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Objective: CD36 regulates biological and pathological processes including inflammation, resolution, phagocytosis, and angiogenesis. Highly expressed in monocytes/macrophages (MM), MM CD36 has been implicated in both detrimental and beneficial effects in acute stroke outcome and recovery. Using myeloid specific deficiency of CD36 (CD36KO MM ) mice, the present study addresses the role of MM CD36 on subset composition of MM in the acute and recovery phases of stroke. Methods: WT (CD36 fl/fl ) and CD36KO MM (M/F, 12w old) mice were subjected to transient focal ischemia. Brain immune cells were collected at 3d, 7d and 2m (n=8-12/timepoint). Infarct volume and hemispheric swelling were measured at 3d (n=13/genotype). In the isolated brain immune cells, CD11b+ cells were further distinguished by CD45 Hi and CD45 Low subsets. The extent of MM trafficking was determined by an intravenous infusion of control GFP+ splenocytes into the stroked WT and CD36KO MM mice 1d prior to sacrifice and MM subset analyses. Results: Compared to WT mice, CD36KO MM mice showed no difference in CD45 Low subset at 3d, 7d and 2m after stroke. However, CD36KO MM mice displayed a significant reduction of CD45 Hi subset, presumably infiltrating cells, at these time points (n=4-8 /group, P MM mice resulted in an increase of GFP+ cells in CD45 Low but a profound reduction in CD45 Hi subset. Despite the near absence of CD45 Hi and increased CD45 Low population in the post-ischemic brain, CD36KO MM mice displayed exacerbation of brain injury (15.1±13.7 vs 25.8±17.5 mm 3 , p Conclusion: The observed reduction of CD45 Hi subset and exacerbation of brain injury in CD36KO MM mice suggests a beneficial role of infiltrated CD45 Hi MMs in limiting stroke injury. The sustained trafficking at 2m indicates potential involvement of MM CD36 in the recovery process. Further studies to assess long-term outcomes of CD36KO MM mice will provide critical insights on the role of macrophage CD36 in stroke recovery.

In light of repeated translational failures with preclinical neuroprotection-based strategies, this preclinical study reevaluates brain swelling as an important pathological event in diabetic stroke and investigates underlying mechanism of the comorbidity-enhanced brain edema formation. Type 2 (mild), type 1 (moderate), and mixed type 1/2 (severe) diabetic mice were subjected to transient focal ischemia. Infarct volume, brain swelling, and IgG extravasation were assessed at 3 days post-stroke. Expression of vascular endothelial growth factor (VEGF)-A, endothelial-specific molecule-1 (Esm1), and the VEGF receptor 2 (VEGFR2) was determined in the ischemic brain. Additionally, SU5416, a VEGFR2 inhibitor, was treated in the type 1/2 diabetic mice, and stroke outcomes were determined. All diabetic groups displayed bigger infarct volume and brain swelling compared to nondiabetic mice, and the increased swelling was disproportionately larger relative to infarct enlargement. Diabetic conditions significantly increased VEGF-A, Esm1, and VEGFR2 expressions in the ischemic brain compared to nondiabetic mice. Notably, in diabetic mice, VEGFR2 mRNA levels were positively correlated with brain swelling, but not with infarct volume. Treatment with SU5416 in diabetic mice significantly reduced brain swelling. The study shows that brain swelling is a predominant pathological event in diabetic stroke and that an underlying event for diabetes-enhanced brain swelling includes the activation of VEGF signaling. This study suggests consideration of stroke therapies aiming at primarily reducing brain swelling for subjects with diabetes.

Background and purpose Although progression of small deep subcortical infarct (PSDI) comprises 12% to 36% of all small deep subcortical infarcts, the therapy for progression is not clear. This study investigated whether induced-hypertension therapy using phenylephrine is a useful therapy for PSDI. Methods A group of 2427 consecutive patients, diagnosed with stroke at a tertiary hospital over a period of 4 years was reviewed retrospectively. We analyzed patients with small deep subcortical infarct using clinical, laboratory, and pulse wave velocity (PWV). PSDI is defined as one or more increase in the motor score according to the NIHSS. Good outcome was designated as a modified Rankin scale of 0 to 2 at discharge. Results Among all 662 patients who had a small deep subcortical infarct, 66 patients experienced motor progression (9.97%). The induced-hypertension therapy group (n = 25) received phenylephrine, and the conventional group (n = 41) received anticoagulation therapy such as heparin, volume expansion, or both. Although there were no significant differences in baseline clinical and laboratory findings, the PSDI group showed a significantly more frequent decrease in blood pressure at progression ( P P = 0.001). The phenylephrine group ( vs the conventional group) had a lower NIHSS score ( P = 0.036) and good outcome at discharge ( P = 0.004). In multiple regression analysis, PWV (OR, 1.004 per 1-cm/s increase; 95% CI, 1.001–1.008; P = 0.018) was an independent predictor of good outcome in the phenylephrine group. A side effect of phenylephrine treatment was dysuria (n = 1). Conclusions The present study suggests that vascular stiffness can be not only a predictor for PSDI but also a predictor of motor improvement after induced-hypertension therapy using phenylephrine in lacunar stroke.

Background and Purpose— Stroke-induced acute severe body weight (BW) loss is associated with a high rate of mortality during a critical poststroke period. Several interventions to reduce weight loss, however, have not been successful. Currently, the biological significance of this extraordinary catabolic process is not well understood. Spleen-derived monocytes/macrophages (MMs) are the major immune cells recruited to the injured brain. The trafficking of MMs has been shown to be important for tissue repair and recovery. The purpose of the study is to investigate whether the BW reduction is essential for MM-mediated immune response for mice to survive and whether a corticosterone-mediated catabolic event underlies the processes. Methods— C57BL/6 male mice (12-week-old) were subjected to transient middle cerebral artery occlusion. BW, total MMs, and their Ly-6C high and Ly-6C low subsets were determined in the spleen, blood, and the brain in poststroke mice. Poststroke survival rate and MM subsets were determined in mice with adrenalectomy, sham-adrenalectomy, and adrenalectomy mice supplemented with corticosterone. Results— Stroke reduced BW with a maximum reduction at day 3 poststroke (17.2±5.2%). The reduction at day 3 was positively linked to injury severity and selective depletion of MMs, but no other types of immune cells, in the spleen. Notably, the splenic MM depletion was significantly greater in mice with severe BW reduction (≥18% at day 3). In the blood, stroke depleted circulating MMs to a similar degree in animals with moderate and severe BW loss. Ly-6C+ monocyte infiltration in the poststroke brain was greater in mice with severe BW loss. Blocking the catabolic process by adrenalectomy significantly increased poststroke mortality, but the mortality was partially rescued by corticosterone supplement in adrenalectomy mice. Conclusions— Stroke-induced BW loss facilitates MM-mediated immune response, and the adrenal corticosterone-mediated catabolic process is necessary for poststroke survival. Visual Overview— An online visual overview is available for this article.

The lack of inclusion of comorbidities in animal models of stroke may underlie the limited development of therapy in stroke. Previous studies in mice deficient of CD36, an immune receptor, indicated its contribution to stroke-induced inflammation and injury in hyperlipidemic conditions. The current study, therefore, tested whether pharmacological inhibition of CD36 provides neuroprotection in hyperlipidemic stroke. The hyperlipidemic mice subjected to stroke showed an exacerbation of infarct size and profound brain swelling. However, post-stroke treatment with CD36 inhibitors did not reduce, and in some cases worsened, acute stroke outcome, suggesting potential benefits of elevated CD36 in the post-stroke brain in a hyperlipidemic condition. On the other hand, chronic treatment of a CD36 inhibitor prior to stroke significantly reduced stroke-induced brain swelling. There was a trend toward infarct reduction, although it did not reach statistical significance. The observed benefit of preventative CD36 inhibition is in line with previously reported smaller infarct volume and swelling in CD36 KO mice. Thus, the current findings suggest that insights gained from the genetic models should be carefully considered before the implementation of pharmacological interventions, as a potential therapeutic strategy may depend on preventative treatment or a post-stroke acute treatment paradigm.

This study aimed to measure serum 25-hydroxyvitamin D [25(OH)D] concentrations, bone mineral density (BMD), and the parameters of bone metabolism in amyotrophic lateral sclerosis (ALS) patients, and their correlation with survival.We retrospectively analysed data of 100 ALS patients who consecutively visited a single referral ALS clinic between January and December 2011. Sex; age and site of symptom onset; and death were recorded. Serum 25(OH)D concentration was dichotomized as10ng/ml and ≥10ng/ml.There was absent relationship between serum 25(OH)D and concentration of bone turnover parameters or between 25(OH)D and BMD. According to the results of the Kaplan-Meier analysis with log-rank test, the survival rates of patients without (≥10ng/ml) and of those with severe vitamin D deficiency (10ng/ml) were not significantly different. Cox regression analysis showed that a poor prognosis was most correlated with older age at onset and bulbar onset after adjustment for all the clinical factors.In conclusion, vitamin D levels were not correlated to other bone markers and survival in a clinic population of ALS patients.

Background and PurposezzEvaluating respiratory function is important in neuromuscular diseases. This study explored the reference ranges of the maximal inspiratory pressure (MIP), maximal expiratory pressure (MEP), and sniff nasal inspiratory pressure (SNIP) in healthy adults, and applied them to amyotrophic lateral sclerosis (ALS) patients. MethodszzMIP, MEP, and SNIP were measured in 67 healthy volunteers aged from 21 to 82 years. Reference ranges were evaluated by multivariate regression analysis using the generalized additive modeling of location, scale, and shape method. Thirty-six ALS patients were reviewed retrospectively, and abnormal values of MIP, MEP, and SNIP were determined according to the reference ranges. ResultszzMIP, MEP, and SNIP were abnormal in 57.1%, 51.4%, and 25.7% of the ALS patients, respectively. MIP and SNIP were significantly correlated with the degree of restrictive pattern and respiratory symptoms. The ALS Functional Rating Scale-Revised score was cor related with SNIP. ConclusionszzThis study has provided the reference range of respiratory muscle strength in healthy adults. This range is suitable for evaluating respiratory function in ALS patients. Key Wordszz maximal inspiratory pressure, maximal expiratory pressure, sniff nasal inspiratory pressure, amyotrophic lateral sclerosis.

Objective: Post-stroke remote limb conditioning (PSLC) has been shown protective effect in preclinical and clinical studies. However, underlying protective mechanisms are multiple but not clearly defined. Although inflammation exacerbates stroke injury, studies showed inflammation also plays a key role in conditioning-mediated protection. This study investigated if PSLC induces changes of monocyte/macrophage (MM) subsets and the changes contribute to PSLC-induced functional benefits. Method: C57BL/6 male mice (12-week-old) were subjected to transient middle cerebral artery occlusion (MCAO) for 30 min. PSLC was induced 2h after MCAO by applying 5 cycles of 5 min inflation (200 mmHg) and 5 min deflation with a cuff on left hind limb. Mononuclear cells were isolated from the spleen, blood and brain at 3D and analyzed by flow cytometer. Acute injury size was measured at 3D. Motor and gait functions were assessed up to 4-month using Rotarod and Catwalk. Additionally, naïve spleen cells were treated sera obtained from sham-conditioned or PSLC animals, and MM subset shifts were determined. Results: PSLC significantly decreased anti-inflammatory (Ly-6C low ) monocytes without changing pro-inflammatory (Ly-6C high ) subset in blood, while there were no changes in the spleen (Fig A). Similarly, PSLC decreased Ly-6C low and increased Ly-6C high subset in the stroked brain (Fig B). The shift to a pro-inflammatory MM subset did not attenuate brain injury size but was associated with improved motor and gait function (Fig D). PSLC serum reduced Ly6-C low and increased Ly-6C high subset in splenocytes (Fig C). There was bigger MM shift and greater behavioral benefits of PSLC in animals with bigger injury (Fig E&F). Conclusion: The study demonstrates a contributing role of pro-inflammatory monocytes to PSLC-induced functional benefits in chronic stroke. This study provides a potential application of PSLC as a neuroimmune-based strategy for ischemic stroke patients.

There is a general consensus that animal models do not perfectly represent human conditions. This is not derived from any specific event, such as an ischemic episode, but rather reflects inherent differences in the physiology, anatomy, and metabolism among different species. Disparities among species also arise from differences in assessing functional recovery in stroke. Humans place a priority in regaining function in speech, cognition, and limb control, but these functions are not always readily translatable in animal models. Moreover, because functional recovery depends on the stroke type and location, induction of an ischemic lesion in a predefined area of an animal model, at best, reflects only a subtype of human stroke. Nevertheless, certain clinical pathophysiology features of stroke, such as acute outcomes and inflammatory/immune responses, are shared in animal models.1 Combined with complementary knowledge from in vitro studies, animal models can provide valuable insight into stroke pathology at a systems level. Analyses of translational inefficiencies in stroke model systems have led to the identification of underlying issues, which include, but are not limited to, rigor in experimental design, internal and external validity, negative bias, and insufficient statistical power. Together, these inefficiencies have steered collaborative international efforts in preclinical research to overcome these challenges.2 An additional issue of importance is the insufficient understanding of stroke pathophysiology beyond the acute phase and the primary reliance on infarct volume in reporting acute outcomes. Together with a lack of inclusion of comorbidities in animal models of stroke, the development of clinical protocols based on findings from normal metabolic animals may contribute to translational failures. In light of a recent concerted effort to address the future of translational stroke research,3 this review focuses on underlying translational issues and discusses insights gained from stroke models inclusive of comorbidities in the preclinical literature. ### Stroke Pathology: A Moving Target With Multiple Components Despite …

Methotrexate (MTX) is a necessary antimetabolite for the treatment of oncological disorders in all age groups. It was first introduced in 1948 for treatment of childhood lymphocytic leukemia [1]. Intrathecal methotrexate is used for prophylaxis and treatment of meningeal leukemia. Neurological adverse effects of methotrexate are not uncommon. Chronic leukoencephalopathy is the most serious neurotoxicity, particularly when it is administered intrathecally in combination with cranial radiation therapy, and is characterized by insidious evolution of dementia, pseudobulbar palsy or ataxia with widespread white matter changes. On the other hand, acute neurological toxicity is rare and not well understood. The diagnosis of acute focal symptoms from MTX treatment is difficult and sometimes challenging in patients who show restriction on diffusion-weighted image. Herein, we report on a case of acuteMTX leukoencephalopathywith a unique radiological finding.

Background and Purpose: Longitudinal imaging studies in stroke demonstrated that infarct development is a dynamic process and it matures over several days, even weeks depending on the severity of stroke. However, due to high cost and multiple exposure of anesthesia associated with repeated imaging, infarct volume has been typically measured at a fixed time point in preclinical studies, which prevent further assessment of behaviors during stroke recovery. In identifying a non-invasive physiological parameter that predicts the extent of stroke-induced brain injury, the study investigated whether acute body weight loss predicts the extent of brain injury and swelling. Methods: C57 male mice (10-12 wk old, 25-30 gr) were subjected to the proximal middle cerebral artery (MCA) occlusion for 30 min. Body weight was recorded daily. Infarct volume corrected with swelling (IVInd) and percent hemispheric swelling (%SW) were determined at 1, 3 and 7d after MCAO. In a retrospective study, we analyzed correlations between 3d %body weight reduction (%BWred) and stroke outcomes (IVInd & %SW) in C57 male animals that were subjected to MCAO by 3 different individuals over 8 years (n=91-96). Results: Stroke induced acute BW loss (%BWred; 1d, 88.9±3.3; 3d, 84.5±8.6; 5d, 86.8±10.6; 7d, 88.9±8.9). Correlation analyses in the post-ischemic time points showed IVind and %SW were significantly correlated with only at 3d BW reduction, but not 1d and 7d (Fig.1). A retrospective analysis in C57 mice also showed a significant correlation between 3d %BWred and IVInd & %SW (Fig.2). Conclusions: The study showed that acute post-stroke BWred is a simple and suitable index to predict the extent of stroke injury in C57 mice. Whether the prediction can be generalized across different strains and in comorbid conditions warrant further investigation.

Background and Purpose: Although postconditioning following stroke has indicated neuroprotective mechanisms, little is known about the mechanisms that lead to neuroprotection. Using flagellin, NF-kB activators and agonists of TLR5, we tested whether TLR5 plays an integral role in NF-kB activation and postconditioning-induced neuroprotection. Method: For the postconditioning group, 30 min of MCAO was induced, then 5 min of reperfusion was performed, after which the MCA was again occluded for 5 min. Using an anaerobic chamber, bEnd.3 and BV-2 cells in the postconditioning group were subjected to 180 min OGD (oxygen and glucose deprivation) and were allowed to recover for 10 min before being subjected to 10 more min of OGD. The control group was subjected to 180 min of OGD. Flagellin, an agonist of TLR5, was used to clarify the association between TLR5 and postconditioning in vivo (ICV; 50, 100 ng/mouse) and in vitro (0.1, 0.25, 0.5, 1 ng/ ml) experiments. Immunoprecipitation and immunoblotting were used to determine the interactions of TLR5, MyD88, p-Akt, and NF-κB. Results: Animals treated with postconditioning showed significant differences in infarct volumes compared with the MCAO group up to 60.51%. Postconditioning elevated NF-κB translocation to the nuclei (pin vivo and in vitro postconditioning. Additionally, inhibition of Akt phosphorylation by Akt inhibitor IV decreased NF-κB translocation after postconditioning. To clarify the role of NF-κB in postconditioning, flagellin was used as a postconditioning mimicking agent. The treatment of flagellin reduced hypoxic-ischemic injury and increased nuclear accumulation of NF-κB and activation of Akt up to 4 hr. Conclusions: Postconditioning has neuroprotective effects by activating NF-κB and Akt survival pathway under TLR5 after stroke. Agonist of TLR5, flagellin simulated NF-κB and Akt survival pathways in cerebral ischemia. Our results suggested new mechanisms of postconditioning through TLR-5 and NF-κB, which leads to the possibility of development of a postconditioning mimicking agent using NF-κB activators such as flagellin.

Background and Purpose: Hyperglycemia is frequently observed in patients with acute ischemic stroke, and conversely, diabetic patients showed a high frequency of stroke. This correlation indicates shared mechanisms in pathology. Glucagon like peptide-1 (GLP-1) stimulates glucose-dependent insulin secretion and is used to treat type 2 diabetes mellitus. Recently, beyond the glucose-lowering effect, GLP-1 has been reported to possess neuroprotective effects. We therefore investigated the neuroprotective mechanism of GLP-1 receptor (GLP-1R) agonist exendin-4 (ex-4) after cerebral ischemia-reperfusion injury. Methods: Male Sprague-Dawley rats were subjected to 60 min of middle cerebral artery occlusion (MCAO) with intracerebroventricular (i.c.v) pretreatment of ex-4. Oxygen glucose deprivation was induced to primary neuron cultures in an anaerobic chamber. Changes of genes were further confirmed by QPCR and Western blot, and siRNA was used to ascertain the mechanism. Results: Ischemia-reperfusion injury reduced the expressions of GLP-1R by 46.3% (p; higher oxidative stress in SOD2 KO mice induced lower expression of GLP-1R, but higher in SOD1 Tg mice. Down regulated GLP-1R by ischemic injury was 70% restored by GLP-1R agonist, ex-4 (p2 terminal kinase (JNK) signaling that stimulates activation of Cox-2 was 36% inhibited by the i.c.v injection of ex-4 at 24 h (p Conclusions: GLP-1R activation by ex-4 resulted in reduction of Cox-2 through increasing IB1, which led to anti-inflammatory neuroprotection in stroke. Our study suggests that anti-inflammatory action of GLP-1 could be a new strategy for the treatment of stroke accompanied by hyperglycemia.

It is common that physicians are faced with patients who have severe steno-occlusion of intracranial arteries in the absence of clinical symptoms. This study is to determine whether a 7T magnetic resonance angiogram (MRA) could provide the improved depiction of microvessels of asymptomatic patients with steno-occlusive middle cerebral artery (MCA) and possible clues for diagnosis of their clinical symptoms. All the patients who were identified as severe MCA steno-occlusions underwent a 7T MRA study. Three of these also underwent conventional angiography (TFCA). The vascular densities around the steno-occlusive MCA and posterior cerebral artery (PCA) branches as control vessels were measured and the difference between the median values of 7T and 3T MRA data was also analyzed. The results of 7T MRA revealed numerous microvessels not visible by conventional MRA. These 7T MRA images were also comparable to those obtained by an invasive conventional angiography (TFCA). The median values of vascular densities observed by 7T MRA were significantly higher than those by 3T MRA (5.28 at 7T vs. 1.45 at 3T for steno-occlusive MCA, p = 0.012, while 3.39 at 7T vs. 3.32 at 3T for normal PCA, p = 0.093). Ultra-high-field 7T MRA is a totally noninvasive angiographic technique that is capable of visualizing microvascular circulation that is usually difficult with conventional MRA. This visualization technique, therefore, could provide an important avenue on the diagnosis of steno-occlusion as well as the search of the roles of the microvessel, especially collaterals, in the preservation of normal blood circulation in patients with asymptomatic MCA steno-occlusion.

BackgroundzzCluster headache is a primary headache disorder characterized by periodic episodes of intense headache accompanied by autonomic symptoms. We report an unusual clinical presentation of cluster headache that was preceded by myoclonus and accompanied by hemiparesis. Case ReportzzA 26-year-old man visited hospital due to recurrent jerky movements on the left side of his face and neck area lasting 3 days. These jerky movements had disappeared spontaneously without specific treatment. On the 10th day after onset of the jerky movements, the patient developed a series of unilateral severe headaches that were accompanied by autonomic symptoms lasting 1-2 hours. According to the second edition of The International Classification of Headache Disorders, he was diagnosed as having cluster headache. Two of the 16 severe headache attacks this patient suffered were accompanied by dysarthria and hemiparesis. Electroencephalography performed during hemiparesis revealed diffuse lateralized slow activity on the ipsilateral hemisphere of the headache side. The headache and accompanying hemiparesis disappeared after medical treatment for cluster headache. ConclusionszzWe describe a case of cluster headache accompanied by hemiparesis, which was preceded by myoclonus. We also outline the possible mechanisms underlying this case. J Clin Neurol 2012;8:83-86

The IkappaB kinase (IKK) complex is a central component in the classic activation of the nuclear factor-kappaB (NF-kappaB) pathway. It has been reported to function in physiologic responses, including cell death and inflammation. We have shown that IKK is regulated by oxidative status after transient focal cerebral ischemia (tFCI) in mice. However, the mechanism by which oxidative stress influences IKKs after tFCI is largely unknown. Nuclear accumulation and phosphorylation of IKKalpha (pIKKalpha) were observed 1 h after 30 mins of tFCI in mice. In copper/zinc-superoxide dismutase knockout mice, levels of NF-kappaB-inducing kinase (NIK) (an upstream kinase of IKKalpha), pIKKalpha, and phosphorylation of histone H3 (pH3) on Ser10 were increased after tFCI and were higher than in wild-type mice. Immunohistochemistry showed nuclear accumulation and pIKKalpha in mouse brain endothelial cells after tFCI. Nuclear factor-kappaB-inducing kinase was increased, and it enhanced pH3 by inducing pIKKalpha after oxygen-glucose deprivation (OGD) in mouse brain endothelial cells. Both NIK and pH3 interactions with IKKalpha were confirmed by coimmunoprecipitation. Treatment with IKKalpha small interfering RNA significantly reduced cell death after OGD. These results suggest that augmentation of NIK, IKKalpha, and pH3 in response to oxidative stress is involved in cell death after cerebral ischemia (or stroke).