脳卒中後の脳へのヒドロゲルバイオマテリアル足場の注入

The reproducible injection of hydrogel scaffolds into a rodent ischemic stroke model allows for the study of how materials and other deliver therapeutics affect brain repair, regeneration and behavioral improvement. The main advantages of using the photothrombotic stroke model to study brain material interactions are the reproducible location and size. Which allow injection of materials into the stroke core without imaging.

The photothrombotic stroke model is designed to be a relevant model for ischemic stroke, in a region of the brain, the motor cortex, that is commonly affected by stroke in humans. And to provide a relevant behavioral readout for stroke, which is motor control of the limb. So as to evaluate a candidate therapeutic, like a biopolymer hydrogel.

Confirm a lack of response to toe pinch in an anesthetized mouse. Then weigh the mouse to determine how much Rose bengal dye to inject, and secure it to a stereotaxic device equipped with a laser. After shaving and disinfecting the surgical area, use forceps to tent the skin between the ears and eyes, and insert a blade into the raised skin to make a lateral one to two centimeter midline incision in the skin.

Separate the skin and press the forceps lightly onto the parietal bones to locate the bregma. Use a surgical marker to label the bregma with a dot. And wearing laser safety goggles, move the laser over the skull of the mouse, fixing the stereotaxic device at a 90 degree angle.

Select the 10 milli watt preset and turn on the laser. Adjust to the X and Y axis until the laser is directly over the bregma. Reset the X and Y on the digital display console, and move the laser 1.8 millimeters left of the bregma.

Bring the laser as close to the skull as possible without letting the laser touch the skull. When the laser is in position, turn it off and use a disposable 29 gauge needle to intra peritoneally inject the appropriate volume of Rose bengal dye. Immediately start a timer to allow the dye to circulate systemically for seven minutes.

While the dye is circulating, select the preset for 40 milliwatts without turning on the laser. When the timer goes off, turn on the laser and set the timer for 13 minutes. When the timer goes off again, change the laser back to 10 milliwatts.

Mark this spot with the surgical marker and turn off the laser. Lift the laser and unlock it from the 90 degree angle, so it can be moved away from the mouse. Use cotton to apply sterile saline to the surgical area as necessary, and drill at the mark in small bursts, perpendicular to the surface of the skull.

Place a small wipe next to the mouse and use the forceps to pull the skin close together and upward. Dab any large drops of surgical glue at the end of the tip on the wipe, and despairingly apply surgical glue to the skin. Hold skin together with forceps for 10 seconds, then continue to apply glue until no additional openings are visible in the skin.

For hydro gel injection into the stroke region, attach injection pumps to the stereotaxic device. Then set the flow rate to one microliter per minute and volume to the desired injection amount in microliters. Reopen the incision over the brain, and use saline soaked cotton to clean any debris from the skull.

A white, yellow circle of tissue, and the burr hole from the drill will be visible. Lock the injection pump at 90 degrees over the mouse. Clean the glass syringe with sterile saline.

And remove the needle. Use a 25 microliter positive displacement pipette to back load the syringe with 10 microliters of hydrogel, and use the plunger to push the gel all the way to the front of the syringe. Reassemble the syringe.

And continue pushing until the gel visibly exudes from the needle. Load the syringe onto the pump, and move the pump and the X and Y directions to orient the syringe over the burr hole. Move the pump in the Z direction until the needle touches the top of the hole.

And reset the Z on the digital display console. Move the syringe 0.75 millimeters in the Z direction and press start on the pump console to inject four to six microliters of the hydrogel at a one microliter per minute flow rate. When all of the hydro gel has been delivered, set a five minute timer to allow the gel to begin cross-linking.

After five minutes, turn the Z knob to slowly pull up the syringe. When the needle is far enough away from the skull, unlock and remove the pump. Then close the skin over the skull, and place the mouse in a clean cage with monitoring until it has recovered from the anesthesia.

At the appropriate experimental end point, use blunt thin forceps to carefully remove pieces of the skull to expose the brain. Using small surgical scissors to cut away the bone over the stroke site as necessary. After harvesting, place the brain in 10 to 15 milliliters of 4%para formaldehyde overnight for 16 to 24 hours, before placing the tissue into 30%sucrose.

The brain will be ready for cryo section when it sinks to the bottom of the tube. Five days after a stroke induction, the infarct can be visualized during surgery and by TTC and immunohistochemically stained slides imaging. An increase in the laser diameter with a two X lens leads to a visual increase in stroke lesion that spans over 2.5 millimeters versus a single one millimeter section using the laser only.

Hyaluronic acid based hydrogel microparticle injection into the stroke lesion five days after a stroke, results in the formation of micro and annealed particles with a porous scaffold that facilitates cell migration into the stroke core. The tissue that are as scaffolds can be retrieved and used for molecular cellular or tissue level analysis. So it's a single cell RNA-seq.

Flow cytometry or clarity. Also behavioral analysis can be performed to assess behavioral improvement. This approach delivers a candidate therapeutic, a biopolymer hydrogel, into the stroke cavity.

Which is a potential cavity in stroke. An area of dead tissue that can accept a transplant. Additionally, this cavity is adjacent to the area of the brain that undergoes the most plasticity after stroke, the peri infarct tissue.

So a candidate therapeutic like a biopolymer hydrogel can deliver cells or small molecules to the part of the brain that is really the target for recovery and stroke.