Ischemic Core Area Research Articles

The Impact of VEGF-C-Induced Dural Lymphatic Vessel Growth on Ischemic Stroke Pathology.

Timely relief of edema and clearance of waste products, as well as promotion of anti-inflammatory immune responses, reduce ischemic stroke pathology, and attenuate harmful long-term effects post-stroke. The discovery of an extensive and functional lymphatic vessel system in the outermost meningeal layer, dura mater, has opened up new possibilities to facilitate post-stroke recovery by inducing dural lymphatic vessel (dLV) growth via a single injection of a vector encoding vascular endothelial growth factor C (VEGF-C). In the present study, we aimed to improve post-stroke outcomes by inducing dLV growth in mice. We injected mice with a single intracerebroventricular dose of adeno-associated viral particles encoding VEGF-C before subjecting them to transient middle cerebral artery occlusion (tMCAo). Behavioral testing, Gadolinium (Gd) contrast agent-enhanced magnetic resonance imaging (MRI), and immunohistochemical analysis were performed to define the impact of VEGF-C on the post-stroke outcome. VEGF-C improved stroke-induced behavioral deficits, such as gait disturbances and neurological deficits, ameliorated post-stroke inflammation, and enhanced an alternative glial immune response. Importantly, VEGF-C treatment increased the drainage of brain interstitial fluid (ISF) and cerebrospinal fluid (CSF), as shown by Gd-enhanced MRI. These outcomes were closely associated with an increase in the growth of dLVs around the region where we observed increased vefgc mRNA expression within the brain, including the olfactory bulb, cortex, and cerebellum. Strikingly, VEGF-C-treated ischemic mice exhibited a faster and stronger Gd-signal accumulation in ischemic core area and an enhanced fluid outflow via the cribriform plate. In conclusion, the VEGF-C-induced dLV growth improved the overall outcome post-stroke, indicating that VEGF-C has potential to be included in the treatment strategies of post-ischemic stroke. However, to maximize the therapeutic potential of VEGF-C treatment, further studies on the impact of an enhanced dural lymphatic system at clinically relevant time points are essential.

Neuroprotective Effects of Tryptanthrin-6-Oxime in a Rat Model of Transient Focal Cerebral Ischemia.

The activation of c-Jun N-terminal kinase (JNK) plays an important role in stroke outcomes. Tryptanthrin-6-oxime (TRYP-Ox) is reported to have high affinity for JNK and anti-inflammatory activity and may be of interest as a promising neuroprotective agent. The aim of this study was to investigate the neuroprotective effects of TRYP-Ox in a rat model of transient focal cerebral ischemia (FCI), which involved intraluminal occlusion of the left middle cerebral artery (MCA) for 1 h. Animals in the experimental group were administered intraperitoneal injections of TRYP-Ox 30 min before reperfusion and 23 and 47 h after FCI. Neurological status was assessed 4, 24, and 48 h following FCI onset. Treatment with 5 and 10 mg/kg of TRYP-Ox decreased mean scores of neurological deficits by 35-49 and 46-67% at 24 and 48 h, respectively. At these doses, TRYP-Ox decreased the infarction size by 28-31% at 48 h after FCI. TRYP-Ox (10 mg/kg) reduced the content of interleukin (IL) 1β and tumor necrosis factor (TNF) in the ischemic core area of the MCA region by 33% and 38%, respectively, and attenuated cerebral edema by 11% in the left hemisphere, which was affected by infarction, and by 6% in the right, contralateral hemisphere 24 h after FCI. TRYP-Ox reduced c-Jun phosphorylation in the MCA pool at 1 h after reperfusion. TRYP-Ox was predicted to have high blood-brain barrier permeability using various calculated descriptors and binary classification trees. Indeed, reactive oxidant production was significantly lower in the brain homogenates from rats treated with TRYP-Ox versus that in control animals. Our data suggest that the neuroprotective activity of TRYP-Ox may be due to the ability of this compound to inhibit JNK and exhibit anti-inflammatory and antioxidant activity. Thus, TRYP-Ox may be considered a promising neuroprotective agent that potentially could be used for the development of new treatment strategies in cerebral ischemia.

Spatial transcriptomics approach to hypercontractile parenchymal arteries associated with ischemia-reperfusion

Background: Despite vessel recanalization, up to 67% of ischemic-stroke patients experience futile reperfusion increasing the risk of neurological morbidity and death. Arterial hypercontractility has been suggested to underlie the no-flow phenomenon, however, this remains to be fully elucidated. Parenchymal arteries in the area surrounding the ischemic core (penumbra) display an elevated vascular tone due to increased intracellular Ca 2+ sensitivity. Ischemia-reperfusion (IR) reduces Na + , K + -ATPase activity and is inversely proportional to Src activation, a tyrosine kinase, which is known to sensitize the contractile machinery to Ca 2+ via myosin light chain kinase (MLCK) activation. We hypothesize that IR reduces Na + , K + -ATPase activity and increases Src with consequent Ca 2+ sensitization and hypercontractility of parenchymal arteries. Materials and Methods: Five 3-month-old C57BL/6J male mice were exposed to one-hour transient middle cerebral artery occlusion by a filament followed by 24-hour reperfusion. Mice were anesthetized with isoflurane (3% induction, 1.5–2% maintenance) and intraperitoneally injected with Temgesic (0.1 mg/kg) 0.5- and 6 hours post-operative. Nissl staining was used to detect infarct areas. Spatial transcriptomics (ST) of coronal slices was performed. Infarct size was corrected for edema and calculated as the percentage of the whole hemisphere. ST was cell-predicted, and the cortex region was specified using gene markers from a single-cell RNA sequencing dataset. Pearson’s correlation coefficient was used to predict cell-cell correlation in the ST spots. Cells of interest were used for average gene expression analysis of scaled data. The expression levels were compared between the two hemispheres using t-tests. Data are presented as mean ± SD. Results: The ischemic core area was 0.67 ± 0.34 cm 2 corresponding to 18.11 ± 8.28% of the whole hemisphere. An interrelationship between cell types from the cerebral blood vessels and astrocytes was found, and these cell types were pooled. When comparing the average gene expressions between the contralateral and ipsilateral hemisphere, the Na + , K + -ATPase α 1 and α 2 isoforms were downregulated in the ipsilateral cortex area (p=2.7e -6 and p=3.3e -6 , respectively). Src (p=2.3e -4 ) and MLCK were downregulated in the ipsilateral cortex (4.8e -2 ). All genes for the myosin light chain phosphatase (MLCP) were downregulated. Conclusion: The data suggest that potentiated hypercontractility of parenchymal arteries is not due to Na + , K + -ATPase, and Src kinase-associated activation of the Ca 2+ -dependent MLCK. However, hypercontractility of parenchymal arteries may be due to a downregulated MLCP. Funding: This study was funded by the Lundbeck Foundation (R344-2020-952). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Injectable Supramolecular Hybrid Hydrogel Delivers IL-1β-Stimulated Exosomes to Target Neuroinflammation

Long-term neuroinflammation is a major barrier to neurological recovery after cerebral ischemia-reperfusion injury (CIRI). Here, a thermosensitive injectable supramolecular hybrid hydrogel is developed to sustainably deliver exosomes derived from interleukin-1β-stimulated bone marrow stromal cells (BMSCs) (βExos) with improved exosome production and anti-inflammatory capacity for neuroinflammation inhibition and neurological recovery. The supramolecular hydrogel displays self-healing and injectable features, along with high biocompatibility and tissue-like softness. The βExos effectively reduce the lipopolysaccharide-induced inflammatory responses in the immortalized mouse microglia (BV2) cell line, and the in situ formed hydrogel improves the exosome retention in the ischemic core area. More remarkably, in the middle cerebral artery occlusion in vivo model, glial scar formation and neuronal loss are significantly reduced by regulating neuroinflammation using the released βExos. Therefore, the combination of interleukin-1β-stimulated exosomes with injectable supramolecular hydrogel provides an appealing strategy for treating central nervous system diseases.

Protective Effect of Buyang Huanwu Decoction on Cerebral Ischemia Reperfusion Injury by Alleviating Autophagy in the Ischemic Penumbra.

Objectives To evaluate the protective effect of Buyang Huanwu Decoction (BHD) against cerebral ischemia reperfusion and investigate whether autophagy is involved in its mechanism of action. Methods Adult male Sprague Dawley rats were randomly divided into three groups: the sham, cerebral ischemia reperfusion (I/R), and I/R + BHD groups. A rat model of cerebral I/R injury was established via middle cerebral artery occlusion (MCAO) for 2 h, followed by 1, 3, and 7 d of reperfusion. Neurological scores and regional cerebral blood flow were assessed to determine whether the model was successfully established. Brain infarct volume was determined by 2,3,5-triphenyl tetrazolium chloride (TTC) staining. The apoptosis rate was detected using TdT-mediated dUTP Nick-End Labeling (TUNEL) staining, and neuronal damage was evaluated by Nissl staining. The Beclin-1 and LC3 protein levels in the ischemic core, penumbra, and contralateral area were analysed by Western blotting. The occurrence of autophagy in the penumbra was observed by transmission electron microscopy (TEM). Results BHD treatment alleviated the cerebral infarct volume, neuronal apoptosis rate, and neuronal damage 3 and 7 d after cerebral I/R injury. Furthermore, 3 d after reperfusion, we observed that the Beclin-1 levels were significantly decreased in the core in the I/R group, whereas transformation of LC3 I to LC3 II exhibited no obvious differences between the sham and I/R groups. In the penumbra, the Beclin-1 levels and transformation of LC3 I to LC3 II in the I/R group were significantly increased compared with that in the sham group. However, no significant difference in the contralateral area was noted between the two groups. BHD significantly inhibited the expression of Beclin-1 and the transformation of LC3 I to LC3 II in the penumbra after cerebral I/R injury but yielded no significant changes in the core and contralateral area. Conclusions BHD exerts a neuroprotective effect by inhibiting autophagy in neurons in the penumbra after cerebral I/R injury.

Color-coded multiphase computed tomography angiography may predict outcome in anterior circulation acute ischemic stroke

PurposeTo evaluate whether arterial and venous color-coded mCTA score may predict clinical outcome in anterior circulation acute ischemic stroke. MethodsConsecutive patients referred to the emergency department with anterior circulation acute ischemic stroke (AIS) were retrospectively reviewed at our center. All patients underwent multimodal brain computed tomography (CT) imaging, including non-contrast CT (NCCT) and multiphase computed tomography angiography (mCTA). Baseline collateral scores of color-coded mCTA, also known as ColorViz, and conventional mCTA were recorded. mCTA was assessed by a 6-point scale whereas color-coded mCTA was assessed by a 3-point scale. In the Color-coded maps, a different color is assigned to intracranial vessels based on the arrival time of the contrast medium and on a per-person adaptive threshold technique. We compared the radiological and clinical features of a group of patients who reached independency (defined as modified Rankin Scale score ≤ 2) with those of patients who did not. A multivariate logistic regression model was then used to assess the potential of color-coded mCTA scores to predict patients' outcome after AIS. ResultsA total of 86 patients (36 M, 50 F) were enrolled in the study. Multivariate logistic regression showed that score 3 at Color-coded mCTA was a good predictor of favorable outcome (p = 0.003). Moreover, NIHSS at onset (p = 0.004) and discharge (p < 0.001) along with ischemic core area (p = 0.011) were significant predictors of favorable prognosis. Conclusionour data confirm that ColorViz is a useful and easily understandable neuroimaging tool that might have a predictive role in assessing the outcome of anterior circulation acute ischemic stroke patients regardless of revascularization therapy.

A Novel Fast CT Perfusion Core-Penumbra Mismatch Score

Advanced neuroimaging can identify patients who can most benefit from reperfusion treatment, discriminating between ischemic core and penumbra area in a quick and accurate manner. Despite core-penumbra mismatch being an independent prognostic factor, computed tomography perfusion (CTP) assessment is still debated in hyperacute decision-making. The authors aimed to study a novel CTP mismatch score in emergency settings and to investigate its relation with clinical outcome in acute ischemic stroke patients treated with intravenous thrombolysis (IVT). Neuroimaging and clinical data of 226 consecutive acute ischemic stroke patients were analyzed. The study population was divided into 5 different CTP scores: (0) without perfusion deficit, (1) only penumbra, (2) penumbra > core, (3) core ≥ penumbra, (4) only core. For differences in outcome between treated and nontreated patients, and among CTP core-penumbra groups to be assessed, the authors have evaluated the outcome in terms of National Institutes of Health Stroke Scale (NIHSS) and modified Rankin scale (mRS) at discharge and symptomatic intracerebral hemorrhage. A decrease in NIHSS was statistically greater in IVT-treated patients compared to nontreated patients showing only penumbra (ΔNIHSS%: 80.0% vs. 50.0%; P=0.0023) or no perfusion deficit (ΔNIHSS%: 89.4% vs. 61.5%; P=0.027) on CTP maps. The same trend was found in other groups without significant difference. A significant correlation was found in IVT patients between core/penumbra score and outcome in terms of ΔNIHSS (Kendall τ=-0.19; P=0.004). The authors proposed a novel immediate CTP assessment to score perfusion mismatch in emergency settings to guide clinicians' decision-making for aggressive treatment and to prevent stroke-related disability.

Investigation of a new type of microglia appearing in the ischemic core area

Investigation of a new type of microglia appearing in the ischemic core area

Transthyretin expression in the postischemic brain.

The unknown role of the carrier protein transthyretin (TTR) in mechanisms of functional recovery in the postischemic brain prompted us to study its expression following experimental stroke. Male C57/B6 mice (age 9 to 10 weeks) were subjected to permanent focal ischemia induced by photothrombosis (PT) and brain tissues were analyzed for ttr expression and TTR levels at 24 hours, 48 hours, 7 days and 14 days following the insult by RT-PCR, Western blot and immunohistochemistry. Fourteen days after PT, non-specific TTR-like immunoreactive globules were found in the ischemic core and surrounding peri-infarct region by immunohistochemistry that could not be allocated to DAPI positive cells. No TTR immunoreactivity was found when stainings were performed with markers for neurons (Neuronal Nuclei, NeuN), reactive astrocytes (glial fibrillary acidic protein, GFAP) or microglia (cluster of differentiation 68, CD68). In addition, we could not find TTR by immunoblotting in protein extracts obtained from the ischemic territory nor ttr expression by RT-PCR at all time points following PT. In all experiments, ttr expression in the choroid plexus and TTR in the mouse serum served as positive controls and recombinant legumain peptide as negative control. Together, our results indicate that TTR is not synthesized in brain resident cells in the ischemic infarct core and adjacent peri-infarct area. Thus, it seems unlikely that in situ synthesized TTR is involved in mechanisms of tissue reorganization during the first 14 days following PT.

Influence of occlusion site and baseline ischemic core on outcome in patients with ischemic stroke.

We assessed patient clinical outcomes based on occlusion location, focusing on distal occlusions to understand if occlusion location was an independent predictor of outcome, and tested the relationship between occlusion location and baseline ischemic core, a known predictor of modified Rankin Scale (mRS) score at 90 days. We analyzed a prospectively collected cohort of thrombolysis-eligible ischemic stroke patients from the International Stroke Perfusion Imaging Registry who underwent multimodal CT pretreatment. For the primary analysis, logistic regression was used to predict the effect of occlusion location and ischemic core on the likelihood of excellent (mRS 0-1) and favorable (mRS 0-2) 90-day outcomes. This study included 945 patients. The rates of excellent and favorable outcome in patients with distal occlusion (M2, M3 segment of middle cerebral artery, anterior cerebral artery, and posterior cerebral artery) were higher than M1 occlusions (mRS 0%-1%, 55% vs 37%; mRS 0%-2%, 73% vs 50%, p < 0.001). Vessel occlusion location was not a strong predictor of outcomes compared to baseline ischemic core (area under the curve, mRS 0-1, 0.64 vs 0.83; mRS 0-2, 0.70 vs 0.86, p < 0.001). There was no interaction between occlusion location and ischemic core (interaction coefficient 1.00, p = 0.798). Ischemic stroke patients with a distal occlusion have higher rate of excellent and favorable outcome than patients with an M1 occlusion. The baseline ischemic core was shown to be a more powerful predictor of functional outcome than the occlusion location, but the relationship between ischemic core and outcome does not different by occlusion locations.

Neuronal Regeneration after Electroacupuncture Treatment in Ischemia-Reperfusion-Injured Cerebral Infarction Rats.

Adult neuronal cells which can regenerate have been reported. The present study investigated whether acupuncture enhances neuronal regeneration in ischemic stroke rats. We established an ischemic stroke rat model by occluding the cerebral blood flow of the right middle cerebral artery for 15 minutes and then allowing reperfusion in Sprague–Dawley rats. The results indicated that, in these rats, 2 Hz electroacupuncture (EA) at both Zusanli (ST36) and Shangjuxu (ST37) acupoints reduced the infarction/hemisphere ratio 8 days after reperfusion and reduced the modified neurological severity score (mNSS) and increased the rotarod test time 4 and 8 days after reperfusion, respectively. In addition, 2 Hz reduced nestin immunoreactive cells in the penumbra area and the ischemic core area; 2 Hz EA also reduced Ki67 immunoreactive cells and increased glial fibrillary acidic protein immunoreactive cells in the penumbra area. These findings suggest that 2 Hz EA at the ST36 and ST37 acupoints has a neuroprotective role. However, additional studies are needed to further investigate these preliminary results.

Combined morphine and limb remote ischemic perconditioning provides an enhanced protection against myocardial ischemia/reperfusion injury by antiapoptosis

BackgroundBoth morphine and limb remote ischemic perconditioning (RIPer) can protect against myocardial ischemia/reperfusion injury (IRI). This experiment was designed to assess whether combined morphine and limb RIPer could provide and enhanced protection against myocardial IRI in an in vivo rat model. MethodsOne hundred male Sprague–Dawley rats were randomly allocated to six groups: sham, ischemia/reperfusion (IR), ischemic preconditioning, RIPer, morphine (M), and combined morphine and remote ischemic perconditioning (M + RIPer). Ventricular arrhythmias that occurred during ischemia and early reperfusion were scored, and serum creatine kinase isoenzyme and cardiac troponin I levels were assayed. The infarct size was determined by Evans blue and triphenyl tetrazolium chloride staining. The apoptosis in the myocardial ischemic core, ischemic border, and nonischemic areas was assessed through real-time polymerase chain reaction for Bax and Bcl-2 and with the transferase-mediated deoxyuridine triphosphate-biotin nick end labeling assay. ResultsThe infarct size, serum cardiac troponin I level, incidence, and score of the arrhythmias during the initial reperfusion were significantly reduced in the M + RIPer group compared with the IR group but did not differ significantly between the ischemic preconditioning and M + RIPer groups. Transferase-mediated deoxyuridine triphosphate-biotin nick end labeling–positive cells were significantly decreased, and the Bcl-2/Bax ratio was significantly increased in the M + RIPer group compared with the IR group. ConclusionsThis experiment demonstrates that combined morphine and limb RIPer provides an enhanced protection against myocardial IRI by the Bcl-2-linked apoptotic signaling pathway.

Differences in the profile of protection afforded by TRO40303 and mild hypothermia in models of cardiac ischemia/reperfusion injury

The mode of protection against cardiac reperfusion injury by mild hypothermia and TRO40303 was investigated in various experimental models and compared to MitoQ in vitro.In isolated cardiomyocytes subjected to hypoxia/reoxygenation, TRO40303, MitoQ and mild hypothermia delayed mPTP opening, inhibited generation of mitochondrial superoxide anions at reoxygenation and improved cell survival. Mild hypothermia, but not MitoQ and TRO40303, provided protection in a metabolic starvation model in H9c2 cells and preserved respiratory function in isolated rat heart mitochondria submitted to anoxia/reoxygenation. In the Langendorff-perfused rat heart, only mild hypothermia provided protection of hemodynamic function and reduced infarct size following ischemia/reperfusion. In biopsies from the left ventricle of pigs subjected to in vivo occlusion/reperfusion, TRO40303 specifically preserved respiratory functions in the peri-infarct zone whereas mild hypothermia preserved both the ischemic core area and the peri-infarct zones. Additionally in this pig model, only hypothermia reduced infarct size.We conclude that mild hypothermia provided protection in all models by reducing the detrimental effects of ischemia, and when initiated before occlusion, reduced subsequent reperfusion damage leading to a smaller infarct. By contrast, although TRO40303 provided similar protection to MitoQ in vitro and offered specific protection against some aspects of reperfusion injury in vivo, this was insufficient to reduce infarct size.

Impact of aging on spreading depolarizations induced by focal brain ischemia in rats

Spreading depolarization (SD) contributes to the ischemic damage of the penumbra. Although age is the largest predictor of stroke, no studies have examined age dependence of SD appearance. We characterized the electrophysiological and hemodynamic changes in young (6 weeks old, n = 7), middle-aged (9 months old, n = 6), and old (2 years old, n = 7) male Wistar rats during 30 minutes of middle cerebral artery occlusion (MCAO), utilizing multimodal imaging through a closed cranial window over the ischemic cortex: membrane potential changes (with a voltage-sensitive dye), cerebral blood volume (green light reflectance), and cerebral blood flow (CBF, laser-speckle imaging) were observed. The initial CBF drop was similar in all groups, with a significant further reduction during ischemia in old rats (p < 0.01). Age reduced the total number of SDs (p < 0.05) but increased the size of ischemic area displaying prolonged SD (p < 0.01). The growth of area undergoing prolonged SDs positively correlated with the growth of ischemic core area (p < 0.01) during MCAO. Prolonged SDs and associated hypoperfusion likely compromise cortical tissue exposed to even a short focal ischemia in aged rats.

Impulsivity moderates the relation between age and increases in adolescent alcohol use

Spreading depolarization (SD) contributes to the ischemic damage of the penumbra. Although age is the largest predictor of stroke, no studies have examined age dependence of SD appearance. We characterized the electrophysiological and hemodynamic changes in young (6 weeks old, n = 7), middle-aged (9 months old, n = 6), and old (2 years old, n = 7) male Wistar rats during 30 minutes of middle cerebral artery occlusion (MCAO), utilizing multimodal imaging through a closed cranial window over the ischemic cortex: membrane potential changes (with a voltage-sensitive dye), cerebral blood volume (green light reflectance), and cerebral blood flow (CBF, laser-speckle imaging) were observed. The initial CBF drop was similar in all groups, with a significant further reduction during ischemia in old rats (p < 0.01). Age reduced the total number of SDs (p < 0.05) but increased the size of ischemic area displaying prolonged SD (p < 0.01). The growth of area undergoing prolonged SDs positively correlated with the growth of ischemic core area (p < 0.01) during MCAO. Prolonged SDs and associated hypoperfusion likely compromise cortical tissue exposed to even a short focal ischemia in aged rats.

Large-area surface-enhanced Raman spectroscopy imaging of brain ischemia by gold nanoparticles grown on random nanoarrays of transparent boehmite.

Although SERS spectroscopy, which is sensitive to molecular vibration states, offers label-free visualization of molecules, identification of molecules and their reliable large-area imaging remains to be developed. Limitation comes from difficulties in fabricating a SERS-active substrate with homogeneity over a large area. Here, we overcome this hurdle by utilizing a self-assembled nanostructure of boehmite that is easily achieved by a hydrothermal preparation of aluminum as a template for subsequent gold (Au) deposition. This approach brought about random arrays of Au-nanostructures with a diameter of ∼125 nm and a spacing of <10 nm, ideal for the hot-spots formation. The substrate, which we named "gold nanocoral" (GNC) after its coral reef-like shape, exhibited a small variability of signal intensities (coefficient value <11.2%) in detecting rhodamine 6G molecule when 121 spots were measured over an area of 10 × 10 mm(2), confirming high uniformity. The transparent nature of boehmite enabled us to conduct the measurement from the back-side of the substrate as efficiently as that from the front-side. We then conducted tissue imaging using the mouse ischemic brain adhered on the GNC substrate. Through nontargeted construction of two-dimensional-Raman-intensity map using differential bands from two metabolically distinct regions, that is, ischemic core and contralateral-control areas, we found that mapping using the adenine ring vibration band at 736 cm(-1) clearly demarcated ischemic core where high-energy adenine phosphonucleotides were degraded as judged by imaging mass spectrometry. Such a detection capability makes the GNC-based SERS technology especially promising for revealing acute energy derangement of tissues.

Reperfusion-induced mitochondrial dysfunction in the porcine heart is reduced by TRO40303 in the area at risk predominantly through preservation of outer mitochondrial membrane intactness

Mitochondria are considered to play critical roles in cell death pathways following ischemia-reperfusion injury. The objective of the present study was to perform a functional assessment of mitochondria following ischemia-reperfusion injury of the porcine heart as well as to evaluate mitochondrial effects of hypothermia and the outer membrane translocator protein (TSPO) ligand TRO40303. Pigs were subjected to 40 min occlusion of the left anterior descending artery followed by 4 hours of reperfusion. Three groups of pigs were treated either by administration of 15 mg/kg TRO40303 or the same volume of saline (1 ml/kg) at normothermia 5 min before reperfusion, or by hypothermia (32°C) initiated prior to occlusion, n=8 for all groups. Transmural needle biopsies were taken from the non-ischemic area in the left lateral wall, from the area at risk in the midventricular anterior wall and from the ischemic core area in the apical anterior wall. Mitochondrial respiratory function was evaluated polarographically in skinned heart fibers using specific substrates and inhibitors. In the control group, heart fibers from both ischemic areas demonstrated a general reduction of respiratory states. However, respiration linked to respiratory complex I was more affected than that to complex II indicating loss of soluble matrix components such as NAD (H). Addition of exogenous cytochrome c (CytC) increased the level of respiration several fold in both ischemic areas indicating increased permeability of the outer membrane and that CytC loss contributed to the reduced levels of respiration. These changes were diminished by hypothermia in both ischemic areas. TRO40303 attenuated inhibition of respiration involving complex II and reduced the stimulatory effects of CytC in the area at risk, but did not significantly reduce the altered ratio of complex I- and II-mediated respiration, and was without effect in the ischemic core area. It is concluded that mitochondria in both the ischemic core area and the area at risk undergo significant alterations in respiratory function following ischemia-reperfusion injury consistent with both inner and outer mitochondrial membrane permeabilization which can be inhibited by hypothermia initiated prior to occlusion. Administration of TRO40303 prior to reperfusion appears to reduce reperfusion-induced mitochondrial dysfunction in the area at risk mainly by preserving outer membrane intactness and limiting CytC release.

Cerebral endothelial expression of Robo1 affects brain infiltration of polymorphonuclear neutrophils during mouse stroke recovery

Increased brain infiltration of polymorphonuclear neutrophils (PMNs) occurs early after stroke and is important in eliciting brain inflammatory response during stroke recovery. In order to understand the molecular mechanism of PMN entry, we investigated the expression and requirement for Slit1, a chemorepulsive guidance cue, and its cognate receptor, Robo1, in a long-term recovery mouse model of cerebral ischemia. The expression levels of Robo1 were significantly decreased bilaterally at 24h following reperfusion. Robo1 expression levels remained suppressed in the ipsilateral cortex until 28d post MCAO–reperfusion, while the levels of Robo1 in the contralateral cortex recovered to the level of sham-operated mouse by 7d reperfusion. Circulating PMNs express high levels of Slit1, but not Robo1. Influx of PMNs into the ischemic core area occurred early (24h) after cerebral ischemia, when endothelial Robo1 expression was significantly reduced in the ischemic brain, indicating that Robo1 may form a repulsive barrier to PMN entry into the brain parenchyma. Indeed, blocking Slit1 on PMNs in a transwell migration assay in combination with an antibody blocking of Robo1 on human umbilical vein endothelial cells (HUVEC) significantly increased PMN transmigration during oxygen glucose deprivation, an in vitro model of ischemia. Collectively, in the normal brain, the presence of Slit1 on PMNs, and Robo1 on cerebral endothelial cells, generated a repulsive force to prevent the infiltration of PMNs into the brain. During stroke recovery, a transient reduction in Robo1 expression on the cerebral endothelial cells allowed the uncontrolled infiltration of Slit1-expressing PMNs into the brain causing inflammatory reactions.

Effect of Oral Administration of Pheretima Aspergillum (Earthworm) in Rats with Cerebral Infarction Induced by Middle-Cerebral Artery Occlusion

We investigated the curative effect of Pheretima aspergillum (earthworm, PA) on rats with middle cerebral artery occlusion (MCAo). The MCAo-induced cerebral infarction was established and its underlying mechanisms by counting the infarction areas and evaluating the rats' neurological status. Immunostaining was used to test the expression of NeuN, and glial fibrillary acidic (GFAP), S100B, and brain-derived neurotrophic factor (BDNF) proteins. Our results showed that oral administration of PA for two weeks to rats with MCAo successfully reduced cerebral infarction areas in the cortex and striatum, and also reduced scores of neurological deficit. The PA-treated MCAo rats showed greatly decreased neuronal death, glial proliferation, and S100B proteins in the penumbra area of the cortex and in the ischemic core area of the cortex, but BDNF did not changed. These results demonstrated novel and detailed cellular mechanisms underlying the neuroprotective effects of PA in MCAo rats.

Abstract 2308: Timing Of Intra-arterial Cell Transplantation Drastically Affects The Behavior Of The Transplanted Cells And Functional Recovery After Stroke

Background and Purpose- Intra-arterial delivery of human bone marrow stromal cells(hBMSCs)results in a migration of the greater number of cells compared to intra-venous delivery in an acute stage after stroke. However, it remains unclear how the timing of intra-arterial administration affects the cell migration, repair process and functional recovery. In this study, we test the hypothesis that the timing of intra-arterial cell transplantation changes the behavior of the transplanted cells and host environment, which is very important to achieve the optimal effect of transplantation. Methods- Sprague-Dawley rats (8week, 270∼300g) received 1X10 6 hBMSCs injected intra-arterially via polyethylene catheter at 1, 4 and 7 days (Day1,4 and 7 group) after 75 min middle cerebral artery occlusion following reperfusion. At the time point of transplantation, regional cerebral blood flow (rCBF) of ischemic core and periinfarct area was examined with transcranial laser Doppler flowmetry (LDF). Animals were sacrificed at 3 and 21 days after transplantation to assess the distribution of the cells, vascular density and final infarct volume with immunohistochemistry. Behavior assessment was also performed until 21 days after stroke. Results- In the periinfarct area of cerebral cortex, the number of hBMSCs was significantly greater in Day4 group than that in Day 1 group ( p =0.0001). On the other hand, there was no significant difference between Day1 and 4 groups in the periinfarct area of striatum. Fewer cells were found in both areas in Day 7 group. In Day1 group, the majority of hBMSCs migrated at ischemic core of cerebral cortex and striatum. rCBF of the ischemic core in cortex was the greatest in Day1 group. Otherwise, rCBF of the periinfarct area in cortex was the greatest in Day4 group. The number of hBMSCs was correlated with rCBF of each area. In Day1 and 4 groups, animals showed a significant increase in vascular density in the periinfarct area of cerebral cortex. However, a reduction of infarct volume and neurological recovery were seen in only Day1 group, but not in Day4 and Day7 group. Conclusions- Our results suggest that the change of host cerebral hemodynamics after stroke drastically affect the distribution of hBMSCs, which delivered intra-arterially. It is important to establish the optimal timing of intra-arterial delivery of stem cells after stroke.