Budbazar Enkhjargal

Recombinant Osteopontin Stabilizes Smooth Muscle Cell Phenotype via Integrin Receptor/Integrin-Linked Kinase/Rac-1 Pathway After Subarachnoid Hemorrhage in Rats

Background and Purpose— Recombinant osteopontin (rOPN) has been reported to be neuroprotective in stroke animal models. The purpose of this study is to investigate a potential role and mechanism of nasal administration of rOPN on preserving the vascular smooth muscle phenotype in early brain injury after subarachnoid hemorrhage (SAH). Methods— One hundred and ninety-two male adult Sprague-Dawley rats were used. The SAH model was induced by endovascular perforation. Integrin-linked kinase small interfering RNA was intracerebroventricularly injected 48 hours before SAH. The integrin receptor antagonist fibronectin-derived peptide Gly-Arg-Gly-Asp-Ser-Pro (GRGDSP), focal adhesion kinase inhibitor Fib-14, and Rac-1 inhibitor NSC23766 were administered 1 hour before SAH induction. rOPN was administered via the intracerebroventricular and nasal route after SAH. SAH grade, neurological scores, brain water content, brain swelling, hematoxylin and eosin staining, India ink angiography, Western blots, and immunofluorescence were used to study the mechanisms of rOPN on the vascular smooth muscle phenotypic transformation. Results— The marker proteins of vascular smooth muscle phenotypic transformation &agr;-smooth muscle actin decreased and embryonic smooth muscle myosin heavy chain (SMemb) increased significantly at 24 and 72 hours in the cerebral arteries after SAH. rOPN prevented the changes of &agr;-smooth muscle actin and SMemb and significantly alleviated neurobehavioral dysfunction, increased the cross-sectional area and the lumen diameter of the cerebral arteries, reduced the brain water content and brain swelling, and improved the wall thickness of cerebral arteries. These effects of rOPN were abolished by GRGDSP, integrin-linked kinase small interfering RNA, and NSC23766. Intranasal application of rOPN at 3 hours after SAH also reduced neurological deficits. Conclusions— rOPN prevented the vascular smooth muscle phenotypic transformation and improved the neurological outcome, which was possibly mediated by the integrin receptor/integrin-linked kinase/Rac-1 pathway.

Intranasal administration of vitamin D attenuates blood–brain barrier disruption through endogenous upregulation of osteopontin and activation of CD44/P-gp glycosylation signaling after subarachnoid hemorrhage in rats:

In this study, we investigated the role of vitamin D3 (VitD3) on endogenous osteopontin (OPN), a neuroprotective glycoprotein, after subarachnoid hemorrhage (SAH). The endovascular perforation SAH model in Sprague-Dawley rats was used to study the effect of intranasal VitD3 (30 ng/kg) before (Pre-SAH + VitD3) and after (Post-SAH + VitD3) subarachnoid hemorrhage. Vitamin D3 (30, 60, 120 ng/kg/day) increased more than one fold endogenous OPN expression in astrocytes and endothelial cells of rat brain. Vitamin D3 significantly decreased brain edema and Evans blue extravasation. In addition, neurobehavioral scores were significantly higher in Pre-SAH + VitD3, but partly higher in Post-SAH + VitD3, group compared with SAH group. These protective effects of vitamin D3 were completely attenuated by intracerebroventricular injection of transcription inhibitor Actinomycin D and significantly inhibited by small interfering ribonucleic acid (siRNA) for vitamin D receptor and OPN in Pre-SAH + VitD3 rats. OPN expression was significantly higher in Pre-SAH + VitD3 rats, specifically A and C, but not B, isomers were upregulated in the astrocytes, leading to CD44 splicing, and P-gp glycosylation in brain endothelial cells. The results show that intranasal vitamin D3 attenuates blood–brain barrier (BBB) disruption through endogenous upregulation of OPN and subsequent CD44 and P-gp glycosylation signals in brain endothelial cells. Furthermore, this study identifies a novel strategy for the cost-effective management of subarachnoid hemorrhage.

Intranasal administration of recombinant Netrin-1 attenuates neuronal apoptosis by activating DCC/APPL-1/AKT signaling pathway after subarachnoid hemorrhage in rats

ABSTRACT Neuronal apoptosis is a crucial pathological process in early brain injury after subarachnoid hemorrhage (SAH). The effective therapeutic strategies to ameliorate neuronal apoptosis are still absent. We intended to determine whether intranasal administration of exogenous Netrin‐1 (NTN‐1) could attenuate neuronal apoptosis after experimental SAH, specifically via activating DCC‐dependent APPL‐1/AKT signaling cascade. Two hundred twenty‐five male Sprague‐Dawley rats were subjected to the endovascular perforation model of SAH. Recombinant human NTN‐1 (rNTN‐1) was administered intranasally. NTN‐1 small interfering RNA (siRNA), APPL‐1 siRNA, and AKT inhibitor MK2206 were administered through intracerebroventricular (i.c.v.) injection. SAH grade, neurological score, neuronal apoptosis assessed by cleaved caspase‐3 (CC‐3) expression and Fluoro‐Jade C (FJC) staining, double immunofluorescence staining, and Western blot were examined. Our results revealed that endogenous NTN‐1 level was increased after SAH. Administration of rNTN‐1 improved neurological outcomes at 24 h and 72 h after SAH, while knockdown of endogenous NTN‐1 worsened neurological impairments. Furthermore, exogenous rNTN‐1 treatment promoted APPL‐1 activation, increased phosphorylated‐AKT and Bcl‐2 expression, as well as decreased apoptotic marker CC‐3 expression and the number of FJC‐positive neurons, thereby alleviated neuronal apoptosis. Conversely, APPL‐1 siRNA and MK2206 abolished the anti‐apoptotic effect of exogenous rNTN‐1 at 24 h after SAH. Collectively, intranasal administration of exogenous rNTN‐1 attenuated neuronal apoptosis and improved neurological function in SAH rats, at least in apart via activating DCC/APPL‐1/AKT signaling pathway. HIGHLIGHTSSubarachnoid hemorrhage (SAH) resulted in neuronal apoptosis and neurological deficits in a rat model.Intranasal administration of exogenous NTN‐1 significantly attenuated neuronal apoptosis and improved neurological functions.The mechanisms of rNTN‐1‐mediated anti‐apoptosis may be mediated by activating DCC‐dependent APPL‐1.The downstream signaling of NTN‐1/DCC/APPL‐1 in subarachnoid hemorrhage may be related to AKT, Bcl‐2 and Caspase‐3.rNTN‐1 may be an attractive candidate for anti‐apoptosis treatment in early brain injury after SAH.

Netrin‐1 Preserves Blood‐Brain Barrier Integrity Through Deleted in Colorectal Cancer/Focal Adhesion Kinase/RhoA Signaling Pathway Following Subarachnoid Hemorrhage in Rats

Background Netrin‐1 (NTN‐1) has been established to be a novel intrinsic regulator of blood‐brain barrier (BBB) maintenance. This study was carried out to investigate the potential roles of exogenous NTN‐1 in preserving BBB integrity after experimental subarachnoid hemorrhage (SAH) as well as the underlying mechanisms of its protective effects. Methods and Results A total of 309 male Sprague‐Dawley rats were subjected to an endovascular perforation model of SAH. Recombinant NTN‐1 was administered intravenously 1 hour after SAH induction. NTN‐1 small interfering RNA or Deleted in Colorectal Cancer small interfering RNA was administered intracerebroventricular at 48 hours before SAH. Focal adhesion kinase inhibitor was administered by intraperitoneal injection at 1 hour prior to SAH. Neurological scores, brain water content, BBB permeability, RhoA activity, Western blot, and immunofluorescence staining were evaluated. The expression of endogenous NTN‐1 and its receptor Deleted in Colorectal Cancer were increased after SAH. Administration of exogenous NTN‐1 significantly reduced brain water content and BBB permeability and ameliorated neurological deficits at 24 and 72 hours after SAH. Exogenous NTN‐1 treatment significantly promoted phosphorylated focal adhesion kinase activation and inhibited RhoA activity, as well as upregulated the expression of ZO‐1 and Occludin. Conversely, depletion of endogenous NTN‐1 aggravated BBB breakdown and neurological impairments at 24 hours after SAH. The protective effects of NTN‐1 at 24 hours after SAH were also abolished by pretreatment with Deleted in Colorectal Cancer small interfering RNA and focal adhesion kinase inhibitor. Conclusions NTN‐1 treatment preserved BBB integrity and improved neurological functions through a Deleted in Colorectal Cancer/focal adhesion kinase/RhoA signaling pathway after SAH. Thus, NTN‐1 may serve as a promising treatment to alleviate early brain injury following SAH.

Pathophysiology and the Monitoring Methods for Cardiac Arrest Associated Brain Injury

Cardiac arrest (CA) is a well-known cause of global brain ischemia. After CA and subsequent loss of consciousness, oxygen tension starts to decline and leads to a series of cellular changes that will lead to cellular death, if not reversed immediately, with brain edema as a result. The electroencephalographic activity starts to change as well. Although increased intracranial pressure (ICP) is not a direct result of cardiac arrest, it can still occur due to hypoxic-ischemic encephalopathy induced changes in brain tissue, and is a measure of brain edema after CA and ischemic brain injury. In this review, we will discuss the pathophysiology of brain edema after CA, some available techniques, and methods to monitor brain oxygen, electroencephalography (EEG), ICP (intracranial pressure), and microdialysis on its measurement of cerebral metabolism and its usefulness both in clinical practice and possible basic science research in development. With this review, we hope to gain knowledge of the more personalized information about patient status and specifics of their brain injury, and thus facilitating the physicians’ decision making in terms of which treatments to pursue.

Exendin-4 attenuates neuronal death via GLP-1R/PI3K/Akt pathway in early brain injury after subarachnoid hemorrhage in rats

ABSTRACT Neuronal apoptosis is considered to be a crucial therapeutic target against early brain injury (EBI) after subarachnoid hemorrhage (SAH). Emerging evidence indicates that Exendin‐4 (Ex‐4), a glucagon‐like peptide 1 receptor (GLP‐1R) agonist, plays a neuroprotective role in cerebrovascular disease. This study was conducted in order to verify the neuroprotective role of EX‐4 in EBI after SAH in rats. The endovascular perforation model of SAH was performed in Sprague‐Dawley rats (n = 153). Ex‐4 was intraperitoneally injected 1 h after SAH induction in the rats (SAH + Ex‐4). To elucidate the underlying molecular mechanism, small interfering ribonucleic acid (siRNA) for GLP‐1R and a specific inhibitor of PI3K, LY294002, were injected intracerebroventricularly into SAH + Ex‐4 rats before induction of SAH (n = 6 per group). SAH grading evaluation, immunohistochemistry, Western blots, neurobehavioral assessment, and Fluoro‐Jade C (FJC) staining experiments were performed. Expression of GLP‐1R was significantly increased and mainly expressed in neurons at 24 h after SAH induction. Administration of Ex‐4 significantly improved both short‐ and long‐term neurobehavior in SAH + Ex‐4 group compared to SAH + Vehicle group after SAH. Ex‐4 treatment significantly increased the expression of GLP‐1R, PI3K, p‐Akt, Bcl‐xl, and Bcl‐2, while at the same time was found to decrease expression of Bax in the brain. Effects of Ex‐4 were reversed by the intervention of GLP‐1R siRNA and LY294002 in SAH + Ex‐4+GLP‐1R siRNA and SAH + Ex‐4+LY294002 groups, respectively. In conclusion, the neuroprotective effect of Ex‐4 in EBI after SAH was mediated by attenuation of neuronal apoptosis via GLP‐1R/PI3K/Akt signaling pathway, therefore EX‐4 should be further investigated as a potential therapeutic agent in stroke patients. HIGHLIGHTSEx‐4 significantly improved neurological function after SAH.GLP‐1R mainly expressed in neurons after SAH.Ex‐4 did not increased risk of hypoglycemia after SAH in SD rats.Ex‐4 improved spatial learning capacity and reference memory after SAH.The anti‐apoptotic effect of Ex‐4 was mediated via GLP‐1R/PI3K/Akt pathway.

Critical role of EphA4 in early brain injury after subarachnoid hemorrhage in rat

&NA; Early brain injury (EBI) is reported as a primary cause of mortality in subarachnoid hemorrhage (SAH) patients. Eph receptor A4 (EphA4) has been associated with blood‐brain barrier integrity and pro‐apoptosis. We aimed to investigate a role of EphA4 in EBI after SAH. One hundred and seventy‐nine male adult Sprague‐Dawley rats were randomly divided into sham versus endovascular perforation model of SAH groups. SAH grade, neurological score, Evans blue dye extravasation, brain water content, mortality, Fluoro‐Jade staining, immunofluorescence staining, and western blot experiments were performed after SAH. Small interfering RNA (siRNA) for EphA4, recombinant Ephexin‐1 (rEphx‐1), and Fasudil, a potent ROCK2 inhibitor, were used for intervention to study a role of EphA4 on EBI after SAH. The expression of EphA4, Ephexin‐1, RhoA, and ROCK2 significantly increased after SAH. Knockdown of EphA4 using EphA4 siRNA injection intracerebroventricularly (i.c.v) reduced Evans blue extravasation, decreased brain water content, and alleviated neurobehavioral dysfunction after SAH. Additionally, the expression of Ephexin‐1, RhoA, ROCK2 and cleaved caspase‐3 were decreased. Tight junction proteins increased, and apoptotic neuron death decreased. The effects of EphA4 siRNA were abolished by rEphx‐1. In contrast, Fasudil abolished the effects of rEphx‐1. These results suggest that EphA4, a novel and promising target for treatment, exacerbates EBI through an Ephexin‐1/ROCK2 pathway after SAH.

Dihydrolipoic Acid Inhibits Lysosomal Rupture and NLRP3 Through Lysosome-Associated Membrane Protein-1/Calcium/Calmodulin-Dependent Protein Kinase II/TAK1 Pathways After Subarachnoid Hemorrhage in Rat

Background and Purpose— The NLRP3 (nucleotide binding and oligomerization domain-like receptor family pyrin domain-containing 3) inflammasome is a crucial component of the inflammatory response in early brain injury after subarachnoid hemorrhage (SAH). In this study, we investigated a role of dihydrolipoic acid (DHLA) in lysosomal rupture, NLRP3 activation, and determined the underlying pathway. Methods— SAH was induced by endovascular perforation in male Sprague–Dawley rats. DHLA was administered intraperitoneally 1 hour after SAH. Small interfering RNA for lysosome-associated membrane protein-1 and CaMKII&agr; (calcium/calmodulin-dependent protein kinase II &agr;) was administered through intracerebroventricular 48 hours before SAH induction. SAH grade evaluation, short- and long-term neurological function testing, Western blot, and immunofluorescence staining experiments were performed. Results— DHLA treatment increased the expression of lysosome-associated membrane protein-1 and decreased phosphorylated CaMKII&agr; and NLRP3 inflammasome, thereby alleviating neurological deficits after SAH. Lysosome-associated membrane protein-1 small interfering RNA abolished the neuroprotective effects of DHLA and increased the level of phosphorylated CaMKII&agr;, p-TAK1 (phosphorylated transforming growth factor-&bgr;-activated kinase), p-JNK (phosphorylated c-Jun-N-terminal kinase), and NLRP3 inflammasome. CaMKII&agr; small interfering RNA downregulated the expression of p-TAK1, p-JNK, and NLRP3 and improved the neurobehavior after SAH. Conclusions— DHLA treatment improved neurofunction and alleviated inflammation through the lysosome-associated membrane protein-1/CaMKII/TAK1 pathway in early brain injury after SAH. DHLA may provide a promising treatment to alleviate early brain injury after SAH.

ErbB4 protects against neuronal apoptosis via activation of YAP/PIK3CB signaling pathway in a rat model of subarachnoid hemorrhage

Neuronal apoptosis is a central pathological process in subarachnoid hemorrhage (SAH)-induced early brain injury. Previous studies indicated that ErbB4 (EGFR family member v-erb-b2 avian erythroblastic leukemia viral oncogene homolog 4) is essential for normal development and maintenance of the nervous system. In this study, we explored the neuroprotective effects of ErbB4 and its downstream YAP (yes-associated protein)/PIK3CB signaling pathway in early brain injury after SAH in a rat model using the endovascular perforation method. Rats were neurologically evaluated with the Modified Garcia Scale and beam balance test at 24h and 72h after SAH. An ErbB4 activator Neuregulin 1β1 (Nrg 1β1), ErbB4 siRNA and YAP siRNA were used to explore this pathway. The expression of p-ErbB4 and YAP was significantly increased after SAH. Multiple immunofluorescence labeling experiments demonstrated that ErbB4 is mainly expressed in neurons. Activation of ErbB4 and its downstream signals improved the neurological deficits after SAH and significantly reduced neuronal cell death. Inhibition of ErbB4 reduced YAP and PIK3CB expression, and aggravated cell apoptosis. YAP knockdown reduced the PIK3CB level and eliminated the anti-apoptotic effects of ErbB4 activation. These findings indicated that ErbB4 plays a neuroprotective role in early brain injury after SAH, possibly via the YAP/PIK3CB signaling pathway.

Recombinant Netrin-1 Binding UNC5B Receptor Attenuates Neuroinflammation and Brain Injury via PPARγ/NFκB Signaling Pathway after Subarachnoid Hemorrhage in Rats

Neuroinflammation is an essential mechanism involved in the pathogenesis of subarachnoid hemorrhage (SAH)-induced brain injury. Recently, Netrin-1 (NTN-1) is well established to exert anti-inflammatory property in non-nervous system diseases through inhibiting infiltration of neutrophil. The present study was designed to investigate the effects of NTN-1 on neuroinflammation, and the potential mechanism in a rat model of SAH. Two hundred and ninety-four male Sprague Dawley rats (weight 280-330 g) were subjected to the endovascular perforation model of SAH. Recombinant human NTN-1 (rh-NTN-1) was administered intravenously. Small interfering RNA (siRNA) of NTN-1 and UNC5B, and a selective PPARγ antagonist bisphenol A diglycidyl ether (BADGE) were applied. Post-SAH evaluations included neurobehavioral function, brain water content, Western blot analysis, and immunohistochemistry. Our results showed that endogenous NTN-1 and its receptor UNC5B level were increased after SAH. Administration of rh-NTN-1 reduced brain edema, ameliorated neurological impairments, and suppressed microglia activation after SAH, which were concomitant with PPARγ activation, inhibition of NFκB, and decrease in TNF-α, IL-6, and ICAM-1, as well as myeloperoxidase (MPO). Knockdown of endogenous NTN-1 increased expression of pro-inflammatory mediators and MPO, and aggravated neuroinflammation and brain edema. Moreover, knockdown of UNC5B using specific siRNA and inhibition of PPARγ with BADGE blocked the protective effects of rh-NTN-1. In conclusion, our findings indicated that exogenous rh-NTN-1 treatment attenuated neuroinflammation and neurological impairments through inhibiting microglia activation after SAH in rats, which is possibly mediated by UNC5B/PPARγ/NFκB signaling pathway. Exogenous NTN-1 may be a novel therapeutic agent to ameliorating early brain injury via its anti-inflammation effect.