Stereological and Flow Cytometry Characterization of Leukocyte Subpopulations in Models of Transient or Permanent Cerebral Ischemia

The overall goal of this procedure is to characterize mouse myeloid cell subsets of the ischemic brain by two different methodologies. First, a permanent model of cerebral ischemia is introduced by occluding, both the common carotid artery or CCA and the distal trunk of the middle cerebral artery or MCA generating infarct that mainly affect the brain cortex. Then for the stereo logical quantification of the myeloid cell subsets, serial coronal sections of the brain are immuno stain for the cells of interest, and then the infiltrated cells are quantified by the optical fractionator approach.

Alternatively, the myeloid cells can be isolated from fresh brain tissue by mechanical dissociation, and then stained and analyzed by flow cytometry. Ultimately an accurate quantification and detailed qualitative characterization of the specific myeloid cell subsets that infiltrate the mouse brain after cerebral ischemia can be achieved by stereo quantification and multiparametric flow cytometry respectively. The main advantage of this ischemia serial model over other existing is that our technique has a significantly lower mortality rates than other methods minimizing the number of animals required for the study.

This area is gimme a method can help answer key questions in the stroke field, such as what are the roles of micro reactivation, extravasation of hematogenous macrophages, or neutrophils in brain injury? They accurate theological quantification on the multiparametric cytometry approaches demonstrated in this paper can provide insight into the number phenotype and function of the myeloid cells population in both innate immune response to CE ischemia. To ligate the CCA first, make a one centimeter midline incision around the ventral surface of the neck of the animal under a surgical microscope.

Once dissected, use a six zero nylon suture to occlude the left CCA with a permanent knot to ligate the distal MCA. Next, make a horizontal incision on the temporal muscle from the right to the left. Then make a second vertical incision on the right side of the temporal muscle.

Taking care to avoid cutting the temporal vein. Pull the temporal muscle apart from the skull and hold it with a suture to maintain a clean skull surface. Then clean the skull with cold sterile saline to detect the exact position of the left MCA via skull transparency.

Once the MCA has been identified, use a stainless steel burr to perform a one millimeter round craniotomy in the frontal lobe between the zygomatic arch and the squamal bone. Carefully remove the skull exposing the MCA. Then use a nine zero nylon suture to occlude the left MCA in the distal trunk just before the bifurcation between the frontal and posterior MCA branches with a permanent knot.

To remove the brain, decapitate the euthanized mouse just above the spinal cord and make a posterior anterior incision in the skin to expose the skull. Then make two lateral cuts at the junction of the lateral walls and the base of the skull and remove the small piece of bone. Next, make a cut through the skull along the sagittal suture and use a forceps to remove the skull overlying each hemisphere to expose the brain.

Use a spatula to separate the brain from the skull and transfer it into a 4%PFA solution. After three hours, remove the PFA and incubate the brain in a 30%sucrose solution to cryo protect the brain. After 48 hours, remove the sucrose solution and freeze the brain quickly in negative 40 degrees Celsius isop pentane then use a freezing microtome to make 30 micrometer coronal brain sections.

Collecting 10 serial sets in a cryo preservative solution. Each serial set is composed of around 14 to 16 coronal slices with an inter slice distance of 300 micrometers. To ensure adequate sampling of the mouse brain between 1.94 and negative 2.46 posterior to the bgma to estimate the total number of infiltrated neutrophils.

After MCA occlusion by an optical fractionator first immunostain the neutrophils with the rat anti LY six G antibody and the appropriate secondary antibody on floating sections by conventional immunofluorescence technique. Then mount the brain sections onto super frosts microscope slides with the infarcted area placed on the right and open the stereo investigator software. Next, click on the tools serial section manager menu and add a new section with the new section button.

Set the evaluation interval to 10 for a one 10th slice sampling fraction. And then from the contour down menu, select the infarcted area contour tool. Draw a contour to outline the infarcted area under the 10 x objective.

Then switch to the 100 x objective on the microscope and under the objective menu. To quantify the neutrophils under the probes menu, now select the optical fractionator and set the XY placement of counting frames to 230 for both the X and Y grid sizes. Now select the neutrophil button from the marker bar and mark the stained neutrophils in the infarcted area.

Export the results of the quantification to a spreadsheet file to dissociate the brain into a single cell suspension. After removing the brain as just demonstrated, dissect out the core and per infarct areas of the ipsilateral cortex from the brain of a brain ischemic mouse. Weigh the brain tissue and place it into five milliliters of ice cold RPMI per call for normalization between the experimental groups, and then use a tissue grinder with Teflon pestles to dissociate the tissue into a single cell suspension.

Transfer the cell suspensions to a 50 milliliter ultracentrifuge tube and add five more milliliters of RPMI per call solution to the samples. Then after centrifuging the cell suspensions for 30 minutes at 7, 800 GS and 25 degrees Celsius. Ensure a white colored layer corresponding to the myelin appears at the top of the solution.

Carefully remove the myelin layer and filter the whole cell suspension, including the pelleted cells through a 40 micrometer nylon mesh strainer. Wash the strainer with 10 milliliters of RPMI to ensure that all the cells are filtered through the mesh, and then transfer the solution to a 50 milliliter tube. Add 20 milliliters of RPMI to the cells and then after spinning down the cells resuspend the leukocyte pellet.

In one milliliter of PBS incubate the cells for two to three minutes in four milliliters of lysis buffer at room temperature to lyce the erythrocytes and then spin down the cells. Label the cells in 200 microliters of PBS containing 1%BSA and the appropriate antibodies on ice. Then after 45 minutes, wash the samples with one milliliter of cold PBS and Resus resuspend the pellets in 100 microliters of fax flow buffer for flow cytometric analysis.

This model is characterized by highly reproducible infarct volumes at 24 hours after middle cerebral artery occlusion as estimated by the cavalieri method. In nial stained sections in agreement with previous studies, neutrophil infiltration is directly correlated with the infarct size. Additionally, leukocyte isolation and flow cytometric characterization allows the isolation of myeloid cells from the cortex of the IPS lesional hemisphere of ischemic mice, which comprise 10 to 30%of the total events found in the cell suspension and present a low forward scatter parameter that is associated with cellular debris.

The CD 11 B positive cells have a higher forward scatter value suggesting that cell debris is not labeled with this marker and as previously indicated, that it can be excluded from further analysis by setting the FSC threshold at 200 neutrophils characterized as LY six G positive cells are the most numerous infiltrated cell population found at 24 hours after MCA occlusion in the ischemic mouse brain using this model of cerebral ischemia and comprise 70 to 80%of the CD 11 B positive CD 45 high cells. The rest of the cells are mostly a subpopulation of CD 11 B, positive CD 45, high LY six G, negatively six C high pro-inflammatory monocytes, which are found in elevated numbers in the ischemic brain areas from 24 to 48 hours after middle cerebral artery occlusion. This P-M-C-A-O model can be performed in 30 minutes if it is performed properly Following the flow cytometry procedure.

Other methods like sorting of a specific cell subset can be performed to answer additional questions like what are the expression patterns, metabolic changes or acetic activities of myeloid cells in this model system. After watching this video just to have a good understanding of how to create a ischemia model and most important, how to characterize the number and phenotype of the infiltrating myeloid cells by two different approaches in a mouse brain injury or tpe after cere ischemia model.