Post-stroke cognitive impairment and brain hemorrhage are augmented in hypertensive mice.

Hypertension is a major risk factor for both stroke and cognitive impairment, but it is unclear whether it may specifically affect post-stroke cognitive impairment. We assessed the effect of hypertension and/or stroke on brain injury, cognitive outcome, and the brain transcriptomic profile. C57BL/6J mice (n = 117; 3-5 mo.) received s.c. infusion of either saline or angiotensin II followed by sham surgery or photothrombotic stroke targeting the prefrontal cortex seven days later. Cognitive function was assessed with the Barnes maze and RNA sequencing was used to quantify transcriptomic changes in the brain. Angiotensin II treatment produced spontaneous hemorrhaging after stroke. In the Barnes maze, hypertensive mice that received stroke surgery had an increased escape latency compared to other groups (day 3: hypertensive + stroke = 166.6 ± 6.0 s vs. hypertensive + sham = 122.8 ± 13.8 s vs. normotensive + stroke = 139.9 ± 10.1 s vs. normotensive + sham = 101.9 ± 16.7 s), consistent with impaired cognition. RNA sequencing revealed >1500 differentially expressed genes related to neuroinflammation in hypertensive + stroke vs. normotensive + stroke, which included genes associated with apoptosis, microRNAs, autophagy, anti-cognitive biomarkers and Wnt signaling. Overall, we show that the combination of hypertension and stroke resulted in greater learning impairment and brain injury.

Deletion of a conserved genomic region associated with adolescent idiopathic scoliosis leads to vertebral rotation in mice.

Adolescent idiopathic scoliosis (AIS) is the most common form of scoliosis, in which spinal curvature develops in adolescence, and 90% of patients are female. Scoliosis is a debilitating disease that often requires bracing or surgery in severe cases. AIS affects 2%-5.2% of the population; however, the biological origin of the disease remains poorly understood. In this study, we aimed to determine the function of a highly conserved genomic region previously linked to AIS using a mouse model generated by CRISPR-CAS9 gene editing to knockout this area of the genome to understand better its contribution to AIS, which we named AIS_CRMΔ. We also investigated the upstream factors that regulate the activity of this enhancer in vivo, whether the spatial expression of the LBX1 protein would change with the loss of AIS-CRM function, and whether any phenotype would arise after deletion of this region. We found a significant increase in mRNA expression in the developing neural tube at E10.5, and E12.5, for not only Lbx1 but also other neighboring genes. Adult knockout mice showed vertebral rotation and proprioceptive deficits, also observed in human AIS patients. In conclusion, our study sheds light on the elusive biological origins of AIS, by targeting and investigating a highly conserved genomic region linked to AIS in humans. These findings provide valuable insights into the function of the investigated region and contribute to our understanding of the underlying causes of this debilitating disease.

N-Terminomic Changes in Neurons During Excitotoxicity Reveal Proteolytic Events Associated With Synaptic Dysfunctions and Potential Targets for Neuroprotection.

Excitotoxicity, a neuronal death process in neurological disorders such as stroke, is initiated by the overstimulation of ionotropic glutamate receptors. Although dysregulation of proteolytic signaling networks is critical for excitotoxicity, the identity of affected proteins and mechanisms by which they induce neuronal cell death remain unclear. To address this, we used quantitative N-terminomics to identify proteins modified by proteolysis in neurons undergoing excitotoxic cell death. We found that most proteolytically processed proteins in excitotoxic neurons are likely substrates of calpains, including key synaptic regulatory proteins such as CRMP2, doublecortin-like kinase I, Src tyrosine kinase and calmodulin-dependent protein kinase IIß (CaMKIIß). Critically, calpain-catalyzed proteolytic processing of these proteins generates stable truncated fragments with altered activities that potentially contribute to neuronal death by perturbing synaptic organization and function. Blocking calpain-mediated proteolysis of one of these proteins, Src, protected against neuronal loss in a rat model of neurotoxicity. Extrapolation of our N-terminomic results led to the discovery that CaMKIIα, an isoform of CaMKIIß, undergoes differential processing in mouse brains under physiological conditions and during ischemic stroke. In summary, by identifying the neuronal proteins undergoing proteolysis during excitotoxicity, our findings offer new insights into excitotoxic neuronal death mechanisms and reveal potential neuroprotective targets for neurological disorders.

Optimization of thermoresponsive chitosan/ß-glycerophosphate hydrogels for injectable neural tissue engineering application.

Regeneration of neural tissue and recovery of lost functions following an accident or disease to the central nervous system remains a major challenge worldwide, with limited treatment options available. The main reason for the failure of conventional therapeutic techniques to regenerate neural tissue is the presence of blood-brain barrier separating nervous system from systemic circulation and the limited capacity of self-regeneration of the nervous system. Injectable hydrogels have shown great promise for neural tissue engineering given their suitability for minimally invasive in situ delivery and tunable mechanical and biological properties. Chitosan (CS)/ß-glycerophosphate (ß-GP) hydrogels have been extensively investigated and shown regenerative potential in a wide variety of tissues such as bone and cartilage tissue engineering. However, the potential of CS/ß-GP hydrogels has never been tested for injectable neural tissue engineering applications. In the present study, CS/ß-GP hydrogels, consisting of 0.5-2% CS and 2-3% ß-GP, were prepared and characterized to investigate their suitability for injectable neural tissue engineering applications. The resulting CS/ß-GP-hydrogels showed a varying range of properties depending on the CS/ß-GP blend ratio. In particular, the 0.5%:3% and 0.75%:3% CS/ß-GP hydrogels underwent rapid gelation (3 min and 5 min, respectively) at physiological temperature (37 °C) and pH (7.4). They also had suitable porosity, osmolality, swelling behavior and biodegradation for tissue engineering. The biocompatibility of hydrogels was determined in vitro using PC12 cells, an immortalized cell line with neuronal cell-like properties, revealing that these hydrogels supported cell growth and proliferation. In conclusion, the thermoresponsive 0.5%:3% and 0.75%:3% CS/ß-GP hydrogels had the greatest potential for neural tissue engineering.

Recovery of Post-Stroke Spatial Memory and Thalamocortical Connectivity Following Novel Glycomimetic and rhBDNF Treatment.

Stroke-induced cognitive impairments remain of significant concern, with very few treatment options available. The involvement of glycosaminoglycans in neuroregenerative processes is becoming better understood and recent advancements in technology have allowed for cost-effective synthesis of novel glycomimetics. The current study evaluated the therapeutic potential of two novel glycomimetics, compound A and G, when administered systemically five-days post-photothrombotic stroke to the PFC. As glycosaminoglycans are thought to facilitate growth factor function, we also investigated the combination of our glycomimetics with intracerebral, recombinant human brain-derived neurotrophic factor (rhBDNF). C56BL/6J mice received sham or stroke surgery and experimental treatment (day-5), before undergoing the object location recognition task (OLRT). Four-weeks post-surgery, animals received prelimbic injections of the retrograde tracer cholera toxin B (CTB), before tissue was collected for quantification of thalamo-PFC connectivity and reactive astrogliosis. Compound A or G treatment alone modulated a degree of reactive astrogliosis yet did not influence spatial memory performance. Contrastingly, compound G+rhBDNF treatment significantly improved spatial memory, dampened reactive astrogliosis and limited stroke-induced loss of connectivity between the PFC and midline thalamus. As rhBDNF treatment had negligible effects, these findings support compound A acted synergistically to enhance rhBDNF to restrict secondary degeneration and facilitate functional recovery after PFC stroke.

Neuroprotective activity of new Δ3-N-acylethanolamines in a focal ischemia stroke model.

N-acylethanolamines (NAE, also called ethanolamides) are significant lipid signaling molecules with anti-inflammatory, pain-relieving, cell-protective, and anticancer properties. Here, we present the use of a hitherto unreported group of Δ3-NAE and also some Δ4- and Δ5-NAE, in in vitro and in vivo assays to gain a better understanding of their structure-bioactivity relationships. We have developed an efficient synthetic method to rapidly produce novel unlabeled and 13 C-labeled Δ3-NAE (NAE-18:5n-3, NAE-18:4n-6) and Δ4-NAE (NAE-22:5n-6). The new NAE with shorter carbon backbone structures confers greater neuroprotection than their longer carbon backbone counterparts, including anandamide (Δ5-NAE-20:4n-6) in a focal ischemia mouse model of stroke. This study highlights structure-dependent protective effects of new NAE following focal ischemia, in which some of the new NAE, administered intranasally, lead to significantly reduced infarct volume and improved recovery of limb use. The relative affinity of the new NAE toward cannabinoid receptors was assessed against anandamide, NAE-22:6n-3 and NAE-20:5n-3, which are known cannabinoid receptor ligands with high-binding constants. Among the newly synthesized NAE, Δ4-NAE-22:5n-6 shows the greatest relative affinity to cannabinoid receptors hCB1 and hCB2 , and inhibition of cyclic adenosine monophosphate activity through hCB2 compared to anandamide.

Obesity Has a Systemic Effect on Immune Cells in Naïve and Cancer-Bearing Mice.

Obesity is a major risk factor for developing cancer, with obesity-induced immune changes and inflammation in breast (BC) and colorectal cancer (CRC) providing a potential link between the two. This study investigates systemic effects of obesity on adaptive and innate immune cells in healthy and tumour-bearing mice. Immune cells from lean and obese mice were phenotyped prior to implantation of either BC (C57mg and EO771.LMB) or CRC (MC38) cells as tumour models. Tumour growth rate, tumour-infiltrating lymphocytes (TIL) and peripheral blood immune cell populations were compared between obese and lean mice. In vitro studies showed that naïve obese mice had higher levels of myeloid cells in the bone marrow and bone marrow-derived dendritic cells expressed lower levels of activation markers compared to cells from their lean counterparts. In the tumour setting, BC tumours grew faster in obese mice than in lean mice and lower numbers of TILs as well as higher frequency of exhausted T cells were observed. Data from peripheral blood showed lower levels of myeloid cells in tumour-bearing obese mice. This study highlights that systemic changes to the immune system are relevant for tumour burden and provides a potential mechanism behind the effects of obesity on cancer development and progression in patients.

The Gliopeptide ODN, a Ligand for the Benzodiazepine Site of GABAA Receptors, Boosts Functional Recovery after Stroke.

Following stroke, the survival of neurons and their ability to reestablish connections is critical to functional recovery. This is strongly influenced by the balance between neuronal excitation and inhibition. In the acute phase of experimental stroke, lethal hyperexcitability can be attenuated by positive allosteric modulation of GABAA receptors (GABAARs). Conversely, in the late phase, negative allosteric modulation of GABAAR can correct the suboptimal excitability and improves both sensory and motor recovery. Here, we hypothesized that octadecaneuropeptide (ODN), an endogenous allosteric modulator of the GABAAR synthesized by astrocytes, influences the outcome of ischemic brain tissue and subsequent functional recovery. We show that ODN boosts the excitability of cortical neurons, which makes it deleterious in the acute phase of stroke. However, if delivered after day 3, ODN is safe and improves motor recovery over the following month in two different paradigms of experimental stroke in mice. Furthermore, we bring evidence that, during the subacute period after stroke, the repairing cortex can be treated with ODN by means of a single hydrogel deposit into the stroke cavity.SIGNIFICANCE STATEMENT Stroke remains a devastating clinical challenge because there is no efficient therapy to either minimize neuronal death with neuroprotective drugs or to enhance spontaneous recovery with neurorepair drugs. Around the brain damage, the peri-infarct cortex can be viewed as a reservoir of plasticity. However, the potential of wiring new circuits in these areas is restrained by a chronic excess of GABAergic inhibition. Here we show that an astrocyte-derived peptide, can be used as a delayed treatment, to safely correct cortical excitability and facilitate sensorimotor recovery after stroke.

Frontal cortex stroke-induced impairment in spatial working memory on the trial-unique nonmatching-to-location task in mice.

Stroke-induced cognitive impairments are of significant concern, however mechanisms that underpin these impairments remain poorly understood and researched. To further characterise cognitive impairments in our frontal cortex stroke model, and to align our assessments with what is used clinically, we tested young C57BL/6J mice trained in operant touchscreen chambers to complete the trial-unique nonmatched-to-location (TUNL) task. Based on baseline performance, animals were given either stroke (n = 12) or sham (n = 12) surgery using a photothrombosis model, bilaterally targeting the frontal cortex. Upon recovery, post-stroke spatial working memory was assessed by varying the degree of separation and delay within TUNL trials. Seven weeks after surgery, animals received a prelimbic injection of the retrograde tracer cholera toxin B (CTB) to access thalamo-PFC connectivity. Tissue was then processed histologically and immunohistochemically to assess infarct volume, astrogliosis and thalamocortical connectivity. Assessment of TUNL probes revealed sensitivity to a frontal cortex stroke (separation: p = 0.0003, delay: p < 0.0001), with stroke animals taking significantly longer (p = 0.0170) during reacquisition of the TUNL task, relative to shams. CTB-positive cell counts revealed a stroke-induced loss of thalamo-PFC connectivity. In addition, quantification of reactive astrogliosis revealed a positive correlation between the degree of astrogliosis expanding into white matter tracts and the development of cognitive impairments. This study reveals a stroke-induced impairment in mice completing the TUNL task. Our findings also demonstrate a significant loss of thalamo-PFC connections and a correlation between white matter reactive astrogliosis and cognitive impairment. Future experiments will investigate therapeutic interventions in the hope of promoting functional improvement in cognition.

Visible light mediated PVA-tyramine hydrogels for covalent incorporation and tailorable release of functional growth factors.

The translation of growth factors (GFs) into clinical applications is limited by their low stability in physiological environments. Controlled GF delivery through biomaterial vehicles provides protection from proteases, targeted delivery, and longer term release profiles. However, current methods used to incorporate GFs into biomaterials still present limitations. While direct adsorption and encapsulation result in burst release, covalent incorporation provides a tailorable release profile but generally requires more complicated processes and chemical modification of the GFs. Bioaffinity methods provide long-term release profiles but fail in their specificity, resulting in GF-dependent applicability and release profiles. In the present study, we introduce tyraminated poly-vinyl-alcohol (PVA-Tyr) as a GF-delivery vehicle that can covalently incorporate native GFs through a photo-initiated cross-linking process via formation of bi-phenol bonds. Mass loss and release studies revealed that protein-loaded PVA-Tyr hydrogels had highly tailorable degradation times from 7 to 92 days, during which the covalently incorporated proteins were released in a linear fashion. The incorporation of bovine serum albumin (BSA), vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), or brain-derived growth factor (BDNF) resulted in similar incorporation efficiencies and release profiles, demonstrating the low specificity and versatility of the system. Furthermore, functional studies demonstrated that VEGF, bFGF and BDNF released from the PVA-Tyr hydrogels retained the ability to increase the metabolic activity, migration, and 3D vessel formation of endothelial cells and mesenchymal stem cells. Taken together, this demonstrates that PVA-Tyr shows high potential as a highly tailorable GF delivery tool for a range of different regenerative medicine applications.

Silk fibroin nanoscaffolds for neural tissue engineering.

The nervous system is a crucial component of the body and damages to this system, either by injury or disease, can result in serious or potentially lethal consequences. An important problem in neural engineering is how we can stimulate the regeneration of damaged nervous tissue given its complex physiology and limited regenerative capacity. To regenerate damaged nervous tissue, this study electrospun three-dimensional nanoscaffolds (3DNSs) from a biomaterial blend of silk fibroin (SF), polyethylene glycol (PEG), and polyvinyl alcohol (PVA). The 3DNSs were characterised to ascertain their potential suitability for direct implant into the CNS. The biological activity of 3DNSs was investigated in vitro using PC12 cells and their effects on reactive astrogliosis were assessed in vivo using a photothrombotic model of ischaemic stroke in mice. Results showed that the concentration of SF directly affected the mechanical characteristics and internal structure of the 3DNSs, with formulations presenting as either a gel-like structure (SF ≥ 50%) or a nanofibrous structure (SF ≤ 40%). In vitro assessment revealed increased cell viability in the presence of the 3DNSs and in vivo assessment resulted in a significant decrease in glial fibrillary acidic protein (GFAP) expression in the peri-infarct region (p < 0.001 for F2 and p < 0.05 for F4) after stroke, suggesting that 3DNSs could be suppressing reactive astrogliosis. The findings enhanced our understanding of physiochemical interactions between SF, PEG, and PVA, and elucidated the potential of 3DNSs as a potential therapeutic approach to stroke recovery, especially if these are used in conjunction with drug or cell treatment.

The Delta-Subunit Selective GABA A Receptor Modulator, DS2, Improves Stroke Recovery via an Anti-inflammatory Mechanism.

Inflammatory processes are known to contribute to tissue damage in the central nervous system (CNS) across a broad range of neurological conditions, including stroke. Gamma amino butyric acid (GABA), the main inhibitory neurotransmitter in the CNS, has been implicated in modulating peripheral immune responses by acting on GABA A receptors on antigen-presenting cells and lymphocytes. Here, we investigated the effects and mechanism of action of the delta-selective compound, DS2, to improve stroke recovery and modulate inflammation. We report a decrease in nuclear factor (NF)-κB activation in innate immune cells over a concentration range in vitro. Following a photochemically induced motor cortex stroke, treatment with DS2 at 0.1 mg/kg from 1 h post-stroke significantly decreased circulating tumor necrosis factor (TNF)-α, interleukin (IL)-17, and IL-6 levels, reduced infarct size and improved motor function in mice. Free brain concentrations of DS2 were found to be lower than needed for robust modulation of central GABA A receptors and were not affected by the presence and absence of elacridar, an inhibitor of both P-glycoprotein and breast cancer resistance protein (BCRP). Finally, as DS2 appears to dampen peripheral immune activation and only shows limited brain exposure, we assessed the role of DS2 to promote functional recovery after stroke when administered from 3-days after the stroke. Treatment with DS2 from 3-days post-stroke improved motor function on the grid-walking, but not on the cylinder task. These data highlight the need to further develop subunit-selective compounds to better understand change in GABA receptor signaling pathways both centrally and peripherally. Importantly, we show that GABA compounds such as DS2 that only shows limited brain exposure can still afford significant protection and promote functional recovery most likely via modulation of peripheral immune cells and could be given as an adjunct treatment.

Stroke Induces a BDNF-Dependent Improvement in Cognitive Flexibility in Aged Mice.

Stroke remains a leading cause of disability worldwide. Recently, we have established an animal model of stroke that results in delayed impairment in spatial memory, allowing us to better investigate cognitive deficits. Young and aged brains show different recovery profiles after stroke; therefore, we assessed aged-related differences in poststroke cognition. As neurotrophic support diminishes with age, we also investigated the involvement of brain-derived neurotrophic factor (BDNF) in these differences. Young (3-6 months old) and aged (16-21 months old) mice were trained in operant touchscreen chambers to complete a visual pairwise discrimination (VD) task. Stroke or sham surgery was induced using the photothrombotic model to induce a bilateral prefrontal cortex stroke. Five days poststroke, an additional cohort of aged stroke animals were treated with intracerebral hydrogels loaded with the BDNF decoy, TrkB-Fc. Following treatment, animals underwent the reversal and rereversal task to identify stroke-induced cognitive deficits at days 17 and 37 poststroke, respectively. Assessment of sham animals using Cox regression and log-rank analyses showed aged mice exhibit an increased impairment on VD reversal and rereversal learning compared to young controls. Stroke to young mice revealed no impairment on either task. In contrast, stroke to aged mice facilitated a significant improvement in reversal learning, which was dampened in the presence of the BDNF decoy, TrkB-Fc. In addition, aged stroke control animals required significantly less consecutive days and correction trials to master the reversal task, relative to aged shams, an effect dampened by TrkB-Fc. Our findings support age-related differences in recovery of cognitive function after stroke. Interestingly, aged stroke animals outperformed their sham counterparts, suggesting reopening of a critical window for recovery that is being mediated by BDNF.

Acute or Delayed Systemic Administration of Human Amnion Epithelial Cells Improves Outcomes in Experimental Stroke.

BACKGROUND AND PURPOSE: Human amnion epithelial cells (hAECs) are nonimmunogenic, nontumorigenic, anti-inflammatory cells normally discarded with placental tissue. We reasoned that their profile of biological features, wide availability, and the lack of ethical barriers to their use could make these cells useful as a therapy in ischemic stroke. METHODS: We tested the efficacy of acute (1.5 hours) or delayed (1-3 days) poststroke intravenous injection of hAECs in 4 established animal models of cerebral ischemia. Animals included young (7-14 weeks) and aged mice (20-22 months) of both sexes, as well as adult marmosets of either sex. RESULTS: We found that hAECs administered 1.5 hours after stroke in mice migrated to the ischemic brain via a CXC chemokine receptor type 4-dependent mechanism and reduced brain inflammation, infarct development, and functional deficits. Furthermore, if hAECs administration was delayed until 1 or 3 days poststroke, long-term functional recovery was still augmented in young and aged mice of both sexes. We also showed proof-of-principle evidence in marmosets that acute intravenous injection of hAECs prevented infarct development from day 1 to day 10 after stroke. CONCLUSIONS: Systemic poststroke administration of hAECs elicits marked neuroprotection and facilitates mechanisms of repair and recovery.

Vitamin D3 Supplementation Reduces Subsequent Brain Injury and Inflammation Associated with Ischemic Stroke.

Acute inflammation can exacerbate brain injury after ischemic stroke. Beyond its well-characterized role in calcium metabolism, it is becoming increasingly appreciated that the active form of vitamin D, 1,25-dihydroxyvitamin D3 (1,25-VitD3), has potent immunomodulatory properties. Here, we aimed to determine whether 1,25-VitD3 supplementation could reduce subsequent brain injury and associated inflammation after ischemic stroke. Male C57Bl6 mice were randomly assigned to be administered either 1,25-VitD3 (100 ng/kg/day) or vehicle i.p. for 5 day prior to stroke. Stroke was induced via middle cerebral artery occlusion for 1 h followed by 23 h reperfusion. At 24 h post-stroke, we assessed infarct volume, functional deficit, expression of inflammatory mediators and numbers of infiltrating immune cells. Supplementation with 1,25-VitD3 reduced infarct volume by 50% compared to vehicle. Expression of pro-inflammatory mediators IL-6, IL-1ß, IL-23a, TGF-ß and NADPH oxidase-2 was reduced in brains of mice that received 1,25-VitD3 versus vehicle. Brain expression of the T regulatory cell marker, Foxp3, was higher in mice supplemented with 1,25-VitD3 versus vehicle, while expression of the transcription factor, ROR-γ, was decreased, suggestive of a reduced Th17/γδ T cell response. Immunohistochemistry indicated that similar numbers of neutrophils and T cells were present in the ischemic hemispheres of 1,25-VitD3- and vehicle-supplemented mice. At this early time point, there were also no differences in the impairment of motor function. These data indicate that prior administration of exogenous vitamin D, even to vitamin D-replete mice, can attenuate infarct development and exert acute anti-inflammatory actions in the ischemic and reperfused brain.

Incorporation of an Endogenous Neuromodulatory Lipid, Oleoylethanolamide, into Cubosomes: Nanostructural Characterization.

Oleoylethanolamide (OEA) is an endogenous lipid with neuroprotective properties and the fortification of its concentration in the brain can be beneficial in the treatment of many neurodegenerative disorders. However, OEA is rapidly eliminated by hydrolysis in vivo, limiting its therapeutic potential. We hypothesize that packing OEA within a nanoparticulate system such as cubosomes, which can be used to target the blood-brain barrier (BBB), will protect it against hydrolysis and enable therapeutic concentrations to reach the brain. Cubosomes are lipid-based nanoparticles with a unique bicontinuous cubic phase internal structure. In the present study, the incorporation and chemical stability of OEA in cubosomes was investigated. Cubosomes containing OEA had a mean particle size of less than 200 nm with low polydispersity (polydispersity index <0.25). Infrared spectroscopy and high-performance liquid chromatography showed chemical stability and the encapsulation of OEA within cubosomes. Cryo-TEM and SAXS measurements were used to probe the influence of the addition of OEA on the internal structure of the cubosomes. Up to 30% w/w OEA (relative to phytantriol) could be incorporated into phytantriol cubosomes without any significant disruption of the nanostructure of the cubosomes. Combined, the results indicate that OEA-loaded cubosomes have the potential for application as a colloidal carrier for OEA, potentially preventing hydrolysis in vivo.

Sonic hedgehog stimulates neurite outgrowth in a mechanical stretch model of reactive-astrogliosis.

Although recovery following a stroke is limited, undamaged neurons under the right conditions can establish new connections and take on-board lost functions. Sonic hedgehog (Shh) signaling is integral for developmental axon growth, but its role after injury has not been fully examined. To investigate the effects of Shh on neuronal sprouting after injury, we used an in vitro model of glial scar, whereby cortical astrocytes were mechanically traumatized to mimic reactive astrogliosis observed after stroke. This mechanical trauma impaired neurite outgrowth from post-natal cortical neurons plated on top of reactive astrocytes. Addition of Shh to the media, however, resulted in a concentration-dependent increase in neurite outgrowth. This response was inhibited by cyclopamine and activated by oxysterol 20(S)-hydroxycholesterol, both of which modulate the activity of the Shh co-receptor Smoothened (Smo), demonstrating that Shh-mediated neurite outgrowth is Smo-dependent. In addition, neurite outgrowth was not associated with an increase in Gli-1 transcription, but could be inhibited by PP2, a selective inhibitor of Src family kinases. These results demonstrate that neurons exposed to the neurite growth inhibitory environment associated with a glial scar can be stimulated by Shh, with signaling occurring through a non-canonical pathway, to overcome this suppression and stimulate neurite outgrowth.

Combined ampakine and BDNF treatments enhance poststroke functional recovery in aged mice via AKT-CREB signaling.

Cerebral ischemia results in damage to neuronal circuits and lasting impairment in function. We have previously reported that stimulation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors with the ampakine, CX1837, increases brain-derived neurotrophic factor (BDNF) levels and affords significant motor recovery after stroke in young mice. Here, we investigated whether administration of CX1837 in aged (24 months old) mice was equally effective. In a model of focal ischemia, administration of CX1837 from 5 days after stroke resulted in a small gain of motor function by week 6 after stroke. Mice that received a local delivery of BDNF via hydrogel implanted into the stroke cavity also showed a small gain of function from 4 to 6 weeks after stroke. Combining both treatments, however, resulted in a marked improvement in motor function from 2 weeks after insult. Assessment of peri-infarct tissue 2 weeks after stroke revealed a significant increase in p-AKT and p-CREB after the combined drug treatment. Using the pan-AKT inhibitor, GSK-690693, or deletion of CREB from forebrain neurons using the CREB-flox/CAMKii-cre mice, we were able to block the recovery of motor function. These data suggest that combined CX1837 and local delivery of BDNF are required to achieve maximal functional recovery after stroke in aged mice, and is occurring via the AKT-GSK3-CREB signaling pathway.

Melatonin treatment following stroke induction modulates L-arginine metabolism.

The efficacy of melatonin treatment in experimental stroke has been established. Some of the neuroprotective properties have been attributed to its anti-oxidant and anti-inflammatory effects. Nitric oxide synthases (NOS) and cyclooxygenases (COX) are considered to have a significant role in the inflammatory milieu occurring in acute stroke. While previous reports have shown that pretreatment with melatonin in a stroke model can modulate NOS isoforms, the effect of post-treatment with melatonin on l-arginine metabolism has not been investigated. This study initially examined the effect of melatonin (1 nm-1 mm) on l-arginine metabolism pathways in human fibrosarcoma fibroblasts (HT-1080) fibroblasts. Evidence of neuroprotection with melatonin was evaluated in rats subjected to middle cerebral artery occlusion (MCAO). Animals were treated with three daily doses of 5 mg/kg i.p., starting 1 hr after the onset of ischemia. Constitutive NOS activity but not expression was significantly increased by in vitro exposure (72 hr) to melatonin. In addition, melatonin treatment increased arginase activity by increasing arginase II expression. In vivo studies showed that melatonin treatment after MCAO significantly inhibited inducible NOS activity and attenuated expression of the inducible isoform, resulting in decreased total NOS activity and tissue nitrite levels. COX activity was significantly reduced with melatonin treatment. The neuroprotective anti-inflammatory effects of melatonin were consistent with the substantial reduction in infarct volume throughout the cortex and striatum and recovery of mitochondrial enzyme activities. The evidence presented here suggests that modulation of l-arginine metabolism by melatonin make it a valuable neuroprotective therapy for stroke.

Angiogenesis as a predictive marker of neurological outcome following hypoxia-ischemia.

Cerebral ischemia induces angiogenesis within and around infarcted tissue. The protection of existing and growth of new blood vessels may contribute to a more favorable outcome. The present study assessed whether angiogenesis can be used as a marker for neurodegeneration/neuroprotection in a model of hypoxia-ischemia (HI). Increased CD31 immunoreactivity 7 days post-HI indicated increased angiogenesis compared to controls (P<0.001). Treatment with the GABA(A) receptor modulator, clomethiazole (CMZ; 414 mg/kg/day), normalized the level of angiogenesis compared to HI + saline (P<0.001). Conversely, the non-selective nitric oxide synthase (NOS) inhibitor, L-NAME (5 mg/kg/day), markedly decreased angiogenesis compared to controls (P<0.001). Circulating plasma levels of IL-1alpha, IL-1beta and GM-CSF were significantly elevated post-HI. CMZ treatment attenuated these increases while also stimulating IL-10 levels. L-NAME treatment did not alter IL-1alpha or IL-1beta levels, but decreased endogenous IL-10 levels and exacerbated the ischemic lesion (P<0.001). CMZ treatment has been shown to increase NOS levels, while L-NAME halted the HI-induced increase in NOS activity (P<0.001). We conclude that angiogenesis can be used as a marker of neurodegeneration/neuroprotection for cerebral HI and is correlated to NOS activity and circulating inflammatory mediators.