Evaluating Cell Death Signaling by Immunofluorescence in a Rat Model of Ischemic Stroke

Ischemic stroke is the leading cause of death and disability worldwide. Our work is focused on drug targets for stroke. We are interested in several emerging targets, such as RUNX1 and cathepsins. To evaluate these targets, we developed a rat model of ischemic stroke by occluding the middle cerebral artery.

We model ischemic stroke in rats by inserting a filament to occlude the middle cerebral artery. The middle cerebral artery occluding model is the most commonly used rodent model for preclinical stroke studies combined with other techniques, such as TTC, immunofluorescence, and the TUNEL staining. We can explore the pathophysiology of stroke in an integrated approach. There are some technical challenges for immunofluorescence, such as the specificity of antibodies minimizing background of fluorescence and optimizing the signal-to-noise ratio by harnessing the properties of flourescent labels and the specific antibodies. Immunofluorescence allows us to understand the intricate landscape of cellular architecture, and the molecular interactions with the middle cerebral artery occluding model.

A number of brain tissue is potentially salvageable with an appropriate time window of the stroke. Our results show that the expression of cathepsin B is activated in the number of rat brains after middle cerebral artery occlusion. It is accompanied by increased apoptosis. Further studies on mechanism underlying cell death signaling may avail new target for drug.

In our future study, we will continue to explore potential molecular targets in inflammatory pathways and cell death signaling. We hope some of these targets discovered by experimental studies can be translated into clinical therapy, thereby improving survival and the quality of life for stroke patients.

[Narrator] To begin, place the anesthetized rat in a supine position and maintain its body temperature at 36.5 °C using a regulated heat mat and a rectal temperature monitor probe. Shave the skin on the neck and disinfect it using an iodophor disinfecting solution. Next, make a 2 cm midline incision in the neck and retract the soft tissues to expose the carotid vessels. Using forceps, isolate the right common carotid artery, or CCA, external carotid artery, ECA, and internal carotid artery, ICA. Ligate the proximal right CCA using sutures. Then clamp the right ICA and ECA at their origin with a microclip. Insert a nylon monofilament coated with a round silicone tip into the lumen of the CCA through a small incision and tie a knot in the CCA to prevent bleeding and dislodging of the monofilament. Open the right ICA microclip and insert the monofilament into the right middle cerebral artery, or MCA, through the ICA stump until the silicone tip occludes the origin of the MCA. Cut off the exposed part of the nylon monofilament and close the midline neck incision. After two hours of MCA occlusion, open the neck incision, and untie the knot in the CCA. Then withdraw the nylon monofilament to reperfuse the MCA. Following surgery, observe the rat during recovery from anesthesia in a heated cage maintained with a temperature regulated heating mat. Using the Zaya Long Behavioral Rating Scale, evaluate neurological function at two hours post middle cerebral artery occlusion, or MCAO, and 22 hours after reperfusion. After isolating the whole brain from the MCAO model rat, freeze the brain at -20 °C for 20 minutes. Cut the frozen brain into six coronal slices with a 2 mm distance between slices. Stain the slices with 2% TTC for 15 minutes at 37 °C in the dark. Then fix the brain slices with 4% paraformaldehyde for 24 hours at room temperature. Photograph the stained slices using a digital camera set to auto-mode for closeup objects. Transfer the images to a computer and use ImageJ software to measure the infarct size. Calculate the infarct size as the ratio of the sum of the infarct area in all six slices to the sum of the total brain area in the same slices. Significant infarct areas were observed in the MCAO group compared to no infarct in the sham group. For immunofluorescence analysis, fix the intact brain from the MCAO model rat in 4% paraformaldehyde for 24 hours. Then dehydrate the brain in a series of sucrose solution gradients at 10%, 15% and 30% concentrations, at 10%, 15% and 30% concentrations, continuing until the tissue sinks. Embed the dehydrated brain in optimal cutting temperature compound and remove any air bubbles. Snap freeze the brain in liquid nitrogen. On a cryostat, section the brain at a thickness of 5 µm. After equilibrating the frozen brain sections at room temperature, wash them three times with sterile PBS for 5 minutes each. Outline the tissue with an immunohistochemistry pen before incubating with 20 µg per mL of proteinase K and 0.3% Triton X-100 at room temperature for 10 minutes. After washing the sections with PBS, apply 3% BSA to the outlined tissue and incubate at room temperature for one hour for blocking. Replace the blocking solution with the appropriate primary antibody and incubate the sections overnight in the dark at 4 °C. The next day, wash the sections three times with sterile PBS containing 0.1% Tween-20 for 5 minutes each. Add the appropriate secondary antibody conjugated to the required fluorophore and incubate the sections in a humidified chamber for one hour, protecting them from light. After washing the sections with PBS containing Tween-20, stain them with a mounting medium containing DAPPI and cover with cover slips. On a fluorescence microscope, set the excitation wavelength to 488 nm, the emission wavelength to 520 nm, and the exposure time to 500 ms to capture stained brain section images. Cathepsin B immunoreactivity was significantly increased in the ischemic core of the MCAO group compared to the sham group. Cathepsin B immunoreactivity was significantly elevated in the penumbra cortex of the MCAO group compared to the sham group. No significant difference in cathepsin B immunoreactivity was observed between the ischemic core and the penumbral cortex. To begin, dehydrate the paraformaldehyde fixed brain slices from the MCAO model rat in increasing ethanol concentration for 35 to 50 minutes each. Then treat the brain slices with turpentine oil type transparent biological agent 1 for 35 to 50 minutes, followed by clearing agent 2 for 35 to 50 minutes until the tissue is fully transparent. Now immerse brain slices in embedding mediums 1, 2, 3, and 4 sequentially 60 minutes each. Place brain slices in an embedding cassette, seal them tightly, and store them in cold storage for solidification. Using the microtome, trim the tissue block at 10 µm then slice at 5 µm. Allow the tissue sections to float in a water bath at 45 °C for 30 seconds and then transfer them onto slides. After drying, deparaffinize the brain sections in a clearing agent for two cycles of 10 minutes each. Then rehydrate the brain sections in decreasing ethanol concentrations. Now rinse the section three times in PBS for five minutes each. After removing the excess moisture, add 100 μL of proteinase K to each section and incubate at 37 °C for 20 minutes. After PBS washes, apply 100 μL of terminal deoxynucleotidyl transferase equilibration buffer to each sample, and incubate in a humidified chamber at 37 °C for 10 to 30 minutes. Remove the buffer using absorbent paper and add 50 μL of labeling working solution from the TUNEL assay kit. Next, apply the DAPPI working solution to the sections and incubate in the dark for five minutes to counterstain the nuclei. Rinse the samples in PBS four times for 5 minutes each. Remove excess liquid and seal the slide with an anti-fade mounting medium. Scan the stained slide using the Vectra Polaris multispectral imaging system. TUNEL staining showed a significant increase in apoptosis, with more TUNEL-positive cells in the penumbral cortex of the MCAO group compared to the sham group.