I.M. Macrae

Neuroprotective efficacy of ebselen, an anti-oxidant with anti-inflammatory actions, in a rodent model of permanent middle cerebral artery occlusion

1 The aim of this study was to investigate whether delayed treatment with the anti‐oxidant and anti‐inflammatory agent ebselen reduces the volume of infarction in a rodent model of permanent focal cerebral ischaemia. 2 Ebselen (10 or 30 mg kg−1) or vehicle was administered by gavage 30 min and 12 h after the induction of cerebral ischaemia by permanent occlusion of the left middle cerebral artery (MCA). Animals were killed 24 h following MCA occlusion, and the volumes of ischaemic damage in the ebselen and control groups were evaluated by quantitative histopathology. 3 Ebselen was quickly absorbed following oral (gavage) administration and reached peak levels in the plasma by 1 h post‐administration (plasma selenium level of 0.68±0.04 and 0.84±0.1 μg ml−1 for 10 and 30 mg kg−1, respectively, compared to control level of 0.51±0.02 μg kg−1). 4 Treatment with the lower dose of ebselen (10 mg kg−1) significantly (P<0.01) reduced the volume of infarction in the cerebral hemisphere and cerebral cortex (by 31.8% and 36.7%, respectively compared with the placebo group). 5 The neuroprotective efficacy of the higher dose ebselen (30 mg kg−1) was less than that of the lower dose ebselen (10 mg kg−1). The volume of ischaemic damage in the cerebral hemisphere was reduced by 23.7% (P<0.02), and cerebral cortex by 27.5% (P<0.01). 6 Both doses of ebselen (10, 30 mg kg−1) had no therapeutic efficacy on the caudate nucleus, where ischaemia was most severe, in this model. 7 Free radical‐mediated injury is normally associated with reperfusion of ischaemic tissue. The present results suggest that oxidative injury is also a significant contributor to brain damage in models of maintained (permanent) ischaemia and that ebselen is effective in attenuating this free radical‐induced damage.

The neuroprotective efficacy of ebselen (a glutathione peroxidase mimic) on brain damage induced by transient focal cerebral ischaemia in the rat

The neuroprotective efficacy of the hydroperoxide scavenger ebselen was assessed in a model of transient focal ischaemia that utilises the potent vasoconstrictor peptide endothelin-1 to induce temporary occlusion of the middle cerebral artery (MCA). Pretreatment with ebselen (10 or 30 mg/kg p.o., 40 min pre-MCA occlusion) dose dependently reduced the volume of ischaemic damage assessed 4 h post-endothelin-1 application in the anesthetised rat. The lower dose of ebselen (10 mg/kg) resulted in a non-significant 35% reduction in the total volume of ischaemic damage compared with the vehicle control. In contrast the higher dose of ebselen (30 mg/kg) significantly reduced the volume of ischaemic damage in the cerebral hemisphere and cerebral cortex by 48% and 53%, respectively. The marked reduction in brain damage achieved with ebselen cannot be attributed to drug-induced alterations in blood pressure, body temperature or arterial blood gases since these physiological variables were closely monitored and were not significantly altered by ebselen treatment. Thus ebselen is an effective neuroprotective agent against acute focal ischaemic-reperfusion injury.

Preclinical stroke research – advantages and disadvantages of the most common rodent models of focal ischaemia

This review describes the most commonly used rodent models and outcome measures in preclinical stroke research and discusses their strengths and limitations. Most models involve permanent or transient middle cerebral artery occlusion with therapeutic agents tested for their ability to reduce stroke‐induced infarcts and improve neurological deficits. Many drugs have demonstrated preclinical efficacy but, other than thrombolytics, which restore blood flow, none have demonstrated efficacy in clinical trials. This failure to translate efficacy from bench to bedside is discussed alongside achievable steps to improve the ability of preclinical research to predict clinical efficacy: (i) Improvements in study quality and reporting. Study design must include randomization, blinding and predefined inclusion/exclusion criteria, and journal editors have the power to ensure statements on these and mortality data are included in preclinical publications. (ii) Negative and neutral studies must be published to enable preclinical meta‐analyses and systematic reviews to more accurately predict drug efficacy in man. (iii) Preclinical groups should work within networks and agree on standardized procedures for assessing final infarct and functional outcome. This will improve research quality, timeliness and translational capacity. (iv) Greater uptake and improvements in non‐invasive diagnostic imaging to detect and study potentially salvageable penumbral tissue, the target for acute neuroprotection. Drug effects on penumbra lifespan studied serially, followed by assessment of behavioural outcome and infarct within in the same animal group, will increase the power to detect drug efficacy preclinically. Similar progress in detecting drug efficacy clinically will follow from patient recruitment into acute stroke trials based on evidence of remaining penumbra.

Inhibition of nitric oxide synthesis does not reduce infarct volume in a rat model of focal cerebral ischaemia

The effect of the nitric oxide (NO) synthesis inhibitor Ng-nitro-L-arginine methylester (L-NAME) on ischaemic brain damage was determined in a rat model of focal cerebral ischaemia. Ischaemia was induced by permanent occlusion of the left middle cerebral artery (MCA) and infarction assessed 4 h post-occlusion by quantitative histopathology. L-NAME (30 mg/kg s.c.), administered 30 min pre- and 30 min post-MCA occlusion, did not significantly alter the volume of ischaemic damage in the cerebral hemisphere, neocortex or caudate nucleus compared with saline controls. This result provides no support for the view that NO generation is a key component in the post-ischaemic cascade leading to acute neuronal death.

Brain aromatase expression after experimental stroke: Topography and time course

Brain aromatase has been shown to be increased in expression after neurotoxic damage and to exert neuroprotection via generation of local oestrogens. The present study investigates the topography and time course of brain aromatase expression after experimental stroke (middle cerebral artery occlusion (MCAO)). Ovariectomised stroke prone spontaneously hypertensive rats underwent distal MCAO by electrocoagulation. Immunohistochemistry revealed increased brain aromatase expression at 24h and 8 days in the cortical penumbra/peri-infarct zones with no increase evident at 2h or 30 days post-MCAO. Double label studies indicate that some of the increased aromatase expression is associated with astrocytic processes. Thus, this is the first evidence that aromatase protein is increased after MCAO and the location (peri-infarct), time course (within 24h) and cellular localisation (astrocytic) indicate the potential for aromatase to promote the survival of cells in the penumbra after experimental stroke by local synthesis of oestrogens.

Reduction of local cerebral blood flow to pathological levels by endothelin-1 applied to the middle cerebral artery in the rat

Endothelin-1 (1 nmol) was applied to the exposed left middle cerebral artery (MCA) in anaesthetised adult male Sprague-Dawley rats. Local cerebral blood flow (1CBF), using [14C]iodoantipyrine and quantitative autoradiography, was measured in 27 anatomically defined structures, 10 min after topical application of endothelin-1. In those areas supplied by the MCA, 1CBF was markedly reduced beyond the threshold for ischaemic damage (e.g. dorsolateral caudate nucleus reduced from 131 +/- 3 to 29 +/- 25 ml.100 g-1.min-1, sensorimotor cortex from 109 +/- 5 to 31 +/- 21 ml.100 g-1.min-1). Distant areas were not affected.

Inhibition of Nitric Oxide Synthesis: Effects on Cerebral Blood Flow and Glucose Utilisation in the Rat

The consequences of inhibition of nitric oxide synthesis on local CBF and glucose utilisation have been studied in the conscious rat using the specific nitric oxide synthase inhibitor Ng-nitro-l-arginine methyl ester (l-NAME; 30 mg kg−1 i.v.). Local CBF and glucose utilisation were assessed with the [14C]iodoantipyrine and the 2-deoxy-d-[14C]glucose autoradiographic techniques, respectively. l-NAME induced prolonged (>3 h) reductions in local CBF throughout the CNS with concomitant increases in arterial blood pressure. For example, 1 h post l-NAME, CBF dropped from 79 ± 4 to 45 ± 1 ml 100 g−1 min−1 in cerebellum, from 76 ± 4 to 47 ± 2 ml 100 g−1 min−1 in medulla oblongata, and from 117 ± 6 to 72 ± 2 ml 100 g−1 min−1 in cortex. l-NAME produced sustained elevations (e.g., 46 ± 2 mm Hg at 1 h after bolus administration) in mean arterial blood pressure throughout the period evaluated. Despite evidence implicating nitric oxide in neuronal signalling, l-NAME did not significantly influence CNS functional activity, as measured by local rates of glucose utilisation, in any neuroanatomical region examined. Consequently, the normal ratio of blood flow to glucose use throughout the brain was significantly reduced in the presence of l-NAME, although the hierarchy of blood flow levels in different neuroanatomical regions was preserved. These results are consistent with the involvement of nitric oxide in the tonic control of cerebral tissue perfusion.

Anti‐ischaemic efficacy of a nitric oxide synthase inhibitor and a N‐methyl‐d‐aspartate receptor antagonist in models of transient and permanent focal cerebral ischaemia

1 We have recently developed a new model of transient focal ischaemia in the rat utilising topical application of endothelin‐1 to the left middle cerebral artery (MCA). In order to validate this approach the present study assessed the neuroprotective efficacy of the NMDA receptor antagonist dizocilpine (MK‐801) in the endothelin‐1 model. The anti‐ischaemic efficacy of the nitric oxide (NO) synthase inhibitor NG‐nitro‐l‐arginine methyl ester (l‐NAME) was subsequently evaluated, and contrasted with its efficacy against permanent focal ischaemia, to determine the utility of the endothelin‐1 model for identification of novel pharmacoprotective agents. 2 MK‐801 (0.12 mg kg−1 bolus, 108 μg kg−1 h−1 infusion i.v., either 1 or 2.5 h pre‐transient MCA occlusion (MCAO)) induced hypotension that persisted for approximately 1.5 h so that mean arterial blood pressure (MABP) at the time of MCAO was significantly lower in the 1 h group compared with control (MABP: 86 ± 11, 68 ± 6 and 84 ± 4 mmHg (mean ± s.d.) for saline, 1h MK‐801 and 2.5 h MK‐801 groups respectively). The 2.5 h pretreatment schedule resulted in significant reduction (71%) in the volume of hemispheric damage (assessed 4 h post onset of ischaemia) while the 1 h pretreatment schedule did not (volumes of hemispheric damage: 59 ± 38, 51 ± 51 and 17 ± 28 mm3 for saline, 1 h and 2.5 h MK‐801 groups). 3 Thus the considerable neuroprotective effect of MK‐801 in the endothelin‐1 model of transient focal cerebral ischaemia was highly sensitive to drug‐induced hypotension. This result is in contrast to previous studies of permanent MCAO where MK‐801‐induced hypotension did not compromise its neuroprotective action. 4 l‐NAME (3 mg kg−1, i.v. 30 min pre‐MCAO) moderately, but significantly, reduced (16%) the volume of ischaemic damage 4 h post‐permanent MCA occlusion, whereas the 29% reduction in volume of damage achieved in the model of transient focal ischaemia did not attain significance due to the greater variability associated with this model. l‐NAME did not significantly alter MABP in either model. 5 The modest neuroprotection achieved with NO synthase inhibition suggests NO is of relatively minor importance as a mediator of neurotoxicity following permanent focal cerebral ischaemia. In addition the comparable efficacy of l‐NAME against transient focal ischaemia suggests the presence of reperfusion does not enhance the contribution of NO to neuronal injury in the acute (4 h) phase following a focal ischaemic insult.

Effects of intracisternal endothelin-1 injection on blood flow to the lower brain stem

The central effects of endothelin-1 (Et-1, 10-30 pmol in 2.5 microliters injected intracisternally) have been investigated in the conscious rat. With 10 and 20 pmol Et-1, no significant change in blood pressure was observed. With 30 pmol Et-1, mean arterial blood pressure rose by 40 +/- 10 mm Hg with an accompanying modest, short-lived bradycardia at 2 min post-injection. Cerebral blood flow [( 14C]iodoantipyrine autoradiography), measured simultaneously with the hypertensive response, was markedly reduced throughout the caudal medulla and cerebellum (by up to 85%), while significant hyperaemia was evident in a number of forebrain structures (e.g. an increase of 78% in sensorimotor cortex). These observations have relevance to two distinct scientific areas. Concerning the significant effect of Et-1 in central cardiovascular control, these results caution against drawing conclusions from ventricular application with knowledge only of cardiovascular parameters. These results also illustrate the profound effects of Et-1 which is uniquely capable of overriding cerebral autoregulatory mechanisms.

Specific expression of the cell cycle regulation proteins, GADD34 and PCNA, in the peri-infarct zone after focal cerebral ischaemia in the rat.

Cell cycle proteins play key roles in cell survival or death under pathological conditions. Expression of growth arrest and DNA damage‐inducible protein, GADD34 and proliferating cell nuclear antigen (PCNA) have been investigated in the core and peri‐infarct zone at 2 and 24 h after middle cerebral artery occlusion (MCAO). At these times after MCAO, numerous GADD34‐positive cells were present, particularly in the peri‐infarct zone (e.g. 24 ± 4 and 52 ± 6 immunopositive cells/0.25 mm2 at 2 and 24 h, respectively, in cortex). PCNA‐immunopositive cells were barely detectable in the peri‐infarct zone at 2 h; however, numerous PCNA‐immunopositive cells were present in this zone by 24 h (0.7 ± 0.3 and 10.6 ± 1.5 immunopositive cells/0.25 mm2, respectively) as well as in the adjacent cortex and in the contralateral cingulate cortex. Most GADD34‐immunopositive cells coexpressed the neuronal marker Neu‐N with a smaller number coexpressing the microglial marker, Mrf‐1. Evidence of morphologically ‘abnormal’ and ‘normal’ GADD34 immunopositive neurons was found within the peri‐infarct zone. The majority of PCNA immunopositive cells were Mrf‐1 positive with a smaller number Neu‐N positive. Double‐labelling revealed colocalization of GADD34 and PCNA in some cells within the peri‐infarct zone and in the ependymal cells lining the ventricles. The presence of GADD34 and PCNA in a key anatomical location pertinent to the evolving ischaemic lesion indicates that GADD34, either alone or in combination with PCNA, has the potential to influence cell survival in ischaemically compromised tissue.