Background and Purpose Many stroke models in rats are based on occlusion of the middle cerebral artery, which supplies a significant portion of multifunctional cortical and deep structures in the cerebral hemisphere. The purpose of this study was to develop a model for direct observation in real time of blood flow in and around focal ischemic regions of the cortex of known function.
Methods Cranial windows were placed over the parietal cortex of adult Wistar and Sprague-Dawley rats anesthetized with ketamine and xylazine. Whisker barrel cortex responding to stimulation of the contralateral whiskers was identified by an intrinsic optical signal. Transits of vital dyes were recorded by videomicroscopy before and after ligation of three to six branches and major collaterals of the middle cerebral artery through the dura. Infarcts were demonstrated with triphenyltetrazolium chloride staining; their relation to barrel cortex was determined by Nissl and cytochrome oxidase histology.
Results Reduced blood flow in small ischemic regions was outlined by patent blue violet in the surrounding nonischemic area; arteriovenous latencies increased more than four times in ischemic cortex. Infarcts, typically 3 mm or less, were seen at 24 hours in 8 of 16 Wistar and 9 of 9 Sprague-Dawley rats. The ministrokes were confirmed by histology to be in the somatosensory cortex.
Conclusions This model of local ischemia, produced deliberately in the functionally defined barrel cortex in rats, leads to ministrokes. Changes can be followed by videomicroscopy as they develop, and processes of recovery can potentially be monitored. Infarcts are confirmed by histology for their location and extent in the somatic representation.
Reliable models of stroke in rats have provided much background information on local cerebral ischemia and its neuropathologic consequences.1 2 This information has been used to develop strategies for early intervention in “brain attacks.”3 Furthermore, rodent stroke models help to correlate cellular events in vitro and in vivo,4 to follow changing cerebrovascular dynamics, and to evaluate stroke-related neural reorganization. A stroke model would be particularly useful and informative if it permitted careful hemodynamic evaluation of events in evolving strokes and provided a neurological context in which to evaluate functional consequences in a specific system.5 6 Here we describe such a model in which localized cerebral ischemia and stroke are produced by deliberate ligation of multiple branches of the middle cerebral artery (MCA) and identified collaterals to functionally defined whisker barrel cortex in rats.
Materials and Methods
Female Wistar (weight, 270 to 350 g; age, >90 days) or male Sprague-Dawley (weight, 400 to 500 g; age, >90 days) rats were anesthetized with ketamine (100 mg/kg) plus xylazine (50 mg/kg IP), supplemented with one third the initial dose as required, and maintained at 38°C on a heating pad. The right external carotid artery was cannulated with heparinized poly ethylene tubing (PE 10) for dye injections. Then 6- to 8-mm cranial windows were made over the right somatosensory cortex with dura intact and bathed with artificial cerebrospinal fluid at 37°C. Barrel cortex was identified by an intrinsic optical signal during natural stimulation of the contralateral whiskers from images through a fluorescein emission filter (520 to 560 nm).7 Small boluses (10 to 20 μL) of the vital dye patent blue violet (10 mmol/L; Sigma) in normal saline were injected into the right external carotid artery to demonstrate transits through surface arteries, capillaries, and veins of the cortex. From video scans of the surface vessels, three to six proximal and distal branches of the MCA around the barrel cortex were selected and ligated with 10-0 sutures through the dura. Afterward the bolus injections and dye transits were repeated.
For survival studies the external carotid artery was not cannulated to minimize possible secondary vascular complications. The whisker barrel cortex was located by intrinsic signals as described above, critical arterial branches in the window were identified and ligated as described above, and the scalp was closed with 6-0 silk. For some Wistar rats the ipsilateral common carotid artery was temporarily occluded for 1 to 2 hours in addition to the permanent ligation of MCA branches. Each rat was returned to its cage with a heat lamp to maintain body temperature. After 24 hours some rats were reanesthetized, the brain was quickly dissected, and fresh 2-mm frontal slices were incubated in 10% triphenyltetrazolium chloride for mitochondrial dehydrogenase activity.8 Neuropathology was confirmed at 24 hours or longer by reanesthetizing rats for transcardial perfusion with 4% buffered paraformaldehyde, and 50-μm frozen sections were stained with 0.125% thionine for Nissl substance or were stained for cytochrome oxidase activity.9 Both procedures also demonstrated the whisker barrels in the somatosensory cortex.10 11
The brain surface and its vasculature are well visualized through a cranial window, even through the dura. Dividing one averaged image at rest by another taken at rest at another time ought to give a totally blank field if no optical changes occurred. Images of surface vessels from brain movements during pulse and respiration provided useful landmarks (Fig 1A⇓). Repetitive stimulation of all the large whiskers outlined their representation to guide ligation of the supplying branches of the MCA (Fig 1B⇓).
Intravascular dye transits through the MCA in controls before ligation were always rapid, direct, and nearly uniform through its branches (Fig 1C⇑). The return flow to surface venules was prompt: the mean difference in latencies of surface arterioles and venules between visible onsets of patent blue dye was 0.7±0.1 seconds (±SEM, n=9 rats) in initial controls. Control studies also demonstrated smaller surface arterioles that also could be ligated.
After several branches of the MCA were tied (Fig 1D⇑), the speed of dye transits was several times slower in the ischemic region. The poorly perfused ischemic regions (pink) contrasted sharply with the nonischemic surround in which patent blue violet passed rapidly through arterioles into parenchymal capillaries and then to venules. Latencies from arterioles outside the ischemic region to venules inside increased more than four times to 3.1±0.4 seconds (mean±SEM, n=9 rats, as above; P<.001, Student’s t test). Previously unrecognized collaterals dilated and could also be ligated for further reduction of flow.
Histology of the ischemic regions 24 hours after multiple MCA ligations demonstrated small focal infarcts (ministrokes) that were typically smaller than 3 mm in diameter and mainly confined to cortical gray matter (Fig 2A⇓ and 2B⇓). Cytochrome oxidase staining in layer IV of the somatosensory cortex after 60 days showed loss of activity in the infarcted region (Fig 2C⇓). All adult Sprague-Dawley rats developed infarcts after the multiple ligations (Table⇓). Infarcts developed at 24 hours in only 50% of Wistar rats, but the incidence increased to 88% with survivals of 16 days or more. The incidence of infarcts in Wistar rats at 24 hours increased to 100% with 1- to 2-hour temporary occlusion of the ipsilateral common carotid artery.
The present model of local cerebral ischemia in rats is focused on the functionally identified whisker barrel cortex. The changes in microcirculatory patterns are observed and recorded directly through cranial windows before and after multiple ligations of small arterial branches and collaterals. By selection of arterial branches for ligation and rats of different strains and ages, local ischemic regions remain at risk (a penumbral model) or proceed to necrosis under experimental control.
Intrinsic optical signals were used to identify the functional barrel cortex before ligations. These intrinsic signals arise from a number of sources, but at the wavelengths for this study the signal is likely dominated by red cell hemoglobin volumes.12 Other experiments have demonstrated the correspondence of patterns of optical changes evoked by mechanical stimulation of single whiskers to the appropriate barrels in layer IV.7 Neuroanatomical markers for somatic cortex are evident with Nissl stains or cytochrome oxidase histochemistry. These can be used to determine the extent to which the ischemic lesions are confined to identified regions of the somatic representation, which is of importance for exploring behavioral, neurophysiological, and neuroanatomical aspects of developing strokes and potential for recovery. In both the short term and the longer term the barrel pattern (Fig 2C⇑) provides a context for evaluating tissue responses to this insult.
One important variable in stroke after MCA occlusion is the strain of rat, which ranked in order from most resistant to most sensitive is Wistar<Sprague-Dawley<spontaneously hypertensive<stroke-prone spontaneously hypertensive.1 Older (heavier) rats are more stroke prone than younger ones. Coyle13 has related sensitivity to infarctions with the diameters of medial collaterals between the anterior and middle cerebral arterial trees and their ability to dilate and to provide collateral flow after the proximal MCA is occluded. Of interest here is that local ligations in two strains of rats result in different vulnerability similar to that reported for MCA occlusion.
This stroke model provides direct, quantitative, high-resolution spatial information on regions of localized function by videomicroscopic techniques. The experimenter can select time intervals before, during, and after ligation in and around a region of ischemia to characterize the development of infarcts and their modification by intervention. This local stroke model, like more global ones,1 can be modulated by degrees of ligation, survival time, and strain and age of rats to produce results of differing severity. The principal advantages of locating these strokes in the barrel field are that they are confined to accessible cortex responding to whisker stimulation and are subsequently verified structurally to provide a context of detailed neurophysiology and neuroanatomy.14 Furthermore, although the animals had no overt behavioral impairments, elegant behavioral methods can be used to evaluate impairment and recovery.15
This study was supported by National Institutes of Health grants NS-07057, NS-28781, and HL-41075; the McDonnell Center for Studies of Higher Brain Function; and an award from the Spastic Paralysis Foundation of the Illinois-Eastern Iowa District of the Kiwanis International.
Presented in preliminary form at the 24th Annual Meeting of the Society for Neuroscience, Miami Beach, Fla, November 13-18, 1994.
- Received January 20, 1995.
- Revision received April 5, 1995.
- Accepted April 25, 1995.
- Copyright © 1995 by American Heart Association
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