Age-dependence of sensorimotor and cerebral electroencephalographic asymmetry in rats subjected to unilateral cerebrovascular stroke

Background: The human population mostly affected by stroke is more than 65 years old. This study was designed to meet the recommendation that models of cerebral ischemia in aged animals are more relevant to the clinical setting than young animal models. Until now the majority of the pre-clinical studies examining age effects on stroke outcomes have used rats of old age. Considering the increasing incidence of stroke among younger than old human population, new translational approaches in animal models are needed to match the rejuvenation of stroke. A better knowledge of alterations in stroke outcomes in middle-aged rats has important preventive and management implications providing clues for future investigations on effects of various neuroprotective and neurorestorative drugs against cerebrovascular accidents that may occur before late senescence. Methods: We evaluated the impact of transient focal ischemia, induced by intracerebral unilateral infusion of endothelin-1 (Et-1) near the middle cerebral artery of conscious rats, on volume of brain damage and asymmetry in behavioral and electroencephalographic (EEG) output measures in middle-aged (11-12 month-old) rats. Results: We did not find any age-dependent difference in the volume of ischemic brain damage three days after Et-1 infusion. However, age was an important determinant of neurological and EEG outcomes after stroke. Middle-aged ischemic rats had more impaired somatosensory functions of the contralateral part of the body than young ischemic rats and thus, had greater left-right reflex/sensorimotor asymmetry. Interhemispheric EEG asymmetry was more evident in middle-aged than in young ischemic rats, and this could tentatively explain the behavioral asymmetry.Conclusions: With a multiparametric approach, we have validated the endothelin model of ischemia in middle-aged rats. The results provide clues for future studies on mechanisms underlying plasticity after brain damage and motivate investigations of novel neuroprotective strategies against cerebrovascular accidents that may occur before late senescence. © 2013 Moyanova et al.; licensee BioMed Central Ltd

Contribution of ipsilesional versus contralesional pyramidal tract plasticity

Schlaganfall trägt zu erhöhter Mortalität und Morbidität trotz akut intensivmedizinischer Behandlung. Trotz vielversprechenden Ergebnisse von Seite der Akutbehandlung, die preklinische und klinische Versuche, die die Morbidität und Mortalität beim Schlaganfall in die postakute bis chronische Phase zu reduzieren versuchen, bleiben frustrierend. Nach dem Schlaganfall, das Hirnparenchym ist einem Reorganisationsprozess ausgesetzt indem vertiefte interzelluläre und zellulär-extrazelluläre Interaktionen eine wichtige Rolle spielen. Das Ontogenese Software wird „restarted“ und damit die endogene Plastizität nach dem Schlaganfall steigt. In wie fern wir diese Eigenschaft für therapeutische Zwecke „ausbeuten“ können bleibt bis jetzt unklar, allerdings man beobachtet bereits in viele Studien eine breitere Therapiefenster mit verlängertem Effekt und Erholungspotenzial nach dem Schlaganfall. In unserem Projekt haben wir mittels mehreren Readouts die Effekten von Erythropoietin (EPO) und Vascular endothelial growth factor (VEGF) auf die Neuroplastizität der langen Bahnen nach dem Schlaganfall untersucht. VEGF ist ein Wachstumsfaktor mit pleiotropen Effekte nach dem Schlaganfall und ist vor allem in die Astroglia und Mikroglia zusammen mit dem Rezeptoren VEGFR1 und VEGFR2 zu finden. In die akute Schlaganfall Phase VEGF ist für verschiedene Effekten in die Penumbra Region zuständig wie Förderung des neuronalen Überlebens, der Angiogenese Prozessen, der Proliferation von neuralen Stamzellen, deren Migration und Differenzierung. Anhalt neuen Studien wurde den VEGFR2 in den kontralesionalen Motorkortex nach dem Schlaganfall identifiziert . Diese Entdeckung weisst indirekt auf einer funktionelle Rolle der VEGF auf die kontralesionale Hemisphäre. Bis jetzt bleibt allerdings die Rolle der VEGF Therapie in die axonale Plastizität der langen Bahnen nach dem Schlaganfall unerforscht und dadurch auch seiner möglichen therapeutischen Rolle in die funktionelle Erholung. EPO ist ein Wachstumsfaktor mit einem breiten Spektrum der schon für klinische Verwendung zugelassen ist und damit zu einer schnelleren Translationsphase von preklinischen zu klinischen Experimenten geeignet ist. Die erste klinische Studie in der Erythropoietin nach dem Schlaganfall untersucht worden ist, zeigte eine Verbesserung der neurologischen Outcome mit Verkleinerung der ischämischen Schaden. In eine weitere Studie wurde dann der Effekt von EPO in 522 Patienten untersucht und zeigte dass die Verabreichung des EPO zusammen mit recombinant tissue-plasminogen activator (rt-PA) akut nach dem Schlaganfall zu einer signifikanten Nachblutung, Hirnödem und thromboembolische Ereignisse führte. Eine Falschinterpretation dieser Studie wird in die Zukunft für eine Ablehnung der EPO Therapie in Schlaganfall führen, obwohl eine alleinige Verabreichung des Epo eine Verbesserung zeigte. Unsere Studie untersucht die Effekte der Epo Therapie verabreicht drei Tage nach dem Schlaganfall mit Fokus auf die axonale Plastizität und funktionelle Erholung. Als Schlaganfall Model wurde eine 30 minutige Okklusion der A. cerebri media (MCAO) linkshemisphärisch durchgeführt. Die Verabreichung der Wachstumsfaktoren erfolgte am dritten postoperativen Tag mittels Alzet Pumpen die intraventrikulär implantiert wurden und eine kontinuierliche Verabreichung für verschiedene Zeitspannen gewährleistete. Als Kontrolle wurden die Pumpen mit NaCl 0.9% gefüllt und gemäss dem gleichen Protokoll intraventrikulär verabreicht. Sechs Wochen nach dem Schlaganfall und Therapie wurden die motorische Kortexanteile ipsi- und kontralesional mittels anterograden Tracttracers [Biotinyliertes Dextrane amid (BDA) in den kontralesionalen Motorkortex und Cascade Blue (CB) in den ipsilesionalen Motorkortex] markiert. Zwei Monate Postischämie wurden die Mäuse in Narkose getötet und das Gewebe für weitere molekularbiologische, biochemische, enzymologische, zytologische und genetische Untersuchungen verwendet. Die funktionelle Erholung als Korrelat zu Neurorehabilitation nach dem Schlaganfall wurde mittels RotaRod test, Grips strength test und Anxiety Test untersucht. Gemäss unseren Ergebnisse, VEGF und EPO Therapie verabreicht drei Tage nach dem Schlaganfall unterstützen die funktionelle Erholung durch koordinierte jedoch unterschiedliche Mechanismen die die Plastizität der langen Bahnen in der kontralesionalen Hemisphäre stimulieren. EPO zeigte vor allem eine vermehrte Translation der Plastizitätsgenen in der kontralesionalen Hemisphäre nach dem Schlaganfall die für eine erhöhte axonale Plastizität kontralesional verantwortlich waren. VEGF therapierten Tieren zeigten auch Erhöhung der axonalen Plastizität nach dem Schlaganfall kontralesional aber auf molekularer Ebene war das Effekt am besten durch eine Unterregulation von Plastizität hemmenden Substanzen in der Extrazellulären Matrix zu erklären.Stroke incidence is increasing due to the rapidly aging population in developed countries. Whereas untreated acute middle cerebral artery occlusion (MCAO) causes death in 20% of patients and long-term disability in more than 70% of patients, acute stroke therapy with rapid vessel recanalization significantly reduces mortality without influencing functional recovery beyond the acute stroke phase. This lack of functional recovery suggests a need for innovative therapies that can restore function after stroke. The purpose of these studies was to examine the effects of delayed administration of the growth factors erythropoietin (Epo) and vascular endogenous growth factor (VEGF) on functional neurological recovery and pyramidal tract plasticity in mice. The first study investigated how subacute delivery of Epo, starting at 3 days after stroke onset and continuing for 30 days (1 I.U. /day or 10 I.U. /day; via mini osmotic pump), influenced neuronal survival, axonal sprouting and neurological function recovery in C57Bl6/j mice submitted to 30-min MCAO. Epo administered in a 10 I.U. /day dose, in contrast to the 1 I.U. /day administration, showed a significant increase in neuronal survival and CD31 + newly-formed capillaries. This vascular growth enabled further neuroregeneration processes. Functional behavioral tests showed a significant improvement of motor coordination (RotaRod test) and grip strength (Grips strength test) among mice with 10 I.U. /day Epo administration, with no improvement for the low dose group. To investigate the neurological changes underlying these results, two anterograde tract tracers (dextrane amines) were injected in the motor cortex ipsilateral and contralateral to the ischemic lesion, in mice treated with the higher Epo dose. Histological evaluation of the tracers, both at the level of rubral and facial nucleus, showed that functional recovery in these animals was due to an increase of contralateral projections, accompanied by a compensatory decrease of ipsilateral projections. In the second study, VEGF was investigated due to its dual actions on vessels and neurons, which have potential for promoting long distance axonal plasticity in the ischemic brain. Mice were submitted to 30 minutes MCAO, followed by the intraventricular delivery of normal saline or VEGF (0.004 or 0.02 µg/day) starting 3 days post-ischemia. The outcome parameters were functional neurological recovery, long distance axonal plasticity by anterograde tract tracing and cellular and molecular responses examined by histochemistry, RT-PCR and Western blots. VEGF promoted neurological recovery when administered at the higher dosage, by stimulating long distance axonal plasticity in the contralesional but not ipsilesional pyramidal tract system. This observation was accompanied by deactivation of matrix metalloproteinase-9 (MMP9) in the ipsilesional brain tissue and downregulation of axonal growth inhibitors and guidance molecules in the contralesional brain tissue. The results support the concept that brain plasticity is consistent with coordinated axonal growth responses both ipsilateral and contralateral to the site of stroke. Considering that Epo is well tolerated in humans, clinical studies are now conceivable in which Epo is applied in patients in the post-acute stroke phase

Identification of prognostic biomarkers of cortical stroke in mouse model

According to the World Health Organization (WHO), worldwide, 15 million people suffer stroke each year. Stroke, i.e. the sudden and severe reduction of blood flow to a brain region, is the second leading cause of death and the third leading cause of disability. Stroke is also a leading cause of dementia and depression. A focal brain damage inevitably causes a drastic alteration of the whole complex neural network that characterizes the affected area. Although stroke damage can be devastating, many patients survive the initial event and display a spontaneous recovery, which can be further increased by rehabilitation therapy. Recovery is possible due to a reorganization of spared areas and connections, i.e. neuroplasticity. Functional recovery is highly variable in stroke patients and strongly depends on many factors (lesion location and volume, etc.). Currently, there are no ways of predicting either the degree or time course of recovery in individual subjects. For these reasons, the identification of biomarkers is crucial in the design and interpretation of stroke rehabilitation trials. Therefore, the aim of this work is the development of new prognostic and therapeutic tools in preclinical models. In this study I exploit a mouse model of stroke, the Middle cerebral artery occlusion (MCAO), that shows a higher variability and is thus closer to the human condition. I conducted experiments to evaluate the occurrence of motor deficits using a battery of behavioral tasks: gridwalk test, skilled reaching test, and retraction task in the M-platform (a robotic device that permits to quantitatively evaluate several kinetic/kinematic parameters related to forelimb movement). Moreover, the ischaemic lesion and electrophysiological alterations was analysed by means of histology and electroencephalographic signals (EEG) respectively. I studied how these mechanisms are altered by stroke, combining the data all these parameters, in order to define possible biomarkers that predict long-term motor recovery. The results obtained, permit new opportunities for therapeutic approaches after stroke allowing the definition of more effective rehabilitation paradigms that can be translated into clinical practice

Clinical Recovery from CNS Damage

After decades of focusing on how to alleviate and prevent recurrence of acute CNS injuries, the emphasis has finally shifted towards repairing such devastating events and rehabilitation. This development has been made possible by substantial progress in understanding the scientific underpinnings of recovery as well as by novel diagnostic tools, and most importantly, by emerging therapies awaiting clinical trials. In this publication, several international experts introduce novel areas of neurological reorganization and repair following CNS damage. Principles and methods to monitor and augment neuroplasticity are explored in depth and supplemented by a critical appraisal of neurological repair mechanisms and possibilities to curtail disability using computer or robotic interfaces. Rather than providing a textbook approach of CNS restoration, the editors selected topics where progress is most imminent in this labyrinthine domain of medicine

Basic characteristics of epileptiform discharges triggered by lindane in rats

Introduction: EEG is a widely used method of epilepsy examination. In order to quantitatively inspect ictal EEG findings, a number of mathematical models have been developed over the years, one of them being the Fast Fourier Transform (FFT). It transforms the signal from time domain into frequency domain, giving information about their power spectral densities (PSD). Lindane is a well-established neurotoxic agent often used in experimental studies as a model of generalized epilepsy. This study aims to quantitatively examine the characteristics of ictal EEG activity in rats on model of generalized epilepsy induced by lindane. Materials and Methods: Wistar albino rats were used for the study. Electrodes were surgically implanted over the frontal, parietal and occipital cortices of each animal for EEG recording purposes prior to lindane administration in convulsive dose. An 8-channel EEG apparatus was used, combined with a software developed in the Laboratory (NeuroSciLaBG). Ictal EEG epochs were extracted from the original signal and FFT analysis performed to obtain information considering PSD in predefined frequency bands. Amplitude histogram feature of the software was used to differentiate ictal spikes based on their voltage. Results: FFT analysis has yielded important information regarding spectral powers in frequency domain. Ictal EEG showed considerable stratification, theta frequency band (4-7 Hz) being markedly dominant. Amplitude histogram showed the majority of spikes to be in the voltage ranges up to 250 μV, while higher voltage spikes were rarely observed. Conclusion: FFT is capable of giving important information about ictal period characteristics. Ictal periods induced by lindane are characterized by dominancy of theta rhythm and spiking activity mostly in amplitude bins up to 250 μV. FFT and amplitude histograms can be of critical importance in the future pharmacological and toxicity studies

Towards a unifying, systems biology understanding of large-scale cellular death and destruction caused by poorly liganded iron: Parkinson’s, Huntington’s, Alzheimer’s, prions, bactericides, chemical toxicology and others as examples

Exposure to a variety of toxins and/or infectious agents leads to disease, degeneration and death, often characterised by circumstances in which cells or tissues do not merely die and cease to function but may be more or less entirely obliterated. It is then legitimate to ask the question as to whether, despite the many kinds of agent involved, there may be at least some unifying mechanisms of such cell death and destruction. I summarise the evidence that in a great many cases, one underlying mechanism, providing major stresses of this type, entails continuing and autocatalytic production (based on positive feedback mechanisms) of hydroxyl radicals via Fenton chemistry involving poorly liganded iron, leading to cell death via apoptosis (probably including via pathways induced by changes in the NF-κB system). While every pathway is in some sense connected to every other one, I highlight the literature evidence suggesting that the degenerative effects of many diseases and toxicological insults converge on iron dysregulation. This highlights specifically the role of iron metabolism, and the detailed speciation of iron, in chemical and other toxicology, and has significant implications for the use of iron chelating substances (probably in partnership with appropriate anti-oxidants) as nutritional or therapeutic agents in inhibiting both the progression of these mainly degenerative diseases and the sequelae of both chronic and acute toxin exposure. The complexity of biochemical networks, especially those involving autocatalytic behaviour and positive feedbacks, means that multiple interventions (e.g. of iron chelators plus antioxidants) are likely to prove most effective. A variety of systems biology approaches, that I summarise, can predict both the mechanisms involved in these cell death pathways and the optimal sites of action for nutritional or pharmacological interventions

Pathophysiological role of RhoA/Rho-kinase under oxygen-glucose deprivation/reperfusion and hyperglycaemia

Introduction: Oxygen-glucose deprivation (OGD)±reperfusion and hyperglycaemia exacerbate the ischaemic cerebral injuries during or after a stroke. The key biochemical events associated with these pathologies include excessive cytoskeletal remodelling, modulation of tight junction proteins and the induction of oxidative stress. Recently, the overactivities of protein kinase C (PKC), RhoA/Rho-kinase, and pro-oxidant NADPH oxidase have been shown to account for the development of these events and the consequent disruption of human blood-brain barrier (BBB) integrity. Objectives: This thesis focused on the putative roles of RhoA/Rho-kinase signalling in OGD and OGD+reperfusion-evoked modulation of cytoskeletal remodelling, tight junction proteins and oxidative stress in human brain microvascular endothelial cells (HBMEC). The effects of hyperglycaemia-mediated PKC overactivities in modulating the RhoA/Rho-kinase pathway with reference to the aforementioned parameters i.e. cytoskeletal remodelling and tight junction protein expression and localisation have also been the focus of this thesis. Methods: For the OGD studies, the HBMEC were exposed to normoxia (controls), OGD (4, 20 hours) alone and followed by reperfusion (20 hours). The HBMEC-human astrocyte (HA) cocultures were established to mimic human BBB before exposing them to the experimental conditions. The integrity and function of HBMEC-HA cocultures were measured by transendothelial electrical resistance (TEER) and flux of permeability markers sodium fluorescein (NaF) and Evan’s blue-labelled albumin (EBA), respectively. For the hyperglycaemia studies, the HBMEC monolayers and the cocultures were exposed to normoglycaemia (5.5 mM D-glucose), hyperglycaemia (25 mM D-glucose), and hyperglycaemia with inhibitors of Rho-kinase, PKC, PKC-α, PKC-β, PKC-βII, PKC-δ; and the BBB integrity and function were measured by the TEER and flux studies, respectively. Fold differences in the protein expression or activity of RhoA, Rho-kinase-2, mono- and di-phosphorylated myosin light chain-2 (MLC2), total MLC2, gp91-phox (a pivotal NADPH oxidase subunit), catalase, occludin, claudin-5, zonula occludens-1 (ZO-1), β-catenin, and vinculin were either measured by in-cell or ordinary Western analyses. Results from the OGD studies: OGD compromised the barrier integrity as observed by decreases in TEER values and concomitant increases in flux of EBA and NaF across the cocultures. Transfection of HBMEC with constitutively active RhoA also decreased the TEER and increased the NaF paracellular permeability, whereas inactivation of RhoA by anti-RhoA-IgG electroporation exerted barrier protective effects. Moreover, OGD alone and after constitutively active RhoA transfection introduced stress fibres in HBMEC, which were abrogated by inactivation of RhoA and the specific inhibition of its main effector Rho-kinase by Y-27632. In addition, dramatic increases in the protein expressions of RhoA-GTP, Rho-kinase-2, gp91-phox, and antioxidant catalase were observed in HBMEC exposed to OGD+reperfusion conditions. These along with increases in the NADPH oxidase activity and total superoxide anion levels confirmed the oxidative stress in HBMEC under these experimental conditions. A marked rise in the protein expressions of claudin-5 and β-catenin observed after OGD (20 hours) alone and followed by reperfusion may represent the effects of oxidative stress on tight and adherens junction proteins stability, respectively. These results also concurred with marked decreases in TEER and concomitant increases in the flux of EBA across the in vitro models of human BBB exposed to OGD±reperfusion conditions when compared with the controls. Cotreatment with Y-27632 under OGD±reperfusion normalised the protein expressions of RhoA, Rho-kinase-2, gp91-phox, claudin-5, catalase; activities of RhoA and NADPH oxidase; and total superoxide anions levels, alongside improving the expression of occludin and the coculture integrity under the OGD±reperfusion conditions. Results from the hyperglycaemia studies: Hyperglycaemia also increased RhoA-GTP, Rho-kinase-2, mono- and di-phosphorylated MLC2 protein levels and total PKC activity. These changes were consistent with the actin stress fibre formations, ZO-1 and occludin redistribution from HBMEC periphery. Hyperglycaemia-mediated endothelial-barrier dysfunction was further characterised by reduction in TEER and elevation in flux of EBA. Glucose normalisation, RhoA neutralisation by anti-RhoA-IgG electroporation and Rho-kinase-2 inhibition by Y-27632 normalised all abovementioned protein expressions, restored actin and tight junction protein localisations and barrier integrity. Cotreatment of HBMEC with hyperglycaemia and a general PKC inhibitor namely, bisindolylmaleimide-I normalised the Rho-kinase-2, mono- and di-phosphorylated MLC2 levels. Moreover, specific inhibitors of PKC-α (Ro-32-0432), PKC-β (LY333531), PKC-βII (CGP53353) attenuated the PKC overactivity, normalised all protein expressions, restored actin localisation and improved barrier integrity. In addition, the PKC-α and PKC-β siRNA transfections mimicked the effects of the specific inhibitors and attenuated the hyperglycaemia-evoked RhoA-GTP, mono- and di-phosphorylated MLC2 protein levels and stress fibre formations. Conclusions: The RhoA/Rho-kinase overactivities compromise the endothelial-barrier integrity, in part, by modulating the cytoskeletal remodelling and inducing the NADPH oxidase-evoked oxidative stress under OGD±reperfusion pathology. Moreover, hyperglycaemia-mediated increases in PKC-α and PKC-β activities exacerbate the endothelial-barrier dysfunction by modulating RhoA/Rho-kinase signalling pathway. Summary: These findings support the hypothesis that OGD±reperfusion and hyperglycaemia perturb BBB integrity through regulation of RhoA/Rho-kinase activity and modulation of cytoskeletal reorganisation, oxidative stress and tight junction protein expressions or localisations

Pathophysiological role of RhoA/Rho-kinase under oxygen-glucose deprivation/reperfusion and hyperglycaemia

Introduction: Oxygen-glucose deprivation (OGD)±reperfusion and hyperglycaemia exacerbate the ischaemic cerebral injuries during or after a stroke. The key biochemical events associated with these pathologies include excessive cytoskeletal remodelling, modulation of tight junction proteins and the induction of oxidative stress. Recently, the overactivities of protein kinase C (PKC), RhoA/Rho-kinase, and pro-oxidant NADPH oxidase have been shown to account for the development of these events and the consequent disruption of human blood-brain barrier (BBB) integrity. Objectives: This thesis focused on the putative roles of RhoA/Rho-kinase signalling in OGD and OGD+reperfusion-evoked modulation of cytoskeletal remodelling, tight junction proteins and oxidative stress in human brain microvascular endothelial cells (HBMEC). The effects of hyperglycaemia-mediated PKC overactivities in modulating the RhoA/Rho-kinase pathway with reference to the aforementioned parameters i.e. cytoskeletal remodelling and tight junction protein expression and localisation have also been the focus of this thesis. Methods: For the OGD studies, the HBMEC were exposed to normoxia (controls), OGD (4, 20 hours) alone and followed by reperfusion (20 hours). The HBMEC-human astrocyte (HA) cocultures were established to mimic human BBB before exposing them to the experimental conditions. The integrity and function of HBMEC-HA cocultures were measured by transendothelial electrical resistance (TEER) and flux of permeability markers sodium fluorescein (NaF) and Evan’s blue-labelled albumin (EBA), respectively. For the hyperglycaemia studies, the HBMEC monolayers and the cocultures were exposed to normoglycaemia (5.5 mM D-glucose), hyperglycaemia (25 mM D-glucose), and hyperglycaemia with inhibitors of Rho-kinase, PKC, PKC-α, PKC-β, PKC-βII, PKC-δ; and the BBB integrity and function were measured by the TEER and flux studies, respectively. Fold differences in the protein expression or activity of RhoA, Rho-kinase-2, mono- and di-phosphorylated myosin light chain-2 (MLC2), total MLC2, gp91-phox (a pivotal NADPH oxidase subunit), catalase, occludin, claudin-5, zonula occludens-1 (ZO-1), β-catenin, and vinculin were either measured by in-cell or ordinary Western analyses. Results from the OGD studies: OGD compromised the barrier integrity as observed by decreases in TEER values and concomitant increases in flux of EBA and NaF across the cocultures. Transfection of HBMEC with constitutively active RhoA also decreased the TEER and increased the NaF paracellular permeability, whereas inactivation of RhoA by anti-RhoA-IgG electroporation exerted barrier protective effects. Moreover, OGD alone and after constitutively active RhoA transfection introduced stress fibres in HBMEC, which were abrogated by inactivation of RhoA and the specific inhibition of its main effector Rho-kinase by Y-27632. In addition, dramatic increases in the protein expressions of RhoA-GTP, Rho-kinase-2, gp91-phox, and antioxidant catalase were observed in HBMEC exposed to OGD+reperfusion conditions. These along with increases in the NADPH oxidase activity and total superoxide anion levels confirmed the oxidative stress in HBMEC under these experimental conditions. A marked rise in the protein expressions of claudin-5 and β-catenin observed after OGD (20 hours) alone and followed by reperfusion may represent the effects of oxidative stress on tight and adherens junction proteins stability, respectively. These results also concurred with marked decreases in TEER and concomitant increases in the flux of EBA across the in vitro models of human BBB exposed to OGD±reperfusion conditions when compared with the controls. Cotreatment with Y-27632 under OGD±reperfusion normalised the protein expressions of RhoA, Rho-kinase-2, gp91-phox, claudin-5, catalase; activities of RhoA and NADPH oxidase; and total superoxide anions levels, alongside improving the expression of occludin and the coculture integrity under the OGD±reperfusion conditions. Results from the hyperglycaemia studies: Hyperglycaemia also increased RhoA-GTP, Rho-kinase-2, mono- and di-phosphorylated MLC2 protein levels and total PKC activity. These changes were consistent with the actin stress fibre formations, ZO-1 and occludin redistribution from HBMEC periphery. Hyperglycaemia-mediated endothelial-barrier dysfunction was further characterised by reduction in TEER and elevation in flux of EBA. Glucose normalisation, RhoA neutralisation by anti-RhoA-IgG electroporation and Rho-kinase-2 inhibition by Y-27632 normalised all abovementioned protein expressions, restored actin and tight junction protein localisations and barrier integrity. Cotreatment of HBMEC with hyperglycaemia and a general PKC inhibitor namely, bisindolylmaleimide-I normalised the Rho-kinase-2, mono- and di-phosphorylated MLC2 levels. Moreover, specific inhibitors of PKC-α (Ro-32-0432), PKC-β (LY333531), PKC-βII (CGP53353) attenuated the PKC overactivity, normalised all protein expressions, restored actin localisation and improved barrier integrity. In addition, the PKC-α and PKC-β siRNA transfections mimicked the effects of the specific inhibitors and attenuated the hyperglycaemia-evoked RhoA-GTP, mono- and di-phosphorylated MLC2 protein levels and stress fibre formations. Conclusions: The RhoA/Rho-kinase overactivities compromise the endothelial-barrier integrity, in part, by modulating the cytoskeletal remodelling and inducing the NADPH oxidase-evoked oxidative stress under OGD±reperfusion pathology. Moreover, hyperglycaemia-mediated increases in PKC-α and PKC-β activities exacerbate the endothelial-barrier dysfunction by modulating RhoA/Rho-kinase signalling pathway. Summary: These findings support the hypothesis that OGD±reperfusion and hyperglycaemia perturb BBB integrity through regulation of RhoA/Rho-kinase activity and modulation of cytoskeletal reorganisation, oxidative stress and tight junction protein expressions or localisations