Most Promising Approaches to Improve Stroke Outcomes: The Stroke Treatment Academic Industry Roundtable XII Workshop.

The Stroke Treatment Academic Industry Roundtable XII included a workshop to discuss the most promising approaches to improve outcome from acute stroke. The workshop brought together representatives from academia, industry, and government representatives. The discussion examined approaches in 4 epochs: pre-reperfusion, reperfusion, post-reperfusion, and access to acute stroke interventions. The participants identified areas of priority for developing new and existing treatments and approaches to improve stroke outcomes. Although many advances in acute stroke therapy have been achieved, more work is necessary for reperfusion therapies to benefit the most possible patients. Prioritization of promising approaches should help guide the use of resources and investigator efforts.

Use of Botulinum Toxin for Limb Immobilization for Rehabilitation in Rats with Experimental Stroke.

Motor rehabilitation strategies after unilateral stroke suggest that the immobilization of the healthy, unimpaired limb can promote the functional recovery of a paretic limb. In rodents, this has been modeled using casts, harnesses, and other means of restricting the use of the non-paretic forelimb in models of experimental stroke. Here, we evaluated an alternative approach, using botulinum toxin injections to limit the function of the non-paretic forelimb. Adult male rats were subjected to permanent ligation of the left distal middle cerebral artery, resulting in right forelimb paresis. The rats were then subjected to: (1) no treatment; (2) botulinum toxin injections 1 day post stroke; or (3) cast placement 5 days post stroke. Casts were removed after 5 weeks, while the botulinum toxin injection effectively immobilized subjects for approximately the same duration. Rats with bilateral forelimb impairment due to the stroke plus casting or botulinum injections were still able to feed and groom normally. Both immobilization groups showed modest recovery following the stroke compared to those that did not receive immobilization, but the casting approach led to unacceptable levels of animal stress. The botulinum toxin approach to limb immobilization had both advantages and disadvantages over traditional physical limb immobilization. The major advantage was that it was far less stress-inducing to the subject animals and appeared to be well tolerated. A disadvantage was that the paresis took roughly 10 weeks to fully resolve, and any degree of residual paresis could confound the interpretation of the behavioral assessments.

Therapeutic hypothermia for stroke: Unique challenges at the bedside.

Therapeutic hypothermia has shown promise as a means to improving neurological outcomes at several neurological conditions. At the clinical level, it has been shown to improve outcomes in comatose survivors of cardiac arrest and in neonatal hypoxic ischemic encephalopathy, but has yet to be convincingly demonstrated in stroke. While numerous preclinical studies have shown benefit in stroke models, translating this to the clinical level has proven challenging. Major obstacles include cooling patients with typical stroke who are awake and breathing spontaneously but often have significant comorbidities. Solutions around these problems include selective brain cooling and cooling to lesser depths or avoiding hyperthermia. This review will cover the mechanisms of protection by therapeutic hypothermia, as well as recent progress made in selective brain cooling and the neuroprotective effects of only slightly lowering brain temperature. Therapeutic hypothermia for stroke has been shown to be feasible, but has yet to be definitively proven effective. There is clearly much work to be undertaken in this area.

Sexual dimorphism in immune cell responses following stroke.

Recent bodies of work in regard to stroke have revealed significant sex differences in terms of risk and outcome. While differences in sex hormones have been the focus of earlier research, the reasons for these differences are much more complex and require further identification. This review covers differences in sex related immune responses with a focus on differences in immune cell composition and function. While females are more susceptible to immune related diseases, they seem to have better outcomes from stroke at the experimental level with reduced pro-inflammatory responses. However, at the clinical level, the picture is much more complex with worse neurological outcomes from stroke. While the use of exogenous sex steroids can replicate some of these findings, it is apparent that many other factors are involved in the modulation of immune responses. As a result, more research is needed to better understand these differences and identify appropriate interventions and risk modification.

Cerebral small vessel disease alters neurovascular unit regulation of microcirculation integrity involved in vascular cognitive impairment.

Cerebral small vessel disease (CSVD) is a generic term used for intracranial vascular disorders caused by the structural changes of cerebral microvessels, including the small arteries, arterioles, capillaries and venules. CSVD exhibits various neuroimaging features and is associated clinical characteristics. Although CSVD is recognized as the leading cause of vascular cognitive impairment (VCI), the underlying mechanism(s) remains elusive. Growing evidence indicates a significant association between altered neurovascular unit (NVU) functioning and the pathophysiology of evolving CSVD-induced VCI. Therefore, research is required to understand how NVU dysregulation contributes to cognitive impairment due to CSVD. In this review, we describe the link between the neuroimaging focal lesions and cognitive alterations. We also discuss the potential pathological role of NVU dysregulation in the entry of pathogens from the blood into the parenchyma by altering the blood-brain barrier (BBB), affecting the cerebral microvascular and consequently cause VCI. Next, we review the coupling of neural activity with cerebral blood flow to control the microvascular perfusion; and the disrupted clearance of metabolic byproducts with CSF-ISF exchange via perivascular pathways and glymphatic system. Finally, we discussed the possible therapeutic interventions in CSVD.

Fibrinogen is an Independent Risk Factor for White Matter Hyperintensities in CADASIL but not in Sporadic Cerebral Small Vessel Disease Patients.

The relationship between fibrinogen and white matter hyperintensities (WMHs) are inconsistent. Whether there are different relationships between WMHs and fibrinogen in disparate subtypes of cerebral small vessel disease (CSVD) remains unknown. Here, we investigated the roles of plasma fibrinogen in sporadic CSVD (sCSVD) and Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy (CADASIL) patients. We performed a cross-sectional study that included 74 CSVD patients (19 CADASIL and 55 sporadic) and 74 age- and gender-matched healthy controls (HCs). Plasma fibrinogen was determined, and the severity of WMHs in CSVD patients was rated according to Fazekas scales. Univariate analysis and ordinal logistic regression were performed to evaluate the relationship between fibrinogen and the severity of WMHs in CSVD. Both CADASIL and sCSVD patients showed significantly higher plasma fibrinogen levels than HCs. No significant difference in the plasma fibrinogen level was observed between CADASIL and sCSVD. Univariate analysis and ordinal logistic regression indicated that fibrinogen is an independent risk factor for the severity of WMHs in CADASIL patients (odds ratio [OR] =1.064; 95% Confidence interval (CI, 1.004-1.127); p =0.037). However, age (odds ratio [OR] =1.093; 95% CI (1.033-1.156); P = 0.002), but not fibrinogen (odds ratio [OR] =1.004; 95% CI (0.997-1.011); P=0.262), is an independent risk factor for the severity of WMHs in sCSVD patients. Our results suggest that high levels of plasma fibrinogen are associated with the severity of WMHs in CADASIL but not in sCSVD patients, indicating that the role of fibrinogen may be different in disparate subtypes of CSVD. A better understanding of fibrinogen may yield insights into the pathogenesis of CSVD.

Clinical perspectives on ischemic stroke.

Treatments for acute stroke have improved over the past years, but have largely been limited to revascularization strategies. The topic of neuroprotection, or strategies to limit brain tissue damage or even reverse it, has remained elusive. Thus, the clinical mainstays for stroke management have focused on prevention. The lack of clinical translation of neuroprotective therapies which have shown promise in the laboratory may, in part, be due to a historic inattention to comorbidities suffered by a majority of stroke patients. With the advent of more stroke models that include one or more relevant comorbidities, it may be possible to identify effective treatments that may translate into new treatments at the clinical level. In the meantime, we review comorbidities in stroke patients, modification of stroke risk factors and available acute stroke treatments in the clinic.

Cystatin C is a potential predictor of unfavorable outcomes for cerebral ischemia with intravenous tissue plasminogen activator treatment: A multicenter prospective nested case-control study.

BACKGROUND AND PURPOSE: The aim of this study was to explore whether cystatin C (CysC) could be used as a potential predictor of clinical outcomes in acute ischemic stroke (AIS) patients treated with intravenous tissue plasminogen activator (IV-tPA). METHODS: We performed an observational study including a retrospective analysis of data from 125 AIS patients with intravenous thrombolysis. General linear models were applied to compare CysC levels between groups with different outcomes; logistic regression analysis and receiver-operating characteristic curves were adopted to identify the association between CysC and the therapeutic effects. RESULTS: Compared with the "good and sustained benefit" (GSB) outcome group (defined as ≥4-point reduction in National Institutes of Health Stroke Scale or a score of 0-1 at 24 h and 7 days) and the "good functional outcome" (GFO) group (modified Rankin Scale score 0-2 at 90 days), serum CysC baseline levels were increased in the non-GSB and non-GFO groups. Logistic regression analysis found that CysC was an independent negative prognostic factor for GSB (odds ratio [OR] 0.010; p = 0.005) and GFO (OR 0.011; p = 0.021) after adjustment for potential influencing factors. Receiver-operating characteristic curves showed the CysC-involved combined models provided credible efficacy for predicting post-90-day favorable clinical outcome (area under the curve 0.86; p < 0.001). CONCLUSIONS: Elevated serum CysC is independently associated with unfavorable clinical outcomes after IV-tPA therapy in AIS. Our findings provide new insights into discovering potential mediators for neuropathological process or treatment in stroke.

Multimodal analysis of gene expression from postmortem brains and blood identifies synaptic vesicle trafficking genes to be associated with Parkinson's disease.

OBJECTIVE: We aimed to identify key susceptibility gene targets in multiple datasets generated from postmortem brains and blood of Parkinson's disease (PD) patients and healthy controls (HC). METHODS: We performed a multitiered analysis to integrate the gene expression data using multiple-gene chips from 244 human postmortem tissues. We identified hub node genes in the highly PD-related consensus module by constructing protein-protein interaction (PPI) networks. Next, we validated the top four interacting genes in 238 subjects (90 sporadic PD, 125 HC and 23 Parkinson's Plus Syndrome (PPS)). Utilizing multinomial logistic regression analysis (MLRA) and receiver operating characteristic (ROC), we analyzed the risk factors and diagnostic power for discriminating PD from HC and PPS. RESULTS: We identified 1333 genes that were significantly different between PD and HCs based on seven microarray datasets. The identified MEturquoise module is related to synaptic vesicle trafficking (SVT) dysfunction in PD (P < 0.05), and PPI analysis revealed that SVT genes PPP2CA, SYNJ1, NSF and PPP3CB were the top four hub node genes in MEturquoise (P < 0.001). The levels of these four genes in PD postmortem brains were lower than those in HC brains. We found lower blood levels of PPP2CA, SYNJ1 and NSF in PD compared with HC, and lower SYNJ1 in PD compared with PPS (P < 0.05). SYNJ1, negatively correlated to PD severity, displayed an excellent power to discriminating PD from HC and PPS. CONCLUSIONS: This study highlights that SVT genes, especially SYNJ1, may be promising markers in discriminating PD from HCs and PPS.

Heat Shock Protein 70 (HSP70) Induction: Chaperonotherapy for Neuroprotection after Brain Injury.

The 70 kDa heat shock protein (HSP70) is a stress-inducible protein that has been shown to protect the brain from various nervous system injuries. It allows cells to withstand potentially lethal insults through its chaperone functions. Its chaperone properties can assist in protein folding and prevent protein aggregation following several of these insults. Although its neuroprotective properties have been largely attributed to its chaperone functions, HSP70 may interact directly with proteins involved in cell death and inflammatory pathways following injury. Through the use of mutant animal models, gene transfer, or heat stress, a number of studies have now reported positive outcomes of HSP70 induction. However, these approaches are not practical for clinical translation. Thus, pharmaceutical compounds that can induce HSP70, mostly by inhibiting HSP90, have been investigated as potential therapies to mitigate neurological disease and lead to neuroprotection. This review summarizes the neuroprotective mechanisms of HSP70 and discusses potential ways in which this endogenous therapeutic molecule could be practically induced by pharmacological means to ultimately improve neurological outcomes in acute neurological disease.

Vascular, inflammatory and metabolic risk factors in relation to dementia in Parkinson's disease patients with type 2 diabetes mellitus.

There are limited data on vascular, inflammatory, metabolic risk factors of dementia in Parkinson's disease (PD) with type 2 diabetes mellitus (DM) (PD-DM). In a study of 928 subjects comprising of 215 PD with DM (including 31 PD-DM with dementia, PD-DMD), 341 PD without DM (including 31 PD with dementia, PDD) and 372 DM without PD (including 35 DM with dementia, DMD) patients, we investigated if vascular, inflammatory, metabolic, and magnetic resonance imaging (MRI) markers were associated with dementia in PD-DM. Lower fasting blood glucose (FBG<5mmol/L, OR=4.380; 95%CI: 1.748-10.975; p=0.002), higher homocysteine (HCY>15µmol/L, OR=3.131; 95%CI: 1.243-7.888; p=0.015) and hyperlipidemia (OR=3.075; 95%CI: 1.142-8.277; p=0.026), increased age (OR=1.043; 95%CI: 1.003-1.084; p=0.034) were the most significant risk factors in PDD patients. Lower low-density lipoprotein cholesterol (LDL-C<2mmol/L, OR=4.499; 95%CI: 1.568-12.909; p=0.005) and higher fibrinogen (>4g/L, OR=4.066; 95%CI: 1.467-11.274; p=0.007) were the most significant risk factors in PD-DMD patients. The area under the curve (AUC) for fibrinogen and LDL-C was 0.717 (P=0.001), with a sensitivity of 80.0% for the prediction of PD-DMD.In summary, we identified several factors including LDL-C and fibrinogen as significant risk factors for PD-DMD and these may have prognostic and treatment implications.

Heat shock protein signaling in brain ischemia and injury.

Heat shock proteins (HSPs) are chaperones that catalyze the refolding of denatured proteins. In addition to their ability to prevent protein denaturation and aggregation, the HSPs have also been shown to modulate many signaling pathways. Among HSPs, the inducible 70 kDa HSP (HSP70) has especially been shown to improve neurological outcome in experimental models of brain ischemia and injury. HSP70 can modulate various aspects of the programmed cell death pathways and inflammation. This review will focus on potential mechanisms of the neuroprotective effects of HSP70 in stroke and brain trauma models. We also comment on potential ways in which HSP70 could be translated into clinical therapies.

Models of poststroke depression and assessments of core depressive symptoms in rodents: How to choose?

Our previous studies have indicated that depression and declined cognition have been involved in some neurodegenerative diseases including Stroke, Parkinson's diseases and Vascular Parkinsonism. Post-stroke depression (PSD) is the most common psychiatric disorder following a stroke and has high morbidity and mortality. Studies on PSD are increasingly common, but the specific mechanisms remain unknown. Current research mainly includes clinical and animal aspects. Questionnaires and peripheral blood examination are two of the most common methods used to study clinical PSD. The results of questionnaires are influenced by multiple factors such as disease history, education background, occupation, economic status, family relationships and social support. There are certain limitations to blood sample testing; for example, it is influenced by cerebrovascular diseases and some other disruptions of the internal environment. It is difficult for either method to fully clarify the pathophysiological mechanism of PSD. Animal models provide alternative methods to further understand the pathophysiological mechanisms of PSD, such as the involvement of neuronal circuits and cytokines. More than ten animal models of PSD have been developed, and new models are constantly being introduced. Therefore, it is important to choose the appropriate model for any given study. In this paper, we will discuss the characteristics of the different models of PSD and comment on the advantages and disadvantages of each model, drawing from research on model innovation. Finally, we briefly describe the current assessment methods for the core symptoms of PSD models, point out the shortcomings, and present the improved sucrose preference test as a rational evaluation of anhedonia.

Plasma Lipoprotein-associated Phospholipase A2 and Superoxide Dismutase are Independent Predicators of Cognitive Impairment in Cerebral Small Vessel Disease Patients: Diagnosis and Assessment.

Lipoprotein-associated phospholipase A2 (Lp-PLA2) and superoxide dismutase (SOD) are linked to regulating vascular/neuro-inflammation and stroke. Using a retrospective design, we investigated whether circulating Lp-PLA2 and SOD in cerebral small vessel disease (CSVD) patients were associated with cognitive impairment. Eighty-seven CSVD patients were recruited. Plasma Lp-PLA2 and SOD were determined, and cognitive status was measured by the Mini-Mental State Examination (MMSE) and Montreal Cognitive Assessment (MoCA). The severity of white matter hypoerintensities (WMHs) in CSVD patients was rated according to Fazekas scales, and Lp-PLA2/SOD levels and MMSE/MoCA were compared. Multiple linear regressions were used to evaluate the relationship between Lp-PLA2 and SOD and the cognitive impairment. Ordinal logistic regression and generalized linear models (OLRGLMs) were applied to confirm whether Lp-PLA2 and SOD are independent risk factors for cognitive impairment in CVSD. Lp-PLA2 and SOD with mild or severe cognitive impairment were lower than those with normal congnition. Lp-PLA2 and SOD in CSVD patients with severe WMHs were significantly lower than those with mild or moderate WMH lesions. We noted positive linear associations of Lp-PLA and SOD with cognitive impairment in CSVD, independent of LDL-C. OLRGLMs confirmed that Lp-PLA2 and SOD were independent risk factors of cognitive impairment in CSVD. Lp-PLA2 and SOD are independently associated with cognitive impairment and WMH lesion, and may be useful for the rapid evaluation of cognitive impairment in CSVD. Lp-PLA2/SOD are modifiable factors that may be considered as therapeutic targets for preventing cognitive impairment in CSVD.

The role of NOX inhibitors in neurodegenerative diseases.

Oxidative stress is a key player in both chronic and acute brain disease due to the higher metabolic demand of the brain. Among the producers of free radicals, NADPH-oxidase (NOX) is a major contributor to oxidative stress in neurological disorders. In the brain, the superoxide produced by NOX is mainly found in leukocytes. However, recent studies have reported that it can be found in several other cell types. NOX has been reported to regulate neuronal signaling, memory processing, and central cardiovascular homeostasis. However, overproduction of NOX can contribute to neurotoxicity, CNS degeneration, and cardiovascular disorders. Regarding the above functions, NOX has been shown to play a crucial role in chronic CNS diseases like Parkinson's disease (PD), Alzheimer's disease (AD), Huntington's disease (HD), multiple sclerosis (MS), and amyotrophic lateral sclerosis (ALS), and in acute CNS disorders such as stroke, spinal cord injury, traumatic brain injury (TBI), and related cerebrovascular diseases. NOX is a multi-subunit complex consisting of two membrane-associated and four cytosolic subunits. Thus, in recent years, inhibition of NOX activity has drawn a great deal of attention from researchers in the field of treating chronic and acute CNS disorders and preventing secondary complications. Mounting evidence has shown that NOX inhibition is neuroprotective and that inhibiting NOX in circulating immune cells can improve neurological disease conditions. This review summarizes recent studies on the therapeutic effects and pharmacological strategies regarding NOX inhibitors in chronic and acute brain diseases and focuses on the hurdles that should be overcome before their clinical implementation.

Therapeutic Hypothermia and Neuroprotection in Acute Neurological Disease.

Therapeutic hypothermia has consistently been shown to be a robust neuroprotectant in many labs studying different models of neurological disease. Although this therapy has shown great promise, there are still challenges at the clinical level that limit the ability to apply this routinely to each pathological condition. In order to overcome issues involved in hypothermia therapy, understanding of this attractive therapy is needed. We review methodological concerns surrounding therapeutic hypothermia, introduce the current status of therapeutic cooling in various acute brain insults, and review the literature surrounding the many underlying molecular mechanisms of hypothermic neuroprotection. Because recent work has shown that body temperature can be safely lowered using pharmacological approaches, this method may be an especially attractive option for many clinical applications. Since hypothermia can affect multiple aspects of brain pathophysiology, therapeutic hypothermia could also be considered a neuroprotection model in basic research, which would be used to identify potential therapeutic targets. We discuss how research in this area carries the potential to improve outcome from various acute neurological disorders.

Cofilin-actin rod formation in experimental stroke is attenuated by therapeutic hypothermia and overexpression of the inducible 70 kD inducible heat shock protein (Hsp70).

BACKGROUND AND PURPOSE: Cofilin-actin rods are covalently linked aggregates of cofilin-1 and actin. Under ischemic conditions, these rods have been observed in neuronal dendrites and axons and may contribute to the loss of these processes. Hypothermia (Hypo) and the 70 kD inducible heat shock protein (Hsp70) are both known to improve outcomes after stroke, but the mechanisms are uncertain. Here, we evaluated the effect of these factors on cofilin-actin rod formation in a mouse model of stroke. MATERIALS AND METHODS: Mice were subjected to distal middle cerebral artery occlusion (dMCAO) and treated with Hypo using a paradigm previously shown to be neuroprotective. We similarly studied mice that overexpressed transgenic (Tg) or were deficient knockout (Ko) in the inducible 70 kDa heat shock protein (Hsp70), also previously shown to be protective by our group and others. Cofilin-actin rod formation was assessed by histological analysis at 4 and 24 h after dMCAO. Its expression was analyzed in three different regions, namely, infarct core (the center of the infarct), middle cerebral artery (MCA) borderzone (the edge of the brain regions supplied by the MCA), and the ischemic borderzone (border of ischemic lesion). Ischemic lesion size and neurological deficits were also assessed. RESULTS: Both Hypo-treated and Hsp70 Tg mice had smaller lesion sizes and improved neurological outcomes, whereas Hsp70 Ko mice had larger lesion sizes and worsened neurological outcomes. Cofilin-actin rods were increased after stroke, but were reduced by therapeutic Hypo and in Hsp70 Tg mice. In contrast, cofilin-actin rods were increased in ischemic brains of Hsp70 Ko mice. CONCLUSIONS: Cofilin-actin rod formation was suppressed under the conditions of neuroprotection and increased under circumstances where outcome was worsened. This suggests that cofilin-actin rods may act to participate in or exacerbate ischemic pathology and warrants further study as a potential therapeutic target.

Microglial Calcium Release-Activated Calcium Channel Inhibition Improves Outcome from Experimental Traumatic Brain Injury and Microglia-Induced Neuronal Death.

Store-operated Ca2+ entry (SOCE) mediated by calcium release-activated calcium (CRAC) channels contributes to calcium signaling. The resulting intracellular calcium increases activate calcineurin, which in turn activates immune transcription factor nuclear factor of activated T cells (NFAT). Microglia contain CRAC channels, but little is known whether these channels play a role in acute brain insults. We studied a novel CRAC channel inhibitor to explore the therapeutic potential of this compound in microglia-mediated injury. Cultured microglial BV2 cells were activated by Toll-like receptor agonists or IFNγ. Some cultures were treated with a novel CRAC channel inhibitor (CM-EX-137). Western blots revealed the presence of CRAC channel proteins STIM1 and Orai1 in BV2 cells. CM-EX-137 decreased nitric oxide (NO) release and inducible nitric oxide synthase (iNOS) expression in activated microglia and reduced agonist-induced intracellular calcium accumulation in microglia, while suppressing inflammatory transcription factors nuclear factor kappa B (NF-κB) and nuclear factor of activated T cells (NFAT). Male C57/BL6 mice exposed to experimental brain trauma and treated with CM-EX-137 had decreased lesion size, brain hemorrhage, and improved neurological deficits with decreased microglial activation, iNOS and Orai1 and STIM1 levels. We suggest a novel anti-inflammatory approach for managing acute brain injury. Our observations also shed light on new calcium signaling pathways not described previously in brain injury models.