treatment significantly reduced hematoma volume, T2-weighted hemorrhagic lesion volume and improved acute neurological outcomes after ICH. Further, Entinostat significantly reduced the hemin-induced release of proinflammatory cytokines in vitro. Consistently, the expression of proinflammatory microglial/macrophage marker, CD16/32, was remarkably reduced in Entinostat treated group after ICH in comparison to control. Altogether, data implicates the potential of class 1 HDACi, Entinostat, in improving acute neurological function after ICH warranting further investigation.

Recombinant CCL17 Enhances Hematoma Resolution and Activation of CCR4/ERK/Nrf2/CD163 Signaling Pathway After Intracerebral Hemorrhage in Mice.

Hematoma is a crucial factor leading to poor prognosis after intracerebral hemorrhage (ICH). Promoting microglial phagocytosis to enhance hematoma resolution may be an important therapeutic target for recovery after ICH. C-C chemokine receptor 4 (CCR4) is important for regulating immune balance in the central nervous system. However, whether CCR4 activation can attenuate hematoma after ICH remains unknown. We aimed to evaluate whether CCL17 (a specific ligand of CCR4) treatment can promote hematoma resolution through CCR4/ERK/Nrf2/CD163 pathway after ICH. A total of 261 adult male CD1 mice were used. Mice were subjected to intrastriatal injection of autologous blood to induce ICH and randomly assigned to receive recombinant CCL17 (rCCL17) or vehicle which was administered intranasally at 1h after ICH. To elucidate the underlying mechanism, C021, a selective inhibitor of CCR4 and ML385 and a selective inhibitor of Nrf2 were administered 1h prior to ICH induction. Clustered regularly interspaced short palindromic repeats (CRISPR) knockout for CD163 was administered by intracerebroventricular injection at 48h before ICH. Brain edema, short- and long-term neurobehavior evaluation, hematoma volume, hemoglobin content, western blot, and immunofluorescence staining were performed. Endogenous CCL17, CCR4, and CD163 expression increased and peaked at 72h after ICH. CCR4 was expressed by microglia. CCR4 activation with rCCL17 significantly improved neurobehavioral scores and reduced hematoma volume and brain edema compared with vehicle. Moreover, rCCL17 treatment significantly promoted phosphorylation of ERK1/2, increased the expression Nrf2, and upregulated CD163 expression after ICH. The protective effects of rCCL17 were abolished by administration of C021, ML385, and CD163 CRISPR knockout. This study demonstrated that CCR4 activation with rCCL17 promoted hematoma resolution by increasing CD163 expression and CCR4/ERK/Nrf2 pathway activation after ICH, thereby reducing brain edema and improving neurological function. Overall, our study suggests that CCR4 activation may be a potential therapeutic strategy to attenuate hematoma in early brain injury after ICH.

Transient receptor potential vanilloid 4 agonist GSK1016790A improves neurological outcomes after intracerebral hemorrhage in mice

Intracerebral hemorrhage (ICH) is one of the most severe subtypes of stroke with high morbidity and mortality. Although a lot of drug discovery studies have been conducted, the drugs with satisfactory therapeutic effects for motor paralysis after ICH have yet to reach clinical application. Transient receptor potential vanilloid 4 (TRPV4), a Ca2+-permeable cation channel and activated by hypoosmolarity and warm temperature, is expressed in various cell types. The present study investigated whether TRPV4 would participate in the brain damage in a mouse model of ICH. ICH was induced by intrastriatal treatment of collagenase. Administration of GSK1016790A, a selective TRPV4 agonist, attenuated neurological and motor deficits. The inhibitory effects of the TRPV4 agonist in collagenase-injected WT mice were completely disappeared in TRPV4-KO mice. The TRPV4 agonist did not alter brain injury volume and brain edema at 1 and 3 days after ICH induction. The TRPV4 agonist did not show any differences with respect to the increased number of Iba1-positive microglia/macrophages, GFAP-positive astrocytes, and Gr1-positive neutrophils at 1 and 3 days after ICH induction. Quantitative RT-PCR experiments revealed that the TRPV4 agonist significantly upregulated the expression level of c-fos, a marker of neuronal activity, while the agonist gave no effects on the expression level of cytokines/chemokines at 1 day after ICH induction, These results suggest that stimulation of TRPV4 would ameliorate ICH-induced brain injury, presumably by increased neuronal activity and TRPV4 provides a novel therapeutic target for the treatment for ICH.

Different responses after intracerebral hemorrhage between young and early middle-aged mice

Although aging is a major risk factor for intracerebral hemorrhage (ICH), there are very few studies comparing ICH pathology between young and early middle-aged mice. In this study, 8-month old mice (early middle-aged mice) were compared against 2-month old mice (young mice) in neurological and histological changes after ICH induction, such as body weight, lesion volume, astrocytic responses, and motor and cognitive functions. At day 8 after ICH, there was no significant difference in lesion volume between the two groups, and both groups did not exhibit significant cognitive decline, as assessed by spontaneous alternative Y-maze test. On the other hand, 8-month old mice showed delayed recovery from body weight loss, along with reduced astrocytic activation. Interestingly, in the two motor function tests (beam-walking test and corner turn test), 8-month old mice exhibited lower scores only in the beam-walking test, suggesting a partial disturbance in motor recovery after ICH. These results suggest that age-related differences in ICH pathology may already start to appear in early middle-aged brains.

L-3-n-butylphthalide attenuates inflammation response and brain edema in rat intracerebral hemorrhage model.

L-3-n-butylphthalide(NBP), a compound found in Apium graveolens Linn seed extracts, has a therapeutic effect on acute ischemic stroke. The pathological inflammatory pathways and consequent brain edema in intracerebral hemorrhage (ICH) share some similar characteristics with ischemic stroke. We hypothesized that NBP has anti-inflammatory and therapeutic effects on rats with ICH. ICH was induced by an infusion of bacterial collagenase type IV into the unilateral striatum of anesthetized rats. The therapeutic effect of NBP was measured by assessing neurological function, brain water content, blood-brain barrier permeability, and expression of tumor necrosis factor-alpha (TNF-α) and matrix metalloproteinase-9 (MMP-9) around the hematoma 48 hours after surgery. Magnetic resonance imaging was performed 4 and 48 hours after ICH induction, and ICH-induced injured area volumes were measured using T2-weighted images. The NBP treatment group performed better in the neurological function test than the vehicle group. Moreover, in comparison with the vehicle group, NBP group showed a lower expanded hematoma volume, brain water content, blood-brain barrier permeability, and TNF-α/ MMP-9 expression level. Our results indicate that NBP attenuates inflammation and brain edema in rat ICH model. Therefore, our findings also provide a potential therapeutic strategy for the treatment of ICH with NBP.

Functional and morphological consequences after atorvastatin intake in a rat model of intracerebral haemorrhage

Intracerebral haemorrhage (ICH) is a devastating disease for which there are currently no curative treatment options. Hypolipidemia has been recognized as a possible risk factor for ICH. Several studies have demonstrated that low cholesterol is a risk factor for ICH; others have reported that hypercholesterolemia is protective against ICH. The current study was designed to demonstrate the effect of low LDL levels on vascular endothelial cells integrity and subsequently on the development of ICH in rats. Material and methods: This study was carried on male Wistar albino rats divided into 2 groups: Group I: normal Sham-operated rats; Group II: ICH rats, were randomly subdivided into 3 subgroups (17 rat each): rats received 2% gum acacia daily orally; rats received atorvastatin 10 mg/kg daily alone or with L-arginine (143 mg/kg) orally for four weeks before induction of ICH and continued for one week after. Behavioural tests were performed 2, 24, and 48 h after ICH and 7 days later. Serum lipid profile was assessed. Brains were dissected and assessed macroscopically and microscopically for the extent of hematoma, brain water content and endothelin level in the brain homogenate were investigated. Immunoassaying for glial acidic fibrillary protein and Ki67 were performed. Results& Conclusion: Rats received atorvastatin and L-arginine showed a significant decrease in serum cholesterol and low-density lipoprotein (LDL) concentration. ICH led to marked impairment in motor functions. Atorvastatin and L-arginine intake before haemorrhage failed to impose any protection on the motor deficits with corresponding large hematoma size with disruption of nearby tissue.

TREM2 activation attenuates neuroinflammation and neuronal apoptosis via PI3K/Akt pathway after intracerebral hemorrhage in mice

BackgroundNeuroinflammation is an important host defense response to secondary brain injury after intracerebral hemorrhage (ICH). Triggering receptor expressed on myeloid cells 2 (TREM2) confers strong neuroprotective effects by attenuating neuroinflammation in experimental ischemic stroke. Recent studies suggest that apolipoprotein E (apoE) is a novel, high-affinity ligand of TREM2. This study aimed to investigate the effects of TREM2 activation on neuroinflammation and neuronal apoptosis in a mouse model of ICH.MethodsAdult male CD1 mice (n = 216) were subjected to intrastriatal injection of bacterial collagenase. The TREM2 ligand, apoE-mimetic peptide COG1410 was administered intranasally at 1 h after ICH induction. To elucidate the underlying mechanism, TREM2 small interfering RNA (siRNA) and the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 were administered intracerebroventricularly prior to COG1410 treatment. Neurobehavioral tests, brain water content, immunofluorescence, western blotting, and Fluoro-Jade C- and terminal deoxynucleotidyl transferase dUTP nick end labeling staining were performed.ResultsEndogenous TREM2 expression was increased and peaked at 24 h after ICH. TREM2 was expressed on microglia, astrocytes, and neurons. COG1410 improved both short-term and long-term neurological functions, reduced brain edema, inhibited microglia/macrophage activation and neutrophil infiltration, and suppressed neuronal apoptotic cell death in perihematomal areas after ICH. Knockdown of endogenous TREM2 by TREM2 siRNA aggravated neurological deficits and decreased the expression of TREM2 in naïve and ICH mice. COG1410 was associated with upregulation of TREM2, PI3K, phosphorylated-Akt, and Bcl-2 and downregulation of TNF-α, IL-1β, and Bax after ICH. The neuroprotective effects of COG1410 were abolished by both TREM2 siRNA and PI3K inhibitor LY294002.ConclusionsOur finding demonstrated that TREM2 activation improved neurological functions and attenuated neuroinflammation and neuronal apoptosis after ICH, which was, at least in part, mediated by activation of PI3K/Akt signaling pathway. Therefore, activation of TREM2 may be a potential therapeutic strategy for the management of ICH patients.

Targeted Delivery of Iron Oxide Nanoparticle-Loaded Human Embryonic Stem Cell-Derived Spherical Neural Masses for Treating Intracerebral Hemorrhage.

This study evaluated the potential of iron oxide nanoparticle-loaded human embryonic stem cell (ESC)-derived spherical neural masses (SNMs) to improve the transportation of stem cells to the brain, ameliorate brain damage from intracerebral hemorrhage (ICH), and recover the functional status after ICH under an external magnetic field of a magnet attached to a helmet. At 24 h after induction of ICH, rats were randomly separated into three experimental groups: ICH with injection of phosphate-buffered saline (PBS group), ICH with intravenous injection of magnetosome-like ferrimagnetic iron oxide nanocubes (FION)-labeled SNMs (SNMs* group), and ICH with intravenous injection of FION-labeled SNMs followed by three days of external magnetic field exposure for targeted delivery by a magnet-embedded helmet (SNMs*+Helmet group). On day 3 after ICH induction, an increased Prussian blue-stained area and decreased swelling volume were observed in the SNMs*+Helmet group compared with that of the other groups. A significantly decreased recruitment of macrophages and neutrophils and a downregulation of pro-inflammatory cytokines followed by improved neurological function three days after ICH were observed in the SNMs*+Helmet group. Hemispheric atrophy at six weeks after ICH was significantly decreased in the SNMs*+Helmet group compared with that of the PBS group. In conclusion, we have developed a targeted delivery system using FION tagged to stem cells and a magnet-embedded helmet. The targeted delivery of SNMs might have the potential for developing novel therapeutic strategies for ICH.

Punicalagin alleviates brain injury and inflammatory responses, and regulates HO-1/Nrf-2/ARE signaling in rats after experimental intracerebral haemorrhage

Purpose: To investigate the effect of punicalagin, an ellagitannin present in pomegranates, on intracerebral haemorrhage (ICH)-induced inflammatory responses and oxidative stress, and also unravel the underlying mechanism(s) of action.&#x0D; Methods: Collagenase type IV (0.2 U) was used to induce ICH in adult male Sprague-Dawley rats. Punicalagin was given to the rats at doses of 25, 50, and 75 mg/kg body weight via oral gavage for 15 days before ICH induction. The animals were sacrificed 24h following induction of ICH, and their brains were excised immediately and used for analysis. Histological changes were determined with Haematoxylin and Eosin (H&amp;E) staining. Permeability to blood-brain barrier (BBB) was determined by quantifying the extent of extravasation of Evan Blue (EB). Protein expressions of HO-1/Nrf-2/ARE and NF-κB signaling were assayed using immunoblotting and RT-PCR. Levels of reactive oxygen species (ROS) and serum levels of cytokines were also determined.&#x0D; Results: Punicalagin treatment reduced inflammatory cell infiltration and cell damage, improved brain tissue architecture and BBB integrity. The punicalagin treatment increased the activities of antioxidant enzymes, and enhanced antioxidant status via activation of Nrf-2/ARE/HO-1 signaling pathway (p &lt; 0.05). The treatment upregulated the expressions of HO-1 to 174 %, relative to 127 % in ICH control rats. Furthermore, it enhanced NF-κB levels and reversed the ICH injury-induced upregulations of IL-6, IL-18 and IL-1β.&#x0D; Conclusion: These findings indicate that punicalagin exerts neuroprotective effect in rats after experimental ICH through regulation of theHO-1/Nrf-2/ARE signaling pathway. Thus, punicalagin has therapeutic potential for ICH.&#x0D; Keywords: Brain injury, Haemoxygenase-1, Intracerebral haemorrhage, Inflammatory responses, Nrf2/ARE signalling, Punicalagin

A blockade of microRNA-155 signal pathway has a beneficial effect on neural injury after intracerebral haemorrhage via reduction in neuroinflammation and oxidative stress

MicroRNAs (miRNAs) have important contributions to multiple pathophysiological processes for cellular response to stress and are considered as promising therapeutic targets with respect to drug development due to their small size, relative ease of delivery, and sequence specificity. Thus, in the current report, we examined the effects of inhibiting miRNA-155 (miR-155) on the levels of pro-inflammatory cytokines (PICs), oxidative stress products as well as vascular endothelial growth factor (VEGF) in the parietal cortex and hippocampus of rats following intracerebral haemorrhage (ICH). Real time PCR was used to examine the levels of miR-155 in the parietal cortex and hippocampus of rats; and ELISA to measure IL-1β, IL-6 and TNF-α, oxidative 8-iso PGF2α and 8-OHdG, and VEGF. Additionally, modified neurological Severity Score (mNSS) was examined to indicate neurological function in animals. In results, with induction of ICH, the levels of miR-155 were amplified in the parietal cortex and hippocampus and this was accompanied with increases of IL-1β, IL-6 and TNF-α; and 8-iso PGF2α and 8-OHdG. Intracerebroventricular infusion of miR-155 inhibitor attenuated the elevation of PICs and amplification of oxidative stress products. Interestingly, miR-155 inhibitor promoted VEGF levels. Furthermore, inhibition of miR-155 led to improvement of neurological deficits in ICH rats. In conclusion, miR-155 signal in the parietal cortex and hippocampus is engaged in the processes of neural injury during ICH and blocking central miR-155 pathway plays a beneficial role in regulating neurological function via reduction in PICs and products of oxidative stress; and enhancement of VEGF. This has implications to target miR-155 and its downstream signal pathway for neuronal dysfunction and vulnerability related to ICH.

Intravenous administration of human amnion-derived stem cells improves neurobehavioral function in intracerebral hemorrhage mouse models by regulating inflammation and inhibition of apoptosis

Background & Aim Intracerebral hemorrhage (ICH) causes disabilities and its functional recovery by rehabilitations is limited. Recently, various stem cell therapies have been reported in animal model of ICH. Intravenous administration of mesenchymal stem cells was reported to be effective in some animal studies. The possible mechanisms of improvement are anti-inflammation and/or anti-edema effect by macrophage polarization. Here we report the effect of human amnion-derived stem cells (hAMSCs) on neurological improvement using the mouse cerebral hemorrhage model as a preclinical test. Methods, Results & Conclusion Methods: Using seven to nine-week-old male C57BL/6J mice, ICH model was made by injecting 0.4U of collagenase into the brain, which located 2 mm to the left side and 3.5 mm depth from the bregma. Mice were divided into the following four group, The Day (D) 1-hAMSC and D3 hAMSC groups received an intravenous injection of hAMSC (105 cells/ 50ul) 24 and 72 hour after the ICH induction respectively; the D3 Vehicle group received an intravenous injection of 50 ul vehicle. We also prepared the sham group with scalp incision and trepanation. From Day 29 and later on, mice were evaluated for phenotypical differences for the following neurobehavioral tests: wire hang, water maze learning, passive avoidance learning, and open space swimming. Immunostaining assay, TUNEL assay and molecular biology assay was performed for researching the mechanisms. Result The hAMSCs administration into the ICH mice improved the neurological function at the subacute phases, which was shown by neurobehavioral experiments. The improvement is particularly remarkable in the D1-hAMSC groups. Besides, the hAMSCs administration decreased the number of CD11+CD45+ cells, Ly6G+ cells, TUNEL positive cells and expression of EGF, CD11c and IL23p19 in the ICH lesions. Conclusion Intravenous administration of hAMSCs during the acute phase improved ICH-induced neurological deficits at the subacute phase because of the regulation of acute inflammation and suppression of apoptosis.

Histone Deacetylation 10 Alleviates Inflammation After Intracerebral Hemorrhage via the PTPN22/NLRP3 Pathway in Rats

The NOD-like receptor family Pyrin domain-containing 3 (NLRP3) inflammasome has a crucial role in the inflammatory process that occurs during intracerebral hemorrhage (ICH)-induced injury. Histone deacetylase 10 (HDAC10) is a newly identified class II histone deacetylase involved in immune responses. However, how HDAC10 affects the inflammatory response after ICH remains unknown. In this study, we investigated whether HDAC10 relieves ICH injury by suppressing NLRP3 inflammasome activation through the protein tyrosine phosphatase, nonreceptor type 22 (PTPN22) pathway. We induced ICH in Sprague-Dawley rats (healthy, male adult) with a single infusion of autologous blood. To knockdown HDAC10, we injected siRNA into the rats. To further explore the mechanisms underlying the role of HDAC10 in ICH injury, PTPN22 was silenced. HDAC10 levels were upregulated after ICH in humans and rats, and reached peak levels 24 h after ICH induction in rats. HDAC10 silencing aggravated ICH injury, as demonstrated by increased modified neurological severity scores, brain water content, Evans blue extravasation, and number of myeloperoxidase (MPO) cells, and the results of Nissl and H&E staining. Furthermore, HDAC10 knockdown increased the expression of PTPN22 and accentuated inflammatory responses mediated by the NLRP3 inflammasome. HDAC10 silencing increased NLRP3 inflammasome activation, and this was effectively reversed by PTPN22 knockdown using siRNA. Furthermore, HDAC10 silencing also promoted the interaction of PTPN22 and NLRP3. Our study demonstrated that HDAC10 silencing aggravated NLRP3-mediated inflammatory responses after ICH in rats via the PTPN22 pathway. These results suggest that regulating the NLRP3 inflammasome may be a novel method to ameliorate ICH injury.

Laquinimod and 3,3′-diindolylemethane alleviate neuropathological events and neurological deficits in a mouse model of intracerebral hemorrhage

We examined the effects of compounds shown to activate aryl hydrocarbon receptor (AhR) signaling on a mouse model of intracerebral hemorrhage (ICH). Daily oral administration of laquinimod (25 mg/kg) or 3,3′-diindolylmethane (250 mg/kg) from 3 h after ICH induction improved motor functions, prevented the decrease of neurons within the hematoma, and attenuated activation of microglia/macrophages and astrocytes in the perihematomal region as well as infiltration of neutrophils into the hematoma. Elevated expression of AhR was detected in microglia and neutrophils, and both drugs inhibited upregulation of interleukin-6 and CXCL1. These results propose AhR as a therapeutic target for ICH.

Abstract TP344: Human pcMSCs Improved Neurological Deficits in Intracerebral Hemorrhage Rats

Introduction: Stem cell therapy is one of the most promising strategies for treating intracerebral hemorrhage (ICH). Human mesenchymal stem cell (MSC) is a frequently used cell source, however, it is not perfect. The main weakness of MSCs is that they gradually lose the stemness during in vitro expansion (usually fewer than P10 in subculture), which limits their availability in clinical application. We developed a method to isolate MSCs from human term placenta in a serum-free selective culture condition and named these cells as placenta choriodecidual membrane-derived MSCs (pcMSCs). According to our in vitro data, pcMSCs expressed standard MSC cellular markers more than P20 during in vitro expansion. Objective: To evaluate whether pcMSC improves neurological deficits in a rat model of ICH and study the underlying mechanisms. Methods: The ICH rat model was induced by striatal injection of collagenase type VII. One day after ICH induction, pcMSCs were directly injected into the hematoma site. Behavior tests such as rotarod test were repeatedly performed for four weeks. [18F]FEPPA positron emission tomography (PET) and magnetic resonance imaging (MRI) were applied to reveal neuroinflammation and brain atrophy, respectively. Results: The rats in situ injected with pcMSCs showed better performance in the rotarod test which assesses the motor coordination and balance (see Figure). Such neurological recoveries occurred four weeks after treatment. MRI data showed reduced brain atrophy in pcMSC-treated rats at the end of study, while preliminary data of PET suggested smaller signals within one week. Conclusion: Human pcMSCs improved the neurological deficits in ICH rats. The underlying mechanisms may be the reduced neuronal loss (indicated by reduced brain atrophy) associated with the regulated neuroinflammation (indicated by smaller signals). Currently, the therapeutic effects of pcMSCs to ICH rat model are intensively studied.

Abstract WMP77: SUMO1 Plays an Important Role in Cardiac Dysfunction After Intracerebral Hemorrhage in Aged Mice

Background: The conjugation of the small ubiquitin-like modifier1 (SUMO1) plays an important role in numerous biological processes, including DNA repair and signal transduction. SUMO1 also reduces cardiac oxidative stress and hypertrophy as well as induces neuroprotective effects. Intracerebral hemorrhage (ICH) induces cardiac deficit in the absence of primary cardiac diseases in young adult wild type mice. In this study, we tested the hypothesis that SUMO1 plays a key role in regulating brain-heart interaction after ICH, and SUMO1 deficit leads to worse brain and heart deficit after ICH in aged mice. Methods: Aged (16-20 months) female Sumo1-deficient (SUMO1-/-) mice or wild-type (SUMO1+/+) mice were subjected to ICH by injecting collagenase IV into the basal ganglia. Cardiac function was measured by echocardiography before ICH induction and at 7 days after ICH. Modified neurological severity (mNSS) ,foot-fault and adhesive removal tests were performed at 1, 3, 6 days after ICH to investigate neurological function. Cognitive functional tests (odor and novel objective tests) were performed before sacrificing the mice at 10 days after ICH, and then histological and immunohistochemically staining were used to evaluate the mechanisms. Results: Compared to SUMO1+/+ mice, SUMO1-/- mice did not exhibit significant cardiac deficit before ICH in aged mice. Compared to SUMO1+/+ICH mice, SUMO1-/-ICH mice exhibit significantly (p&lt;0.05) increased: 1) brain hemorrhage volume and induced worse neurological and cognitive deficits, 2) cardiac dysfunction identified by decreased cardiac contractile function measured by left ventricular ejection fraction (LVEF) and fractional shortening (FS); 3) cardiac hypertrophy and fibrosis; 4) ionized calcium binding adaptor molecule (IBA1) positive macrophages/microglia and CD45 positive leukocyte infiltration into both heart and brain tissue. Conclusions: Aged SUMO1 deficient mice subjected to ICH not only exhibit increased neurological and cognitive functional deficit, but also significantly increased cardiac dysfunction and inflammatory cell infiltration into heart. These data suggest that SUMO1 plays an important role in brain-heart interaction and SUMO1 impacts brain-cardiac dysfunction after ICH.