Rose Bengal Photothrombosis by Confocal Optical Imaging In Vivo: A Model of Single Vessel Stroke

The overall goal of this procedure is to use a semi invasive optical microscopy approach to induce a rose bele photo thrombotic clot in the somatosensory cortex of a mouse. This is accomplished by anesthetizing the mouse and preparing a thin skull cranial window for imaging. Next, the rose bengal is injected and a laser is used to induce a photo thrombosis.

Ultimately, the effects of the clot made by the rose Bengal photo thrombosis can then be immediately analyzed after recovery from anesthesia. The main advantage of this technique is that it allows in vivo cellular responses to be seen immediately after rose bengal photo thrombosis is induced. Individuals new to this technique may struggle with creating the thin skull cranial window because it is difficult and time consuming.

To begin anesthetize the mouse with two to 3%isof fluorine mixed with oxygen. Take note of a decrease in respiration as the mouse is induced. Then pinch the paw of the mouse.

To confirm the anesthetic state, use a heating pad and temperature probe to maintain the body temperature within half a degree of 37 Celsius throughout the procedure. Then apply ophthalmic ointment to monitor the animal's vitals. Clip an oximetry system to the tail or foot.

Adjust the anesthetic to maintain 50 to 65 breaths per minute and a heart rate between 300 and 450 beats per minute. Oxygen saturation is ideally maintained at 97 to 98%If all these parameters are maintained, long-term survival is possible. Lastly, prepare the skin for surgery.

Shave the hair from the cranium and use repeated scrubbing of Betadine, followed by ethanol to clean the skin. Begin the surgery with a five millimeter incision into the scalp to expose the cranial fissure. Remove the overlying fascia with a cotton swab and locate the bgma.

Next glue. A custom made stainless steel ring to the bone overlying the parietal cortex. Map this position from the bma moving negative one to three millimeters horizontally and positive two to four millimeters laterally.

After two minutes, the glue will have set. Then attach the ring to a stereotaxic holder to stabilize the animal. Now under a surgical grade, dissecting microscope, slowly drill a one to two millimeter section in the cranium over the somatosensory cortex Using a zig sag drilling pattern, be sure to keep the cranium level as it is drilled out to prevent heat build.

Use the drill on a low speed and take frequent breaks as the bone begins to shine. Keep drilling with the zigzag pattern to thin out the skull. A thin cranial window is critical for the laser to reach the rose bengal filled vessel and induce the clot.

When the window is thin enough, use a scalpel to make small linear strokes with light pressure to remove thin layers of bone until the vasculature is clearly visible through the ocular. To image the brain use a microscope that has enough space between the objective and the stage to fit the animal on its platform. An inverted scope is a good choice.

The platform is designed to fit into a customized stage comprised of a jack mounted to a plate with four poles. After the mouse is positioned on the stage, position the objective inverter over the cranial window, then illuminate the cranium with an external light source. If a water-based objective is utilized, artificial CSF should be applied to the cranial window cavity into which the objective can be lowered.

Now focus on the imaging area with visible vasculature. After the mouse is set up at the microscope, prepare fresh rose bengal in artificial CSF at 20 milligrams per milliliter filter, sterilize the mixture and dispose of it after the experiment is completed. It must always be used fresh.

Now while scanning the cranial window with a 561 nanometer laser inject 0.1 milliliters of the mixture via the tail vein. Within five seconds, the dye should easily be seen in the cranial vasculature. After sufficient dye has been injected, select a 40 to 80 micron vessel for thrombosis.

Arteries can be differentiated from veins based on the flow of larger to smaller vessels and vice versa. Now prepare to laser the selected vessel. Increase the dwell time to whatever is appropriate for the system in use.

Increase the laser power to 100%and set the image collection to one frame per second. With maximum scan speed, scan the mouse until a staple clot is formed. Usually this takes about five minutes.

Ideally, only one field of view should be scanned to create the clot, choose the objective accordingly. If no clot forms, try using more dye once the clot has formed. Remove the mouse from the imaging area and prepare to complete the surgery under the dissecting microscope.

First, carefully remove the ring while checking for bleeding. If any bleeding occurs, the experiment must be terminated. Next, using a six oh suture, close the incision, then apply an antibiotic to the incision line for pain relief.

Provide subcutaneous injections of bup printex for three days after administering the first injection of bnx. Place the mouse in a recovery chamber and monitor it until it is ambulatory. Then return it to its home cage, which should be cleaned.

Longitudinal imaging is detailed in the text protocol. Using this protocol, strokes were induced by forming clots with cranial vessels. Clot formation progressed from over several minutes.

Initially, the vessel was entirely white with free flowing rose Bengal gradually irradiation of the dye produced reactive oxygen species and thus activated an immune response that induced clot formation. Complete occlusion of the vessel was noted by accumulation of rose Bengal in the vessel upstream of the clot. To confirm that an ischemic stroke was generated by the blood clot.

TTC staining was used to detect cerebral infarction. Healthy tissue forms red form on products. While infarcted areas do not react and remain white evidence of a stroke was clearly apparent by five days after the procedure.

After watching this video, you should have a good understanding of how to prepare a mouse and induce a rose Bengal photo thrombosis in an intact mouse following this procedure. Other methods such as loading the brain with other imaging dyes such as flow four could help answer additional questions such as How does calcium respond to an ischemic event?