Osteopontin modulates microglial activation states and attenuates inflammatory responses after subarachnoid hemorrhage in rats.

AIMS: Osteopontin (OPN) has demonstrated neuroprotective effects in various stroke models. Its role in neuroinflammation after brain injury remains to be elucidated. This study aims to clarify the effect of OPN on neuroinflammation, particularly on the functional states of microglia after subarachnoid hemorrhage (SAH). METHODS: 77 rats were randomly divided into the following groups: Sham, SAH 24 h, SAH + rOPN, SAH + Vehicle (PBS), SAH + OPN siRNA, and SAH + Scr siRNA, SAH + rOPN+Fib-14 and SAH + rOPN+DMSO. Modified Garcia and beam balance tests were used to evaluate neurobehavioral outcomes. Semi-quantitative immunofluorescence staining was performed to measure expression of myeloperoxidase (MPO) and microglia activation state markers CD16, CD206 after SAH and recombinant OPN treatment. The quantification of microglia activation and functional markers CD16, CD206, TNF-α and IL-10 were further evaluated using Western-blotting. RESULTS: Nasal administration of rOPN improved neurological dysfunction, attenuated neutrophil infiltration, and decreased expression of phenotypic and functional markers of pro-inflammatory microglia CD16 and TNF-α. It also promoted an anti-inflammatory microglial state, as evidenced by increased expression of CD206 and IL-10. Furthermore, after blocking the phosphorylation of FAK signaling, the effects of rOPN on microglial activation states were partially reversed. The downstream pathways of STAT3 and NF-κB also exhibited consistent changes, suggesting the involvement of the STAT3 and NF-κB pathways in OPN's modulation of microglial activation via integrin-FAK signaling. CONCLUSION: OPN attenuates inflammatory responses after SAH by promoting an anti-inflammatory microglial state, potentially mediated through the integrin-FAK-STAT3 and NF-κB signaling pathways.

Exendin-4 Preserves Blood-Brain Barrier Integrity via Glucagon-Like Peptide 1 Receptor/Activated Protein Kinase-Dependent Nuclear Factor-Kappa B/Matrix Metalloproteinase-9 Inhibition After Subarachnoid Hemorrhage in Rat.

In this study, we investigated the role of Exendin-4 (Ex-4), a glucagon-like peptide 1 receptor (GLP-1R) agonist, in blood-brain barrier (BBB) disruption after subarachnoid hemorrhage (SAH) in rats. The endovascular perforation model of SAH was performed in Sprague-Dawley rats. Ex-4 was intraperitoneally injected 1 h after SAH induction. To elucidate the underlying molecular mechanism, small interfering ribonucleic acid (siRNA) for GLP-1R and Dorsomorphin, a specific inhibitor of adenosine monophosphate-activated protein kinase (AMPK), were intracerebroventricularly injected 48 h before induction of SAH correspondingly. Immunofluorescence results supported GLP-1R expressed on the endothelial cells of microvessels in the brain after SAH. Administration of Ex-4 significantly reduced brain water content and Evans blue extravasation in both hemispheres, which improved neurological scores at 24 h after SAH. In the mechanism study, Ex-4 treatment significantly increased the expression of GLP-1R, p-AMPK, IκB-α, Occludin, and Claudin-5, while the expression of p-nuclear factor-kappa B (NF-κB) p65, matrix metalloproteinase-9 (MMP-9), and albumin was significantly decreased. The effects of Ex-4 were reversed by the intervention of GLP-1R siRNA or Dorsomorphin, respectively. In conclusion, Ex-4 could preserve the BBB integrity through GLP-1R/AMPK-dependent NF-κB/MMP-9 inhibition after SAH, which should be further investigated as a potential therapeutic target in SAH.

Recombinant OX40 attenuates neuronal apoptosis through OX40-OX40L/PI3K/AKT signaling pathway following subarachnoid hemorrhage in rats.

Subarachnoid hemorrhage (SAH) is the most devastating form of stroke. Reducing neuronal apoptosis is an important countermeasure against early brain injury (EBI) after SAH. Recent evidence indicates that OX40-OX40L coupling is critical for cell survival and proliferation. Current study was performed to detect the role of recombinant OX40 (ReOX40) against neuronal apoptosis after SAH. The endovascular perforation model of SAH was performed on Sprague-Dawley (SD) rats. ReOX40 was injected intracerebroventricularly (i.c.v) 1 h after SAH induction and the following methods were employed: neurological function evaluation, immunofluorescence staining, fluoro-Jade C staining, and western blot. To study the underlying precise molecular mechanism, small interfering ribonucleic acid (siRNA) for OX40L and a specific inhibitor of PI3K, LY294002, were injected i.c.v. into SAH + ReOX40 rats before induction of SAH. When compared with sham rats, the expression of OX40 and OX40L was seen to decrease in the brain at 24 h after SAH induction. Administration of ReOX40 (5 µg/kg) increased expression of the OX40L, reduced the neuronal apoptosis, and improved short and long-term neurological function deficits. Furthermore, ReOx40 heightened activation of OX40L/PI3K/AKT axis, increased the downstream anti-apoptotic protein (Bcl2, Bcl-XL), and depressed the apoptotic protein (cleaved caspase 3, Bax). However, the protective effects of ReOX40 were abolished by the administration of OX40L siRNA and LY294002, respectively. These results demonstrate that ReOX40 attenuates neuronal apoptosis through OX40-OX40L/PI3K/AKT pathway in EBI after SAH.

Activation of Melanocortin 1 Receptor Attenuates Early Brain Injury in a Rat Model of Subarachnoid Hemorrhage viathe Suppression of Neuroinflammation through AMPK/TBK1/NF-κB Pathway in Rats.

Neuroinflammation plays a vital role in early brain injury (EBI) following subarachnoid hemorrhage (SAH). The hypothesis of this study was that activation of melanocortin 1 receptor (MC1R) with BMS-470539 attenuates EBI by suppression of neuroinflammation after SAH. We utilized BMS-470539, MSG-606, and MRT-68601 to verify the neuroprotective effects of MC1R. We evaluated brain water content, short-term and long-term neurobehavior after SAH. Western blotting and immunofluorescence staining were utilized to assess the changes of protein levels. The results of western blotting suggested that the expressions of MC1R, phosphorylated-adenosine monophosphate-activated protein kinase (p-AMPK), and phosphorylated-TANK binding kinase 1 (p-TBK1) were increased and reached their peak points at 24 h following SAH. Moreover, BMS-470539 treatment notably attenuated neurological deficits caused by SAH, and also notably improved long-term spatial learning and memory abilities after SAH. The underlying mechanisms of the neuroprotection of BMS-470539 involved the suppression of microglia activation, promotion of CD206+ microglia transformation and reduction of neutrophil infiltration by increasing the levels of p-AMPK and p-TBK1 while decreasing the levels of NF-κB, IL-1ß, and TNFα. The neuroprotective effects of BMS-470539 were significantly abolished by MSG-606 and MRT-68601. The activation of MC1R with BMS-470539 notably attenuates EBI after SAH by suppression of microglial activation and neutrophil infiltration via the AMPK/TBK1/NF-κB signaling pathway.

Osteopontin attenuates early brain injury through regulating autophagy-apoptosis interaction after subarachnoid hemorrhage in rats.

AIM: To determine the effect of osteopontin (OPN) on autophagy and autophagy-apoptosis interactions after SAH. METHODS: The endovascular perforation model of SAH or sham surgery was performed in a total of 86 Sprague-Dawley male rats. The temporal expressions of endogenous OPN and autophagy-related proteins (Beclin 1, ATG5, LC3 II to I ratio) were measured in sham and SAH rats at different time points (3, 6, 12, 24, and 72 hours). Rats were randomly divided into three groups: Sham, SAH + Vehicle (PBS, phosphate-buffered saline), and SAH + rOPN (5 µg/rat recombinant OPN). Neurobehavioral tests were performed 24 hours after SAH, followed by the collection of brain samples for assessment of autophagy and apoptosis proteins. These tests assessed whether an autophagy-apoptosis relationship existed on the histological level in the brain. RESULTS: Endogenous OPN and autophagy-related proteins all increased after SAH. rOPN administration improved neurological dysfunction, increased the expression of autophagy-related proteins (Beclin 1, ATG5, LC3 II to I ratio) and antiapoptotic protein Bcl-2, while decreasing the expression of proapoptotic proteins (cleaved Caspase-3 and Bax). rOPN also regulated autophagy-apoptosis interactions 24 hours after SAH. CONCLUSION: rOPN attenuates early brain injury and inhibits neuronal apoptosis by activating autophagy and regulating autophagy-apoptosis interactions.

FGF-2 Attenuates Neuronal Apoptosis via FGFR3/PI3k/Akt Signaling Pathway After Subarachnoid Hemorrhage.

Neuronal apoptosis is a common and critical pathology following subarachnoid hemorrhage (SAH). We investigated the anti-apoptotic property of fibroblast growth factor (FGF)-2 after SAH in rats. A total of 289 rats underwent endovascular perforation to induce SAH or sham operation. Three dosages (3, 9, or 27 µg) of recombinant FGF-2 (rFGF-2) or vehicle was administered intranasally to rats 30 min after SAH induction. The pan-FGF receptor (FGFR) inhibitor PD173074 or vehicle was administered intracerebroventricularly (i.c.v.) 1 h before modeling, in addition to rFGF-2 treatment. Small interfering ribonucleic acid (siRNA) for FGFR1 and FGFR3 or scrambled siRNA was administered i.c.v. 48 h before SAH induction in addition to rFGF-2 treatment. Anti-FGF-2 neutralizing antibody or normal mouse immunoglobulin G (IgG) was administered i.c.v. 1 h before SAH model. Neurobehavioral tests, SAH severity, brain water content, immunofluorescence, Fluoro-Jade C, TUNEL staining, and western blot were evaluated. The expression of FGF-2, FGFR1, and FGFR3 increased after SAH. FGFR1 and FGFR3 were expressed in the neurons. Nine micrograms of FGF-2 alleviated neurological impairments, brain edema, and neuronal apoptosis following SAH. A rFGF-2 treatment improved motor skill learning and spatial memory and increased the number of surviving neurons postinjury to 28 days after SAH. PD173074 abolished the anti-apoptotic effects of rFGF-2 via suppression of the expression of PI3k, phosphorylated Akt (p-Akt), and Bcl-2 leading to enhancement of the expression of Bax. FGFR3 siRNA worsened neurobehavioral function and suppressed the expression of PI3k, p-Akt, and Bcl-2 rather than FGFR1 siRNA in SAH rats treated with rFGF-2. Anti-FGF-2 neutralizing antibody suppressed the expression of PI3k and p-Akt after SAH. FGF-2 may be a promising therapy to reduce post-SAH neuronal apoptosis via activation of the FGFR3/PI3k/Akt signaling pathway.

Hydrogen gas therapy improves survival rate and neurological deficits in subarachnoid hemorrhage rats: a pilot study.

The high morbidity, high mortality, and significant shortage of effective therapies for subarachnoid hemorrhage (SAH) have created an urgency to discover novel therapies. Human studies in Asia have established the safety of hydrogen gas in the treatment of hepatic, renal, pulmonary, and cardiac diseases. Mechanistically, hydrogen gas has been shown to affect oxidative stress, inflammation, and apoptosis. We hypothesized that hydrogen therapy would improve neurological function and increase survival rate in SAH. High dose hydrogen gas (66% at 3 L/min) was administered for 2 hours at 0.5, 8, and 18 hours after SAH. This treatment increased 72-hour survival rate and provided 24-hour neuroprotection after SAH in rats. To our knowledge, this is the first report demonstrating that high dose hydrogen gas therapy reduces mortality and improves outcome after SAH. Our results correlate well with the proposed mechanisms of hydrogen gas therapy within the literature. We outline four pathways and downstream targets of hydrogen gas potentially responsible for our results. A potentially complex network of pathways responsible for the efficacy of hydrogen gas therapy, along with a limited mechanistic understanding of these pathways, justifies further investigation to provide a basis for clinical trials and the advancement of hydrogen gas therapy in humans. This study was approved by the Institutional Animal Care and Use Committee of Loma Linda University, USA (Approval No. 8160016) in May 2016.

Activation of retinoid X receptor by bexarotene attenuates neuroinflammation via PPARγ/SIRT6/FoxO3a pathway after subarachnoid hemorrhage in rats.

BACKGROUND: Subarachnoid hemorrhage (SAH) is a life-threatening subtype of stroke with high mortality and disabilities. Retinoid X receptor (RXR) has been shown to be neuroprotective against ischemia/reperfusion injury. This study aimed to investigate the effects of the selective RXR agonist bexarotene on neuroinflammation in a rat model of SAH. METHODS: Two hundred male Sprague-Dawley rats were used. The endovascular perforation induced SAH. Bexarotene was administered intraperitoneally at 1 h after SAH induction. To investigate the underlying mechanism, the selective RXR antagonist UVI3003 and RXR siRNA or SIRT6 inhibitor OSS128167 was administered via intracerebroventricular 1 h before SAH induction. Post-SAH assessments including SAH grade, neurological score, brain water content, Western blot, and immunofluorescence were performed. RESULTS: The endogenous RXR and sirtuin 6 (SIRT6) protein levels were increased after SAH. Bexarotene treatment significantly reduced brain edema and improved the short-/long-term neurological deficit after SAH. Mechanistically, bexarotene increased the levels of PPARγ and SIRT6; decreased the expression of phosphorylated FoxO3a (p-FoxO3a), IL-6, IL-1ß, and TNF-a; and inhibited the microglia activation and neutrophils infiltration at 24 h after SAH. Either UVI3003, OSS128167, or RXR siRNA abolished the neuroprotective effects of bexarotene and its regulation on protein levels of PPARγ/SIRT6/p-FoxO3a after SAH. CONCLUSIONS: The activation of RXR by bexarotene attenuated neuroinflammation and improved neurological deficits after SAH. The anti-neuroinflammatory effect was at least partially through regulating PPARγ/SIRT6/FoxO3a pathway. Bexarotene may be a promising therapeutic strategy in the management of SAH patients.

LRP1 activation attenuates white matter injury by modulating microglial polarization through Shc1/PI3K/Akt pathway after subarachnoid hemorrhage in rats.

White matter injury (WMI) is associated with motor deficits and cognitive dysfunctions in subarachnoid hemorrhage (SAH) patients. Therapeutic strategy targeting WMI would likely improve the neurological outcomes after SAH. Low-density lipoprotein receptor-related protein-1 (LRP1), a scavenger receptor of apolipoprotein E (apoE), is able to modulate microglia polarization towards anti-inflammatory M2 phenotypes during inflammatory and oxidative insult. In the present study, we investigated the effects of LRP1 activation on WMI and underlying mechanisms of M2 microglial polarization in a rat model of SAH. Two hundred and seventeen male Sprague Dawley rats (weight 280-330 g) were used. SAH was induced by endovascular perforation. LPR1 ligand, apoE-mimic peptide COG1410 was administered intraperitoneally. Microglial depletion kit liposomal clodronate (CLP), LPR1 siRNA or PI3K inhibitor were administered intracerebroventricularly. Post-SAH assessments included neurobehavioral tests, brain water content, immunohistochemistry, Golgi staining, western blot and co-immunoprecipitation. SAH induced WMI shown as the accumulation of amyloid precursor protein and neurofilament heavy polypeptide as well as myelin loss. Microglial depletion by CLP significantly suppressed WMI after SAH. COG1410 reduced brain water content, increased the anti-inflammatory M2 microglial phenotypes, attenuated WMI and improved neurological function after SAH. LRP1 was bound with endogenous apoE and intracellular adaptor protein Shc1. The benefits of COG1410 were reversed by LPR1 siRNA or PI3K inhibitor. LRP1 activation attenuated WMI and improved neurological function by modulating M2 microglial polarization at least in part through Shc1/PI3K/Akt signaling in a rat model of SAH. The apoE-mimic peptide COG1410 may serve as a promising treatment in the management of SAH patients.

AVE 0991 attenuates oxidative stress and neuronal apoptosis via Mas/PKA/CREB/UCP-2 pathway after subarachnoid hemorrhage in rats.

Oxidative stress and neuronal apoptosis have been demonstrated to be key features in early brain injury (EBI) after subarachnoid hemorrhage (SAH). Previous studies have indicated that Mas receptor activation initiates an anti-oxidative and anti-apoptotic role in the brain. However, whether Mas activation can attenuate oxidative stress and neuronal apoptosis after SAH remains unknown. To investigate the beneficial effect of Mas on oxidative stress injury and neuronal apoptosis induced by SAH, a total of 196 rats were subjected to an endovascular perforation model of SAH. AVE 0991 (AVE), a selective agonist of Mas, was administered intranasally 1 h after SAH induction. A779, a selective inhibitor of Mas, and small interfering ribonucleic acid (siRNA) for UCP-2 were administered by intracerebroventricular (i.c.v) injection at 1 h and 48 h before SAH induction respectively. Neurological tests, immunofluorescence, TUNEL, Fluoro-Jade C, DHE staining, and Western blot experiments were performed. We found that Mas activation with AVE significantly improved neurobehavioral scores and reduced oxidative stress and neuronal apoptosis in SAH+AVE group compared with SAH+vehicle group. Moreover, AVE treatment significantly promoted phosphorylation of CREB and the expression UCP-2, as well as upregulated expression of Bcl-2 and downregulation of Romo-1 and Bax. The protective effects of AVE were reversed by i.c.v injection of A779 and UCP-2 siRNA in SAH+AVE+A779 and SAH+AVE+UCP-2 siRNA groups, respectively. In conclusion, our data provides evidence that Mas activation with AVE reduces oxidative stress injury and neuronal apoptosis through Mas/PKA/p-CREB/UCP-2 pathway after SAH. Furthermore, our study indicates that Mas may be a novel therapeutic treatment target in early brain injury of SAH.

Aggf1 attenuates neuroinflammation and BBB disruption via PI3K/Akt/NF-κB pathway after subarachnoid hemorrhage in rats.

BACKGROUND: Neuroinflammation and blood-brain barrier (BBB) disruption are two critical mechanisms of subarachnoid hemorrhage (SAH)-induced brain injury, which are closely related to patient prognosis. Recently, angiogenic factor with G-patch and FHA domain 1 (Aggf1) was shown to inhibit inflammatory effect and preserve vascular integrity in non-nervous system diseases. This study aimed to determine whether Aggf1 could attenuate neuroinflammation and preserve BBB integrity after experimental SAH, as well as the underlying mechanisms of its protective roles. METHODS: Two hundred forty-nine male Sprague-Dawley rats were subjected to the endovascular perforation model of SAH. Recombinant human Aggf1 (rh-Aggf1) was administered intravenously via tail vein injection at 1 h after SAH induction. To investigate the underlying neuroprotection mechanism, Aggf1 small interfering RNA (Aggf1 siRNA) and PI3K-specific inhibitor LY294002 were administered through intracerebroventricular (i.c.v.) before SAH induction. SAH grade, neurological score, brain water content, BBB permeability, Western blot, and immunohistochemistry were performed. RESULTS: Expression of endogenous Aggf1 was markedly increased after SAH. Aggf1 was primarily expressed in endothelial cells and astrocytes, as well as microglia after SAH. Administration of rh-Aggf1 significantly reduced brain water content and BBB permeability, decreased the numbers of infiltrating neutrophils, and activated microglia in the ipsilateral cerebral cortex following SAH. Furthermore, rh-Aggf1 treatment improved both short- and long-term neurological functions after SAH. Meanwhile, exogenous rh-Aggf1 significantly increased the expression of PI3K, p-Akt, VE-cadherin, Occludin, and Claudin-5, as well as decreased the expression of p-NF-κB p65, albumin, myeloperoxidase (MPO), TNF-α, and IL-1ß. Conversely, knockdown of endogenous Aggf1 aggravated BBB breakdown, inflammatory response and neurological impairments at 24 h after SAH. Additionally, the protective roles of rh-Aggf1 were abolished by LY294002. CONCLUSIONS: Taken together, exogenous Aggf1 treatment attenuated neuroinflammation and BBB disruption, improved neurological deficits after SAH in rats, at least in part through the PI3K/Akt/NF-κB pathway.

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

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.

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 1ß1 (Nrg 1ß1), 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.

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.

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

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.

Rho-Kinase Inhibition During Early Cardiac Development Causes Arrhythmogenic Right Ventricular Cardiomyopathy in Mice.

OBJECTIVE: Arrhythmogenic right ventricular cardiomyopathy (ARVC) is characterized by fibrofatty changes of the right ventricle, ventricular arrhythmias, and sudden death. Though ARVC is currently regarded as a disease of the desmosome, desmosomal gene mutations have been identified only in half of ARVC patients, suggesting the involvement of other associated mechanisms. Rho-kinase signaling is involved in the regulation of intracellular transport and organizes cytoskeletal filaments, which supports desmosomal protein complex at the myocardial cell-cell junctions. Here, we explored whether inhibition of Rho-kinase signaling is involved in the pathogenesis of ARVC. APPROACH AND RESULTS: Using 2 novel mouse models with SM22α- or αMHC-restricted overexpression of dominant-negative Rho-kinase, we show that mice with Rho-kinase inhibition in the developing heart (SM22α-restricted) spontaneously develop cardiac dilatation and dysfunction, myocardial fibrofatty changes, and ventricular arrhythmias, resulting in premature sudden death, phenotypes fulfilling the criteria of ARVC in humans. Rho-kinase inhibition in the developing heart results in the development of ARVC phenotypes in dominant-negative Rho-kinase mice through 3 mechanisms: (1) reduction of cardiac cell proliferation and ventricular wall thickness, (2) stimulation of the expression of the proadipogenic noncanonical Wnt ligand, Wnt5b, and the major adipogenic transcription factor, PPARγ (peroxisome proliferator activated receptor-γ), and inhibition of Wnt/ß-catenin signaling, and (3) development of desmosomal abnormalities. These mechanisms lead to the development of cardiac dilatation and dysfunction, myocardial fibrofatty changes, and ventricular arrhythmias, ultimately resulting in sudden premature death in this ARVC mouse model. CONCLUSIONS: This study demonstrates a novel crucial role of Rho-kinase inhibition during cardiac development in the pathogenesis of ARVC in mice.

Characterization of vasoconstrictor-induced relaxation in the cerebral basilar artery.

The vascular endothelium regulates vascular smooth muscle functions by releasing endothelium-derived vasoactive substances. To identify physiological mechanisms mediating the inhibitory effect of the endothelium on vasoconstrictors, the basilar arteries isolated from Wistar rats were used in an organ bath study. In the intact basilar artery (with endothelium), 100 nM serotonin (5-HT) induced phasic contraction (28.7+/-4.1% of 60 mM KCl-induced contraction) followed by profound time-dependent relaxation at 3 min (3.8+/-0.4%). In the denuded artery (without endothelium), the 5-HT-induced contraction was enhanced (51.7+/-16.1%), while the relaxation was abolished. In the intact basilar artery, the contraction was facilitated and the amplitude of the phasic contraction was significantly enhanced (70.1+/-10.3%), but time-dependent relaxation was still manifested at 3 min (25.7+/-10.0%) in the presence of Nomega-nitro-L-arginine methyl ester hydrochloride (L-NAME) and indomethacin. Time-dependent relaxation induced by 5-HT was abolished in Ca(2+)-free and in K(+)-free Krebs-Henseleit buffer (KHB). Furthermore, the 5-HT-induced contraction was enhanced by treatment with ouabain (105.6+/-11.8%), tetraethylammonium chloride (133.2+/-7.9%), charybdotoxin with apamin (145.4+/-6.4%) or BaCl(2) (72.2+/-13.8%) at 3 min; also, time-dependent relaxation was abolished by these blockers in the presence of L-NAME and indomethacin. U46619 (100 nM) induced sustained contraction without time-dependent relaxation in normal KHB, but charybdotoxin with apamin did not affect the contraction. The results suggest that time-dependent relaxation is modulated by endothelial sodium-potassium pump (Na(+)/K(+)-ATPase) and Ca(2+)-activated K(+) channel (K(Ca)) activity, especially small- and intermediate-conductance K(Ca)-prominent ionic mechanisms of the so-called endothelium-derived hyperpolarizing factor.

Characteristics of contractile activity in the renal artery of ovariectomized rats.

The incidence of cardiovascular disease is markedly lower in cycling, pre-menopausal women and post-menopausal women receiving estrogen than in men or untreated post-menopausal women. Clinical studies demonstrate a protective role of estrogen in hormone replacement therapy in terms of reducing cardiovascular risk. However, the benefits of hormone replacement therapy in cardiovascular disease remain unclear. We investigated the effects of estrogen on the contractile responses of the renal artery of ovariectomized Wistar rats (OVX) compared to both ovariectomized 17 beta-estradiol-treated rats (OVXE) and sham-operated (control) rats. Isometric contraction of renal artery was recorded with a strain gauge transducer. The maximum contractile response of the renal artery smooth muscle to KCl (80 mM) in the OVXE group was significantly higher than that in both the control and OVX groups. The phenylephrine (PE) concentration-response curves in all three groups indicated a greater sensitivity at lower concentrations of PE following treatment with 100 microM L-arginine methyl ester (L-NAME). The EC50 values for PE in the three groups were 2 times lower in the presence of L-NAME than those lacking exposure to L-NAME. The EC50 value for PE in the OVX group was approximately 3 times lower in the presence of L-NAME than in those lacking exposure to L-NAME and 100 nM BMY 7378, an alpha 1D-adrenoceptor antagonist. The rate of relaxation of the PE-induced contraction (T1/2) was significantly reduced in the OVX group relative to both the control and OVXE groups. T1/2 values after treatment with 100 microM L-NAME were slower than those lacking exposure to L-NAME in all groups. Further, the T1/2 value of the OVX group was 2 times greater than that of the control; this change was reversed in the OVXE group. In conclusion, our results suggest that estrogen regulates contraction and relaxation in the renal artery via NO synthase activity and alteration of the Ca2+ transport systems.