Using behaviour to predict stroke severity in conscious rats: Post-stroke treatment with 3′, 4′-dihydroxyflavonol improves recovery☆
Introduction
Despite the continued disappointment from clinical trials of potential neuroprotective drugs in acute stroke there are new clues about how to improve testing of new drugs in pre-clinical animal studies (Feuerstein et al., 2008, Hill, 2007). It is now critical to develop more appropriate animal models of ischemic stroke (Feuerstein et al., 2008). In particular the STAIR guidelines highlight the importance of testing drugs in more than one animal model of stroke prior to clinical trial and to use those that mimic most closely the human condition (STAIR-I, 1999).
Occlusion of the middle cerebral artery mimics closely the cause of the majority of strokes in humans (Mohr et al., 1986), and the rat is the species of choice for it shares a similar cranial circulation to humans (Yamori et al., 1976) and rats have a well documented behavioural profile (Hunter et al., 1998). However recent studies indicate problems associated with the most widely accepted and commonly used model of middle cerebral artery occlusion in rats, that produced by an intraluminal filament. Adverse effects include interruption of blood supply to the external carotid artery territory (Dittmar et al., 2003), subsequent hyperthermia (Li et al., 1999), subarachnoid haemorrhage (Schmid-Elsaesser et al., 1998) and damage to the temporalis muscle during surgery resulting in marked weight loss and poorer recovery of motor function (Dittmar et al., 2003). Of particular concern in almost all experimental models is the use of general anaesthesia during induction of stroke, for humans are not usually under anaesthesia when a stroke occurs. Furthermore, barbiturate and inhalational anaesthetics can confound experimental findings due to their own neuroprotective effects (Hagerdal et al., 1978, Bhardwaj et al., 2001), can affect reactive oxygen species (ROS) production (Hagerdal et al., 1978) and cause long-lasting depression of protein synthesis (Fütterer et al., 2004) and ischemic tolerance (Kapinya et al., 2002).
The endothelins are powerful vasoconstrictors that act through 2 receptor subtypes, the endothelin A and endothelin B receptors (Arai et al., 1990). A potent and long lasting constrictor action of endothelin has been described in both animal and human cerebral vasculature, and there is evidence to suggest that it may be involved in the genesis or maintenance of delayed vasospasm following subarachnoid hemorrhage (Zemke et al., 2007), migraine (Tietjen, 2007) and ischemic stroke (Estrada et al., 1994, Patel et al., 1995). Stereotaxic application of endothelin-1 to the middle cerebral artery is now a well established model of focal ischaemic in conscious rats and this model has also been adapted for use in primates (Virley et al., 2004). Although receptors for endothelin are found in non-vascular brain tissue (Nie and Olsson, 1996), endothelin-1 is believed to act at the endothelin A and B receptors on the adventitial surface of cerebral vessels. Thus endothelin-1 injected intraluminally to cerebral arteries induces only minor changes in vessel caliber, unlike abluminal applications (Ogura et al., 1991). Abluminal application of 3 µl of endothelin-1 to the exposed rat middle cerebral artery results in a dose-dependent and reversible vasoconstriction of the artery with up to 80% reduction in cerebral blood flow observed within the first 10 min, similar to that seen with permanent occlusion of the proximal middle cerebral artery — a standard model of focal ischaemia in rats (Macrae et al., 1993, Sharkey et al., 1993, Sharkey and Butcher, 1995). The endothelin model is the only model in which focal cerebral ischemia (‘stroke’) is induced in the absence of anaesthesia, and in which spontaneous reperfusion occurs (Sharkey et al., 1993). However a disadvantage of this model is that it is not practical to measure cerebral blood flow in conscious rats to determine the duration of ischemia (Bogaert et al., 2000), and thus it has been difficult to determine the initial severity of stroke in this model.
Prognostic clinical approaches such as the use of the Scandinavian Stroke Scale, allow prediction of functional outcome and survival in stroke patients in order to support clinical management and to correctly stratify treatment groups in clinical trials (Counsell et al., 2002). The use of a similar approach in experimental animals has not previously been attempted. By administering endothelin-1 to constrict the middle cerebral artery it is possible to observe behavioural responses during the induction of stroke because the rats are conscious. We and others have observed behavioural responses including circling in the direction contralateral to the occluded artery, and clenching and dragging of the contralateral forepaw (Sharkey et al., 1993, Callaway et al., 1999, Bogaert et al., 2000). Based on these responses we have assigned a rating scale of stroke severity. The present study was undertaken to determine the predictive value of behavioural observations and ratings made during induction of stroke using the endothelin-1 model in conscious rats, on both histological and behavioural outcomes.
We have chosen to study flavonols as potential cytoprotective agents. Flavonols are clinically well tolerated, highly lipid soluble antioxidants that easily permeate biological membranes and scavenge intracellular, as well as extracellular reactive oxygen species (Pietta, 2000). We have recently shown that 3′, 4′-dihydroxyflavonol (DiOHF), given before reperfusion of the ischemic myocardium in sheep, greatly reduces infarct size, myocardial injury and helps restore coronary blood flow (Wang et al., 2004). In addition, DiOHF enhances nitric oxide bioavailability and improves vascular function after ischemia and reperfusion injury in rat hindquarters (Chan et al., 2003). We therefore set out to assess the protective potential of DiOHF in stroke using behavioral responses to stratify rats into groups based on stroke severity (as discussed above), prior to assessment of recovery of neurological functions and morphologically mapped brain damage.
Section snippets
Surgical preparation
All experiments were performed in accordance with the guidelines of the National Health & Medical Research Council of Australia Code of Practice for the Care and Use of Animals for Experimental Purposes in Australia, which complies with the Guide for the Care and Use of Laboratory Animals as adopted and promulgated by the United States National Institutes of Health. Male Hooded Wistar rats (280–340 g, n = 69) were anesthetised with pentobarbital sodium in a volume of 0.6 ml (60 mg/kg i.p.). A
Stroke rating
Rats showed neurological behavioural deficits indicative of stroke within 2–10 min of endothelin-1 injection, but not after injection of saline in the sham group. These deficits included specific behavioural responses such as clenching and failure to extend the contralateral forepaw, and circling in the direction contralateral to the occlusion. Behavioural responses observed at the time of stroke could be rated based on their degree and severity (Table 1, Fig. 1). Grooming behaviour preceded
Discussion
Here we demonstrate for the first time that in the conscious rat it is possible to predict accurately both histological and functional outcomes based on behavioural changes observed during infusion of endothelin-1 to constrict the middle cerebral artery. In addition, we have demonstrated the usefulness of stratifying rats into treatment groups using stroke ratings based on initial behaviours, to test the outcomes of synthetic flavonol treatment across all stroke outcomes. Using this prognostic
Acknowledgments
This study was supported in part by grants from the National Health and Medical Research Council of Australia (program # 236805) and Neurosciences Victoria. GJD is Principal Research Fellow of NHMRC (# 400303). We thank Prof John Ludbrook MD DSc ChM BMedSc FRCS FRACS AStat, Director Biomedical Statistical Consulting Service PTY LTD, Carlton North, Victoria, Australia for advice on statistical analyses.
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Grant information: this study was supported in part by grants from the National Health and Medical Research Council of Australia (program # 236805) and Neurosciences Victoria. GJD is Principal Research Fellow of NHMRC (# 400303).
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The first two authors contributed equally to this work.