Devin W. McBride

Neuroprotective Effect of Hydrogen-Rich Saline against Neurologic Damage and Apoptosis in Early Brain Injury following Subarachnoid Hemorrhage: Possible Role of the Akt/GSK3β Signaling Pathway

Backgrounds Early brain injury (EBI) plays a key role in the pathogenesis of subarachnoid hemorrhage (SAH). Neuronal apoptosis is involved in the pathological process of EBI. Hydrogen can inhibit neuronal apoptosis and attenuate EBI following SAH. However, the molecular mechanism underlying hydrogen-mediated anti-apoptotic effects in SAH has not been elucidated. In the present study, we aimed to evaluate whether hydrogen alleviates EBI after SAH, specifically neuronal apoptosis, partially via the Akt/GSK3β signaling pathway. Methods Sprague-Dawley rats (n = 85) were randomly divided into the following groups: sham group (n = 17), SAH group (n = 17), SAH + saline group (n = 17), SAH + hydrogen-rich saline (HS) group (n = 17) and SAH + HS + Ly294002 (n = 17) group. HS or an equal volume of physiological saline was administered immediately after surgery and repeated 8 hours later. The PI3K inhibitor, Ly294002, was applied to manipulate the proposed pathway. Neurological score and SAH grade were assessed at 24 hours after SAH. Western blot was used for the quantification of Akt, pAkt, GSK3β, pGSK3β, Bcl-2, Bax and cleaved caspase-3 proteins. Neuronal apoptosis was identified by double staining of terminal deoxynucleotidyl transferase mediated nick end labeling (TUNEL) staining and NeuN, and quantified by apoptosis index. Immunohistochemistry and immunofluorescent double-labeling staining was performed to clarify the relationships between neuronal apoptosis and pAkt or pGSK3β. Results HS significantly reduced neuronal apoptosis and improved neurological function at 24 hours after SAH. The levels of pAkt and pGSK3β, mainly expressed in neurons, were markedly up-regulated. Additionally, Bcl-2 was significantly increased while Bax and cleaved caspase-3 was decreased by HS treatment. Double staining of pAkt and TUNEL showed few colocalization of pAkt-positive cells and TUNEL-positive cells. The inhibitor of PI3K, Ly294002, suppressed the beneficial effects of HS. Conclusions HS could attenuate neuronal apoptosis in EBI and improve the neurofunctional outcome after SAH, partially via the Akt/GSK3β pathway.

G-CSF attenuates neuroinflammation and stabilizes the blood-brain barrier via the PI3K/Akt/GSK-3β signaling pathway following neonatal hypoxia-ischemia in rats.

OBJECTIVE Neonatal hypoxia occurs in approximately 60% of premature births and is associated with a multitude of neurological disorders. While various treatments have been developed, translating them from bench to bedside has been limited. We previously showed G-CSF administration was neuroprotective in a neonatal hypoxia-ischemia rat pup model, leading us to hypothesize that G-CSF inactivation of GSK-3β via the PI3K/Akt pathway may attenuate neuroinflammation and stabilize the blood-brain barrier (BBB). METHODS P10 Sprague-Dawley rat pups were subjected to unilateral carotid artery ligation followed by hypoxia for 2.5h. We assessed inflammation by measuring expression levels of IKKβ, NF-κB, TNF-α, IL-1β, IL-10, and IL-12 as well as neutrophil infiltration. BBB stabilization was evaluated by measuring Evans blue extravasation, and Western blot analysis of Claudin-3, Claudin-5, ICAM-1, and VCAM-1. MEASUREMENTS AND MAIN RESULTS First, the time course study showed that p-β-catenin/β-catenin, IKKβ, and NF-κB expression levels peaked at 48h post-HI. The knockdown of GSK-3β with siRNA prevented the HI-induced increase of p-β-catenin/β-catenin, IKKβ, and NF-κB expression levels 48h after HI. G-CSF treatment reduced brain water content and neuroinflammation by downregulating IKKβ, NF-κB, TNF-α, IL-1β, and IL-12 and upregulating IL-10, thereby reducing neutrophil infiltration. Additionally, G-CSF stabilizes the BBB by downregulating VCAM-1 and ICAM-1, as well as upregulating Claudins 3 and 5 in endothelial cells. G-CSFR knockdown by siRNA and Akt inhibition by Wortmannin reversed G-CSFs neuroprotective effects. CONCLUSIONS We demonstrate G-CSF plays a pivotal role in attenuating neuroinflammation and BBB disruption following HI by inactivating GSK-3β through the PI3K/Akt pathway.

G-CSF ameliorates neuronal apoptosis through GSK-3β inhibition in neonatal hypoxia-ischemia in rats.

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.

Follistatin-Like 1 Attenuates Apoptosis via Disco-Interacting Protein 2 Homolog A/Akt Pathway After Middle Cerebral Artery Occlusion in Rats

Background and Purpose— Follistatin-like 1 (FSTL1), an extracellular glycoprotein, has been reported to decrease apoptosis in ischemic cardiac diseases, but its effect in ischemic stroke has not been examined. We hypothesized that recombinant FSTL1 attenuates neuronal apoptosis through its receptor disco-interacting protein 2 homolog A (DIP2A) and the Akt pathway after middle cerebral artery occlusion (MCAO) in rats. Methods— One hundred forty male Sprague-Dawley rats were subjected to 2 hours of MCAO followed by reperfusion. In a subset of animals, the time course and location of FSTL1 and DIP2A were detected by Western blot and immunofluorescence double staining. Another set of animals were intracerebroventricularly given either recombinant FSTL1 1 hour after reperfusion or FSTL1-small interfering RNA (siRNA) 48 hours before reperfusion. Additionally, DIP2A was knockdown by siRNA in some animals. Infarction volume and neurological deficits were measured, and the expression of FSTL1, DIP2A, phosphorylated Akt, cleaved caspase-3, and terminal deoxynucleotidyl transferase dUTP nick end labeling were quantified using Western blot. Results— The expression of FSTL1 and DIP2A was increased in neurons and peaked 24 hours after MCAO. Recombinant FSTL1 reduced brain infarction and improved neurological deficits 24 and 72 hours after MCAO via activation of its receptor DIP2A and downstream phosphorylation of Akt. These effects were reversed by DIP2A-siRNA and FSTL1-siRNA. Conclusions— Recombinant FSTL1 decreases neuronal apoptosis and improves neurological deficits through phosphorylation of Akt by activation of its receptor DIP2A after MCAO in rats. Thus, FSTL1 may have potentials as a treatment for patients with ischemic stroke.

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.

Protease-Activated Receptor 1 and 4 Signal Inhibition Reduces Preterm Neonatal Hemorrhagic Brain Injury

Background and Purpose— This study examines the role of thrombin’s protease-activated receptor (PAR)-1, PAR-4 in mediating cyclooxygenase-2 and mammalian target of rapamycin after germinal matrix hemorrhage. Methods— Germinal matrix hemorrhage was induced by intraparenchymal infusion of bacterial collagenase into the right ganglionic eminence of P7 rat pups. Animals were treated with PAR-1, PAR-4, cyclooxygenase-2, or mammalian target of rapamycin inhibitors by 1 hour, and ⩽5 days. Results— We found increased thrombin activity 6 to 24 hours after germinal matrix hemorrhage, and PAR-1, PAR-4, inhibition normalized cyclooxygenase-2, and mammalian target of rapamycin by 72 hours. Early treatment with NS398 or rapamycin substantially improved long-term outcomes in juvenile animals. Conclusions— Suppressing early PAR signal transduction, and postnatal NS398 or rapamycin treatment, may help reduce germinal matrix hemorrhage severity in susceptible preterm infants.

Acute Hyperglycemia Is Associated with Immediate Brain Swelling and Hemorrhagic Transformation After Middle Cerebral Artery Occlusion in Rats

Hemorrhagic transformation occurs in as many as 48 % of stroke patients and is a major contributor to post-insult morbidity and mortality. Experimental models of hemorrhagic transformation are utilized for understanding the mechanisms behind its development, as well as for investigating potential therapeutics for prevention and reduction of bleeding. Thoroughly studying animal models of hemorrhagic transformation is critically important for testing novel treatments. Thus far, no study has examined the progression of brain swelling and hemorrhagic transformation after transient middle cerebral artery occlusion (MCAO). Herein, we investigate the development of infarction, brain swelling, and hemorrhagic transformation following MCAO in hyperglycemic rats. Twenty-five Sprague-Dawley rats were subjected to either 1.5 h of MCAO or sham surgery 15 min after induction of hyperglycemia. Animals were sacrificed at 0.25, 1, 3, or 24 h after reperfusion for measurement of infarct volume, brain swelling, and hemoglobin volume. Within 15 min of reperfusion, the infarct volume was significantly larger than in sham animals and did not increase in size over the 24 h. However, both brain swelling and hemorrhagic transformation, which began immediately after reperfusion, increase over 24 h after reperfusion.

An experimental model of vasovagal syncope induces cerebral hypoperfusion and fainting-like behavior in awake rats

Vasovagal syncope, a contributing factor to elderly falls, is the transient loss of consciousness caused by decreased cerebral perfusion. Vasovagal syncope is characterized by hypotension, bradycardia, and reduced cerebral blood flow, resulting in fatigue, altered coordination, and fainting. The purpose of this study is to develop an animal model which is similar to human vasovagal syncope and establish an awake animal model of vasovagal syncope. Male Sprague-Dawley rats were subjected to sinusoidal galvanic vestibular stimulation (sGVS). Blood pressure, heart rate, and cerebral blood flow were monitored before, during, and post-stimulation. sGVS resulted in hypotension, bradycardia, and decreased cerebral blood flow. One cohort of animals was subjected to sGVS while freely moving. sGVS in awake animals produced vasovagal syncope-like symptoms, including fatigue and uncoordinated movements; two animals experienced spontaneous falling. Another cohort of animals was preconditioned with isoflurane for several days before being subjected to sGVS. Isoflurane preconditioning before sGVS did not prevent sGVS-induced hypotension or bradycardia, yet isoflurane preconditioning attenuated sGVS-induced cerebral blood flow reduction. The sGVS rat model mimics elements of human vasovagal syncope pathophysiology (hypotension, bradycardia, and decreased cerebral perfusion), including behavioral symptoms such as fatigue and altered balance. This study indicates that the sGVS rat model is similar to human vasovagal syncope and that therapies directed at preventing cerebral hypoperfusion may decrease syncopal episodes and reduce injuries from syncopal falls.

Precision Stroke Animal Models: the Permanent MCAO Model Should Be the Primary Model, Not Transient MCAO

An argument for preclinical stroke research to make more use of the permanent middle cerebral artery occlusion (MCAO) model, rather than transient MCAO, is presented. Despite STAIR recommending permanent MCAO as the primary model, preclinical stroke research has not been listened. In 2012, Hossmann reported that 64% of the treatment studies for MCAO used prompt transient MCAO models and only 36% of the studies used permanent MCAO or gradual transient MCAO (i.e., embolic stroke model). Then, in 2014 and 2015, 88% of published basic science studies on large vessel occlusion used the transient MCAO model. However, this model only represents 2.5–11.3% of large vessel stroke patients. Therefore, the transient MCAO model, which mimics stroke with reperfusion, does not accurately reflect the majority of clinical stroke cases. Thus, once again, the argument for studying permanent MCAO as a primary model is made and supported.

Crotalus atrox venom preconditioning increases plasma fibrinogen and reduces perioperative hemorrhage in a rat model of surgical brain injury

Perioperative bleeding is a potentially devastating complication in neurosurgical patients, and plasma fibrinogen concentration has been identified as a potential modifiable risk factor for perioperative bleeding. The aim of this study was to evaluate preconditioning with Crotalus atrox venom (Cv-PC) as potential preventive therapy for reducing perioperative hemorrhage in the rodent model of surgical brain injury (SBI). C. atrox venom contains snake venom metalloproteinases that cleave fibrinogen into fibrin split products without inducing clotting. Separately, fibrinogen split products induce fibrinogen production, thereby elevating plasma fibrinogen levels. Thus, the hypothesis was that preconditioning with C. atrox venom will produce fibrinogen spilt products, thereby upregulating fibrinogen levels, ultimately improving perioperative hemostasis during SBI. We observed that Cv-PC SBI animals had significantly reduced intraoperative hemorrhage and postoperative hematoma volumes compared to those of vehicle preconditioned SBI animals. Cv-PC animals were also found to have higher levels of plasma fibrinogen at the time of surgery, with unchanged prothrombin time. Cv-PC studies with fractions of C. atrox venom suggest that snake venom metalloproteinases are largely responsible for the improved hemostasis by Cv-PC. Our findings indicate that Cv-PC increases plasma fibrinogen levels and may provide a promising therapy for reducing perioperative hemorrhage in elective surgeries.