Simultaneous Photothrombosis and Fiber Photometry to Induce and Monitor Ischemic Stroke in Behaving Mice

We're interested in mechanisms of memory impairment, and resilience following stroke. Most preclinical models that investigate stroke related neurophysiology require anesthetic or head fixation, which makes behavioral assessment limited. This approach addresses those issues.

To begin, use a digital scale to weigh the mouse. Transfer the anesthetized mouse to the nose cone in the stereotaxic frame to maintain anesthesia. Using an electric razor, remove the hair from the top of the head, starting near the nape and extending towards the eyes.

Next, secure mouse in ear bars. Then use a cotton tipped applicator to apply lubricating eye ointment to both eyes. Then clean the dorsal surface of the mouse's head with three serial applications of iodine, followed by 70%ethanol.

Now, pinch the toe and observe for any withdrawal reflex and check for blinking to verify sufficient anesthetic depth. Afterward, inject a buprenorphine solution intraperitoneally at a dose of 0.01 milligrams per kilogram. Then make an approximately three centimeter anterior to posterior incision along the scalp to expose the skull.

Using a cotton tipped applicator, apply 5%hydrogen peroxide to the dorsal skull to remove the periosteum. Then adjust the stereotaxic frame and ear bars to level the skull in both the dorsal ventral and medial lateral planes. Now, position the tip of the stereotaxic drill above bregma and adjust its anterior, posterior, and medial lateral location to the desired hippocampal coordinates.

Lower the stereotaxic drill to the skull and drill a small burr hole using short pulses. Apply ice cold saline intermittently to prevent heating of the skull and brain surface. Repeat until the burr hole is through the skull and the surface of the brain is exposed.

Lower the optical fiber until it just contacts the surface of the brain without penetration. Slowly lower the optical fiber just above the CA1 region. Using a precision applicator, apply a quick air dry dental cement to cover the entire exposed skull and the lower one third of the fiber optic cannula to secure it in place.

Next, apply UV cured cement to support stability of the implanted fiber. Suture the skin surrounding the implanted fiber optic cannula and hardened dental cement. After turning off the isoflurane vaporizer and loosening the nose piece, transfer the mouse to a heated recovery cage.

Turn on the 561-nanometer fiber coupled laser and toggle to TTL mode. Using a digital handheld optical power meter, adjust the 561-nanometer laser power to six milliwatts. Now, turn on the fiber photometry console, the light emitting diode drivers and the detectors.

Open the fiber photometry acquisition software. Click on the device selection tab and open NC500 console. Under the add channels tab, add lock-in modulation channels for 465 nanometer and 405 nanometer LEDs using preset frequencies.

Match the output voltage of the 405 nanometer LED to approximate the voltage of the 465 nanometer LED. Add a digital input output channel to configure an eight-minute-long square pulse for the TTL input to the 561-nanometer laser. With a 10-minute starting delay.

Add a camera channel to set the overhead behavioral camera to be initiated simultaneous with photometry at 20 hertz. Adjust the overhead camera to ensure the entire behavioral arena is clearly visible in the frame. Weigh the mouse using a digital scale.

Using a lint-free wipe sprayed with 70%ethanol, scruff the mouse and clean the fiber optic cannula by wiping away any debris. For stroke mice, inject 0.5%rose bengal solution via intraperitoneal injection. While holding the mouse by the scruff, connect the fiber optic patch cord to the implanted feral by twisting the sleeve gently while applying even pressure.

Place the mouse into the recording arena and press record to initiate synchronized data collection. After 40 minutes, stop the recording in the photometry acquisition software. Turn off the 561-nanometer laser.

Pick up the mouse and carefully detach the fiber optic patch cord by securing the head, then holding the sleeve and using a gentle twisting motion. A sustained increase in neuronal calcium concentration was observed during terminal depolarization in mice injected with rose bengal and subjected to photothrombotic illumination with subsequent infarct formation Under the fiber optic tip. In sham mice injected with saline instead of rose bengal, there was no increase in neuronal calcium and no infarct was detected by TTC staining.

The absence of a biosensor, such as in cases of incorrect genotype or misplaced fiber, was indicated by little to no photobleaching at the start of the photometry recording. In contrast, recordings with successful biosensor expression showed an initial downward slope consistent with photobleaching. An optimal hippocampal recording displayed a stable baseline with spontaneous signal transient.

The absence of spontaneous transience in the photometry trace suggested biosensor expression failure or incorrect fiber placement. Mice subjected to lower power and shorter photothrombotic illumination showed only transient calcium influx, while those exposed to longer and higher intensity illumination displayed a sustained increase. Both control mice injected with vehicle and exposed to illumination and mice injected with rose bengal but not illuminated, showed no appreciable changes in GCaMP6f signal and no infarct.

We identified that depolarization spread through the hippocampus during acute stroke and impair spatial navigation as well as memory. We can now identify the relationship between neurophysiological changes in the hyperacute phase of stroke and how they relate to behavior and recovery. This protocol introduces the ability to simultaneously monitor brain activity dynamics and stroke symptomology in freely behaving mice.