Deborah Dawson

Inhibition of tumor necrosis factor-alpha reduces focal cerebral ischemic injury in the spontaneously hypertensive rat

Tumor necrosis factor-alpha (TNF-alpha) is acutely expressed following focal cerebral ischemia, but its pathophysiological role remains to be extensively characterized. In this study we determined the effect of inhibiting TNF-alpha on the microvascular perfusion impairment and ischemic injury induced by permanent middle cerebral artery occlusion (MCAO). TNF-alpha activity was inhibited with recombinant type I soluble TNF receptor (TNFbp; 1 mg/kg i.v., 0.5 h pre- or post-MCAO). TNFbp significantly attenuated the microvessel perfusion impairment observed in vehicle treated rats, particularly in perifocal/penumbral regions of cortex, and significantly reduced (by 34-38%) the total volume of ischemic injury. These results demonstrate that TNF-alpha contributes to focal ischemic injury and that inhibition of TNF-alpha can confer dramatic neuroprotection. The association of the neuroprotective effect of TNFbp with improved microvascular perfusion suggests that inflammatory and vascular responses to TNF-alpha contribute to its pathological action.

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.

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.

Cerebrovascular hemodynamics and ischemic tolerance : Lipopolysaccharide-induced resistance to focal cerebral ischemia is not due to changes in severity of the initial ischemic insult, but is associated with preservation of microvascular perfusion

Lipopolysaccharide (LPS), administered 72 hours before middle cerebral artery (MCA) occlusion, confers significant protection against ischemic injury. For example, in the present study, LPS (0.9 mg/kg intravenously) induced a 31% reduction in infarct volume (compared with saline control) assessed 24 hours after permanent MCA occlusion. To determine whether LPS induces true tolerance to ischemia, or merely attenuates initial ischemic severity by augmenting collateral blood flow, local CBF was measured autoradiographically 15 minutes after MCA occlusion. Local CBF did not differ significantly between LPS- and saline-pretreated rats (e.g., 34 ± 10 and 29 ± 15 mL·100 g−1·min−1 for saline and LPS pretreatment in a representative region of ischemic cortex), indicating that the neuroprotective action of LPS is not attributable to an immediate reduction in the degree of ischemia induced by MCA occlusion, and that LPS does indeed induce a state of ischemic tolerance. In contrast to the similarity of the initial ischemic insult between tolerant (LPS-pretreated) and nontolerant (saline-pretreated) rats, microvascular perfusion assessed either 4 hours or 24 hours after MCA occlusion was preserved at significantly higher levels in the LPS-pretreated rats than in controls. Furthermore, the regions of preserved perfusion in tolerant animals were associated with regions of tissue sparing. These results suggest that LPS-induced tolerance to focal ischemia is at least partly dependent on the active maintenance of microvascular patency and hence the prevention of secondary ischemic injury.

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.

A comparative assessment of the efficacy and side-effect liability of neuroprotective compounds in experimental stroke.

There are many examples of compounds showing neuroprotective efficacy in animal models of stroke but not in clinical trials. It is possible that some or all of these compounds possess poor therapeutic ratios, which results in the administration of sub-efficacious doses in order to avoid the emergence of side-effects. In order to explore this possibility, this study compared the therapeutic ratios of a number of neuroprotective agents that have undergone clinical trials. Neuroprotective efficacy was established using the mouse permanent (24 h) middle cerebral artery occlusion model. Side-effect liability was determined by assessment of motor coordination using the rotarod test. The therapeutic ratio was calculated as the ratio between the minimum effective dose (MED) for significant impairment in rotarod performance and the MED for significant neuroprotection. Compounds were administered i.p. 30 min prior to rotarod testing or onset of ischemia. Drugs such as Ifenprodil, Cerestat and Selfotel, that have failed in clinical trials, were found to have very low therapeutic ratios of < or = 1, whereas compounds with more tolerable clinical side-effect profiles were found to have higher therapeutic ratios (2, 10 and 10 for Sipatrigine, Remacemide and sPBN, respectively). It is concluded that the lack of efficacy of a number of neuroprotectants in clinical trials may well be a consequence of their poor therapeutic ratios.

Tau protein is altered by focal cerebral ischaemia in the rat : an immunohistochemical and immunoblotting study

Breakdown of the cytoskeleton may be involved in the evolution of ischaemic brain damage and alterations in microtubule-associated proteins may play an important role in this process. In the present study, tau, a microtubule-associated protein predominantly located in axons, was examined after 2 or 6 h of focal cerebral ischaemia in the rat. Immunohistochemistry revealed increased Tau1 staining in the neuropil, some perikarya and in glial cells throughout the dorsolateral caudate nucleus and ventrolateral neocortex in the ipsilateral hemisphere at both 2 and 6 h after occlusion of the middle cerebral artery. Contrastingly, immunostaining of another tau antibody, TP70, was unchanged in the neuropil, but was increased specifically in glial cells in these regions. Immunoblotting revealed the presence of additional tau bands in tissue extracts of the caudate nucleus and ventrolateral neocortex ipsilateral to the occluded middle cerebral artery as detected by both tau antibodies after either 2 or 6 h. The results suggest that tau is dephosphorylated and/or degraded in axons and some neuronal perikarya in response to focal cerebral ischaemia. In contrast to the response in neurons, increased immunoreactivity of both tau antibodies in glial cells indicates a differential response of neuronal and glial tau to focal cerebral ischaemia.

Pharmacological modification of glutamate neurotoxicity in vivo

The ability of five agents (dizocilpine [MK-801], 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(F)-quinoxaline [NBQX], enadoline [CI-977], L-nitroarginine methyl ester [L-NAME] and BW 1003c87) with well defined, distinct pharmacological profiles and with established anti-ischemic efficacy, to modify neuronal damage has been examined in a simple in vivo model of glutamate excitotoxicity. Cortical lesions were produced in physiologically-monitored halothane-anesthetised rats by reverse dialysis of glutamate. The volume of the lesion was quantified histologically by image analysis of approximately 20 sections taken at 200 microm intervals throughout the lesion. The AMPA and NMDA receptor antagonists (NBQX and MK-801) and the inhibitor of nitric oxide synthase (L-NAME) significantly reduced the lesion volume by a similar extent (by approximately 30% from vehicle). Two agents (the kappa opioid agonist, CI-977 and the sodium channel blocker, BW 1003c87) which putatively inhibit the release of endogenous glutamate presynaptically, had dissimilar effects on lesion size. CI-977 failed to alter the amount of damage produced by exogenous glutamate, whereas BW 1003c87 reduced the lesion size by approximately 50%. Using this model, the neuroprotective effects of anti-ischemic drugs can be explored in vivo, uncomplicated in contrast to experimental ischemia by reduced oxygen delivery, drug effects on tissue blood flow and compromised energy generation. In consequence, additional mechanistic insight into anti-ischemic drug action in vivo can be obtained.

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.

Endothelin, Cerebral Ischaemia and Infarction

Endothelin-1 (ET-1) is a 21 aminoacid peptide with potent vasoconstrictor properties. It is synthesised and released by endothelial cells in both the peripheral and cerebral vasculature and is also localised within neurones in discrete brain areas where it may contribute to the central regulation of blood pressure. We have shown that intracisternal ET-1 in conscious rats induces a marked pressor response that is associated with an intense widespread reduction in cerebral blood flow. Subsequent studies with local application of ET-1 to the middle cerebral artery (MCA) revealed a dose dependent reversible vasoconstriction of the artery that resulted in profound reductions in local cerebral blood flow and the development of cerebral infarction. Thus abluminal application of ET-1 to the MCA offers a simple model of reversible focal cerebral ischaemia in the rat that complements the existing models of permanent MCA occlusion. The ET-1 model will help to provide new insights into the mechanisms of cerebral ischaemia and reperfusion injury, and to evaluate the usefulness of novel strategies of neuroprotection.