Stroke In Man Research Articles

Abstract P283: CCL5 Acts As A Vasoconstrictor On Penetrating Arterioles In The Cerebral Circulation By Enhancing 20-HETE Activity

20-HETE is synthesized from arachidonic acid by cytochrome P450 (CYP) enzymes 4A and 4F. Inactivating mutations in the CYP enzymes that produce 20-HETE are associated with hypertension and stroke in man. We previously revealed that inactivating variants of CYP4A/F enzymes are associated with dementia in the Atherosclerosis Risk in Communities Neurocognitive Study (ARIC-NS) population. 20-HETE is involved with sodium regulation in the kidney and is a powerful vasoconstrictor. It was recently discovered that CCL5 and 20-HETE share the same receptor, GPR75. We previously found that 20-HETE constricts and augments the myogenic response (MR) of the middle cerebral artery (MCA) and renal afferent arteriole. However, whether CCL5 has any effect on penetrating arterioles (PAs) and interacts with 20-HETE is unknown. We found that GPR75 is expressed in PAs and pericytes in the brain. CYP4A is also expressed in pericytes and is inversely proportional to levels of GPR75 in the brain. In the present study, we found that 20-HETE contributes to the basal myogenic tone of PAs in SD rats. Administration of HET0016, a 20-HETE synthesis inhibitor, dilated the PA by 34 ± 3% (n = 6) under 10 mmHg perfusion pressure. Administration of WIT003, a 20-HETE agonist, constricted the vessel by 23 ± 4% (n = 6) under the same perfusion pressure. We found that CCL5 also reduced PA diameter by 20 ± 4% (n = 7) in SD rats under 10 mmHg perfusion pressure. Moreover, we compared the response to CCL5 in SS rats that are 20-HETE deficient and SS.CYP4A1 transgenic rats in which 20-HETE production is restored. PAs isolated from SS rats treated with 0.1 nM CCL5 constricted by 9 ± 5% (n = 6) while those treated with 10 nM constricted by 12 ± 3% (n = 6). CCL5 had a greater response in PAs from the SS.CYP4A1 strain, and the diameter of the PAs constricted by 14 ± 2% (n = 5) and 24 ± 5% (n = 5) in response to 0.1 and 10 nM CCL5, respectively. These results demonstrate that CCL5 has a direct effect on PAs similar to 20-HETE that acts via the GPR75 receptor. However, further study is needed to determine how CCL5 and 20-HETE interact to promote vasoconstriction. These studies would help further understand the involvement of 20-HETE in disease and potentially identify novel drug targets.

Relationships between selective neuronal loss and microglial activation after ischaemic stroke in man.

Modern ischaemic stroke management involves intravenous thrombolysis followed by mechanical thrombectomy, which allows markedly higher rates of recanalization and penumbral salvage than thrombolysis alone. However, <50% of treated patients eventually enjoy independent life. It is therefore important to identify complementary therapeutic targets. In rodent models, the salvaged penumbra is consistently affected by selective neuronal loss, which may hinder recovery by interfering with plastic processes, as well as by microglial activation, which may exacerbate neuronal death. However, whether the salvaged penumbra in man is similarly affected is still unclear. Here we determined whether these two processes affect the non-infarcted penumbra in man and, if so, whether they are inter-related. We prospectively recruited patients with (i) acute middle-cerebral artery stroke; (ii) penumbra present on CT perfusion obtained <4.5 h of stroke onset; and (iii) early neurological recovery as a marker of penumbral salvage. PET with 11C-flumazenil and 11C-PK11195, as well as MRI to map the final infarct, were obtained at predefined follow-up times. The presence of selective neuronal loss and microglial activation was determined voxel-wise within the MRI normal-appearing ipsilateral non-infarcted zone and surviving penumbra masks, and their inter-relationship was assessed both across and within patients. Dilated infarct contours were consistently excluded to control for partial volume effects. Across the 16 recruited patients, there was reduced 11C-flumazenil and increased 11C-PK11195 binding in the whole ipsilateral non-infarcted zone (P = 0.04 and 0.02, respectively). Within the non-infarcted penumbra, 11C-flumazenil was also reduced (P = 0.001), but without clear increase in 11C-PK11195 (P = 0.18). There was no significant correlation between 11C-flumazenil and 11C-PK11195 in either compartment. This mechanistic study provides direct evidence for the presence of both neuronal loss and microglial activation in the ipsilateral non-infarcted zone. Further, we demonstrate the presence of neuronal loss affecting the surviving penumbra, with no or only mild microglial activation, and no significant relationship between these two processes. Thus, microglial activation may not contribute to penumbral neuronal loss in man, and its presence in the ipsilateral hemisphere may merely reflect secondary remote degeneration. Selective neuronal loss in the surviving penumbra may represent a novel therapeutic target as an adjunct to penumbral salvage to further improve functional outcome. However, microglial activation may not stand as the primary therapeutic approach. Protecting the penumbra by acutely improving perfusion and oxygenation in conjunction with thrombectomy for example, may be a better approach. 11C-flumazenil PET would be useful to monitor the effects of such therapies.

Abstract 2584: Inhibition of Mitochondrial Permeability Transition with NIM811 Improves Functional Recovery after Focal Cerebral Ischemia

Introduction: Mitochondrial dysfunction plays a seminal role in acute ischemic neuronal degeneration, the signature injury in stroke. A selective inhibitor of mitochondrial permeability transition (mPT) may prevent collapse of the mitochondrial membrane potential, thereby inhibiting the intrinsic pathway to neuronal death. Hypothesis: A novel mPT inhibitor, NIM811, will reduce infarct volume and improve functional recovery after focal cerebral ischemia. Methods: NIM811 was obtained as a gift from Novartis Pharma Ltd. (Basel, Switzerland). Focal cerebral ischemia was induced by unilateral carotid and middle cerebral artery occlusion for 2 hrs in male spontaneously hypertensive rats (SHRs). During post-ischemic reperfusion, animals received NIM811 (50 mg/kg at 1- and 24-hrs of post-ischemic reperfusion) or drug-free vehicle. Infarct volume was measured at 24 hrs or 7 days. Functional performance was assessed with 2 measures, fall latency time (FLT) in a Rotarod device and Removal Delay (RD) in a tape removal task, over 28 days. Results: When given after 1 hr of post-ischemic reperfusion, 50 mg/kg NIM811 reduced 24-hr infarct volume (63.6 ± 18.7 [SD] v. 89.3 ± 12.5 mm 3 in controls; n = 8 - 10/group; p ≤ 0.01; unpaired t -test). Serial dosing of NIM811 lowered 7-day infarct volume compared to controls (51.9 ± 7.4 v. 61.9 ± 10.3 mm 3 ; n = 13 - 16/group; p ≤ 0.01; unpaired t -test). In the Rotarod task, NIM811-treated animals had prolonged FLT over controls at all examination times up to 28 days (n = 7 per group; p ≤ 0.0001; ANOVA/Scheffe’s test). NIM811-treated SHRs also had shorter RD for discarding tape applied to the paretic forelimb, compared to controls (n = 7 per group; p ≤ 0.002; ANOVA/Scheffe’s test). Conclusions: Inhibition of mPT with NIM811 significantly reduced infarct volume, when given within a clinically relevant post-ischemic interval. Serial dosing of NIM811 achieved neuroprotective resilience at 7 days. Reductions in infarct volume by NIM811 were complemented by improved functional performance up to 28 days after ischemia. These promising results highlight the potential of NIM811 for neuroprotective treatment of stroke in man.

Clinical factors in patients with ischemic versus hemorrhagic stroke in East China.

Stroke is one of the leading causes of mortality and morbidity of vascular diseases, and its incidence maintains at a high level around the world. In China, stroke has been a major public health problem. Because the pathogenesis of ischemic stroke is different from that of hemorrhagic stroke, their clinical factors would not be the same. Therefore to investigate the different effects of various effect factors on ischemic versus hemorrhagic stroke and then to enhance the prevention are crucial to decrease the incidence. A total of 692 patients, consisting of 540 ischemic stroke patients and 152 hemorrhagic stroke patients from East China, were included in this study. The related factors of stroke subtypes were collected and analyzed. The factors significantly associated with ischemic stroke as opposed to hemorrhagic stroke were family history of stroke, obesity, atherosclerotic plaque of the common carotid artery, atrial fibrillation, hyperfibrinogenemia, transient ischemic attack (TIA), atherosclerotic plaque of the internal carotid artery, coronary heart, lower high-density lipoproteins (lower HDL), increasing age, diabetes mellitus, and gender (male) (P<0.05). Leukocytosis, hypertension and family history of hypertension were the significant factors associated with hemorrhagic stroke versus ischemic stroke. Smoking, drinking, kidney diseases and lower HDL-C were the significant factors contributing to ischemic stroke in man. Obesity, family history of hypertension, family history of stroke, hypercholesteremia and myocardial ischemia were the significant factors for females with ischemic stroke. The most prominent factors for overall stroke in East China were hypertension, followed by higher pulse pressure and hypercholesteremia. The factors for ischemic and hemorrhagic stroke are not the same. Different effects of risk factors on stroke are found in male and female patients.

Both black and green tea consumption associated with reduced risk of stroke

Animal experiments show tea extracts to reduce the infarct volume in a dose dependent fashion. To examine whether tea consumption in man is associated with a reduced risk of cerebral infarction in man we conducted a research librarian supported literature search and a meta analysis of all published human clinical and epidemiologic research on tea and stroke. Risk estimates in each study were calibrated to estimate the effect on stroke associated with consumption of 3 cups of tea. Of 59 citations, 10 studies were identified with original data on either fatal or non‐fatal ischemic stroke in relationship to tea consumption. All of these studies provided risk point estimates below 1.0. The meta analysis demonstrated no significant heterogeneity across studies. The pooled, fixed effect relative risk was 0.79 with a 95% confidence interval of 0.73‐0.85. These effects were quite consistent for black tea (RR=0.76, 0.67‐0.86) and green tea (RR=0.79, 0.72‐0.86). We found that, regardless of country of origin or type of tea consumed, the additional consumption of 3 cups of tea per day is associated with an average 21% lower risk of ischemic stroke, suggesting that the onset of ischemic stroke in man may be prevented through daily tea consumption.

Leukaemia inhibitory factor is over‐expressed by ischaemic brain tissue concomitant with reduced plasma expression following acute stroke

Leukaemia inhibitory factor (LIF) is a glycoprotein of the interleukin-6 family, which has potent pro-inflammatory properties and is involved in regulation of neuronal differentiation. We have previously identified its upregulation in gene microarrays following acute ischaemic stroke in man. LIF expression and localization was measured in human ischaemic stroke autopsy specimens, in a rat model of middle cerebral artery occlusion (MCAO) and in human foetal neural cell cultures following oxygen-glucose deprivation (OGD) by Western blotting and immunohistochemistry. Circulating LIF was determined in the plasma of patients in the hyper-acute stroke phase using a multiplex enzyme-linked-immunosorbent serologic assay system. Patients demonstrated an increase in LIF expression in peri-infarcted regions with localization in neurons and endothelial cells of microvessels surrounding the infarcted core. The rat MCAO model showed similar upregulation in neurons with a peak increase at 90 min. Circulating serum LIF expression was significantly decreased in the hyper-acute phase of stroke. Brain-derived neurons and glia cultured in vitro demonstrated an increase in gene/protein and protein expression respectively following exposure to OGD. Increased LIF expression in peri-infarcted regions and sequestration from the peripheral circulation in acute stroke patients are characteristic of the pathobiological response to ischaemia and tissue damage.

Changes in hyaluronan production and metabolism following ischaemic stroke in man

The extent of recovery from stroke is dependent on the survival of neurons, particularly in peri-infarcted regions. Angiogenesis is critical for the development of new microvessels and leads to re-formation of collateral circulation, reperfusion and better recovery. Hyaluronan (HA) is an important component of the brain extracellular matrix and a regulator of cellular differentiation, migration, proliferation and angiogenesis. We have found that the production of total HA and low molecular mass 3-10 disaccharides of HA (o-HA) was increased in post-mortem tissue and in the serum of patients 1, 3, 7 and 14 days (peaking at 7 days) after ischaemic stroke. Hyaluronidase activity was also increased in serum samples (peaking after 3 days), which might explain the subsequent increase in o-HA. Affinity-histochemical staining was performed using a HA-specific biotinylated binding protein, and it showed enhanced deposition of HA in blood vessels and intracellularly as well as in the nuclei of peri-infarcted neurons. Western blotting and immunohistochemistry demonstrated upregulation of HA synthases (HAS1 and 2) and hyaluronidases (HYAL1 and 2) in inflammatory cells from both stroke and peri-infarcted regions of the brain. HYAL1 was upregulated in microvesssels and intracellularly in neurons, whilst HAS2 became translocated into the nuclei of neurons in peri-infarcted areas. Receptor for HA-mediated motility was observed intracellularly and in the nuclei of neurons, in the tunica media of larger blood vessels and in the endothelial cells of microvessels in stroke-affected tissue, whilst expression of other receptors for HA, CD44 and tumour necrosis factor-stimulated gene 6 (TSG-6) were mainly increased in infiltrating mononuclear cells from inflammatory regions. The data presented here demonstrate that HA breakdown is a feature of the acute stage of stroke injury. Increased o-HA production soon after stroke may be detrimental through enhancement of the inflammatory response, whilst activation of HA and/or o-HA-induced cellular signalling pathways in neurons and microvessels may impact on the remodelling process by stimulating angiogenesis and revascularization, as well as the survival of susceptible neurons.

Expression of cyclin‐dependent kinase 5 mRNA and protein in the human brain following acute ischemic stroke

Cyclin-dependent kinase 5 (Cdk5) is expressed in neurons, and growing evidence suggests that it can promote neuronal death. Little has been published on Cdk5 expression after stroke in man. In this study, RT-PCR, western blotting and immunohistochemistry have been employed to study Cdk5 expression in human post-mortem stroke tissue, and in human neurons, astrocytes and brain endothelial cells exposed to oxygen-glucose deficiency and reperfusion. Since Cdk5 activation results both from binding to its regulatory subunit p35 and from phosphorylation, expression of both p35 and phosphorylated Cdk5 (p-Cdk5) was investigated. An increase in the number of neurons, astrocytes and microvessels stained for Cdk5, p-Cdk5 and p35 occurred in the infarct and penumbra of patients after stroke. Staining became irregular and clumped in the cytoplasm and nuclear translocation occurred. Association of Cdk5 with apoptosis after stroke was found, as Cdk5 colocalized with TUNEL positive neurons and p-Cdk5 was found in the nucleus of active caspase-3 stained cells. Similar upregulation in human cerebral cortical foetal neurons, astrocytes and human brain microvascular endothelial cells (HBMEC) subjected to oxygen-glucose deficiency (OGD) was seen. These results provide evidence for a role of Cdk5 in the events associated with response to ischemic injury. Financial support for this study came from HEFCE and RIHSC.

Desmoteplase (DSPA) does not interact with or cleave the NMDA receptor NR1 subunit: Possible molecular basis for improved tolerability in treatment of acute ischemic stroke

Over the last decade, tissue type plasminogen activator (tPA) has been implicated in a variety of brain functions. It is secreted from growth cones or extending neurites, modulates neurite outgrowth and promotes neuronal migration in vitro. tPA has also been involved in glutamatergic-dependent processes such as synaptic plasticity and long term potentiation. For example, tPA deficient mice display alterations in learning paradigms and memory processes. Similarly, tPA has been shown to play an important role in the pathogenesis of seizures, multiple sclerosis, trauma and ischemic brain injury 1. Thrombolysis by tissue type plasminogen activator (tt-PA) is currently the only approved treatment in acute ischemic stroke (NINDS, 1995)2. However, experimental models suggest that rt-PA could have deleterious effects by potentiating ischemic neuronal death. We have previously demonstrated that rt-PA promotes excitotoxic neuronal death by interacting with and cleaving the N-terminus of the NMDA receptor NR1 subunit, at arginine 260, thereby augmenting NMDA receptor-signalling 3-4. DSPA is a related plasminogen activator derived from bat saliva and lacks the Kringle2 domain. More recently, DSPA administered 3 to 9 hours after acute ischemic stroke in man, has been shown to produce high reperfusion rates and an improved clinical outcome with a favorably low bleeding risk 5. In this study, we demonstrate that DSPA, unlike rt-PA, neither interacts with nor cleaves the NR1 subunit. These data are consistent with the observation that, in contrast to rt-PA, DSPA does not potentiate NMDA-induced neuronal death in murine models 6. They provide a molecular basis that possibly explains why DSPA has a more favorable profile compared to rt-PA in the treatment of acute ischemic stroke.

Differential role of synaptic an extrasynaptic NMDA receptors in glutamate mediated neuronal injury

It has been known for 30 years that high concentrations of glutamate induce cell death in vitro and that similar extracellular concentrations are present in the rodent brain during ischaemia. It was subsequently shown that NMDA receptors mediate most of the glutamate-induced cell death in vitro and in vivo. Taken together, these data suggested that administration of NMDA antagonists in human beings could prevent cell death and confer neuroprotection after stroke. However, discouraging news started to accumulate as the clinical trials were terminated. Despite these developments, the theory of glutamate-induced excitotoxicity, the major power that forced NMDA receptor antagonists into human trials, has not been questioned. So it is possible that a fundamental misunderstanding of the pathophysiological roles of NMDA receptors may have been at the origin of absence of efficacy of glutamate antagonists in the treatment of stroke in Man. In the present study, we investigate the influence of NMDA receptors cellular locations on excitotoxicity.

Expression of Basic Fibroblast Growth Factor mRNA and Protein in the Human Brain following Ischaemic Stroke

Our previous work has demonstrated that angiogenesis occurs in the damaged brain tissue of patients surviving acute ischaemic stroke and increased microvessel density in the penumbra is associated with longer patient survival. The brain is one of the richest sources of FGF-2 and several studies have noted its angiogenic and neuroprotective effects in the nervous system. These findings led us to investigate the expression and localisation of both FGF-2 mRNA and protein in brain tissue collected within 12 h of death from 10 patients who survived for between 24 h and 43 days after acute stroke caused by thrombosis or embolus. Western blot analysis demonstrated increased FGF-2 protein expression in both grey and white matter in the infarcted core and the penumbra region compared to the normal contralateral hemisphere of all 10 patients studied. Using indirect immunoperoxidase staining of paraffin embedded sections, we observed the presence of FGF-2 in neurones, astrocytes, macrophages and endothelial cells. In situ hybridisation was used to localise and quantify mRNA expression in ischaemic brain tissue of the same 10 patients. The expression of FGF-2 in the penumbra of all patients was significantly raised compared with infarcted tissue and normal-looking contralateral hemisphere. In addition, serum FGF-2 was significantly increased between 1 and 14 days (P<0.001) in many patients with both ischaemic stroke (n=28) and intra-cerebral haemorrhage (n=16) compared with age-matched control subjects undergoing routine medical examinations (n=20). We suggest that up-regulation of FGF-2 is one of the mechanisms that leads to angiogenesis and neuro-protection in the penumbra region after acute stroke in man.

Transforming growth factor-beta and ischemic brain injury.

1. Necrosis and apoptosis are the two fundamental hallmarks of neuronal death in stroke. Nevertheless, thrombolysis, by using the recombinant serine protease t-PA, remains until now the only approved treatment of stroke in man. 2. Over the last years, the cytokine termed Transforming Growth Factor-beta1 (TGF-beta1) has been found to be strongly up-regulated in the central nervous system following ischemia-induced brain damage. 3. Recent studies have shown a neuroprotective activity of TGF-beta1 against ischemia-induced neuronal death. In vitro, TGF-beta1 protects neurons against excitotoxicity by inhibiting the t-PA-potentiated NMDA-induced neuronal death through a mechanism involving the up-regulation of the type-1 plasminogen activator inhibitor (PAI-1) in astrocytes 4. In addition, TGF-beta1 has been recently characterized as an antiapoptotic factor in a model of staurosporine-induced neuronal death through a mechanism involving activation of the extracellular signal-regulated kinase 1/2 (Erk1/2) and a concomitant increase phosphorylation of the antiapoptotic protein Bad. 5. Altogether, these observations suggest that either TGF-beta signaling or TGF-beta1-modulated genes could be good targets for the development of new therapeutic strategies for stroke in man.

A novel guinea pig model with cyclic flow reductions following thrombotic cerebral ischemia

Cyclic flow reductions (CFRs), a phenomenon indicated with repeating rethrombosis following thrombolysis in clotted vessel has been observed in coronal, carotid and popliteal arteries in various species when they were occluded with vessel damage, suggesting the presence of CFRs in the acute phase of stroke in man. Although much effort has been directed toward rethrombosis or ischemia–reperfusion injuries in acute strokes, a therapy for CFRs in strokes has not been established because of the lack of ideal animal models. We have established a novel guinea pig model with CFRs in the middle cerebral artery (MCA) using a photothrombotic technique, in which the MCA was spontaneously recanalized within 20 min after the first occlusion with subsequent CFRs. We also investigated the effects of antiplatelet agents and the anticoagulant heparin using this model. All tested antiplatelet agents inhibited CFRs, whereas an anticoagulant did not. These results show that this model is unique with respect to its development of MCA cyclic flow reductions, and may be suitable for investigating mechanisms and therapeutic reagents of CFRs in the MCA.

Protective effects of PJ34, a novel, potent inhibitor of poly(ADP-ribose) polymerase (PARP) in in vitro and in vivo models of stroke

Focal cerebral ischemia activates the nuclear protein poly(ADP-ribose) polymerase (PARP) by single DNA strand breaks which leads to energy depletion and cell necrosis. Deletion or inhibition of PARP protects against ischemic brain injury. Here we examined the neuroprotective effect of PJ34, a novel potent inhibitor of PARP in vitro and in vivo. Serum-free primary neuronal cultures derived from rat cortex (E15-17) and kept in culture for 10 days were exposed to oxygen glucose deprivation (OGD) in vitro. Neuronal injury was quantified by LDH release after 24 h. Pretreatment with 30-1000 nM PJ34 significantly protected from OGD-induced cell injury in a dose-dependent manner. For in vivo experiments SV/129 mice were treated with PJ34 (50 microg) by intraperitoneal injection 2 h before 1 h middle cerebral artery occlusion (MCAo) and again 6 h later. Twenty-three h after reperfusion ischemic injury was significantly decreased compared to vehicle-treated controls (infarct volume reduction of 40%, p<0.05). Similarly, in a rat model of MCAo (2 h occlusion followed by up to 22 h reperfusion), PJ34 administration (10 mg/kg i.v.) significantly reduced infarct size, and the effect of the drug was maintained even if it was given as late as 10 min prior to reperfusion time. PJ34 significantly protected in a 4 h, but not in a 24 h permanent occlusion model. In conclusion, PJ34, a novel, potent inhibitor of PARP exerts massive neuroprotective agents, with a significant therapeutic window of opportunity. The present work strengthens the concept that pharmacological PARP inhibition may be a suitable approach for the treatment of acute stroke in man.

Delayed Intrahemispheric Remote Hypometabolism

Although ‘intrahemispheric diaschisis’ (i.e. a hypometabolism affecting the cerebral hemisphere ipsilateral to but remote from the infarct) may classically exacerbate acute-stage neurological deficit and influence early recovery, it has been studied only rarely. Out of a series of 30 patients with first-ever middle cerebral artery (MCA) territory stroke, we analyzed the data from 19 survivors investigated by ¹⁵O positron emission tomography (PET) both in the acute (within 5–18 h of clinical onset) and subacute (approximately 3 weeks later) stage, and for whom chronic-stage CT coregistered with PET was available to assess infarct topography and size. Orgogozo’s MCA scale was used to assess neurological deficits at the time of, and recovery between, the PET studies. Oxygen consumption was obtained for both PET sessions for the whole ipsilateral hemisphere (excluding ventricles and infarct), as well as for the thalamus and the occipital and mesial-prefrontal cortex (i.e. potentially deafferented tissue outside the MCA territory). In all regions except the occipital cortex, the oxygen consumption significantly decreased between the first and the second session, without significant correlation with the concomitant changes in MCA scores. However, acute-stage mesial-prefrontal metabolism was significantly correlated with neurological recovery. Also, both the hemisphere and the mesial-prefrontal metabolism at the second session were significantly correlated with both infarct size and concomitant MCA scores, but the latter relationship became insignificant when infarct size was taken into account. This study reveals no evidence of acute intrahemispheric diaschisis after MCA territory stroke in man. However, it documents for the first time a phenomenon of delayed intrahemispheric remote hypometabolism developing while the patients clinically recover. Because the degree of this secondary phenomenon is a function of infarct size, a mechanism of degeneration of the damaged neuron terminals is likely. Finally, contrary to other reports, neurological recovery was not a function of thalamic hypometabolism, but appeared to be influenced by acute-stage mesial-frontal metabolism, perhaps because this region is part of a network that has an important compensatory role in motor recovery.

Clomethiazole protects against hemineglect in a primate model of stroke

Permanent occlusion of the M1 segment of the middle cerebral artery (pMCAO) in the marmoset, a New World species of monkey, produces unilateral functional deficits, including motor neglect with the contralesional arm and contralesional spatial hemineglect. In this study we examined whether clomethiazole, a drug which modulates the γ-aminobutyric acid A receptor, reduced the severity of the hemineglect and other deficits in this primate model of stroke. Nine monkeys received pMCAO; 1 h later four of the nine were administered clomethiazole by intraperitoneal injection and subcutaneous implantation of osmotic mini-pumps, which released clomethiazole for 48 h. The monkeys had been trained and tested on a number of behavioral tasks prior to surgery and were re-tested 3 and 10 weeks later. Three weeks after pMCAO, monkeys treated with clomethiazole had a significantly reduced degree of spatial neglect compared to untreated controls. Clomethiazole was not effective against the severe contralesional motor impairment in the current study, although it ameliorated a somewhat less severe motor deficit in a previous study in which the more distal, M2 segment of the middle cerebral artery had been occluded. Postmortem analysis of the brains showed that clomethiazole treatment had significantly reduced the area of damage in part of the parietal cortex. These data suggest that clomethiazole may reduce the neglect that can be a debilitating consequence of right-sided stroke in man.

Inducible Nitric Oxide Production and Expression of Transforming Growth Factor-β1 in Serum and CSF after Cerebral Ischaemic Stroke in Man

A residual blood supply to the ischaemic brain is a crucial determinant for tissue survival. Early changes in the vascular network and subsequent angiogenesis may be mediated by short-lived molecules like nitric oxide (NO) or growth factors such as transforming growth factor-β1 (TGF-β1). Although TGF-β1 can inhibit NO production, this interaction has not been studied after ischaemia in humans. Serum samples were taken from patients at 24 h and 6 months and cerebrospinal fluid (CSF) samples at 24 h and 1 week later for possible correlation between the two factors. Tissue expression of TGF-β1 and of the inducible isoform of NO synthase (NOS2) was assessed by immunohistochemistry. CSF levels of [fig] as well as total (active + latent) TGF-β1 were higher in stroke patients as compared to controls 24 h after the stroke. Both [fig] and TGF-β1 were lower 6 months after the stroke compared to 24 h. Levels of [fig]correlated with levels of TGF-β1 within the time points (P= 0.041, Kendall correlation coefficient). There was a strong staining for NOS2 in brain tissue sections in neurones, reactive astrocytes, infiltrating white blood cells, and endothelial cells of larger microvessels. TGF-β1 expression was mainly limited to neurones and reactive astrocytes. These findings suggest that the interaction between TGF-β1 and NOS2 might be important for angiogenesis after cerebral ischaemia and may indicate that TGF-β1 is upregulated as a negative feedback response to elevated levels of NO.

Progressive impairment of brain oxidative metabolism reversed by reperfusion following middle cerebral artery occlusion in anaesthetized baboons

A better understanding of the temporospatial evolution of ischaemic brain tissue towards necrosis would be of crucial value to establish and validate therapeutic strategies for stroke in man. By means of sequential positron emission tomographic (PET) studies performed through the acute to the chronic stages of infarction, we addressed the question of the effect of 6 h temporary occlusion of the middle cerebral artery (MCAO) on the evolution of the volume of severely hypometabolic tissue in anaesthetized baboons and compared it to that reported previously in permanently occluded baboons. Thirteen anaesthetized baboons underwent serial PET ( 15O steady-state technique) examinations before and 1, 4, 7, 24–48 h and 15–62 days following transorbital MCAO. Reperfusion, at 6 h post-occlusion, was assessed by Doppler sonography and cerebral blood flow (CBF) values after clip removal. In each baboon, the infarct volume was calculated by standard histological procedures 20–91 days after MCAO. The severely hypometabolic tissue volume, as defined by a threshold of oxidative metabolism, showed a progressive increase for up to 24–48 h in a not dissimilar manner to that found in baboons with permanent occlusion. However, these hypometabolic volumes regressed in the chronic stage ( p<0.05). Permanent and temporary occluded baboons, when taken together, showed a highly significant relationship between histological and chronic hypometabolic volumes ( r=0.84; p<0.001). Moreover, the final hypometabolic volume where cerebral metabolic rate of oxygen (CMRO 2) was below 40% of contralateral metabolism corresponded well to that of histological infarction volume. We conclude that, in anaesthetized baboons, restoration of blood flow will reverse (even if not immediately) the progressive derangement of metabolism after MCAO and markedly limit the final volume of consolidated infarction.

Aminoacid recovery via microdialysis and photoinduced focal cerebral ischemia in brain cortex of rats

A photochemical method using the Rose Bengal dye as thrombogenic agent was employed to induce focal cerebral ischemia in frontoparietal cortex of rats. A transcerebral microdialysis probe was used to collect samples from ischemic cortical area. An increase in glutamate (6-fold) and in taurine (4-fold) within the first hour occurred. Neuropathological investigations demonstrate a reproducible damaged area surrounded by a thin peripheral area showing neuronal apoptotic phenomena. The method represents a reproducible model of focal cerebral ischemia with neuropathological aspects superimposable to those characteristic of thrombogenic stroke in man. This method could also be relevant in the study of neurotransmitters during the evolution of ischemia. Furthermore, the presence of apoptotic phenomena in the perilesional halo confirms an ischemic penumbra suggesting the significance of preclinical pharmacological trials.

SB 201823-A, a neuronal Ca 2+ antagonist is neuroprotective in two models of cerebral ischaemia

We have characterised the Ca 2+ channel blocking properties of a new non-peptide Ca 2+ channel antagonist, SB 201823-A, in cultures of rat sensory neurones. The IC 50 for SB 201823-A against total Ca 2+ current in sensory neurones was 4.9 μM. SB 201823-A showed little selectivity for sub-types of neuronal Ca 2+ channel but was selective for Ca 2+ channels over Na + and K + channels. Efficacy against other types of cation channel such as agonist gated channels was not assessed. SB 201823-A was neuroprotective in vivo when administered post-ischaemia in one focal and one global model of neuronal ischaemia. In the rat photothrombotic focal lesion model, SB 201823-A administered i.p. 10 min post-ischaemia resulted in a dramatic reduction in lesion volume. In the gerbil bilateral carotid artery occlusion global model, SB 201823-A dosed i.p. 30 min post-occlusion resulted in both histological and functional improvements when compared to vehicle treated animals. These data suggest that such novel neuronal Ca 2+ channel antagonists may have potential in ameliorating both the pathological and functional consequences of stroke in man.