Focal Brain Ischemia Research Articles

Dickkopf-3 Causes Neuroprotection by Inducing Vascular Endothelial Growth Factor.

Dickkopf-3 (Dkk3) is an atypical member of the Dkk family of Wnt inhibitors, which has been implicated in the pathophysiology of neurodegenerative disorders. However, the role of Dkk3 in mechanisms of cell degeneration and protection is unknown. We used Dkk3 knockout mice to examine how endogenous Dkk3 influences ischemic brain damage. In addition, we used primary cultures of astrocytes or mixed cultures of astrocytes and neurons to investigate the action of Dkk3 on cell damage and dissect the underlying molecular mechanisms. In a model of focal brain ischemia induced by permanent middle cerebral artery (MCA) occlusion (MCAO) Dkk3−/− mice showed a significantly greater infarct size with respect to their wild-type counterparts at all time points investigated (1, 3 and 7 days after MCAO). Immunohistochemical analysis showed that Dkk3 expression was enhanced at the borders of the ischemic focus, and was predominantly detected in astrocytes. This raised the possibility that Dkk3 produced by astrocytes acted as a protective molecule. We tested this hypothesis using either primary cultures of cortical astrocytes or mixed cortical cultures containing both neurons and astrocytes. Genetic deletion of Dkk3 was permissive to astrocyte damage induced by either oxidative stress or glucose deprivation. In addition, application of human recombinant Dkk3 (hrDkk3) was highly protective against oxidative stress in cultured astrocytes. We tested the hypothesis that the protective activity of Dkk3 was mediated byvascular endothelial growth factor (VEGF). Interestingly, glucose deprivation up-regulated both Dkk3 and VEGF in cultured astrocytes prepared from wild-type mice. VEGF induction was not observed in astrocytes lacking Dkk3 (i.e., in cultures prepared from Dkk3−/− mice). In mixed cultures of cortical cells, excitotoxic neuronal death induced by a brief pulse with N-methyl-D-aspartate (NMDA) was significantly enhanced when Dkk3 was lacking in astrocytes, whereas post-NMDA addition of hrDkk3 was neuroprotective. Neuroprotection by hrDkk3 was significantly reduced by pharmacological blockade of type-2 VEGF receptors and was mimicked by hrVEGF. These data offer the first evidence that Dkk3 protects both neurons and astrocytes against a variety of toxic insults, and at least in culture, protection involves VEGF induction.

Headache in transient ischemic attacks

BackgroundHeadache is a common feature in acute cerebrovascular disease but no studies have evaluated the prevalence of specific headache types in patients with transient ischemic attacks (TIA). The purpose of the present study was to analyze all headaches within the last year and the last week before TIA and at the time of TIA.MethodsEligible patients with TIA (n = 120, mean age 56.1, females 55%) had focal brain or retinal ischemia with resolution of symptoms within 24 h without presence of new infarction on MRI with DWI (n = 112) or CT (n = 8). All patients were evaluated within one day of admission by a single neurologist. As a control group we used patients (n = 192, mean age 58.7, females 64%) admitted with diagnoses “lumbago”, “lumbar spine osteochondrosis” or “gastrointestinal ulcer”.ResultsOne-year prevalence of migraine without aura was significantly higher in TIA patients than in controls: 20.8% and 7.8% respectively (p = 0.002, OR 3.1, 95% CI 1.6–6.2). 22 patients (18.3%) had sentinel or warning headache within the last week before TIA. At the time of TIA a new type of headache was observed in 16 patients (13.3%). No controls had a new type of headache. 12 of these 16 patients had migraine-like headache, 8 patients had tension-type-like headache and one patient thunderclap headache. Posterior circulation TIA was associated with headaches within last week before TIA and at the time of TIA much more frequently than anterior circulation TIA.ConclusionsThe one year prevalence of migraine was significantly higher in TIA patients than in controls and so was the prevalence of headache within the last week before TIA and at the time of TIA. A previous headache that worsens and a new type of headache can be a warning of impending TIA.

A Herbal Formulation of Danshen and Gegen Protects Rat Brains from Injuries Induced by Focal and Global Ischaemia

Ischaemic stroke is a disease that affects many people in developed countries. The occurrence of this disease is also rising globally. Danshen and Gegen are two commonly used Chinese herbal medicines for the treatment of cardiovascular diseases. This study investigates the efficacy of a combined formulation of Danshen and Gegen (DG) in protecting the rat brains from damages induced by global and focal brain ischaemia.

Rat’s behaviour in eight-arms maze after modeling of focal transient cerebral ischemia with subsequent activation of striatal microglia

It is well known that cognitive disorders at cerebral ischemia-reperfusion are followed by neuronal death. The inflammation of nervous tissue occurs, but the role of microgliocytes in neuroprotection is poorly understood. The aim of our work was to study microgliocytes in rats with cognitive disorders in delayed postischemic period after focal transient cerebral ischemia. In an experiment we used male rats (n=15), weighing 200-250. The model of focal brain ischemia was performed in anaesthetized rats (intraperitonealy, chloralhydrate 450 mg/kg). Microsurgical introduction monofilament to the left middle cerebral artery was made by J. Koizumi technique. Time of middle cerebral artery (MCA) occlusion was 30 minutes with the subsequent reperfusion period. Testing in eight-arm maze was performed within seven days twice with a two-day interval during which each animal carried out a 5-minute session 3 times with a 3-minute interval. For identification of microgliocytes used polyclonal goat antibodies to Iba1 antigen. At behavior assessment in an eight-arm maze there were errors of working memory revealed and prolongation of time of task performance. Cognitive disorders at cerebral ischemia, perhaps, serve as the neuroinflammation indicator. Microglia is likely involved in mechanisms of neuroprotection and compensation for cognitive disorders.

Diagnostic criteria for transient ischemic attack

The diagnosis of transient ischemic attack (TIA) is fraught with problems; particularly this concerns the differentiation of TIA with its mimicking conditions. Explicit diagnostic criteria can improve the accuracy of TIA recognition. The authors of this paper propose new TIA diagnostic criteria Objective : to elaborate TIA diagnostic criteria and to determine their sensitivity in a group of patients with TIA, as well as their specificity in those who suffered from migraine with aura from Russia and Denmark. Patients and methods . TIA diagnostic criteria were developed using the International Classification of Headache Disorders (ICHD) and the data available in the literature on migraine with aura and on the clinical characteristics and diagnosis of TIA. The sensitivity of the criteria was tested in a prospective study of 120 patients who developed TIA before the elaboration of these criteria. The patients were questioned in the acute period of the disease through detailed semi-structured interviews. Eligible patients had focal brain or retinal ischemia with resolution of symptoms within 24 hours without acute brain tissue damage, as evidenced by diffusion-weighted magnetic resonance imaging (n=112) or computed tomography (n=8). These criteria were also tested for specificity in Danish (n=1390) and Russian (n=152) patients suffering from migraine with aura, the diagnosis of which was established according to ICHD-3 beta. Results and discussion . The sensitivity of the proposed criteria in TIA patients was 99%. The specificity in the Danish and Russian patients with migraine with aura was 95% and 96%, respectively. Conclusion . The new TIA diagnostic criteria are characterized by high sensitivity and specificity. These will be able to improve the diagnosis of TIA. It is recommended that the testing of these criteria in patients with TIA should be continued in future investigations.

Enhancement of bradykinin-induced relaxation by focal brain ischemia in the rat middle cerebral artery: Receptor expression upregulation and activation of multiple pathways.

Focal brain ischemia markedly affects cerebrovascular reactivity. So far, these changes have mainly been related to alterations in the level of smooth muscle cell function while alterations of the endothelial lining have not yet been studied in detail. We have, therefore, investigated the effects of ischemia/reperfusion injury on bradykinin (BK)-induced relaxation since BK is an important mediator of tissue inflammation and affects vascular function in an endothelium-dependent manner. Focal brain ischemia was induced in rats by endovascular filament occlusion (2h) of the middle cerebral artery (MCA). After 22h reperfusion, both MCAs were harvested and the response to BK studied in organ bath experiments. Expression of the BK receptor subtypes 1 and 2 (B1, B2) was determined by real-time semi-quantitative RT-qPCR methodology, and whole mount immunofluorescence staining was performed to show the B2 receptor protein expression. In control animals, BK did not induce significant vasomotor effects despite a functionally intact endothelium and robust expression of B2 mRNA. After ischemia/reperfusion injury, BK induced a concentration-related sustained relaxation in all arteries studied, more pronounced in the ipsilateral than in the contralateral MCA. The B2 mRNA was significantly upregulated and the B1 mRNA displayed de novo expression, again more pronounced ipsi- than contralaterally. Endothelial cells displaying B2 receptor immunofluorescence were observed scattered or clustered in previously occluded MCAs. Relaxation to BK was mediated by B2 receptor activation, abolished after endothelium denudation, and largely diminished by blocking nitric oxide (NO) release or soluble guanylyl cyclase activity. Relaxation to BK was partially inhibited by charybdotoxin (ChTx), but not apamin or iberiotoxin suggesting activation of an endothelium-dependent hyperpolarization pathway. When the NO-cGMP pathway was blocked, BK induced a transient relaxation which was suppressed by ChTx. After ischemia/reperfusion injury BK elicits endothelium-dependent relaxation which was not detectable in control MCAs. This gain of function is mediated by B2 receptor activation and involves the release of NO and activation of an endothelium-dependent hyperpolarization. It goes along with increased B2 mRNA and protein expression, leaving the functional role of the de novo B1 receptor expression still open.

Neuroprotective Effects of Guanosine Administration on In Vivo Cortical Focal Ischemia in Female and Male Wistar Rats.

Guanosine (GUO) has neuroprotective effects in experimental models of brain diseases involving glutamatergic excitotoxicity in male animals; however, its effects in female animals are poorly understood. Thus, we investigated the influence of gender and GUO treatment in adult male and female Wistar rats submitted to focal permanent cerebral ischemia in the motor cortex brain. Female rats were subdivided into non-estrogenic and estrogenic phase groups by estrous cycle verification. Immediately after surgeries, the ischemic animals were treated with GUO or a saline solution. Open field and elevated plus maze tasks were conducted with ischemic and naïve animals. Cylinder task, immunohistochemistry and infarct volume analyses were conducted only with ischemic animals. Female GUO groups achieved a full recovery of the forelimb symmetry at 28-35days after the insult, while male GUO groups only partially recovered at 42days, in the final evaluation. The ischemic insult affected long-term memory habituation to novelty only in female groups. Anxiety-like behavior, astrocyte morphology and infarct volume were not affected. Regardless the estrous cycle, the ischemic injury affected differently female and male animals. Thus, this study points that GUO is a potential neuroprotective compound in experimental stroke and that more studies, considering the estrous cycle, with both genders are recommended in future investigation concerning brain diseases.

A novel cell-penetrating peptide protects against neuron apoptosis after cerebral ischemia by inhibiting the nuclear translocation of annexin A1.

Nuclear translocation of annexin A1 (ANXA1) has recently been reported to participate in neuronal apoptosis after cerebral ischemia. Prevention of the nuclear translocation of ANXA1 should therefore inhibit neuronal apoptosis and protect against cerebral stroke. Here, we found that, in the repeat III domain of ANXA1, the amino-acid residues from R228 to F237 function as a unique nuclear translocation signal (NTS) and are required for nuclear translocation of ANXA1. Intriguingly, we synthesized a cell-penetrating peptide derived by conjugating the trans-activator of transcription (Tat) domain to the NTS sequence. This Tat-NTS peptide specifically blocked the interaction of ANXA1 with importin β and, consequently, the nuclear translocation of ANXA1 without affecting the nucleocytoplasmic shuttling of other proteins. The Tat-NTS peptide inhibited the transcriptional activity of p53, decreased Bid expression, suppressed activation of the caspase-3 apoptosis pathway and improved the survival of hippocampal neurons subjected to oxygen-glucose deprivation and reperfusion in vitro. Moreover, using a focal brain ischemia animal model, we showed that the Tat-NTS peptide could be efficiently infused into the ischemic hippocampus and cortex by unilateral intracerebroventricular injection. Injection of the Tat-NTS peptide alleviated neuronal apoptosis in the ischemic zone. Importantly, further work revealed that administration of the Tat-NTS peptide resulted in a dramatic reduction in infarct volume and that this was correlated with a parallel improvement in neurological function after reperfusion. Interestingly, the effects of Tat-NTS were injury specific, with little impact on neuronal apoptosis or cognitive function in sham-treated nonischemic animals. In conclusion, based on its profound neuroprotective and cognitive-preserving effects, it is suggested that the Tat-NTS peptide represents a novel and potentially promising new therapeutic candidate for the treatment of ischemic stroke.

Plasma Exosomal miRNA-122-5p and miR-300-3p as Potential Markers for Transient Ischaemic Attack in Rats.

Background: Differentiation of transient ischaemic attack (TIA) from ischaemic stroke within the thrombolysis time window is difficult. Although TIA may be diagnosed within this window, the latest imaging technologies are complex and costly. Serum markers, which are non-invasive, rapid and economic, are used for diagnosis and prognosis of various diseases. Exosome-derived miRNA markers for TIA are unknown.Methods: We examined focal brain ischaemia produced by occlusion of the middle cerebral artery (MCAo) for 5 min, 10 min, and 2 h in rats. Exosomal miRNAs with consistent trends in cerebrospinal fluid (CSF) and plasma were identified by deep sequencing and quantitative real-time polymerase chain reaction (qRT-PCR). The areas under the curve (AUC) of the receiver operating characteristic (ROC) curve were used to evaluate the diagnostic accuracy of these miRNAs for TIA in rats.Results: Rno-miR-122-5p and rno-miR-300-3p were selected. Plasma exosomal rno-miR-122-5p was significantly downregulated in 10 min ischaemic rats compared with control and 5 min plasma. Plasma exosomal rno-miR-300-3p was significantly upregulated in 5 min ischaemic rats compared with control, 10 min and 2 h rats. Plasma and CSF levels of these miRNAs were correlated. ROC analysis showed high AUC values for rno-miR-122-5p (0.960) and rno-miR-300-3p (0.970) in the 10 and 5 min rats, respectively, compared with controls.Conclusions: Plasma exosomal rno-miR-122-5p and rno-miR-300-3p may be blood-based TIA biomarkers.

Photoacoustic microscopy reveals the hemodynamic basis of sphingosine 1-phosphate-induced neuroprotection against ischemic stroke.

Rationale: Emerging evidence has suggested that sphingosine 1-phosphate (S1P), a bioactive metabolite of sphingolipids, may play an important role in the pathophysiological processes of cerebral hypoxia and ischemia. However, the influence of S1P on cerebral hemodynamics and metabolism remains unclear.Material and Methods: Uniquely capable of high-resolution, label-free, and comprehensive imaging of hemodynamics and oxygen metabolism in the mouse brain without the influence of general anesthesia, our newly developed head-restrained multi-parametric photoacoustic microscopy (PAM) is well suited for this mechanistic study. Here, combining the cutting-edge PAM and a selective inhibitor of sphingosine kinase 2 (SphK2) that can increase the blood S1P level, we investigated the role of S1P in cerebral oxygen supply-demand and its neuroprotective effects on global brain hypoxia induced by nitrogen gas inhalation and focal brain ischemia induced by transient middle cerebral artery occlusion (tMCAO).Results: Inhibition of SphK2, which increased the blood S1P, resulted in the elevation of both arterial and venous sO2 in the hypoxic mouse brain, while the cerebral blood flow remained unchanged. As a result, it gradually and significantly reduced the metabolic rate of oxygen. Furthermore, pre-treatment of the mice subject to tMCAO with the SphK2 inhibitor led to decreased infarct volume, improved motor function, and reduced neurological deficit, compared to the control treatment with a less potent R-enantiomer. In contrast, post-treatment with the inhibitor showed no improvement in the stroke outcomes. The failure for the post-treatment to induce neuroprotection was likely due to the relatively slow hemodynamic responses to the SphK2 inhibitor-evoked S1P intervention, which did not take effect before the brain injury was induced.Conclusions: Our results reveal that elevated blood S1P significantly changes cerebral hemodynamics and oxygen metabolism under hypoxia but not normoxia. The improved blood oxygenation and reduced oxygen demand in the hypoxic brain may underlie the neuroprotective effect of S1P against ischemic stroke.

Oral administration of a novel lipophilic PPARδ agonist is not neuroprotective after rodent cerebral ischemia

Peroxisome proliferator-activated receptors are regulators of inflammatory signaling. This has fostered hope that PPAR agonists might have neuroprotective potential. We hypothesized that PPARδ activation by the novel orally administered lipophilic PPARδ agonist SAR145 may improve short- and long-term outcome after focal brain ischemia. We induced ischemia by transient filamentous middle cerebral artery occlusion (MCAo) in 227 C57BL/6 mice and administered SAR145 in varying doses and time windows post-injury. Outcome was assessed by three functional tests and histologically determining ischemic lesion sizes. In a second experiment, we tested SAR145 treatment in 40 PPARδ-knockout mice using the same procedures. Three independent groups treated with 10 mg/kg bodyweight SAR145 directly after filament removal showed a mean reduction in lesion sizes of 18 ± 10% compared to vehicle-treated groups. We did not observe a consistent improvement in the long-term functional outcome by SAR145-treatment. PPARδ-knockout mice showed a significantly higher mortality after MCAo. As expected, we did not find a reduction of lesion size by SAR145-treatment in PPARδ-knockout mice. In summary, we found no evidence of a long-term neuroprotective effect of post-injury SAR145 treatment in cerebral ischemia. However, PPARδ appears to play a pathophysiologic role in acute infarct development and overall mortality after brain ischemia.

Transplantation of feeder‐free human induced pluripotent stem cell–derived cortical neuron progenitors in adult male Wistar rats with focal brain ischemia

The use of human induced pluripotent stem cells (hiPSCs) eliminates the ethical issues associated with fetal or embryonic materials, thus allowing progress in cell therapy research for ischemic stroke. Strict regulation of cell therapy development requires the xeno-free condition to eliminate clinical complications. Maintenance of hiPSCs with feeder-free condition presents a higher degree of spontaneous differentiation in comparison with conventional cultures. Therefore, feeder-free derivation might be not ideal for developing transplantable hiPSC derivatives. We developed the feeder-free condition for differentiation of cortical neurons from hiPSCs. Then, we evaluated the cells' characteristics upon transplantation into the sham and focal brain ischemia on adult male Wistar rats. Grafts in lesioned brains demonstrated polarized reactivity toward the ischemic border, indicated by directional preferences in axonal outgrowth and cellular migration, with no influence on graft survival. Following the transplantation, forelimb asymmetry was better restored compared with controls. Herein, we provide evidence to support the use of the xeno-free condition for the development of cell therapy for ischemic stroke.

Explicit diagnostic criteria for transient ischemic attacks to differentiate it from migraine with aura

Background The diagnosis of transient ischemic attacks is fraught with problems. The inter-observer agreement has repeatedly been shown to be low even in a neurological setting, and the specificity of the diagnosis is modest to low, reflected in a poor separation of transient ischemic attacks and mimics, particularly migraine with aura with its varied symptomatology. In other disease areas, explicit diagnostic criteria have improved sensitivity and specificity of diagnoses. We therefore present novel explicit diagnostic criteria for transient ischemic attacks tested for sensitivity and for specificity against migraine with aura. Methods The proposed criteria were developed using the format of the international headache classification. We drew upon the existing literature about clinical characteristics and diagnosis of migraine with aura and transient ischemic attacks. We tested the criteria for sensitivity in a prospectively-collected material of 120 patients with transient ischemic attacks diagnosed before we developed the criteria using extensive semi-structured interview forms in the acute phase after admission. Eligible patients had focal brain or retinal ischemia with resolution of symptoms within 24 hours without presence of new infarction on magnetic resonance imaging with diffusion weighted imaging (n = 112) or computed tomography (n = 8). These criteria were also tested for specificity against a Danish (n = 1390) and a Russian (n = 152) material of patients with migraine with aura diagnosed according to the International Classification of Headache Disorders edition 3 (beta). Results The sensitivity of the proposed criteria was 99% in patients with transient ischemic attacks. The specificity was 95% in the Danish material of patients with migraine with aura and 96% in the Russian material. Conclusions Proposed explicit diagnostic criteria for transient ischemic attacks showed both high specificity and sensitivity. They are likely to improve the emergency room diagnosis of transient ischemic attacks. Further testing in unselected materials referred to transient ischemic attacks clinics was beyond the scope of the present study but is recommended for future study.

In Vivo Neuroimaging of Exosomes Using Gold Nanoparticles.

Exosomes are emerging as effective therapeutic tools for various pathologies. These extracellular vesicles can bypass biological barriers, including the blood-brain barrier, and can serve as powerful drug and gene therapy transporters. However, the progress of therapy development is impeded by several challenges, including insufficient data on exosome trafficking and biodistribution and the difficulty to image deep brain structures in vivo. Herein, we established a method for noninvasive in vivo neuroimaging and tracking of exosomes, based on glucose-coated gold nanoparticle (GNP) labeling and computed tomography imaging. Labeling of exosomes with the GNPs was achieved directly, as opposed to the typical and less efficient indirect labeling mode through parent cells. On the mechanistic level, we found that the glucose-coated GNPs were uptaken into MSC-derived exosomes via an active, energy-dependent mechanism that is mediated by the glucose transporter GLUT-1 and involves endocytic proteins. Next, we determined optimal parameters of size and administration route; we demonstrated that 5 nm GNPs enabled improved exosome labeling and that intranasal, compared to intravenous, administration led to superior brain accumulation and thus enhanced in vivo neuroimaging. Furthermore, using a mouse model of focal brain ischemia, we noninvasively tracked intranasally administered GNP-labeled exosomes, which showed increased accumulation at the lesion site over 24 h, as compared to nonspecific migration and clearance from control brains over the same period. Thus, this exosome labeling technique can serve as a powerful diagnostic tool for various brain disorders and could potentially enhance exosome-based treatments for neuronal recovery.

A Magnetic Resonance Imaging Protocol for Stroke Onset Time Estimation in Permanent Cerebral Ischemia

MRI provides a sensitive and specific imaging tool to detect acute ischemic stroke by means of a reduced diffusion coefficient of brain water. In a rat model of ischemic stroke, differences in quantitative T1 and T2 MRI relaxation times (qT1 and qT2) between the ischemic lesion (delineated by low diffusion) and the contralateral non-ischemic hemisphere increase with time from stroke onset. The time dependency of MRI relaxation time differences is heuristically described by a linear function and thus provides a simple estimate of stroke onset time. Additionally, the volumes of abnormal qT1 and qT2 within the ischemic lesion increase linearly with time providing a complementary method for stroke timing. A (semi)automated computer routine based on the quantified diffusion coefficient is presented to delineate acute ischemic stroke tissue in rat ischemia. This routine also determines hemispheric differences in qT1 and qT2 relaxation times and the location and volume of abnormal qT1 and qT2 voxels within the lesion. Uncertainties associated with onset time estimates of qT1 and qT2 MRI data vary from ± 25 min to ± 47 min for the first 5 hours of stroke. The most accurate onset time estimates can be obtained by quantifying the volume of overlapping abnormal qT1 and qT2 lesion volumes, termed 'Voverlap' (± 25 min) or by quantifying hemispheric differences in qT2 relaxation times only (± 28 min). Overall, qT2 derived parameters outperform those from qT1. The current MRI protocol is tested in the hyperacute phase of a permanent focal ischemia model, which may not be applicable to transient focal brain ischemia.

A Magnetic Resonance Imaging Protocol for Stroke Onset Time Estimation in Permanent Cerebral Ischemia.

MRI provides a sensitive and specific imaging tool to detect acute ischemic stroke by means of a reduced diffusion coefficient of brain water. In a rat model of ischemic stroke, differences in quantitative T1 and T2 MRI relaxation times (qT1 and qT2) between the ischemic lesion (delineated by low diffusion) and the contralateral non-ischemic hemisphere increase with time from stroke onset. The time dependency of MRI relaxation time differences is heuristically described by a linear function and thus provides a simple estimate of stroke onset time. Additionally, the volumes of abnormal qT1 and qT2 within the ischemic lesion increase linearly with time providing a complementary method for stroke timing. A (semi)automated computer routine based on the quantified diffusion coefficient is presented to delineate acute ischemic stroke tissue in rat ischemia. This routine also determines hemispheric differences in qT1 and qT2 relaxation times and the location and volume of abnormal qT1 and qT2 voxels within the lesion. Uncertainties associated with onset time estimates of qT1 and qT2 MRI data vary from ± 25 min to ± 47 min for the first 5 hours of stroke. The most accurate onset time estimates can be obtained by quantifying the volume of overlapping abnormal qT1 and qT2 lesion volumes, termed 'Voverlap' (± 25 min) or by quantifying hemispheric differences in qT2 relaxation times only (± 28 min). Overall, qT2 derived parameters outperform those from qT1. The current MRI protocol is tested in the hyperacute phase of a permanent focal ischemia model, which may not be applicable to transient focal brain ischemia.

LOTUS overexpression accelerates neuronal plasticity after focal brain ischemia in mice.

Nogo receptor-1 (NgR1) and its ligands inhibit neuronal plasticity and limit functional recovery after brain damage such as ischemic stroke. We have previously shown that lateral olfactory tract usher substance (LOTUS) antagonizes NgR1-mediated signaling. Here, we investigated whether LOTUS enhances neuronal plasticity and functional recovery after brain focal ischemia in adult mice. Focal ischemic infarcts were induced in wild-type and LOTUS-overexpressing transgenic mice via middle cerebral artery occlusion. Endogenous LOTUS expression was increased in brain and cervical spinal cord of the contralateral side of ischemia in the chronic phase after brain ischemia. LOTUS overexpression accelerated midline-crossing axonal sprouting from the contralateral side to the ipsilateral side of ischemia in the medullar reticular formation and gray matter of denervated cervical spinal cord. Importantly, LOTUS overexpression improved neurological score highly correlated with laterality ratio of corticoreticular fibers of the medulla oblongata, indicating that LOTUS overexpression may overcome the inhibitory environment induced by NgR1 signaling for damaged motor pathway reconstruction after ischemic stroke. Thus, our data suggest that LOTUS overexpression accelerates neuronal plasticity in the brainstem and cervical spinal cord after stroke and LOTUS administration is useful for future therapeutic strategies.

Accumulation of corticosterone and interleukin-1β in the hippocampus after focal ischemic damage of the neocortex: Selective vulnerability of the ventral hippocampus

Most ischemic strokes are caused by the occlusion of the middle cerebral artery (MCAO), which results in focal brain lesions in different areas of the neocortex. Secondary damage develops in brain regions located out of the infarct area, including the hippocampus. Hippocampal lesion may lead to cognitive impairments and post-stroke depression. Here, we studied the time course of changes in the levels of corticosterone and proinflammatory cytokine interleukine-1β (IL-1β) in the blood and hippocampus of rats after transient focal brain ischemia. Activation of the hypothalamo–pituitary–adrenal axis, which causes a release of corticosterone into blood, was observed at the early stage after MCAO and was accompanied by the presence of the stress hormone in the hippocampi of both the ischemic and contralateral hemispheres. We show for the first time that this effect was observed only in the ventral hippocampus (VH) but not in the dorsal hippocampus (DH). MCAO induced accumulation of the proinflammatory cytokine IL-1β, which coexisted with the elevated level of corticosterone at the early and delayed stages after reperfusion and was also observed in the VH of both hemispheres. Our data show that the VH is more vulnerable to remote damage induced by MCAO compared to the DH and corticosteroid response and neuroinflammation may be detected in the VH of both ischemic and contralateral hemispheres.

IMM-H004, A New Coumarin Derivative, Improved Focal Cerebral Ischemia via Blood–Brain Barrier Protection in Rats

IMM-H004 (7-hydroxy-5-methoxy-4-methyl-3-[4-methylpiperazin-1-yl]-2H-chromen-2-one) is a novel coumarin derivative that showed better effect in improving global cerebral ischemia in rats. However, the effects and mechanisms in focal cerebral ischemia were not clear. Blood-brain barrier (BBB) protection is a vital strategy for the treatment of cerebral ischemia. This study is to investigate whether IMM-H004 improves brain ischemia injury via BBB protection. Focal brain ischemia model was induced by middle cerebral artery occlusion for 1 hour and reperfusion (MCAO/R) for 24 hours in rats. IMM-H004 (1.5, 3, 6 mg/kg) and edaravone (positive drug, 6 mg/kg) were administered after 5 minutes of occlusion. Neurological score and TTC staining were used to evaluate the effect of IMM-H004. Evans Blue (EB) staining and electron microscopy were used to assess BBB permeability. Western blot, reverse transcription-polymerase chain reaction, and immunohistochemistry were used to detect the expression of BBB structure-related proteins. Compared with the model group, IMM-H004 in the focal brain ischemia model improved neurological function and reduced cerebral infarction size and edema content. IMM-H004 sharply reduced the EB content and alleviated BBB structure. In addition, IMM-H004 increased the level of zonula occludens (ZO-1) and occluding, decreased the level of aquaporin 4 and matrix metalloproteinase 9, either in cortex or in hippocampus. And all of these changed were related to BBB protection. IMM-H004 improved cerebral ischemia injury via BBB protection. For a potential therapy drug of cerebral ischemia, IMM-H004 merits further study.

SANGUINATE™ (PEGylated Carboxyhemoglobin Bovine) Improves Cerebral Blood Flow to Vulnerable Brain Regions at Risk of Delayed Cerebral Ischemia After Subarachnoid Hemorrhage.

Delayed cerebral ischemia (DCI) after subarachnoid hemorrhage (SAH) has been linked to focal reductions in cerebral blood flow (CBF) and microvascular impairments in oxygen delivery. Effective therapies that restore flow and oxygen transport to vulnerable brain regions are currently lacking. SANGUINATE is a dual-action carbon monoxide-releasing and hemoglobin-based oxygen transfer agent with efficacy in animal models of focal brain ischemia and tolerability in patients with sickle cell disease. We performed a safety and proof-of-principle study in 12 SAH patients at risk of DCI across three escalating doses (160, 240, and 320mg/kg). We used 15O-PET (performed at baseline, after SANGUINATE and at 24h) to evaluate efficacy for improving CBF and restoring flow-metabolism balance (assessed by oxygen extraction fraction [OEF]) to vulnerable regions (defined as baseline OEF≥0.50). SANGUINATE resulted in a transient rise in mean arterial pressure (116±15-127±13mm Hg, p=0.001) that normalized by 24h and allowed three patients with DCI to be weaned off vasopressors. No adverse events were noted during infusion. Global CBF did not rise (43±8-46±9ml/100g/min) although a trend was seen at the highest dose (45±7-51±9, p=0.044). However, a significant 16% rise in regional CBF associated with reduction in OEF was seen in vulnerable regions, but did not persist at 24h. We demonstrated that this novel agent can improve regional CBF and may improve oxygen supply-demand balance. Clinical studies (likely with repeat dosing) are required to evaluate whether this effect can prevent DCI or cerebral infarction.