Rat Model Of Intracerebral Hemorrhage Research Articles

Reducing Suppressors of Cytokine Signaling-3 (SOCS3) Expression Promotes M2 Macrophage Polarization and Functional Recovery After Intracerebral Hemorrhage.

Intracerebral hemorrhage (ICH) is a fatal subtype of stroke, and effective interventions to improve the functional outcomes are still lacking. Suppressor of cytokine signaling 3 (SOCS3) plays critical roles in the inflammatory response by negatively regulating cytokine-Jak–Stat signaling. However, the role of SOCS3 in the regulation of macrophage polarization is highly controversial and the fine regulation exerted by SOCS3 needs further understanding. In this study, rat ICH models were established by infusion of collagenase into the caudate nucleus. To decrease SOCS3 expression into microglia/macrophages in the hemorrhagic lesion area, we injected lentiviral short hairpin RNA (shSOCS3) (Lenti-shSOCS3) into the hematoma cavity at 24 h following ICH. We found that the number of iNOS-positive cells (M1 phenotype) was significantly reduced, whereas arginase-1-positive cells (M2 phenotype) were markedly elevated in animals that received Lenti-shSOCS3 injections compared with those in the Lenti-EGFP and saline groups. The increase in arginase-1-positive cells was associated with a significantly lower pro-inflammatory microenvironment, which included the downregulation of pro-inflammatory cytokines [interleukin (IL)-1β, IL-6, and TNF-α] and concurrent upregulation of anti-inflammatory (IL-10) mediators. In addition, this marked shift toward the M2 phenotype was associated with suppressed NF-κB activation. Furthermore, these changes notably enhanced the neuroprotective effects and functional recovery in Lenti-shSOCS3-injected animals. Our findings indicated that reduction in SOCS3 expression caused a marked bias toward the M2 phenotype and ameliorated the inflammatory microenvironment, which enhanced neuroprotective effects and resulted in notable improvement in functional recovery after ICH.

Protective Effect of miR-340-5p against Brain Injury after Intracerebral Hemorrhage by Targeting PDCD4

Objective: Intracerebral hemorrhage (ICH) is a common cerebrovascular disease. Increasing evidence has documented the crucial role of microRNAs in ICH. The present study aimed to investigate the role and underlying mechanism of miR-340-5p in ICH. Methods: The collagenase-induced ICH rat model was established. The neurological function of rats and the cerebral water content of rat brain tissue were measured to assess the brain injury. BV-2 cells were recruited and treated by LPS to mimic ICH-induced inflammatory response. qRT-PCR was used for the measurement of miR-340-5p. The protein levels of TNF-α, IL-6, and IL-1β were detected using ELISA. Luciferase reporter gene assay was performed to confirm the target gene. Results: Downregulation of miR-340-5p was detected in the serum of ICH patients and the brain tissues of ICH rats. Overexpression of miR-340-5p reversed the influence of ICH on the neurological function score and cerebral water content and inhibited the production of proinflammatory cytokines (TNF-α, IL-6, and IL-1β), which were induced by ICH in vivo. In in vitro study, levels of TNF-α, IL-6, and IL-1β were significantly enhanced in cells after LPS treatment, but these increases were eliminated by overexpression of miR-340-5p. PDCD4 was a direct target gene of miR-340-5p. Conclusion: miR-340-5p protects against brain injury after ICH. miR-340-5p might exert an anti-inflammatory effect during the occurrence of ICH via targeting PDCD4.

Effect of mild hypothermia on behaviors of rats with intracerebral hemorrhage and the possible mechanism

To explore the effect of mild hypothermia on inflammatory response and angiogenesis in brain tissues of rats with intracerebral hemorrhage (ICH) and its possible mechanism for improving behavioral deficits of the rats After ICH. A total of 120 healthy male SD rats were randomly divided into sham operation group, ICH group and mild hypothermia group. Rat models of ICH were established in the latter two groups by stereotactic injection of autogenous blood in the brain, and the rats in the sham operation group received injection of normal saline in the same manner. At 15 min after modeling, the rats in hypothermia group were subjected to mild hypothermia (30-32 ℃) for 8 h followed by rewarming (37-38 ℃); the body temperature was maintained at 37-38 ℃ in the other two groups. At 2, 4, 7, 14 and 21 days after the treatment, Longa scoring, balance beam scoring and Berderson scoring were used to evaluate the behavioral deficits of the rats. Immunohistochemical staining was used to detect the protein expressions of tumor necrosis factor-α (TNF-α) and nuclear factor-κB (NF-κB) in the brain tissue of the rats, and the mRNA expressions of α subunit of hypoxia-inducible factor 1 (HIF1-α) and vascular endothelial growth factor (VEGF) were detected using RT- PCR. At 2, 4, 7, 14 and 21 days after the treatment, the behavioral scores of the rats were significantly higher in ICH group and mild induced hypothermia group than in the sham operation group (P < 0.05 or 0.01). The protein expressions of TNF-α and NF-κB and mRNA expressions of HIF1-α and VEGF were significantly higher in ICH group and mild hypothermia group than in the sham operation group (P < 0.01). The behavioral scores were significantly lower in mild hypothermia group than in ICH group (P < 0.05), and the protein expressions of TNF-α and NF-κB were lower and the mRNA expressions of HIF1- α and VEGF were higher in mild hypothermia group than in ICH group (P < 0.05 or 0.01). Mild hypothermia can improve behavioral deficits in rats with ICH possibly by antagonizing brain inflammation and promoting angiogenesis.

Elevated miR-29a Contributes to Axonal Outgrowth and Neurological Recovery After Intracerebral Hemorrhage via Targeting PTEN/PI3K/Akt Pathway.

Spontaneous intracerebral hemorrhage (ICH) is a clinical challenge with high disability and lacks an effective treatment. miR-29a strongly expressed in the brain has been implicated in various neurological disorders. In this study, we investigated the biological roles of miR-29a in axonal outgrowth and neurological outcomes after ICH and relevant molecular mechanism. The rat model of ICH was established by injection of autologous whole blood into the right basal ganglia. First, a significant decrease in miR-29a level was found in perihematomal brain tissues and cerebrospinal fluid (CSF) after ICH in vivo and hemin-treated neurons in vitro. Further study documented that lentivirus-mediated miR-29a overexpression could remarkably attenuate hemorrhagic brain injury, promoted regenerative outgrowth of injured axons and improved neurobehavioral and cognitive impairments after ICH in rats. In addition, we also identified that overexpression of miR-29a obviously alleviated neuronal damage and mitochondrial dysfunctions, and facilitated neurite outgrowth in cultured neurons exposed to hemin in vitro. Furthermore, luciferase reporter assay showed that miR-29a directly targeted the 3'-UTR region of phosphatase and tensin homolog (PTEN) mRNA and negatively regulated its expression. More importantly, pharmacological inhibition of PTEN has similar neuroprotective effects as miR-29a overexpression involving activation of the PI3K/Akt pathway after hemorrhagic stroke. Collectively, these results suggested that elevated miR-29a could contribute to axonal outgrowth and neurological recovery through targeting PTEN/PI3K/Akt pathway after ICH, thereby providing a potential therapeutic target for patients with ICH.

VEGF-transfected hBMSCs Aggravate Early Brain Edema in Cerebral Hemorrhage Rats

To investigate the effects of human bone marrow mesenchymal stem cells/vascular endothelial growth factor (hBMSCs/VEGF) transplantation on early brain edema in rats with cerebral hemorrhage. Cultured the hBMSCs in vitro, transducted the cells with VEGF recombinant lentivirus vector to abtain the hBMSCs/VEGF cells. Intracerebral hemorrhage (ICH) rat model was established by injection of type Ⅰ collagenase and heparin into the caudate putamen. 2 h after the operation, the rats were evaluated with modified nerve function defect (mNSS) score to ensure whether the model was successfully established. At the third day after the injection, SD rats were randomly divided into sham group (only injected with empty needle), cerebral hemorrhage group, saline group, hBMSCs group and hBMSCs/VEGF group. Saline group, hBMSCs group and hBMSCs/VEGF group mice were injected with normal saline, hBMSCs (2×10 6 cells/kg body mass) and hBMSCs/VEGF (2×10 6 cells/kg body mass) respectively; sham group and model group did not perform intervention. On day 3 and 7 after injection, the rats were evaluated with modified neurological function score (mNSS). Then rats were sacrificed, and brain tissue specimens from the right caudate putamen area were separated. The wet and dry weighing method was used to measure the water content, and HE staining was used to evaluate pathological and functional changes. The expressions of VEGF, matrix metalloproteinase 9 (MMP-9) and aquaporin 4 (AQP-4) proteins were detected by immunofluorescence and Western blot. 2 h after injection, compared with rats in sham operation group, mNSS scores of rats in model group were increased, indicating that the models have successfully established. 3 d and 7 d after intervention, the mNSS score, the content of brain water, the expression level of VEGF, MMP-9 and AQP-4 proteins in model group and NS group were significantly higher than those of sham group (P<0.05), while the above values in hBMSCs group were significantly lower than those in saline group and model group (P<0.001), and the above values in hBMSCs/VEGF group were significantly higher than those of hBMSCs group (P<0.05). There were obvious hemorrhage and infarction in the brain tissue in the model group and NS group rats observed under the light microscope. Besides, the brain tissue developed loose structure and edema change. However, the bleeding and infarction in the brain tissue of hBMSCs group mice were reduced. Compared with the hBMSCs group mice, the brain tissue of hBMSCs/VEGF group mice was looser, more vacuoles occurred, and many neurons showed apoptosis changes such as nuclear deflation. hBMSCs transplantation could improve neurological function and relieve brain edema. But hBMSCs/VEGF will increase the vascular permeability and then aggravate the early cerebral edema in rats with cerebral hemorrhage.

Galectin-9 Promotes Neuronal Restoration via Binding TLR-4 in a Rat Intracerebral Hemorrhage Model

Intracerebral hemorrhage (ICH) is a devastating disease with high rates of mortality and morbidity. Galactose lectin-9 (Gal-9) belongs to the family of β-galactoside-binding lectins, which has been shown to play a vital role in immune tolerance and inflammation. However, the function of Gal-9 in ICH has not been fully studied in details. Several experiments were carried out to explore the role of Gal-9 in the late period of ICH. Primarily, ICH models were established in male adult Sprague Dawley (SD) rats. Next, the relative protein levels of Gal-9 at different time points after ICH were examined and the result showed that the level of Gal-9 increased and peaked at the 7th day after ICH. Then we found that when the content of Gal-9 increased, both the number of M2-type microglia and the corresponding anti-inflammatory factors also increased. Through co-immunoprecipitation (CO-IP) analysis, it was found that Gal-9 combines with Toll-like receptor-4 (TLR-4) during the period of the recovery after ICH. TUNEL staining and Fluoro-Jade B staining (FJB) proved that the amount of cell death decreased with the increase of Gal-9 content. Additionally, several behavioral experiments also demonstrated that when the level of Gal-9 increased, the motor, sensory, learning, and memory abilities of the rats recovered better compared to the ICH group. In short, this study illustrated that Gal-9 takes a crucial role after ICH. Enhancing Gal-9 could alleviate brain injury and promote the recovery of ICH-induced injury, so that Gal-9 mayexploit a new pathway for clinical treatment of ICH.

Semi-Interpenetrating Polymer Network of Hyaluronan and Chitosan Self-Healing Hydrogels for Central Nervous System Repair.

The repair of the central nervous system (CNS) is a major challenge because of the difficulty for neurons or axons to regenerate after damages. Injectable hydrogels have been developed to deliver drugs or cells for neural repair, but these hydrogels usually require conditional stimuli or additional catalysts to control the gelling process. Self-healing hydrogels, which can be injected locally to fill tissue defects after stable gelation, are attractive candidates for CNS treatment. In the current study, the self-healing hydrogel with a semi-interpenetrating polymer network (SIPN) was prepared by incorporation of hyaluronan (HA) into the chitosan-based self-healing hydrogel. The addition of HA allowed the hydrogel to pass through a narrow needle much more easily. As the HA content increased, the hydrogel showed a more packed nanostructure and a more porous microstructure verified by coherent small-angle X-ray scattering and scanning electron microscopy. The unique structure of SIPN hydrogel enhanced the spreading, migration, proliferation, and differentiation of encapsulated neural stem cells in vitro. Compared to the pristine chitosan-based self-healing hydrogel, the SIPN hydrogel showed better biocompatibility, CNS injury repair, and functional recovery evaluated by the traumatic brain injury zebrafish model and intracerebral hemorrhage rat model. We proposed that the SIPN of HA and chitosan self-healing hydrogel allowed an adaptable environment for cell spreading and migration and had the potential as an injectable defect support for CNS repair.

NLRP6 contributes to inflammation and brain injury following intracerebral haemorrhage by activating autophagy.

Inflammation is a crucial factor contributing to secondary brain injury after intracerebral haemorrhage (ICH). NLRP6, a member of nod-like receptors (NLRs) family, has been reported to participate in inflammation and host-defence in multiple diseases. Distinct from the other NLR family members, NLRP6 regulates inflammation in an inflammasome-dependent as well as an inflammasome-independent pathway. However, the role of NLRP6 in regulating signalling pathways during ICH is poorly understood. In the present study, we demonstrated that NLRP6 expression was upregulated after ICH, both in humans and in rats. Subsequently, we developed a rat model of ICH and found that NLRP6 knockdown reduced brain injury, alleviated inflammation, and suppressed autophagy following ICH. Further, results indicated that autophagy involved in NLRP6 mediated inflammation after ICH. Moreover, we found that NLRP6 mediated regulation of autophagy and inflammation was inflammasome-dependent. This study revealed the underlying molecular mechanism of NLRP6 in inflammation and highlights the therapeutic potential of targeting NLRP6 in secondary brain injury after ICH. KEY MESSAGES: • NLRP6 was upregulated following ICH in humans and rats. • NLRP6 knockdown reduced brain injury, alleviated inflammation, and suppressed autophagy following ICH. • NLRP6 aggravated inflammation after ICH by activating autophagy. • NLRP6 regulated inflammation and autophagy after ICH by activating inflammasome pathway.

Hypoxia-inducible Factor 2α Exerts Neuroprotective Effects by Promoting Angiogenesis via the VEGF/Notch Pathway after Intracerebral Hemorrhage Injury in Rats

Angiogenesis after intracerebral hemorrhage (ICH) injury can effectively alleviate brain damage and improve neurological function. Hypoxia-inducible factor 2α (HIF-2α) is an important angiogenic regulator and exhibits protective effects in several neurological diseases; however, its role in ICH has not yet been reported. Hence, in the present study, we explored whether HIF-2α reduces ICH injury by promoting angiogenesis. In addition, we explored the role of the vascular endothelial growth factor (VEGF)/Notch pathway in HIF-2α-mediated angiogenesis. We injected 50 μL of autologous blood taken from the femoral artery into the right striatum of healthy male adult Sprague-Dawley rats to create an autologous-blood-induced rat model of ICH. Lentiviral vectors were injected to both overexpress and knock down HIF-2α expression. VEGF receptor 2 (VEGFR2) and Notch-specific inhibitors were injected intraperitoneally to block VEGFR2- and Notch-mediated signaling after lentiviral injections. Our data showed that HIF-2α overexpression reduced neurological-damage scores and brain-water content, suggesting it had a protective effect on ICH injury. In addition, overexpression of HIF-2α promoted angiogenesis, increased focal cerebral blood flow (CBF), and reduced neuronal damage, whereas HIF-2α knockdown resulted in the opposite effects. Furthermore, we found that HIF-2α-mediated angiogenesis was blocked by a Notch-specific inhibitor. Likewise, the HIF-2α-mediated increase in phospho-VEGFR-2, cleaved-Notch1 and Notch1 expression was reversed via a VEGFR2-specific inhibitor. Taken together, our results indicate that HIF-2α promotes angiogenesis via the VEGF/Notch pathway to attenuate ICH injury. Moreover, our findings may contribute to the development of a novel strategy for alleviating ICH injury via HIF-2α-mediated upregulation of angiogenesis.

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.

Dl-3-n-Butylphthalide promotes neovascularization and neurological recovery in a rat model of intracerebral hemorrhage

BackgroundCerebral stroke occurs following ischemic and hemorrhagic lesions in the brain. Survival and recovery of stroke patients depend on the severity of the initial injury but also the therapeutic approaches applied for emergent lifesaving and continuing post-stroke management. Dl-3-n-Butylphthalide (NBP), an active compound derived from Chinese celery seeds, has shown clinical efficacy in the treatment of ischemic cerebral stroke.ResultsIn the present study we explored the therapeutic effect of NBP in a rat model of intracerebral hemorrhage (ICH), focusing on its potential role in promoting neovascularization in the perihemorrhagic zone. ICH was induced in male Sprague-Dawley rats by unilateral injection of autologous blood into the globus pallidus, with sham-operated (Sham group), vehicle-treated (ICH) and NBP-treated (at 10 and 25 mg/kg/Bid, p.o., ICH + NBP10 and ICH + NBP25, respectively) groups examined behaviorally, macroscopically, histologically and biochemically at 1, 3, 7 and 15 days (d) post operation. Rats in the ICH + NBP10 and ICH + NBP25 groups showed reduced Longa’s motor scores relative to the ICH groups at the 3 and 7d time points, while the hematoma volume was comparable in the two NBP relative to the ICH groups as measured at 7d and 15d. In the perihemorrhagic zone, the numeric density of blood vessels immunolabeled by CD34, an angiogenic marker, was greater in the ICH + NBP10 and ICH + NBP25 than ICH groups, more so in the higher dosage group, at 1, 3, 7 and 15d. Levels of the vascular endothelial growth factor (VEGF) and angiopoietins-2 (Ang-2) proteins were elevated in the NBP groups relative to the sham and vehicle controls in immunoblotting of tissue lysates from the injection region.ConclusionThese results suggest that NBP can alleviate neurological defects following experimentally induced local brain hemorrhage, which is associated with a potential role of this drug in promoting neovascularization surrounding the bleeding loci.

Levetiracetam Reduces Early Inflammatory Response After Experimental Intracerebral Hemorrhage by Regulating the Janus Kinase 2 (JAK2)-Signal Transducer and Activator of Transcription 3 (STAT3) Signaling Pathway.

BackgroundLevetiracetam (LEV) is an antiepileptic drug that promotes recovery of neurological function by alleviating inflammatory reactions. However, it is not known whether it can improve secondary brain injury after intracerebral hemorrhage (ICH). The aim of this study was to determine whether LEV can reduce early inflammatory response after ICH in rats.Material/MethodsAn in vitro model of early inflammation was created by treating microglia cells with lipopolysaccharide (LPS). After exposure to various concentrations of LEV, the expression levels of NF-κB and STAT3 and inflammatory factors such as interleukin (IL)-1β and tumor necrosis factor (TNF)-α in microglia were detected. In vivo, autologous blood was used to induce the rat ICH model. The effects of LEV on post-cerebral hemorrhagic inflammatory response were examined using neurobehavioral tests, FJC staining, brain water content testing, and analysis of protein expression levels of NF-κB, JAK2, STAT3, and inflammatory factors.ResultsLEV treatment significantly reduced the expression of inflammatory factors and protein expression levels of NF-κB and STAT3 in LPS-treated microglia cells (P<0.05). In male Sprague-Dawley (SD) rats, LEV treatment markedly decreased the volume of hematoma and the number of degenerative neurons (P<0.05). It also improved the neurological function and relieved brain edema. The protein expression levels of NF-κB, JAK2, and STAT3 were significantly lower in the ICH+LEV group than in the control group (P<0.05).ConclusionsOur study suggests that treatment with LEV alleviates early inflammatory responses induced by ICH. Mechanistically, LEV inhibited the JAK2-STAT3 signaling pathway and reduced neuronal injury around the hematoma, and ameliorated brain edema, all of which promoted recovery of nerve function after hemorrhage.

Intravenous Human Umbilical Cord-Derived Mesenchymal Stromal Cell Administration in Models of Moderate and Severe Intracerebral Hemorrhage.

Intracerebral hemorrhage (ICH) is as a life-threatening condition that can occur in young adults, often causing long-term disability. Recent preclinical data suggest mesenchymal stromal cell (MSC)-based therapies as promising options to minimize brain damage after ICH. However, therapeutic evidence and mechanistic insights are still limited, particularly when compared with other disorders such as ischemic stroke. Herein, we employed a model of collagenase-induced ICH in young adult rats to investigate the potential therapeutic effects of an intravenous injection of human umbilical cord Wharton's jelly-derived MSCs (hUC-MSCs). Two doses of collagenase were used to cause moderate or severe hemorrhages. Magnetic resonance imaging showed that animals treated with hUC-MSCs after moderate ICH had smaller residual hematoma volumes than vehicle-treated rats, whereas the cell therapy failed to decrease the hematoma volume in animals with a severe ICH. Functional assessments (rotarod and elevated body swing tests) were performed for up to 21 days after ICH. Enduring neurological impairments were seen only in animals subjected to severe ICH, but the cell therapy did not induce statistically significant improvements in the functional recovery. The biodistribution of Technetium-99m-labeled hUC-MSCs was also evaluated, showing that most cells were found in organs such as the spleen and lungs 24 h after transplantation. Nevertheless, it was possible to detect a weak signal in the brain, which was higher in the ipsilateral hemisphere of rats subjected to a severe ICH. These data indicate that hUC-MSCs have moderately beneficial effects in cases of less severe brain hemorrhages in rats by decreasing the residual hematoma volume, and that optimization of the therapy is still necessary.

Intracerebral Hemorrhage Induced Brain Injury Is Mediated by the Interleukin-12 Receptor in Rats.

BackgroundIL-12 inhibition of the endothelial cell functions and angiogenesis is mediated by the cross-talk between the lymphocyte and the endothelial cells, which plays a key role in inhibiting the process of angiogenesis in the eyeballs and in malignant tumors.MethodsWe established the intracerebral hemorrhage (ICH) rat model, and IL-12 receptor beta monoclonal antibody was injected into the ICH rats. Western blot, immunofluorescence and RT-qPCR were used to detect the gene expression. Brain water content, EB staining, Garcia test, Beam walking test and wire hanging test were used to assess the injury of brain in ICH rats.ResultsIL-12 gene was significantly increase in hematoma border tissue of ICH rats, and IL-12 protein mainly localized in monocytes. Anti-IL-12 treatment with IL-12 monoclonal antibodies could not only significantly decrease the brain water content and EB content in brain tissues of ICH rats, but also significantly increase the score of the Garcia, Beam balance and the Wire hanging test in ICH rats. Moreover, anti-IL-12 treatment significantly decrease the expression of pro-inflammatory gene, inflammatory gene, p-JAK2/JAK2 and p-STAT4/STAT4 protein, but significantly increase the expression anti-inflammatory gene and CD31 protein, and M2 macrophage ratio in hematoma border tissues of ICH rats. In vitro, rmIL-12 inhibited the tube formation of brain microvascular endothelial cells (BMVES) in BMVES and bone marrow-derived monocytes (BMDM) co-culture systems, but not work in a separately cultured BMVES system. In addition, Fedratinib not only reduced p-JAK2/JAK2 and p-STAT4/STAT4 protein expression in BMDM after treating with b-FGF and rmIL-12, but also significantly increased the tube formation of BMVES in BMVES and BMDM co-culture systems after treating with b-FGF and rmIL-12.ConclusionBlockade of IL-12 receptor attenuated brain injury after ICH in rat by promoting angiogenesis, and the mechanism might be related to blocking IL-12 could inhibit M2 cell activation via the JAK2/STAT4 pathway.

Danhong Injection Protects Hemorrhagic Brain by Increasing Peroxiredoxin 1 in Aged Rats.

Intracerebral hemorrhage (ICH) is a severe cerebrovascular disease with a high incidence, mortality and disability rate. Danhong injection (DHI) is beneficial for ischemic stroke, but is prohibited for ICH due to risk of bleeding. The present study aims to explore the potential therapeutic time window and molecular mechanism of DHI in a collagenase-induced ICH model in aged rats. DHI administration after ICH could significantly improve body weight and neurological deficits, and reduce the hematoma volume and brain water content when compared to the vehicle control. Furthermore, the protective effect of DHI administration on days 1–3 after ICH was superior to those on days 3–5 or 7–9 after ICH. DHI remarkably increased the Peroxiredoxin 1 (Prx1) expression in astrocytes and reduced the expression of inflammatory factors tumor necrosis factor-α (TNF-α) and interleukin-β (IL-1β) after ICH. The immediate treatment of Prx1 inhibiter chelerythrine (Che) after ICH abolished the protective effect of DHI. Furthermore, the Che treatment reduced the expression of Prx1 in astrocytes, but increased the expression of TNF-α and IL-1β after ICH. DHI treatment could not reverse these changes. Therefore, the earlier DHI is administered, the better the neuroprotective effect. DHI exerts antioxidative and anti-inflammatory function by increasing Prx1 in astrocytes. These present results may change the established understanding of DHI, and reveal a novel treatment approach for ICH.

Downregulation of microRNA-181a-5p Protects Against Intracerebral Hemorrhage by Regulating the Sirtuin 1/Nuclear Factor-κB Signaling Pathway

Intracerebral hemorrhage (ICH) is a common cerebrovascular disease. MicroRNA-181a-5p (miR-181a-5p) has been investigated in various diseases. However, till now, the role of miR-181a-5p in ICH remains unclear. The present study was conduct to analyze the function of miR-181a-5p in ICH. First, an ICH rat model was conducted to assess the level of miR-181a-5p in ICH, and the data showed that miR-181a-5p was significantly up-regulated in ICH rat. Then, TargetScan and the dual luciferase reporter gene assay were applied to confirm the association between miR-181a-5p and sirtuin (SIRT)1, and the findings indicated that miR-181a-5p directly targeted SIRT1. The results also indicated that SIRT1 was downregulated in the ICH rat model. Subsequently, the effect of miR-181a-5p deficiency on ICH rats was investigated. It was observed that the miR-181a-5p inhibitor significantly reduced the neurobehavioral scores, brain edema and nerve cell apoptosis in ICH rats. ELISA results suggested that miR-181a-5p inhibitor significantly inhibited the expression of tumor necrosis factor-α and interleukin-1β in the serum and cerebrospinal fluid of ICH rats. In addition, the results demonstrated that the miR-181a-5p inhibitor significantly reduced serum malondialdehyde content and increased superoxide dismutase activity. Western blot assay revealed that the miR-181a-5p inhibitor suppressed the expression of p-p65 and the p-p65/p65 ratio in the brain tissue of ICH rats. Of note, all these results were reversed by SIRT1-siRNA. In conclusion, this study indicated that the miR-181a-5p inhibitor protected against ICH by targeting SIRT1.

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.

MicroRNA-152 attenuates neuroinflammation in intracerebral hemorrhage by inhibiting thioredoxin interacting protein (TXNIP)-mediated NLRP3 inflammasome activation

Neuroinflammation significantly contributes to brain injury and neurological deterioration following intracerebral hemorrhage (ICH). MicroRNA-152(miR-152) was reported to be downregulated in ICH patients and to possess anti-inflammatory properties in other diseases. In this study, we aimed to explore the role of miR-152 in ICH, and the underlying mechanisms, using a collagenase-induced rat ICH model and hemin-exposure as a cell model. We first confirmed that miR-152 was consistently downregulated in both models. Overexpression of miR-152 in microglial BV2 cells reduced hemin-induced inflammatory response and reactive oxygen species (ROS) generation, thus protecting co-cultured neuronal HT22 cells. Moreover, overexpression of miR-152 by intracerebroventricular lentivirus injection in ICH rats significantly alleviated neurodecifits, brain edema, and hematoma. These changes were associated with a marked reduction in ICH-induced neuronal death, as detected by co-staining of NeuN and TUNEL, and ICH-induced neuroinflammation, as revealed by inflammatory cytokine levels as well as by the number of Iba1 positive-stained cells in the perihematomal region. Mechanistically, miR-152 significantly inhibited ICH-induced TXNIP expression, and its overexpression blocked the interaction between TXNIP and NOD-like receptor pyrin domain containing 3(NLRP3), thus inhibiting NLRP3-driven inflammasome activation to attenuate neuroinflammation in vivo and in vitro. Moreover, the results of si-TXNIP transfection further confirmed that TXNIP inhibition was involved in the reduction of NLRP3 inflammasome activation by the overexpression of miR-152. Collectively, the present study demonstrates that miR-152 confers protection against ICH-induced neuroinflammation and brain injury by inhibiting TXNIP-mediated NLRP3 inflammasome activation, indicating a potential strategy for ICH treatment.

Increased expression of Triad1 is associated with neuronal apoptosis after intracerebral hemorrhage in adult rats

Objective: It has been demonstrated that Triad1 (2 RING fingers and double RING finger linked 1) negatively regulates myeloid cell growth and induces cell apoptosis. However, its functions in intracerebral hemorrhage (ICH) disease have not been conducted. In this study, the role of Triad1 in rat model of ICH was explored.Methods: We observe an increasing expression of Triad1 in areas adjacent to hematoma after ICH. Immunofluorescence shows that Triad1 is colocalized with neurons, while not microglia or astrocyte, indicates its correlation with neuronal activities following ICH.Results: As neuronal apoptosis is the most crucial event in ICH disease, the expression of active caspase-3 and p53 is also enhanced around the hematoma, which is consistent with Triad1 in expression tendency. In turn, Triad1 depletion in primary cortical neurons decreased the apoptosis of neurons after using Triad1 shRNA.Conclusion: We conclude that inhibition of Triad1 expression might protect the brain from secondary damage following ICH.