This model was originally described by Koizumi et al and has since been modified by others 11 . It is the most commonly used of rat models of stroke due to its relative simplicity and noninvasiveness. The MCA is occluded by inserting a monofilament suture into the internal carotid artery (ICA) to block blood flow to the MCA either permanently or transiently by keeping the filament in place or withdrawing it, respectively. Several manuscripts describe in detail the technical and procedural features of this model 11 12 13 14 15 16 .

This model typically induces infarcts in the lateral caudatoputamen and frontoparietal cortex 17 . The infarct is reproducible and there is a significant ischemic penumbra early after MCA occlusion, making this model suitable for testing neuroprotective agents 18 . However, several technical factors may influence infarct size, such as: 1) physical differences in the employed monofilament suture, (2) insertion distance of the suture, and (3) accidental premature reperfusion 19 20 21 . It is therefore essential that standardized surgical and technical procedures be used by adequately trained personnel in order to generate reproducible lesions. There are also some complications with the intraluminal MCA occlusion model such as subarachnoid hemorrhage secondary to suture-induced arterial rupture, spontaneous hyperthermia when the duration of ischemia is longer than 2 h, and mechanical damage of endothelium which can complicate reperfusion 13 22 23 . The complication rates may be reduced by using silicone coated sutures 13 .

The intraluminal MCAO model is suitable for neuroprotection drug experiments because it produces a substantial amount of penumbra (salvageable tissue) in the first 60-90 minutes after onset 16 . Also, the location, volume, and temporal evolution of infarction are similar to those produced by proximal electrocoagulation of the MCA 16 . The suture is easily withdrawn, enabling investigators to study the aspects of reperfusion

The suture MCA occlusion model has recently been modified to induce ischemia in a magnetic resonance imaging (MRI) unit by remotely advancing the suture occluder 16 . This in-bore occlusion method has achieved a high reproducibility rate and enables investigators to monitor in vivo ischemic changes at pre-occlusion, acute, subacute and chronic post-occlusion time points 16 . Combined with multiparametric MRI techniques, the MCA occlusion model enables anatomic, diffusion, perfusion, and functional data to be obtained longitudinally and noninvasively in the same animal, making it a powerful tool for studying the pathophysiology of brain ischemia 23 24 .

Though thromboembolic ischemia can be induced by a photochemical approach, the most commonly used thromboembolic model is blood clot injection, first described in the dog by Hill et al. and later applied to the rat 25 26 27 . This model is of great interest to researchers because of its close resemblance to human ischemic stroke and its utility in evaluating thrombolytic therapies 28 . Thrombolytic therapy with recombinant tissue plasminogen activator (rt-PA) administered intravenously within 3 h of onset of ischemic stroke in select patients is the only FDA-approved treatment for human stroke and has been shown to improve neurological outcome 29 . Recently, there has been heightened interest in studying the efficacy of combining thrombolytic and neuroprotective agents in the treatment of stroke, giving thromboembolic animal models an increasingly important role in this respect 30 .

Several disadvantages were common to the early thromboembolic models, such as diffuse and inhomogenous infarction in the MCA territory from microembolization to peripheral branches 27 . Additionally, spontaneous recanalization frequently occurred, making it difficult to study thrombolytic therapies 16 . Infarct sizes were variable, contralateral strokes were common, and ischemia caused by multiple small clots did not mimic typical clinical ischemic stroke 16 . Later, it was determined that size (length and diameter) and the biological characteristics of the blood clot (fibrin-rich) are crucial to the relevance and reproducibility of this model 16 .

Busch et al developed a rat clot model that surmounted the above issues in which a single fibrin-rich autologous clot was injected to produce reliable occlusion of the proximal MCA, with consistent reduction of cerebral blood flow (CBF) and histological damage in the MCA territory seen 31 . No spontaneous thrombolysis was observed and, in separate experiments, thrombolytic therapy with rt-PA or prourokinase recanalized the occluded MCA 31 32 . Recently, Henninger et al used the embolic model in conjunction with multimodal MRI to investigate the pathophysiological mechanisms underlying the relatively rare clinical phenomenon of "spectacular shrinking deficit" in stroke patients 33 .

In conclusion, the single fibrin-rich clot model induces reproducible infarcts in the MCA territory similar to those produced by the intraluminal MCA occlusion model. The clot model has the added advantages of bearing closer similarity to the mechanism underlying human ischemic stroke and better utility for studying thrombolytic therapy. Combined with modern imaging techniques, thromboembolic models have the potential to take the experimental study of stroke to new frontiers.

Numerous compounds have been used to produce artificial emboli which are typically injected into the ICA, most commonly in rats 34 . Microsphere embolization is the most widely used model, with the severity of ischemic damage related to the number of emboli used 35 . The lesions require longer time to develop (24 hours on average) than in the intraluminal models, allowing for a larger therapeutic window for drug testing in microsphere models. However, the permanency of the ischemia does not simulate most clinical situations which limits the applicability of these models. Also, lesions are multifocal and have low reproducibility, though recent macrosphere models have resulted in more reproducible infarcts by increasing the diameter of the spheres and using less of them 36 .

Numerous techniques have been developed to surgically approach and occlude the MCA, with the rat being the species most widely used 13 . The orbital route is the least traumatic and, compared to procedures requiring craniotomy, results in less blood loss and artifacts 37 . Electrocautarization of the MCA results in permanent occlusion, whereas clipping or ligature snares enable reperfusion 38 . Occlusion of the MCA following a transient hypotension produces a larger infarct area 39 . Tandem occlusion of the distal MCA and ipsilateral CCA results in more reproducible infarcts 40 . Recently, a three-vessel occlusion model has been shown to produce reproducible and selective neocortical infarction 41 . Common to all of these variants, the procedure is always invasive and requires extreme surgical skill which limits their utility.

This model induces a cortical infarct by systemic injection of a photoactive dye in combination with irradiation by a light beam transmitted through the intact skull 42 . Oxidative damage to the endothelium caused by the altered dye leads to platelet aggregation in the irradiated area 43 . This model is used primarily in spontaneously hypertensive rats 44 . A disadvantage of this model is that vasogenic edema and blood-brain barrier breakdown occur within minutes which does not allow for the formation of penumbra--therefore, this model has been considered by many to be unsuitable for preclinical drug studies 44 . However, a new model overcomes this limitation and induces ischemia over a greatly extended time period, consistently producing a penumbra-like region 45 . This, combined with the ability to noninvasively and reproducibly induce infarct in any cortical location, are obvious advantages of the photochemical infarct model. A major disadvantage is the atypical features of the lesion (prominent vascular injury and early vasogenic edema) which are unlike human stroke 13 .

Endothelin-1 (ET-1) is a natural peptide that causes vasoconstriction and several models use this as an agent to induce MCA stroke 13 . Invasive approaches have been largely replaced by stereotactic intracerebral injection of ET-1 adjacent to the MCA, which avoid complications of surgery 46 . When ET-1 is applied to the MCA there is a significant decrease of cerebral blood flow (CBF) in its territory, resulting in an ischemic lesion pattern similar to that of direct surgical MCAO 47 . This model may be useful in restorative drug studies. Notably, after a period (~20 minutes) of severe CBF reduction, there is a slow and progressive return of blood flow to normal with the rate being dose-dependent 47 . This can be disadvantageous source of variability unless the dose is carefully standardized in the experiment.

Many neurological deficits are difficult to assess in animals. Motor deficits are perhaps the easiest to quantify and simple measures of motor function are available in rodent models 49 . Refined tests that assess sensorimotor function include limb placing, beam walking, sticky label test, grid walking, and rotarod 50 . A number of cognitive tests examining learning and short term memory are available for rats, including the Morris water maze 51 . Combining neurological assessment with histological measurements is becoming more critical with the heightened interest in neuroprotective drugs, the effectiveness of which may be reflected more by subtle structural and chemical changes rather than changes in gross infarct volume 52 . This statement is supported by the fact that animal data suggest a poor correlation between reductions in infarct size with neurological or behavioral deficits 13 . Also of importance is the need to extend the period of testing of neurological status for at least 1 month post-insult in animal studies, per the recommendations of the STAIR Committee 14 . However, this may be difficult to implement as survival times are 48 hours or less in many animal stroke models 53 .

In models of focal ischemia, the chief outcome has been the infarct volume, traditionally measured by quantitative histology. Among a number of histopathological methods, 2,3,5-triphenyltetrazolium chloride (TTC) and hematoxylin-eosin (H&E) staining are the two most commonly employed. TTC can be used to stain tissue much more rapidly, easily, and cheaply than H&E 54 . TTC is a colorless chemical that is reduced by mitochondrial enzymes into a compound that stains intact brain regions dark red whereas infarcted regions remain white. Studies show TTC staining to be reliable between 6 and 72 hours post-ischemia 13 . Prior to 6 hours, there may not be sufficient number of damaged mitochondria to create contrast between normal and infarcted tissue, and after 72 hours pathophysiologic inflammatory response often obscures the line of demarcation in the periphery of the damaged area 13 .

Notwithstanding staining techniques, infarcts have complex shapes with sometimes indistinct margins, making it difficult to measure their volumes. Many methods have been developed to deal with these complexities, each with their own advantages and disadvantages. Perhaps one of the most important considerations to account for is the effect of vasogenic edema on infarct volume. Edema in the rat MCAO stroke model typically accounts for 20-30% of the total apparent infarct volume 55 . Separate measurement of "corrected" infarct volume (which accounts for edema) and "uncorrected" volume (which does not account for edema) is important because of the possibility that some interventions may reduce edema but not salvage brain tissue and vice-versa 13 .