Dear Fawad.

In my experience, the infarct area depends on the age, weight, sex, and strain of the rat.  Even when all of these are controlled for, there is still some variation in the damaged area because there is some animal-to-animal variability in the branching patterns of the MCA.  It is important to validate the damage created by your surgical technique in some way, rather than to just rely on the descriptions of the damaged areas in the literature.

What my colleagues and I did was to verify the damaged areas using TTC staining:

J Pharmacol Methods. 1986 Nov;16(3):201-14.

Morphometric evaluation of brain infarcts in rats and gerbils.

Lundy EF, Solik BS, Frank RS, Lacy PS, Combs DJ, Zelenock GB, D'Alecy LG.

The Levine rat preparation, the gerbil stroke model, and appropriate control

animals were used to determine if the 2,3,5-triphenyltetrazolium chloride (TTC)

would selectively identify noninfarcted versus infarcted cerebral tissue. The TTC

is frequently used to quantify infarcted myocardial tissue and has been shown to

have great specificity, reproducibility, and efficacy. The TTC produces a red

product upon reaction with the respiratory enzymes (dehydrogenases) present in

non-infarcted tissues. Irreversibly damaged tissues, lacking dehydrogenases, do

not form red reaction products. Six gerbil brains and seven rat brains were

incubated with the TTC, and the unreacted areas were macroscopically identified.

The brains were fixed and sectioned for routine hematoxylin and eosin staining to

determine the specificity of the TTC. The TTC was found to react selectively only

with non-infarcted cerebral tissue. The gross brain sections were evaluated by

macroscopic morphometric analysis, and the unreacted area was always ipsilateral to ligation and correlated with histologic identification of infarct. The brains from neurologically intact animals demonstrated neither macroscopic nor

histological evidence of infarction. This technique allows macroscopic

quantification of infarct size by planimetry. The average area of infarct for the

neurologically impaired rats was 34.7% and it was 31.4% for the impaired gerbils.

The percentage of surface area of each infarcted slice was found to correlate

with the severity of the neurologic deficit. We conclude that TTC staining is

effective for macroscopically delineating cerebral infarcts in rats and gerbils,

thus permitting quantification of infarct size.

PMID: 2431224 [PubMed - indexed for MEDLINE]

The technique is very easy, since it is performed on live brain tissue rather than fixed.  We invested in a stainless steel brain matrix in order to reproducibly slice the brains into thin sections, and used alternate sections for TTC staining and biochemical analysis.  In order to do this, you must order the brain matrix that matches the weight of your rats.

After the TTC stained sections were photographed, we fixed them in cold paraformaldehyde overnight, cryoprotected them in sucrose, and cut 20-40 um free-floating frozen sections for other types of histology (NOT apoptotic markers).  Once we had this data to confirm the damaged regions, we could then analyze new surgical groups without using the TTC.  We would either perfuse the animals for traditional immunohistochemistry, or snap-freeze fresh brains for frozen sections, depending on the markers we were interested in.

Good Luck!

Jill

Thank you Jill Miotke for your elaborated explanation. I do stained my sections with ttc, and even i have noticed infarcted area after 2 hrs of permanent MCAO, but later on when i processed these tissues for western analysis,  found no apoptotic response from these infarct ed tissues. Infarcted tissues as per literature are necrotic tissues. what i am concerned is, which particular region in somoto-sensory area (fronto-parital cortex) is hit by mcao damage. i collected just surface layer from these cortical regions. i am using SD rats for my experiments  and according to (stroke. 1989;20: 1037-1043, used wistar rats) ciricle of willi/collateral circulation have minimum blood supply to the occluded MCA area (somatosensory cortex). 

Dear Fawad

Why not using photothrombotic ischemia/stroke model? We have it now using Rose Bengal and light shot and the results are very good, because we know (i) the localization of infarct area , (ii) it is much less harmful for whole brain, (iii) the animals can be used for behavioral tests, (iv) easy to process for further immunocytochemical staining and WB, (iv) easy to establish the method and you will not have problems to choose the area.

Also, physiologically the stroke is most times local, while MCO is total hemisphere block of circulation.

For the method , please check this information:

Labat-gest V, Tomasi S. (2013) Photothrombotic ischemia: a minimally invasive and reproducible photochemical cortical lesion model for mouse stroke studies.J Vis Exp. 2013 Jun 9;(76). doi: 10.3791/50370.

Very cordially, Serguei.

Dear Fawad,

I second Serguei's suggestion to switch to photothrombotic stroke models.  We also successfully used Rose Bengal in rats (SD & Wistar).  It is much easier on the animals, and you can be certain that you only damage cortex, and not other areas of the brain. 

Either way, before using a Western blot to look for apoptotic markers, you might want to stain tissue sections with the same marker if possible, to see how many cells might be dying by that mechanism.   Sometimes the number of apoptotic cells is very low after an ischemic insult, since you are looking at a single snapshot in time, and this can be difficult to detect on a Western blot.

So, are you using the method you cite in the 1989 Stroke paper from Nagasawa & Kogure that uses a silicone-coated suture inserted into the vasculature to occlude the MCA?  I have used that method on SD rats for both permanent and reversible occlusion, mostly as a model of reperfusion injury (reversible occlusion).  The data shown in that paper is for reversible occlusion with reperfusion--otherwise, there will be a lot more damage, particularly to the caudate-putamen, with permanent occlusion.   If you are using it for permanent occlusion, that could be why you are not seeing any apoptosis--there will be so much necrosis by 48 hours, you might have to carefully dissect out the penumbra of the infarct to find it.  With TTC staining, I defined the penumbra as the pinkish area between the red healthy tissue and the white infarcted tissue.

I will just note that with the suture method is is important to make sure of the length of your suture for the size of the animal, so that you don't accidentally occlude the ACA, or worse, poke through the vasculature and cause a bleed in anterior cortex.

Good Luck,

Jill