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Disruption of the blood-brain barrier results in the formation of edema and contributes to the loss of neurological function following intracerebral hemorrhage (ICH). This study examined insulin-like growth factor-1 (IGF-1) as a treatment and its mechanism of action for protecting the blood-brain barrier after ICH in mice. 171 Male CD-1 mice were subjected to ICH via collagenase or autologous blood. A dose study for recombinant human IGF-1 (rhIGF-1) was performed. Brain water content and behavioral deficits were evaluated at 24 and 72 h after the surgery, and Evans blue extravasation and hemoglobin assay were conducted at 24 h. Western blotting was performed for the mechanism study and interventions were used targeting the IGF-1R/GSK3β/MEKK1 pathway. rhIGF-1 reduced edema and blood-brain barrier permeability, and improved neurobehavior outcomes. Western blots showed that rhIGF-1 reduced p-GSK3β and MEKK1 expression, thereby increasing occludin and claudin-5 expression. Inhibition and knockdown of IGF-1R reversed the therapeutic benefits of rhIGF-1. The findings within suggest that stimulation of the IGF-1R is a therapeutic target for ICH which may lead to improved neurofunctional and blood-brain barrier protection.

Autophagy activation exerts neuroprotective effects in the ischemic stroke model. Ezetimibe (Eze), a Niemann-Pick disease type C1-Like 1 (NPC1L1) pharmacological inhibitor, has been reported to protect hepatocytes from apoptosis via autophagy activation. In this study, we explored whether Eze could attenuate neuronal apoptosis in the rat model of middle cerebral artery occlusion (MCAO), specifically via activation of the AMPK/ULK1/autophagy pathway. Two hundred and one male Sprague-Dawley rats were subjected to transient MCAO followed by reperfusion. Eze was administered 1 h after MCAO. To elucidate the underlying molecular mechanism, Dorsomorphin, a selective AMPK inhibitor, and 3-methyladenine (3-MA), an autophagy inhibitor, were injected intracerebroventricularly before MCAO. Infarct volume, neurological score, brain cholesterol levels, immunofluorescence staining, Western blot, and Fluoro-Jade C (FJC) staining were used to evaluate the effects of Eze. The endogenous NPC1L1 expression increased and mainly expressed in neurons after MCAO. Intranasal administration of Eze reduced brain infarct volume at 24 and 72 h after MCAO, with improved short and long-term neurological functions after MCAO. Eze reduced brain cholesterol levels (total cholesterol, free cholesterol and cholesteryl esters) and the number of FJC-positive neurons. The expression of phosphorylated AMPK (p-AMPK) and downstream ULK1, Beclin1, LC3BII, Bcl-2, and Bcl-xl increased, while P62 and proapoptotic Bax decreased after treatment with Eze. Pretreatment with Dorsomorphin and 3-MA reversed the beneficial effects of Eze. These findings suggest that intranasal administration of Eze plays neuroprotective role through autophagy activation after MCAO in rats. Lowered cholesterol levels and AMPK activation may act in conjunction to induce autophagy after treatment with Eze. Eze merits further investigation as a potential therapeutic agent in ischemic stroke patients.

Background and purpose Ischemic stroke activates Toll-like receptors (TLRs), triggering rapid inflammatory cytokine production. Axl signaling has multiple roles, including regulating cytokine secretion, clearing apoptotic cells, and maintaining cell survival, however, its role in inflammation after ischemic stroke has not been examined. We hypothesized that activation of Axl by recombinant Growth-arrest-specific protein 6 (rGas6) attenuates neuroinflammation by inhibiting the TLR/TRAF/NF-κB pathway after middle cerebral artery occlusion (MCAO) in rats. Meth Sprague-Dawley rats were subjected to 2 h of MCAO. One hour after reperfusion, the rats were given an intranasal injection of rGas6, vehicle, or R428 (Axl receptor inhibitor). Neurological scores, infarct volumes, immunofluorescence staining, Morris Water Maze, rotarod test and histology alterations were analyzed. The expressions of proinflammatory cytokines, including IL-1β, IL-6, TNF-α, and Gas6, Axl, STAT1, SOCS1, SOCS3 and the TLR/TRAF/NF-κB pathway were quantified using Western blot. Results Endogenous expressions of Gas6 and Axl decreased significantly by 24 h after MCAO. rGas6 reduced brain infarction and improved neurologic deficits scores, and increased expression of Axl and decreased the expressions of TRAF3, TRAF6 and inflammatory factors IL-1β, IL-6, and TNF-α. Four weeks after MCAO, rGas6 improved long-term neurological behavior and memory. Inhibition of the Axl/TLR/TRAF/NF-κB pathway reversed the brain protection by rGas6. Conclusion rGas6 reduced the neurological deficits by inhibiting neuroinflammation via the TLR/TRAF/NF-κB signaling pathway. rGas6 can be used as potential treatment to ischemic stroke.

Germinal matrix hemorrhage is induced by stereotaxic injection of collagenase into the germinal matrix of P7 Sprague-Dawley rats. Hemoglobin assay, western blot, immunofluorescence and neurobehavioral tests were used to test the effects of BLVRA on hematoma resolution and anti-inflammatory response. We showed that BLVRA triggered a signaling cascade that ameliorated post-hemorrhagic neurological deficits in both short-term and long-term neurobehavioral tests in a GMH rat model. Specifically, BLVRA inhibited toll-like receptor 4 (TLR4) expression by translocating to the nucleus in an endothelial nitric oxide (eNOS)/nitric oxide (NO)-dependent manner. BLVRA also induced the upregulation of CD36 scavenger receptor level in microglia/microphages, of which the prominent role is to enhance hematoma resolution. However, the beneficial effects of BLVRA were abolished with the knockdown of eNOS, indicating that the eNOS/NO system is an important downstream factor of BLVRA. Our results demonstrate a mechanism of BLVRA modulating hematoma resolution and suppressing inflammation through eNOS/NO/TLR4 pathway in the GMH rat model.

Subarachnoid hemorrhage (SAH) results in severe neuronal dysfunction and degeneration. Since the nicotinic acetylcholine α(7) receptors (α(7)-AChR) are involved in neuronal function and survival, we investigated if stimulation of α(7)-AChR would promote neuronal survival and improve behavioral outcome following SAH in mice. Male mice subjected to SAH were treated with either galantamine (α(7)-AChR agonist) or vehicle. Neurobehavioral testing was performed 24 hours after SAH, and mice were euthanized for analysis of neuronal cell death or a cell survival (PI3K/Akt) signaling pathway. Neuron cell cultures were subjected to hemoglobin toxicity to assess the direct effects of α(7)-AChR agonism independent of other cells. Treatment with the α(7)-AChR agonist promoted neuronal survival and improved functional outcomes 24 hours post-SAH. The improved outcomes corresponded with increased PI3K/Akt activity. Antagonism of α(7)-AChR or PI3K effectively reversed galantamine’s beneficial effects. Tissue from α(7)-AChR knockout mice confirmed α(7)-AChR’s role in neuronal survival after SAH. Data from the neuronal cell culture experiment supported a direct effect of α(7)-AChR agonism in promoting cell survival. Our findings indicate that α(7)-AChR is a therapeutic target following SAH which can promote neuronal survival, thereby improving neurobehavioral outcome. Thus, the clinically relevant α(7)-AChR agonist, galantamine, might be a potential candidate for human use to improve outcome after SAH.

Brain edema is a major contributor to poor outcome and reduced quality of life after surgical brain injury (SBI). Although SBI pathophysiology is well-known, the correlation between cerebral edema and neurological deficits has not been thoroughly examined in the rat model of SBI. Thus, the purpose of this study was to determine the correlation between brain edema and deficits in standard sensorimotor neurobehavior tests for rats subjected to SBI. Sixty male Sprague-Dawley rats were subjected to either sham surgery or surgical brain injury via partial frontal lobectomy. All animals were tested for neurological deficits 24 post-SBI and fourteen were also tested 72 hours after surgery using seven common behavior tests: modified Garcia neuroscore (Neuroscore), beam walking, corner turn test, forelimb placement test, adhesive removal test, beam balance test, and foot fault test. After assessing the functional outcome, animals were euthanized for brain water content measurement. Surgical brain injury resulted in a significantly elevated frontal lobe brain water content 24 and 72 hours after surgery compared to that of sham animals. In all behavior tests, significance was observed between sham and SBI animals. However, a correlation between brain water content and functional outcome was observed for all tests except Neuroscore. The selection of behavior tests is critical to determine the effectiveness of therapeutics. Based on this study’s results, we recommend using beam walking, the corner turn test, the beam balance test, and the foot fault test since correlations with brain water content were observed at both 24 and 72 hours post-SBI.

Granulocyte-colony stimulating factor (G-CSF), a growth factor, has known neuroprotective effects in a variety of experimental brain injury models. Herein we show that G-CSF administration attenuates neuronal apoptosis after neonatal hypoxia-ischemia (HI) via glycogen synthase kinase-3β (GSK-3β) inhibition. Ten day old Sprague-Dawley rat pups (n=157) were subjected to unilateral carotid artery ligation followed by 2.5h of hypoxia or sham surgery. HI animals received control siRNA, GSK-3β siRNA (4 μL/pup), G-CSF (50 μg/kg), G-CSF combined with 0.1 or 0.4 nM G-CSF receptor (G-CSFR) siRNA, phosphatidylinositol 3-kinase (PI3K) inhibitor Wortmannin (86 ng/pup), or DMSO (vehicle for Wortmannin). Pups were euthanized 48 h post-HI to quantify brain infarct volume. G-CSFR, activated Akt (p-Akt), activated GSK-3β (p-GSK-3β), Cleaved Caspase-3 (CC3), Bcl-2, and Bax were quantified using Western blot analysis and the localizations of each was visualized via immunofluorescence staining. Neuronal cell death was determined using terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL). Our results showed p-GSK-3β increased after HI until its peak at 48 h post-ictus, and both GSK-3β siRNA and G-CSF administration reduced p-GSK-3β expression, as well as infarct volume. p-GSK-3β and CC3 were generally co-localized in neurons. Furthermore, G-CSF increased p-Akt expression and the Bcl-2/Bax ratio and also decreased p-GSK-3β and CC3 expression levels in the ipsilateral hemisphere, which were all reversed by G-CSFR siRNA, Wortmannin, and GSK-3β siRNA. In conclusion, G-CSF attenuated caspase activation and reduced brain injury by inhibiting GSK-3β activity after experimental HI in rat pups. This neuroprotective effect was abolished by both G-CSFR siRNA and Wortmannin.

The free-solvent model has been shown to have excellent predictability of the osmotic pressure for single and binary non-interactive proteins in aqueous solutions. Here the free-solvent model is extended to be more generalized by including the contributions of intra- and inter-protein interactions to the osmotic pressure of a solution in the form of homo- and hetero-multimers. The solute-solvent interactions are considered to be unique for each homo- and hetero-multimer in solution. The effect of the various generalized free-solvent model parameters on the osmotic pressure are examined for a single protein solution with a homo-dimer, a binary protein solution with no protein-protein interactions, and a binary protein solution with a hetero-dimer. Finally, the limitations associated with the generalized free-solvent model are discussed.