Rodent Gymnastics: Neurobehavioral Assays in Ischemic Stroke

Despite years of research, most preclinical trials on ischemic stroke have remained unsuccessful owing to poor methodological and statistical standards leading to “translational roadblocks.” Various behavioral tests have been established to evaluate traits such as sensorimotor function, cognitive and social interactions, and anxiety-like and depression-like behavior. A test’s validity is of cardinal importance as it influences the chance of a successful translation of preclinical results to clinical settings. The mission of choosing a behavioral test for a particular project is, therefore, imperative and the present review aims to provide a structured way to evaluate rodent behavioral tests with implications in ischemic stroke.     

Irisin Peptide Protects Brain Against Ischemic Injury Through Reducing Apoptosis and Enhancing BDNF in a Rodent Model of Stroke

Evidence has shown therapeutic potential of irisin in cerebral stroke. The present study aimed to assess the effects of recombinant irisin on the infarct size, neurological outcomes, blood–brain barrier (BBB) permeability, apoptosis and brain-derived neurotrophic factor (BDNF) expression in a mouse model of stroke. Transient focal cerebral ischemia was established by middle cerebral artery occlusion (MCAO) for 45 min and followed reperfusion for 23 h in mice. Recombinant irisin was administrated at doses of 0.1, 0.5, 2.5, 7.5, and 15 µg/kg, intracerebroventricularly (ICV), on the MCAO beginning. Neurological outcomes, infarct size, brain edema and BBB permeability were evaluated by modified neurological severity score (mNSS), 2,3,5-triphenyltetrazolium chloride (TTC) staining and Evans blue (EB) extravasation methods, respectively, at 24 h after ischemia. Apoptotic cells and BDNF protein were detected by TUNEL assay and immunohistochemistry techniques. The levels of Bcl-2, Bax and caspase-3 proteins were measured by immunoblotting technique. ICV irisin administration at doses of 0.5, 2.5, 7.5 and 15 µg/kg, significantly reduced infarct size, whereas only in 7.5 and 15 µg/kg improved neurological outcome (P?<?0.001). Treatment with irisin (7.5 µg/kg) reduced brain edema (P?<?0.001) without changing BBB permeability (P?>?0.05). Additionally, irisin (7.5 µg/kg) significantly diminished apoptotic cells and increased BDNF immunoreactivity in the ischemic brain cortex (P?<?0.004). Irisin administration significantly downregulated the Bax and caspase-3 expression and upregulated the Bcl-2 protein. The present study indicated that irisin attenuates brain damage via reducing apoptosis and increasing BDNF protein of brain cortex in the experimental model of stroke in mice.     

Cardioembolism and Involvement of the Insular Cortex in Patients with Ischemic Stroke

BackgroundTo evaluate whether topographical characteristics of insular involvement in ischemic stroke are associated with cardioembolism.ConclusionsIn cases of stroke in the MCA territory, involvement of the insular cortex may be associated with a risk of cardioembolism.     

Blueberry-Enriched Diet Protects Rat Heart from Ischemic Damage

Objectives

to assess the cardioprotective properties of a blueberry enriched diet (BD).

Background

Reactive oxygen species (ROS) play a major role in ischemia-related myocardial injury. The attempts to use synthetic antioxidants to block the detrimental effects of ROS have produced mixed or negative results precipitating the interest in natural products. Blueberries are readily available product with the highest antioxidant capacity among fruits and vegetables.

Methods and Results

Following 3-mo of BD or a regular control diet (CD), the threshold for mitochondrial permeability transition (t_MPT) was measured in isolated cardiomyocytes obtained from young male Fischer-344 rats. Compared to CD, BD resulted in a 24% increase (p<0.001) of ROS indexed t_MPT. The remaining animals were subjected to a permanent ligation of the left descending coronary artery. 24 hrs later resulting myocardial infarction (MI) in rats on BD was 22% less than in CD rats (p<0.01). Significantly less TUNEL(+) cardiomyocytes (2% vs 9%) and 40% less inflammation cells were observed in the myocardial area at risk of BD compared to CD rats (p<0.01). In the subgroup of rats, after coronary ligation the original diet was either continued or switched to the opposite one, and cardiac remodeling and MI expansion were followed by serial echocardiography for 10 weeks. Measurements suggested that continuation of BD or its withdrawal after MI attenuated or accelerated rates of post MI cardiac remodeling and MI expansion.

Conclusion

A blueberry-enriched diet protected the myocardium from induced ischemic damage and demonstrated the potential to attenuate the development of post MI chronic heart failure.     

Lesioning of Deep Prepiriform Cortex Protects Against Ischemic Neuronal Necrosis by Attenuating Extracellular Glutamate Concentrations

Abstract: An area of the deep prepiriform cortex is a controlling site for limbic seizures. Focal pharmacologic blockade of NMDA receptors in the deep prepiriform cortex protects against hippocampal cell injury during limbic seizures induced by intravenous kainate and during the excitotoxicity of global ischemia. In the current study, the deep prepiriform cortex was lesioned bilaterally by microinjection of kainate, 3 days before 10 min of global ischemia induced by four-vessel occlusion. Extracellular glutamate concentrations in the hippocampus were measured before, during, and after global ischemia by using in vivo microdialysis technique. Surviving hippocampal neurons were counted 7 days after ischemia. Lesioned animals showed significantly greater numbers of surviving neurons and significantly lower ischemia-induced elevations of extracellular glutamate concentrations than non-lesioned animals. During seizures induced from the deep prepiriform cortex, the immediate early gene cox-2 is expressed in the hippocampus. These results indicate that deep prepiriform cortex can be a modulatory site for ischemic hippocampal injury.     

Five-year Prognosis after Mild to Moderate Ischemic Stroke by Stroke Subtype: A Multi-Clinic Registry Study

Background and Purpose

Mild to moderate ischemic stroke is a common presentation in the outpatient setting. Among the various subtypes of stroke, lacunar infarction (LI) is generally very common. Currently, little is known about the long-term prognosis and factors associated with the prognosis between LI and non-LI. This study aims to compare the risk of death and acute cardiovascular events between patients with LI and non-LI, and identify potential risk factors associated with these outcomes.

Methods

A total of 710 first-ever ischemic stroke patients (LI: 474, non-LI: 263) from 18 clinics were recruited consecutively from 2003 to 2004. They were prospectively followed-up until the end of 2008. Hazard ratios and 95% confidence intervals were calculated using multivariable Cox proportional hazards regression.

Results

After a 5-year follow up, 54 deaths and 96 acute cardiovascular events occurred. Recurrent stroke was the most common cause of death (19 cases, 35.18%) and new acute cardiovascular events (75 cases, 78.13%). There were no significant differences between patients with LI and non-LI in their risks of death, new cardiovascular events, and recurrent stroke after adjusting for age, sex, hypertension, diabetes, cardiac diseases, body mass index, dyslipidemia, smoking, alcohol consumption, ADL dependence, and depressive symptoms. Among the modifiable risk factors, diabetes, hypertension, ADL dependency, and symptoms of depression were independent predictors of poor outcomes in patients with LI. In non-LI patients, however, no modifiable risk factors were detected for poor outcomes.

Conclusion

Long-term outcomes did not differ significantly between LI and non-LI patients. Detecting and managing vascular risk factors and depression as well as functional rehabilitation may improve the prognoses of LI patients.     

Proximal Arterial Occlusion in Acute Ischemic Stroke with Low NIHSS Scores Should Not Be Considered as Mild Stroke

Background

Untreated acute mild stroke patients have substantial 90-day disability rates and worse outcomes than those who are treated with thrombolysis. There is little information regarding which patients with acute mild stroke will benefit from thrombolysis. We sought to investigate factors that are associated with early neurological deterioration (END) and poor prognosis in patients with acute mild stroke.

Methods

This was a retrospective study of consecutively registered patients with acute mild stroke (NIHSS ≤3) at our tertiary stroke center between October 2008 and December 2011. END was defined as an increase in NIHSS ≥2 points between hospital days 0 and 5. Modified Rankin Scale (mRS) scores of 0–1 at 90 days post-stroke were defined as favorable outcomes.

Results

A total of 378 (mean age, 65.9±13.0 years) patients were included in this study. END occurred in 55 patients (14.6%). IV-thrombolysis was performed in only 9 patients. Symptomatic arterial occlusion on the initial MRA was independently associated with END (OR, 2.206; 95% CI, 1.219–3.994; p = 0.009) by multivariate logistic regression. Of the 119 patients with symptomatic arterial occlusion, ICA occlusion was independently associated with END (OR, 8.606; 95% CI, 2.312–32.043; p = 0.001).

Conclusions

This study demonstrates that symptomatic arterial occlusion may be an important predictor of END in patients with acute mild stroke. It may therefore be important to consider that acute ischemic stroke with symptomatic arterial occlusion and low NIHSS scores may not represent mild stroke in acute periods.     

Primary Sensory and Motor Cortex Excitability Are Co-Modulated in Response to Peripheral Electrical Nerve Stimulation

Peripheral electrical stimulation (PES) is a common clinical technique known to induce changes in corticomotor excitability; PES applied to induce a tetanic motor contraction increases, and PES at sub-motor threshold (sensory) intensities decreases, corticomotor excitability. Understanding of the mechanisms underlying these opposite changes in corticomotor excitability remains elusive. Modulation of primary sensory cortex (S1) excitability could underlie altered corticomotor excitability with PES. Here we examined whether changes in primary sensory (S1) and motor (M1) cortex excitability follow the same time-course when PES is applied using identical stimulus parameters. Corticomotor excitability was measured using transcranial magnetic stimulation (TMS) and sensory cortex excitability using somatosensory evoked potentials (SEPs) before and after 30 min of PES to right abductor pollicis brevis (APB). Two PES paradigms were tested in separate sessions; PES sufficient to induce a tetanic motor contraction (30–50 Hz; strong motor intensity) and PES at sub motor-threshold intensity (100 Hz). PES applied to induce strong activation of APB increased the size of the N₂₀-P₂₅ component, thought to reflect sensory processing at cortical level, and increased corticomotor excitability. PES at sensory intensity decreased the size of the P25-N33 component and reduced corticomotor excitability. A positive correlation was observed between the changes in amplitude of the cortical SEP components and corticomotor excitability following sensory and motor PES. Sensory PES also increased the sub-cortical P₁₄-N₂₀ SEP component. These findings provide evidence that PES results in co-modulation of S1 and M1 excitability, possibly due to cortico-cortical projections between S1 and M1. This mechanism may underpin changes in corticomotor excitability in response to afferent input generated by PES.     

ALDH2 Protects Against Ischemic Stroke in Rats by Facilitating 4-HNE Clearance and AQP4 Down-Regulation

Aldehyde dehydrogenase 2 (ALDH2) is a new therapeutic target in the central nervous system. However, the association between ALDH2 and brain edema following ischemic stroke (IS) remains unclear. The present study was investigated to whether active ALDH2 can attenuate brain edema by using a rat model of IS, with the aim of clarifying the underlying mechanisms involved. Rats were administered the ALDH2 agonist Alda-1, vehicle or the ALDH2 inhibitor cyanamide (CYA) 15 min prior to a 1.5 h middle cerebral artery occlusion (MCAO) surgery. The effects of ALDH2 were subsequently investigated 24 h after reperfusion by evaluating neurological function, infarct sizes, brain edema volumes, 4-hydroxy-2-nonenal (4-HNE) levels, and aquaporin 4 (AQP4) protein expression. The results demonstrated that increasing ALDH2 activity significantly improved neurological deficits, reduced infarct sizes, and attenuated brain edema after MCAO. Alda-1 administration led to decreased 4-HNE levels and inhibited AQP4 protein expression in the peri-infarct section of the brain. Whereas, CYA administration increased 4-HNE levels, AQP4 expression, and simultaneously aggravated brain edema following MCAO. In conclusion, increasing ALDH2 activity can improve brain edema, infarct volumes, and reduce neurological impairment in a rat IS model. The therapeutic benefits of ALDH2 are related to 4-HNE clearance and AQP4 down-regulation.     

Intraamniotic Ethyl Docosahexaenoate Administration Protects Fetal Rat Brain from Ischemic Stress

Abstract: Studies were conducted on the prenatal rat given a single intraamniotic injection of ethyl docosahexaenoate (Et-DHA; 9.6–12 mmol per fetus) or subjected to an n-3 fatty acid-deficient diet to assess the role of docosahexaenoate on oxidative stress during episodes of ischemia. A time-dependent decrease in the ability of brain slices from animals treated with Et-DHA to produce thiobarbituric acid-reactive substance (TBARS), most pronounced after 1 day (from 58.1 ± 4.22 to 15.9 ± 1.6 nmol/mg of DNA), was noticed on stimulation with Fe²⁺. Brain slices from fetuses treated for 1 day with Et-DHA and those from untreated fetuses produced TBARS levels of 46.7 ± 6.5 and 114.8 ± 10.8 nmol/mg of DNA, respectively, after a 20-min occlusion of the fetal-maternal circulation at embryonic day 20, suggesting a protective effect of Et-DHA. The protective effect of a single dose of Et-DHA in utero remained high up to 3 days after injection ( p < 0.001) and was long-lasting, yet not significant, up to 3 days following birth. In agreement with a reduction in TBARS production by slices, the endogenous levels of TBARS in brains of Et-DHA-treated animals were lower than in the controls. Et-DHA-injected fetuses exhibited significantly higher levels of esterified DHA than the non-injected controls. n-3-deficient diet given to dams for 2 weeks before birth did not affect the levels of TBARS production in control fetal brain slices but abolished the increase caused by ischemia. Et-DHA administration for 24 h to n-3-deficient fetuses reduced the amount of TBARS produced by the fetal brain slices from 49.1 ± 8.5 to 31.7 ± 4.1 nmol/mg of DNA. A protective effect from oxidative damage after postischemic oxidative stress in fetal brain following DHA supplements is suggested, whereas the effect of n-3 fatty acid deficiency in this regard is more ambiguous.     

Environmental Enrichment Protects the Retina from Early Diabetic Damage in Adult Rats

Diabetic retinopathy is a leading cause of reduced visual acuity and acquired blindness. Available treatments are not completely effective. We analyzed the effect of environmental enrichment on retinal damage induced by experimental diabetes in adult Wistar rats. Diabetes was induced by an intraperitoneal injection of streptozotocin. Three days after vehicle or streptozotocin injection, animals were housed in enriched environment or remained in a standard environment. Retinal function (electroretinogram, and oscillatory potentials), retinal morphology, blood-retinal barrier integrity, synaptophysin, astrocyte and Müller cell glial fibrillary acidic protein, vascular endothelial growth factor, tumor necrosis factor-α, and brain-derived neurotrophic factor levels, as well as lipid peroxidation were assessed in retina from diabetic animals housed in standard or enriched environment. Environmental enrichment preserved scotopic electroretinogram a-wave, b-wave and oscillatory potential amplitude, avoided albumin-Evan''s blue leakage, prevented the decrease in retinal synaptophysin and astrocyte glial fibrillary acidic protein levels, the increase in Müller cell glial fibrillary acidic protein, vascular endothelial growth factor and tumor necrosis factor-α levels, as well as oxidative stress induced by diabetes. In addition, enriched environment prevented the decrease in retinal brain-derived neurotrophic factor levels induced by experimental diabetes. When environmental enrichment started 7 weeks after diabetes onset, retinal function was significantly preserved. These results indicate that enriched environment could attenuate the early diabetic damage in the retina from adult rats.     

Lyophilized Powder of Catalpol and Puerarin Protects Neurovascular Unit from Stroke

Hunting for an effective medicine for brain stroke has been a medical task in neuroscience for decades. The present research showed that the lyophilized Powder of Catalpol and Puerarin (C-P) in all the tested doses (65.4 mg/kg, 32.7 mg/kg, 16.4 mg/kg) significantly reduced the neurological deficiency, infarct volume and apoptotic cells in ischemic/reperfusion (I/R) rats. It also promoted astrocyte processes and prolonged neuron axons in infarct area. Further, it decreased MDA, NO, NF-κB/p65, TNF-α, IL-1β and IL-6 and enhanced the EPOR and GAF-43. 65.4 mg/kg and 32.7 mg/kg C-P could up-regulated EPO and VEGF significantly. In vitro, 49 μg/mL and 24.5 μg/mL C-P decreased the leakage of sodium fluorescein and increased the activity of γ-GTP. Additionally, it increased SOD and decreased MDA, NO, and LDH and decreased NF-κB/p65, TNF-α, IL-1β and IL-6 and unregulated EPO, EPOR, VEGF, and GAP-43. Only the dose of 49 μg/mL increased TEER and Claudin-5 and turned the typically damaged morphologies of neurons, astrocytes and endothelium into a favorable trend. These data imply that C-P improved the recovery of neurological deficiency in motor, sense, balance and reflex, and protected the whole NVU by anti-oxidative stress, anti-inflammation and up-regulating some protective factors. This research provides a candidate medicine for brain stroke and, at the same time, a pattern for drug study targeting NVU in vitro.     

Enriched Environment Protects the Optic Nerve from Early Diabetes-Induced Damage in Adult Rats

Diabetic retinopathy is a leading cause of reduced visual acuity and acquired blindness. Axoglial alterations of the distal (close to the chiasm) optic nerve (ON) could be the first structural change of the visual pathway in streptozotocin (STZ)-induced diabetes in rats. We analyzed the effect of environmental enrichment on axoglial alterations of the ON provoked by experimental diabetes. For this purpose, three days after vehicle or STZ injection, animals were housed in enriched environment (EE) or remained in a standard environment (SE) for 6 weeks. Anterograde transport, retinal morphology, optic nerve axons (toluidine blue staining and phosphorylated neurofilament heavy immunoreactivity), microglia/macrophages (ionized calcium binding adaptor molecule 1 (Iba-1) immunoreactivity), astrocyte reactivity (glial fibrillary acid protein-immunostaining), myelin (myelin basic protein immunoreactivity), ultrastructure, and brain derived neurotrophic factor (BDNF) levels were assessed in non-diabetic and diabetic animals housed in SE or EE. No differences in retinal morphology or retinal ganglion cell number were observed among groups. EE housing which did not affect the STZ-induced weight loss and hyperglycemia, prevented a decrease in the anterograde transport from the retina to the superior colliculus, ON axon number, and phosphorylated neurofilament heavy immunoreactivity. Moreover, EE housing prevented an increase in Iba-1 immunoreactivity, and astrocyte reactivity, as well as ultrastructural myelin alterations in the ON distal portion at early stages of diabetes. In addition, EE housing avoided a decrease in BDNF levels induced by experimental diabetes. These results suggest that EE induced neuroprotection in the diabetic visual pathway.     

Developmental Switch in Neurovascular Coupling in the Immature Rodent Barrel Cortex

Neurovascular coupling (NVC) in the adult central nervous system (CNS) is a mechanism that provides regions of the brain with more oxygen and glucose upon increased levels of neural activation. Hemodynamic changes that go along with neural activation evoke a blood oxygen level-dependent (BOLD) signal in functional magnetic resonance imaging (fMRI) that can be used to study brain activity non-invasively. A correct correlation of the BOLD signal to neural activity is pivotal to understand this signal in neuronal development, health and disease. However, the function of NVC during development is largely unknown. The rodent whisker-to-barrel cortex is an experimentally well established model to study neurovascular interdependences. Using extracellular multi-electrode recordings and laser-Doppler-flowmetry (LDF) we show in the murine barrel cortex of postnatal day 7 (P7) and P30 mice in vivo that NVC undergoes a physiological shift during the first month of life. In the mature CNS it is well accepted that cortical sensory processing results in a rise in regional cerebral blood flow (rCBF). We show in P7 animals that rCBF decreases during prolonged multi-whisker stimulation and goes along with multi unit activity (MUA) fatigue. In contrast at P30, MUA remains stable during repetitive stimulation and is associated with an increase in rCBF. Further we characterize in both age groups the responses in NVC to single sensory stimuli. We suggest that the observed shift in NVC is an important process in cortical development that may be of high relevance for the correct interpretation of brain activity e.g. in fMRI studies of the immature central nervous system (CNS).     

Growing Neural Stem Cells from Conventional and Nonconventional Regions of the Adult Rodent Brain

Recent work demonstrates that central nervous system (CNS) regeneration and tumorigenesis involves populations of stem cells (SCs) resident within the adult brain. However, the mechanisms these normally quiescent cells employ to ensure proper functioning of neural networks, as well as their role in recovery from injury and mitigation of neurodegenerative processes are little understood. These cells reside in regions referred to as "niches" that provide a sustaining environment involving modulatory signals from both the vascular and immune systems. The isolation, maintenance, and differentiation of CNS SCs under defined culture conditions which exclude unknown factors, makes them accessible to treatment by pharmacological or genetic means, thus providing insight into their in vivo behavior. Here we offer detailed information on the methods for generating cultures of CNS SCs from distinct regions of the adult brain and approaches to assess their differentiation potential into neurons, astrocytes, and oligodendrocytes in vitro. This technique yields a homogeneous cell population as a monolayer culture that can be visualized to study individual SCs and their progeny. Furthermore, it can be applied across different animal model systems and clinical samples, being used previously to predict regenerative responses in the damaged adult nervous system.     

Evaluation of Off-Hour Emergency Care in Acute Ischemic Stroke: Results from the China National Stroke Registry

Background and Purpose

The quality of after-hour emergency care of patients with acute ischemic stroke is debatable. We therefore, sought to analyze the performance measures, quality of care and clinical outcomes in these patients admitted during off-hours.

Methods

Our study included 4493 patients from a selected cohort of patients admitted to the hospitals with ischemic stroke in the China National Stroke Registry (CNSR) from September 2007 to August 2008. On-hour presentation was defined as arrival at the emergency department from the scene between 8AM and 5PM from Monday through Friday. Off-hours included the remainder of the on-hours and statutory holidays. The association between off-hour presentation and outcome was analyzed using multivariate logistic-regression models.

Results

Off-hour presentation was identified in 2672 (59.5%) patients with ischemic stroke. Comparison of patients admitted during off-hours with those admitted during on-hours revealed an unadjusted odds ratio of in-hospital mortality of 1.38 (95% confidence interval, 1.04–1.85), which declined to 1.34 (95% confidence interval, 0.93–1.93) after adjusting for patient characteristics (especially, pre-hospital delay). No difference in 30-day mortality, total death or dependence at three, six and 12 months between two groups was observed. No association between off-hour admission and quality of care was found.

Conclusions

In the CNSR database, compared with on-hour patients, off-hour patients with acute ischemic stroke admitted to the emergency departments from scene manifested a higher incidence of in-hospital mortality. However, the difference in incidence and quality of care between the groups disappeared after adjusting for pre-hospital delay and other variables.     

Dendrimers Target the Ischemic Lesion in Rodent and Primate Models of Nonarteritic Anterior Ischemic Optic Neuropathy

IntroductionPolyamidoamine dendrimer nanoparticles (~ 4 nanometers) are inert polymers that can be linked to biologically active compounds. These dendrimers selectively target and accumulate in inflammatory cells upon systemic administration. Dendrimer-linked compounds enable sustained release of therapeutic compounds directly at the site of damage. The purpose of this study was to determine if dendrimers can be used to target the optic nerve (ON) ischemic lesion in our rodent and nonhuman primate models of nonarteritic anterior ischemic optic neuropathy (NAION), a disease affecting >10,000 individuals in the US annually, and for which there currently is no effective treatment.MethodsNAION was induced in male Long-Evans rats (rNAION) and in one adult male rhesus monkey (pNAION) using previously described procedures. Dendrimers were covalently linked to near-infrared cyanine-5 fluorescent dye (D-Cy5) and injected both intravitreally and systemically (in the rats) or just systemically (in the monkey) to evaluate D-Cy5 tissue accumulation in the eye and optic nerve following induction of NAION.ResultsFollowing NAION induction, Cy-5 dendrimers selectively accumulated in astrocytes and circulating macrophages. Systemic dendrimer administration provided the best penetration of the ON lesion site when injected shortly after induction. Systemic administration 1 day post-induction in the pNAION model gave localization similar to that seen in the rats.ConclusionsDendrimers selectively target the ischemic ON lesion after induction of both rNAION and pNAION. Systemic nanoparticle-linked therapeutics thus may provide a powerful, targeted and safe approach to NAION treatment by providing sustained and focused treatment of the cells directly affected by ischemia.