Multiple Soluble Components of the Glycocalyx Are Increased in Patient Plasma After Ischemic Stroke.

Background and Purpose- The GLX (glycocalyx) is a protein/polysaccharide meshwork at the cellular surface. Consisting largely of glycosaminoglycans and proteoglycans, the GLX can shed in response to stress. In this study, we assay 11 components of the GLX in plasma from patients with ischemic stroke from a longitudinal cohort. Methods- Plasma samples from healthy individuals (N=8), and patients with ischemic stroke day ≥3, day 7, and day 90 (N=9-14) were immunoassayed for diverse components of the GLX. Results- Median stroke severity was mild (National Institutes of Health Stroke Scale 2.0 (range, 0-6) at day ≤3). Three (keratan-chondroitin-heparan-sulfate) of 4 glycosaminoglycans and CD44 (proteoglycan) were increased at day 7 and returned to baseline at day 90. Proteoglycan syndecan (Syn)-3 increased and Syn-2 levels decreased, significantly. Conclusions- Individual GLX components are often assayed as stand-alone biomarkers for endothelial health. This study suggests a full assessment of GLX components is more indicative of the endothelial health of an individual and represents a complex GLX signature that may be valuable as a composite biomarker of disease.

Selectivity, efficacy and toxicity studies of UCCB01-144, a dimeric neuroprotective PSD-95 inhibitor.

Inhibition of postsynaptic density protein-95 (PSD-95) decouples N-methyl-d-aspartate (NMDA) receptor downstream signaling and results in neuroprotection after focal cerebral ischemia. We have previously developed UCCB01-144, a dimeric PSD-95 inhibitor, which binds PSD-95 with high affinity and is neuroprotective in experimental stroke. Here, we investigate the selectivity, efficacy and toxicity of UCCB01-144 and compare with the monomeric drug candidate Tat-NR2B9c. Fluorescence polarization using purified proteins and pull-downs of mouse brain lysates showed that UCCB01-144 potently binds all four PSD-95-like membrane-associated guanylate kinases (MAGUKs). In addition, UCCB01-144 affected NMDA receptor signaling pathways in ischemic brain tissue. UCCB01-144 reduced infarct size in young and aged male mice at various doses when administered 30 min after permanent middle cerebral artery occlusion, but UCCB01-144 was not effective in young male mice when administered 1 h post-ischemia or in female mice. Furthermore, UCCB01-144 was neuroprotective in a transient stroke model in rats, and in contrast to Tat-NR2B9c, high dose of UCCB01-144 did not lead to significant changes in mean arterial blood pressure or heart rate. Overall, UCCB01-144 is a potent MAGUK inhibitor that reduces neurotoxic PSD-95-mediated signaling and improves neuronal survival following focal brain ischemia in rodents under various conditions and without causing cardiovascular side effects, which encourages further studies towards clinical stroke trials.

The Hypothermic Influence on CHOP and Ero1-α in an Endoplasmic Reticulum Stress Model of Cerebral Ischemia.

Hypoxia induced endoplasmic reticulum stress causes accumulation of unfolded proteins in the endoplasmic reticulum and activates the unfolded protein response, resulting in apoptosis through CCAAT-enhancer-binding protein homologous protein (CHOP) activation. In an in vitro and in vivo model of ischemic stroke, we investigated whether hypothermia regulates the unfolded protein response of CHOP and Endoplasmic reticulum oxidoreductin-α (Ero1-α), because Ero1-α is suggested to be a downstream CHOP target. The gene expression of CHOP and Ero1-α was measured using Quantitative-PCR (Q-PCR) in rat hippocampi following global cerebral ischemia, and in hypoxic pheochromocytoma cells during normothermic (37 °C) and hypothermic (31 °C) conditions. As a result of ischemia, a significant increase in expression of CHOP and Ero1-α was observed after three, six and twelve hours of reperfusion following global ischemia. A stable increase in CHOP expression was observed throughout the time course (p < 0.01, p < 0.0001), whereas Ero1-α expression peaked at three to six hours (p < 0.0001). Induced hypothermia in hypoxia stressed PC12 cells resulted in a decreased expression of CHOP after three, six and twelve hours (p < 0.0001). On the contrary, the gene expression of Ero1-α increased as a result of hypothermia and peaked at twelve hours (p < 0.0001). Hypothermia attenuated the expression of CHOP, supporting that hypothermia suppress endoplasmic reticulum stress induced apoptosis in stroke. As hypothermia further induced up-regulation of Ero1-α, and since CHOP and Ero1-α showed differential regulation as a consequence of both disease (hypoxia) and treatment (hypothermia), we conclude that they are regulated independently.

Comparison of quantitative estimation of intracerebral hemorrhage and infarct volumes after thromboembolism in an embolic stroke model.

BACKGROUND: Strokes have both ischemic and hemorrhagic components, but most studies of experimental stroke only address the ischemic component. This is likely because investigations of hemorrhagic transformation are hindered by the lack of methods based on unbiased principles for volume estimation. AIMS: We evaluated different methods for estimating the volume of infarcts, hemorrhages, after embolic middle cerebral artery occlusion with or without thrombolysis. METHODS: An experimental thromboembolytic rat model was used in this study. The rats underwent surgery and were placed in two groups. Group 1 was treated with saline, and group 2 was treated with 20 mg/kg recombinant tissue plasminogen activator to promote intracerebral hemorrhages. Stereology, semiautomated computer estimation, and manual erythrocyte counting were used to test the precision and efficiency of determining the size of the infarct and intracerebral hemorrhage. RESULTS: No differences were observed in the infarct volume or amount of bleeding when comparing the three methods of volume estimation. Although semiautomated computer estimation and manual erythrocyte counting provided similar results as the stereological measurements, the stereological method was the most efficient and advantageous. CONCLUSIONS: We found that stereology was the superior method for quantification of hemorrhagic volume, especially for rodent petechial bleeding, which is otherwise difficult to measure. Our results suggest the possibility of measuring both the ischemic and the hemorrhagic components of stroke, two parameters that may be differentially regulated when therapeutic regimens are tested.

Drug-induced hypothermia as beneficial treatment before and after cerebral ischemia.

OBJECTIVES: Hypothermia is still unproven as beneficial treatment in human stroke, although in animal models, conditioning the brain with hypothermia has induced tolerance to insults. Here, we delineate the feasibility of drug-induced mild hypothermia in reducing ischemic brain damage when conditioning before (preconditioning) and after (postconditioning) experimental stroke. METHODS: Hypothermia was induced in rats with a bolus of 6 mg/kg talipexole followed by 20 h continuous talipexole infusion of 6 mg/kg in total. Controls received similar treatment with saline. The core body temperature was continuously monitored. In preconditioning, hypothermia was terminated before either reversible occlusion of the middle cerebral artery (MCAO) for 60 min or global ischemia for 10 min with 2-vessel occlusion and hypotension. In postconditioning, rats experienced 60 min of MCAO before hypothermia was induced either immediately or with 3 h delay. Rats survived ischemia for 2, 7 or 90 days. Infarct volumes were quantified by stereology. Additional experiments of methodological relevance were included in the study. RESULTS: Talipexole induced mild hypothermia (35.1±1.1 to 36.0±0.5°C) for <20 h. Hypothermic pre- and postconditioning reduced infarct sizes by more than 60% as monitored during the first 90 days after experimental stroke (p<0.05). CONCLUSION: Talipexole is registered for use as a dopamine substitute in humans with Parkinson's disease. Although dosages cannot be directly translated to patients, our study exemplifies in an animal model that drug-induced hypothermia in a clinical setting might reduce cerebral ischemic damage before neuro- and cardiac surgical procedures and after stroke.

Effects of buprenorphine and meloxicam analgesia on induced cerebral ischemia in C57BL/6 male mice.

Laboratory mice constitute an extensively used model to study the pathologic and functional outcomes of cerebral ischemic stroke. The middle cerebral artery occlusion (MCAO) model requires surgical intervention, which potentially can result in postsurgical pain and stress. In the present study, we investigated whether buprenorphine and meloxicam, at clinically relevant doses provided pain relief without altering infarct volume in male C57BL/6 mice. Common known side-effects of buprenorphine, including decreased food consumption, were noted after surgery in buprenorphine-treated mice, but these effects were brief and seen only during the treatment period. Fecal corticosterone metabolites did not differ significantly between the groups. In the present study, buprenorphine treatment did not alter infarction volume when compared with that of mice that did not receive analgesia. In contrast, meloxicam treatment significantly reduced infarct volume and may be a confounder if used as an analgesic during MCAO surgery. Furthermore, investigation of behavioral profiles by using an automated behavioral scoring system showed that rearing and sniffing behaviors decreased as infarct volume increased. This suggests that studies of exploratory behavior may aid in developing new markers of short-term stroke-related behavioral deficiencies in laboratory mice.

Remote post-conditioning reduces hypoxic damage early after experimental stroke.

OBJECTIVES: Given that reliable markers for early ischemic brain damage are lacking, we set out to test whether pimonidazole can be used as a reliable tool in the quantification of hypoxic insults, at early time points following experimental stroke. METHODS: We have used semi-quantitative Western blotting detection of pimonidazole adducts in a rat model of reversible middle cerebral artery occlusion (MCAO), treated with remote post-conditioning. RESULTS: First, we demonstrated that a linear relationship exist between pimonidazole binding in the ischemic hemisphere and duration of ischemia, in animals subjected to 5, 15, 30, or 60 minutes of occlusion followed by 120 minutes of reflow. Then we showed a significant reduction in pimonidazole binding in the infarcted hemisphere, when rats with 60 minutes of MCAO, immediately after establishment of cerebral reflow, had 3×15 minutes intermittent hind limb ischemia followed by 24-hour survival. We analysed the middle cerebral arteries from animals with 60 minutes of MCAO and early remote post-conditioning, followed by 30 minutes, 24, or 48 hours of reflow. At 24 hours of reflow increases in phosphorylated protein kinase C-alpha with concomitantly increased levels of p38 phosphorylation were observed. CONCLUSIONS: Our investigation demonstrates that pimonidazole can be used for quantifying ischemic impact in stroke, even after very short survival times. It furthermore shows that early remote post-conditioning reduces ischemic damage, probably through hyperpolarization and reduced reflow vasospasm in the conduit middle cerebral arteries.

Functionally induced changes in water transport in the proximal tubule segment of rat kidneys.

To eliminate freezing artifacts in the proximal tubule cells, two cryotechniques were applied to normal rat kidneys, ie, freeze substitution and special freeze drying. In addition, salt depletion and salt loading were applied to groups of rats to evaluate whether the segmental structure of the proximal tubule could be altered. In the superficial part of the renal cortex of normal kidneys, the typical first segment structure in the proximal tubule was generally present in the early postglomerular fraction of the tubule. However, in the second segment, a special cellular phenomenon was constantly present, comprising a significant intercellular space that was easily identified using a light microscope. In the third segment, in which the presence of basolateral interdigitations is minimal, the small lateral space, which was found to be present in cryopreparations between neighboring cells from the normal kidney, was found to be enlarged by heavy salt loading of short duration. It is concluded that these cryotechniques demonstrate quantitative structural variations between superficial and deep nephrons, as well as the presence of extracellular areas between the cells of the second and the third segment, representing a structural background for the essential transport of water from the proximal tubules to the peritubular capillaries.

Differences in origin of reactive microglia in bone marrow chimeric mouse and rat after transient global ischemia.

Current understanding of microglial involvement in disease is influenced by the observation that recruited bone marrow (BM)-derived cells contribute to reactive microgliosis in BM-chimeric mice. In contrast, a similar phenomenon has not been reported for BM-chimeric rats. We investigated the recruitment and microglial transformation of BM-derived cells in radiation BM-chimeric mice and rats after transient global cerebral ischemia, which elicits a characteristic microglial reaction. Both species displayed microglial hyperplasia and rod cell transformation in the hippocampal CA1 region. In mice, a subpopulation of lesion-reactive microglia originated from transformed BM-derived cells. By contrast, no recruitment or microglial transformation of BM-derived cells was observed in BM-chimeric rats. These results suggest that reactive microglia in rats originate from resident microglia, whereas they have a mixed BM-derived and resident origin in mice, depending on the severity of ischemic tissue damage.

The effect of voluntarily ingested buprenorphine on rats subjected to surgically induced global cerebral ischaemia.

The effect of perioperatively administered buprenorphine analgesia on rats subjected to surgically induced global ischaemia was assessed. Rats supplied with buprenorphine, mixed in nut paste for voluntary ingestion, displayed significant reductions in postoperative excretions of faecal corticosterone, in both magnitude and variance. This is indicative of lowered stress levels and less inter-animal metabolic variation. Although corticosterone has been reported to modulate the extent of cerebral damage, histology of coronal sections exhibited no differences in the extent of the ischaemia in buprenorphine-treated and untreated animals. A part from a slightly higher hyperthermia immediately after surgery and typical opiate-associated behaviour, the buprenorphine treatment had no apparent adverse effects on the experimental model. In contrast, the analgesic treatment improved the model by minimizing stress-associated confounding variables in the experimental animals.

The site of embolization related to infarct size, oedema and clinical outcome in a rat stroke model - further translational stroke research.

BACKGROUND AND PURPOSE: Reliable models are essential for translational stroke research to study the pathophysiology of ischaemic stroke in an effort to find therapies that may ultimately reduce oedema, infarction and mortality in the clinic. The purpose of this study was to investigate the relation between the site of arterial embolization and the subsequent oedema, infarction and clinical outcome in a rat embolic stroke model. METHODS: Thirty-six male Sprague-Dawley rats were thromboembolized into the internal carotid artery. The site of occlusion was demonstrated by arteriography. Following histological preparation and evaluation, the size of the hemispheres and the infarcts were measured by quantitative histology and planimetry. Another parallel stroke model study was subsequently examined to investigate if the conclusions from the first study could be applied to the second study. RESULTS: The median size of the infarct was 40% of the ipsilateral hemisphere in both the 19 animals with occlusion localised to the intracranial part of the internal carotid artery and in the 11 animals where the main trunk of the middle cerebral artery was occluded. In 5 animals, occlusion of the extracranial part of the internal carotid artery resulted in significantly smaller infarcts compared to other groups (p < 0.01). Another independent study re-confirmed these results. Furthermore, significant correlations (R > 0.76, p < 0.0001) were found between 1) cortical, subcortical, and total infarct volumes, 2) oedema in percent of the left hemisphere, 3) clinical score before termination and 4) postoperative weight loss. CONCLUSIONS: Distal occlusions of the intracranial part of the internal carotid or middle cerebral arteries resulted in comparable large sized infarctions and oedema. This indicates that investigators do not need a similar number of such occlusions in each experimental group. Contrary to observations in the clinic, distal internal carotid artery occlusions did not result in worse outcome than middle cerebral stem occlusions, but this finding may be explained by the controlled emboli size in this experimental stroke model.

Actions and interactions of extracellular potassium and kainate on expression of 13 gamma-aminobutyric acid type A receptor subunits in cultured mouse cerebellar granule neurons.

Cerebellar granule neurons in culture are a popular model for studying neuronal signaling and development. Depolarizing concentrations of K(+) are routinely used to enhance cell survival, and kainate is sometimes added to eliminate GABAergic neurons. We have investigated the effect of these measures on expression of mRNA for gamma-aminobutyric acid type A (GABA(A)) receptor alpha1-6, beta1-3, gamma1-3, and delta subunits in cultures of mouse cerebellar granule neurons grown for 7 or 12 days in vitro (DIV) using semiquantitative reverse transcription-PCR. We detected mRNA for the alpha1, alpha2, alpha5, alpha6, beta2, beta3, gamma2, and delta subunits in all the cell cultures, but the expression levels of the alpha5-, alpha6-, and beta2-subunit mRNAs were significantly dependent on the composition of the culture medium. Both an increase of the extracellular K(+) concentration from 5 to 25 mm and the addition of 50 microm kainate immediately depolarized the neurons but prolonged exposure (7-8 DIV)-induced compensatory hyperpolarization. 25 mm K(+) caused a shift from alpha6 to alpha5 expression measured at 7 and 12 DIV, which was mimicked by kainate in 12 DIV cultures. The expression of beta2 was decreased by 25 mm K(+) in 7 DIV cultures and by kainate in 12 DIV cultures. The effects on beta2 expression could not be ascribed to depolarization. Alterations of alpha6 mRNA expression were reflected in altered sensitivity to GABA and furosemide of the resulting receptors. Our study has shown that a depolarizing K(+) concentration as well as kainate in the culture medium significantly disturbs maturation of GABA(A) receptor subunit expression.