We like to acknowledge the National Institutes of Health and the National Institute of Neurological Disorders and Stroke for funding (R01NS079691).

The experiments were conducted in accordance with IUCAC at Duke University and University of California Los Angeles. 8 to 12-week-old male C57Bl/6J mice were used in this study. The animals were housed under controlled temperature (22 &#177; 2 &#176;C), with a 12 h light-dark cycle period and access to pelleted food and water&#160;ad libitum.Analgesia and sedation protocols are described as approved by the IUCAC but might differ from protocols used in other laboratories.
Animals may be prematurely euthanized if they experience excessive restlessness, vocalization (indicating uncontrollable pain), self-trauma, decreased or impaired mobility, physical signs of dermal infection, loss of appetite and/or weight loss greater than 10%-15% as an outcome of the survival surgery. There are no anticipated adverse effects from injection of the gel, as it is a composed of a naturally&#160;occurring polymer within the brain. Animals should be&#160;monitored daily, including weekends and holidays, and reweighed every other day. Studies from our laboratory and others&#39; have found no deficits in grooming, loss of body weight or signs/symptoms of withdrawal or chronic stress in mice after these small cortical or subcortical strokes. If animals exhibit any signs of local head wound infection they should&#160;be euthanized. Animals should also be monitored for poor grooming, wound condition, and evidence of fighting between animals (back wounds). If&#160;there is evidence of fighting or wound dehiscence, animals should&#160;first be treated after contacting the veterinarians. This often involves antibiotic in the water and/or local topical antibiotic application. If these measures fail to produce healing, animals should be&#160;euthanatized. &#160;
1. Preparation of materials and instruments
<ol>
	<li>Prepare photothrombotic dye prior to the surgery. Weigh out 15 mg of Rose Bengal into a 2 mL microcentrifuge tube. 
	CAUTION: Rose Bengal can cause serious eye damage/irritation.
	<ol>
		<li>Dissolve the Rose Bengal in 1.5 mL of sterile filtered 1x PBS to make a working concentration of 10 mg/mL.</li>
		<li>Vortex the tube until no clumps are visible, indicating the dye has fully dissolved, then cover in foil until further use. 
		NOTE: Amount of dye required will depend on the number of mice. Dye is injected intraperitoneally IP at a concentration of 10 &#181;L/g, e.g., 200 &#181;L for a 20 g mouse.</li>
	</ol>
	</li>
	<li>Turn on the heating pad to maintain the mouse body temperature during anesthesia (37 &#176;C).</li>
	<li>Prepare autoclaved scissors, forceps, cotton, vet eye ointment, surgical glue, or suture material, a blue or black surgical marker, and sterile alcohol and beta iodine wipes. Prepare bone drill with sterilized drill bit heads.</li>
	<li>Turn on the bead sterilization to 160 &#176;C to allow for sterilization of instruments between mice surgery. Set aside a 50 mL conical tube of sterile saline or 1x PBS solution for the later use in maintaining a hydrated operation area.</li>
	<li>Attach the laser to the stereotaxic device. Wearing laser safety goggles, measure the power of the laser (mW). 
	CAUTION: Use laser safety goggles whenever the laser is turned on in the procedure.
	<ol>
		<li>Adjust the current on the laser console, following the laser manufacturing instructions, until the laser power output reads 10 mW, and set the current as a pre-set on the home screen console. Then adjust the current again to establish another pre-set where the laser output power reads 40 mW. Now turn the laser off until further use. 
		NOTE: The 10 mW will be used to orient the laser prior to use.</li>
	</ol>
	</li>
	<li>Prepare a clean cage for mice after surgery. Then, place the mouse into the induction chamber with an isoflurane concentration of 4% to anesthetize it until spontaneous movement stops and its breathing comes to a slow, steady rate.</li>
	<li>Once asleep, weigh the mouse to determine how much Rose Bengal dye to inject. Then, transfer and secure the mouse to the stereotaxic device with a maintenance isoflurane concentration of 1.5%. 
	NOTE: Increasing isoflurane concentrations can dilate vessels and prevent sufficient occlusion or consistently sized strokes between mice.</li>
	<li>Apply vet eye ointment to both eyes. 
	NOTE: Watch breathing and temperature throughout the procedure and pinch toes occasionally to ensure that the mouse cannot feel anything.</li>
</ol>
2. Photothrombotic stroke model9
<ol>
	<li>Shave the fur off the mouse&#8217;s head and aseptically prepare the area by using an alcohol wipe followed by a beta iodine wipe. Repeat three times. 
	NOTE: If necessary, use an extra alcohol wipe at the end to clean off all beta iodine as it causes skin irritation which leads the mice to itch their skull after surgery.</li>
	<li>Using small operation scissors, make a lateral incision between the ears to the eyes, about 1-2 cm. Do this by pulling the skin upward with forceps to create a tent, cutting once downwards, then inserting the scissors into the opening and creating a continuous midline incision.</li>
	<li>Separate the skin and press lightly on the parietal bones with the forceps to locate the bregma. Mark the bregma with a dot using the surgical marker. 
	NOTE: Bone fragment movement highlights the frontal border of the parietal bones. The bregma is the center point at which the frontal and parietal bone fragments meet (Figure 1A).</li>
	<li>Wearing laser safety goggles, move the laser over the skull of the mouse, fixing the stereotaxic device at a 90&#176; angle. Select the 10 mW pre-set and turn on the laser.
	<ol>
		<li>Adjust the laser&#8217;s x- and y-axis until it is directly over the bregma. Reset the x and y on the digital display console, and then move the laser 1.8 mm left of the bregma. Now bring the laser as close to the skull as possible without letting it touch the skull.</li>
		<li>Move the laser to 2.2 mm left of the bregma to ensure that it can move without any obstructions. Turn off the laser.</li>
	</ol>
	</li>
	<li>Inject the appropriate amount of Rose Bengal dye intraperitoneally (IP) using a disposable 29 G needle. Immediately start a timer for 7 min to allow dye to circulate systemically. Select the pre-set for 40 mW without turning the laser on. 
	NOTE: Once comfortable with the procedure, the dye can be injected before aseptic preparation of the mouse and finished before the 7 min are over to save time.</li>
	<li>Turn on the laser (40 mW) when the 7 min timer goes off, while wearing laser safety goggles, and set a 10 min timer.
	<ol>
		<li>After 10 min, move the laser to 2.2 mm left of the bregma without turning it off, and set another 10 min timer.</li>
		<li>Once the second 10 min timer has gone off, change the laser back to 10 mW and move the laser to 2.0 mm left of the bregma. Mark this spot with the surgical marker. Then turn off the laser.</li>
		<li>Lift the laser and unlock it from the 90&#176; angle so it can be moved away from the mouse. At this point, apply the sterile saline or PBS with cotton if the surgical area is dry.</li>
	</ol>
	</li>
	<li>Drill in small bursts at the 2.0 mm mark perpendicular to the surface of the skull. 
	NOTE: Be careful to drill slowly to avoid drilling too deeply and hitting the brain. There will be a slight drop/change in resistance once the drill has gone through the skull, and drilling may cause bleeding.
	<ol>
		<li>Use cotton to absorb any excess blood. It is typical to see some blood from nicked arteries in the skull after drilling &#8211; excessive blood means the drill bit hit the brain. If this happens, record the mouse identifier, and move on.</li>
	</ol>
	</li>
	<li>Place a small wipe next to the mouse. Using the forceps, pull the skin close together to prepare for gluing. 
	NOTE: Suturing can be done here instead. Suturing typically requires only 3 sutures.
	<ol>
		<li>Using the forceps, grab the two sides of the skin and pull them together and upward. Dab off any large drops of surgical glue at the end of the tip on the wipe before applying to the mouse.</li>
		<li>Apply the surgical glue sparingly and hold the skin together with forceps for 10 s. Continue until there are no visible openings. 
		NOTE: The glue comes out quickly &#8211; do not squeeze the bottle. Avoid gluing the skin to the skull or getting glue in the eye.</li>
	</ol>
	</li>
	<li>After gluing, or suturing, place the mouse in the new cage to recover from anesthesia. It takes 5-10 min for the animal to recover, and it helps to rest half of the cage on a heating pad.</li>
</ol>
3. Sham stroke operation
<ol>
	<li>Perform all procedures identically to the operation described above in section 2 except inject 1x PBS or saline instead of Rose Bengal dye.</li>
</ol>
4. Injection of hydrogel or other biomaterials
<ol>
	<li>Perform the injection of biomaterials at a user-defined time. In this experiment injections were performed between 5- and 7-days post-stroke. 2 - 6 &#181;L of hydrogel are injected (user-defined).
	<ol>
		<li>Turn on the heating pad to maintain the mouse body temperature during anesthesia (37 &#176;C).</li>
		<li>Prepare autoclaved scissors, forceps, cotton, vet eye ointment, surgical glue or suture material, and sterile alcohol and beta iodine wipes. Prepare the bone drill with sterile drill bit heads and the 25 &#181;L glass Hamilton syringes with metal 30 G needles.</li>
		<li>Turn on the bead sterilization to 160 &#176;C to allow for sterilization of instruments between mice.</li>
		<li>Attach the injection pumps to the stereotaxic device. Set the flow rate to 1 &#181;L/min and set volume to 4 &#181;L.</li>
		<li>Prepare a clean cage for the mice after surgery.</li>
	</ol>
	</li>
	<li>Place the mouse into the induction chamber with an isoflurane concentration of 4% to anesthetize it until spontaneous movement stops and its breathing comes to a slow, steady rate.
	<ol>
		<li>Once asleep, transfer and secure the mouse to the stereotaxic device with a maintenance isoflurane concentration of 1.5%.</li>
		<li>Apply the vet eye ointment to both eyes.</li>
		<li>Shave any fur that may have regrown of the mouse&#8217;s head, and aseptically prepare the area by using an alcohol wipe followed by a beta iodine wipe. Repeat three times. 
		NOTE: If necessary, use an extra alcohol wipe at the end to clean off all the beta iodine as it causes skin irritation and leads the mice to itch their skull after surgery.</li>
	</ol>
	</li>
	<li>Using the small scissors and/or forceps, reopen the skin above the brain. Use cotton with sterile saline or PBS to clean any debris from the skull. A white-yellow circle (the stroke, Figure 1B) and the burr hole from the drill will be visible. If the dead tissue is not visible, likely no stroke occurred. If the burr hole is not centered above the stroke, re-drill to center it.</li>
	<li>Orient the injection pump over the mouse and lock at 90&#176;. Clean the syringe with sterile saline or PBS solution before backloading material.
	<ol>
		<li>Once clean, disassemble the glass syringe so that there is no needle. Backload the syringe using a 25 &#181;L positive displacement pipette with the 10 &#181;L of hydrogel (or other biomaterial here with or without cells).</li>
		<li>Using the syringe plunger, push the gel all the way to the front of the syringe. Then, reassemble the syringe and continue pushing until the gel visibly exudes from the needle.</li>
	</ol>
	</li>
	<li>Place the syringe onto the pump. The back of the pump may need to be adjusted so the syringe fits. Do this by adjusting the flow rate to 30 &#181;L/min, select Withdrawal or Inject depending on the direction it needs to move, and then press Start. Hit Stop when the back of the pump is at the correct location.
	<ol>
		<li>Screw the back of the pump so the syringe is secure. If adjustments were previously made, make sure the flow rate is set back to 1 &#181;L/min and is set to inject.</li>
	</ol>
	</li>
	<li>Move the pump in the x and y direction to orient it over the hole. Move the pump down in the z direction until the needle of the syringe touches the top of the hole.</li>
	<li>Reset the z on the digital display console. Then, move the syringe down in the z direction 0.750 mm. If the syringe bends or there is resistance, the skull either healed or was not completely drilled through and needs to be re-drilled.</li>
	<li>Press Start on the pump console to inject 4 - 6 &#181;L of the hydrogel at a rate of 1 &#181;L/min.</li>
	<li>Set a 5 min timer when the pump stops injecting to allow the gel to begin crosslinking.</li>
	<li>After 5 min, slowly pull up the syringe by turning the z nob. Watch to see if any material is accidently pulled or leaking from the hole. Unlock the pump and move it away from the mouse when the needle is far enough away from the skull.</li>
	<li>Using forceps, surgical glue, or sutures, close the skin above the head. Place the mouse in the clean cage and observe its recovery. It should take about 5 &#8211; 10 min for the mouse to recover from anesthesia.</li>
</ol>
5. Perfusion and sample collection
<ol>
	<li>Anesthetize the mouse using isoflurane.</li>
	<li>Fix the animal in the supine position and open the abdominal cavity with a median cut. Optionally, clamp the descending abdominal aorta to use less fixative and PBS.</li>
	<li>Cut open the diaphragm and move up the sides of the rib cage to open the chest cavity. Insert a perfusion cannula into the left ventricle and cut an opening in the right atrium. Start to slowly perfuse with 10-20 mL of PBS or until the liquid runs semi-clear.</li>
	<li>Once clear, switch to 4% PFA for another 10 &#8211; 20 mL. Unpinning the arms allows them to contract as the PFA infuses. Once they stop moving, wait for another 30 s.</li>
	<li>Decapitate the animal and pull back the skin to expose the skull. Using blunt, thin forceps, carefully remove the pieces of the skull to expose the brain. Special care needs to be taken when removing the skull above the stroke site. Small surgical scissors can be used here to help cut away the skull. The biggest challenge is gel getting stuck to the skull and coming out of the brain as the skull is removed.</li>
	<li>Once the brain is detached, place in 10 &#8211; 15 mL of 4% PFA overnight (no longer than 24 h).</li>
	<li>Move the brains from PFA to 30% sucrose the following day. The brain is ready for cryosection once it sinks to the bottom (about 2 &#8211; 3 days). It can also be left for storage at this point.</li>
</ol>
6. Cryosectioning and staining
<ol>
	<li>Take the brain out of sucrose and place on a glass slide. Cut a small portion of the back of the brain off with a razor blade to create a flat portion to be mounted onto a chuck.
	<ol>
		<li>Place a dollop of Optimal Cutting Temperature Compound (OCT) onto the chuck, and then place the brain, flat side down, onto the chuck in the OCT. Place this specimen on dry ice until frozen or in the cryostat on the freezing block. 
		NOTE: OCT can be added to fully encompass the brain to help prevent the material from separating from the brain during sectioning.</li>
	</ol>
	</li>
	<li>Cut the brains serially on a cryostat at -20 &#176;C at 30 &#181;m thick sections across 10 gelatin coated slides. Take care when sectioning ensuring not to lose the hydrogel. If this part is difficult, place some OCT on the top of the brain over the gel (Figure 3). Store at -80 &#176;C until further use.</li>
	<li>Take slides out of the -80 &#176;C and place on a staining tray to allow them to dry. Slides that have not be frozen but just sectioned can also be stained immediately.</li>
	<li>Using a hydrophobic pen, draw a circle around the brain slices on the slide. Once dry, rehydrate the brain slices with 1x filtered PBS and place on a shaker at room temperature for 15 min. This takes about 1 mL of buffer per slide with 9 &#8211; 12 brain sections.</li>
	<li>Wash the slides with 1x filtered PBS for 5 min at room temperature, repeat three times.</li>
	<li>Block slides for 30 min to an hour with 10% donkey serum in 0.01% PBS-Triton X at room temperature on the belly dancer. This takes about 200 &#181;L per slide.</li>
	<li>Incubate the primary antibody in 10% donkey serum with 0.01% PBS-Triton X overnight at 4&#176;C on a shaker. Test primary antibodies first to determine correct concentrations. Here GFAP (1:400), Iba1 (1:250), Glut1 (1:500) (Figure 4) were used.</li>
	<li>Wash slides with 1x filtered PBS the following day for 5 min at room temperature, repeat three times.</li>
	<li>Incubate with secondary antibody in 10% donkey serum in 0.01% PBS-Triton X and DAPI for 2 h on the lab shaker at room temperature. 1:1,000 was used for all secondaries and DAPI.</li>
	<li>Wash the slides for 5 min with 1x filtered PBS.</li>
	<li>Allow sections to dry at room temperature in the dark. This can take 20 min to 4 h; placing the slides in a desiccator can speed up the process.</li>
	<li>Dehydrate slides in ascending concentrations of alcohol (1 min per solution) of 50%, 70%, 95%, 100%, and 100%.</li>
	<li>De-fat in first bath of xylene for 1 min, then second bath of xylene for 5 min.</li>
	<li>Quickly take the slides out one-by-one and add mounting medium while brain sections are wet with xylene. Quickly add a glass coverslip on top. Use a cover slip that is of correct length and thickness needed for the slide and microscope used for imaging, respectively.</li>
	<li>Get rid of any bubbles under the coverslip by pressing gently with a pipette tip in an outward motion from the center of the slide. Allow DPX to dry for 4 h to overnight in the dark at room temperature.</li>
	<li>Use a razor blade to scrape off any dried mounting medium that spilled onto the slides. Wipe down slides with lens cleaner. Slides can now be imaged with fluorescence microscopy. 
	NOTE: It is best to store slide in the dark or at 4 &#176;C until imaging is finished.</li>
</ol>

<ol>
	<li>National Center for Health Statistics. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Health, United States, 2015: With Special Feature on Racial and Ethnic Health Disparities. , (2016).</li><li>Benjamin, E. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heart+disease+and+stroke+statistics-2017+update:+a+report+from+the+American+Heart+Association.">Heart disease and stroke statistics-2017 update: a report from the American Heart Association.</a> Circulation. 135 (10), 229 (2017).</li><li>Rosamond, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heart+Disease+and+Stroke+Statistics-2007+update:+a+report+from+the+American+Heart+Association+Statistics+Committee+and+Stroke+Statistics+Subcommittee.">Heart Disease and Stroke Statistics-2007 update: a report from the American Heart Association Statistics Committee and Stroke Statistics Subcommittee.</a> Circulation. 115 (5), 69 (2017).</li><li>Ovbiagele, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Forecasting+the+future+of+stroke+in+the+united+states:+A+policy+statement+from+the+American+heart+association+and+American+stroke+association.">Forecasting the future of stroke in the united states: A policy statement from the American heart association and American stroke association.</a> Stroke. 44 (8), 2361-2375 (2013).</li><li>Carmichael, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Themes+and+strategies+for+studying+the+biology+of+stroke+recovery+in+the+poststroke+epoch.">Themes and strategies for studying the biology of stroke recovery in the poststroke epoch.</a> Stroke. 39 (4), (2008).</li><li>Carmichael, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rodent+models+of+focal+stroke:+size,+mechanism,+and+purpose.">Rodent models of focal stroke: size, mechanism, and purpose.</a> NeuroRx: the journal of the American Society for Experimental NeuroTherapeutics. 2 (3), (2005).</li><li>Qin, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+adaptive+role+for+BDNF+Val66Met+polymorphism+in+motor+recovery+in+chronic+stroke.">An adaptive role for BDNF Val66Met polymorphism in motor recovery in chronic stroke.</a> Journal of Neuroscience. 34 (7), 2493-2502 (2014).</li><li>Fluri, F., Schuhmann, M. K., Kleinshnitz, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+ischemic+stroke+and+their+application+in+clinical+research.">Animal models of ischemic stroke and their application in clinical research.</a> Drug Design, Development and Therapy. 9, 3445-3454 (2015).</li><li>Talley Watts, L., Zheng, W., Garling, R. J., Frohlich, V. C., Lechleiter, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rose+Bengal+Photothrombosis+by+Confocal+Optical+Imaging+In+Vivo:+A+Model+of+Single+Vessel+Stroke.">Rose Bengal Photothrombosis by Confocal Optical Imaging In Vivo: A Model of Single Vessel Stroke.</a> Journal of Visualized Experiments. (100), e52794 (2015).</li><li>Tuladhar, A., Payne, S. L., Scoichet, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Harnessing+the+potential+of+biomaterials+for+brain+repair+after+stroke.">Harnessing the potential of biomaterials for brain repair after stroke.</a> Frontiers. 5, 14 (2018).</li><li>Nih, L. R., Sideris, E., Carmicahel, S. T., Segura, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Injection+of+microporous+annealing+particle+(MAP)+hydrogels+in+the+stoke+cavity+reduces+gliosis+and+inflammation+and+promotes+NPC+migration+to+the+lesion.">Injection of microporous annealing particle (MAP) hydrogels in the stoke cavity reduces gliosis and inflammation and promotes NPC migration to the lesion.</a> Advance Materials. 29 (32), (2017).</li><li>Nih, L. R., Gojgini, S., Carmichael, S. T., Segura, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Duel-function+injectable+angiogenic+biomaterial+for+the+repair+of+brain+tissue+following+stroke.">Duel-function injectable angiogenic biomaterial for the repair of brain tissue following stroke.</a> Nature Materials. 17, 642-651 (2018).</li><li>Cook, D. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrogel-delivered+brain-derived+neurotrophic+factor+promotes+tissue+repair+and+recovery+after+stroke.">Hydrogel-delivered brain-derived neurotrophic factor promotes tissue repair and recovery after stroke.</a> Journal of Cerebral Blood Flow &amp; Metabolism. 37, 1030-1045 (2017).</li><li>Lam, J., Lowry, W. E., Carmichael, S. T., Segura, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Delivery+of+iPS-NPCs+to+the+stroke+cavity+within+a+hyaluronic+acid+matrix+promotes+the+differentiation+of+transplanted+cells.">Delivery of iPS-NPCs to the stroke cavity within a hyaluronic acid matrix promotes the differentiation of transplanted cells.</a> Advance Functional Materials. 24, 7053-7062 (2015).</li><li>Nih, L. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineered+HA+hydrogel+for+stem+cell+transplantation+in+the+brain:+Biocompatibility+data+using+a+design+of+experiment+approach.">Engineered HA hydrogel for stem cell transplantation in the brain: Biocompatibility data using a design of experiment approach.</a> Data in Brief. 10, 202-209 (2016).</li><li>Carmichael, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+3+Rs+of+Stroke+Biology:+Radial,+Relayed,+and+Regenerative.">The 3 Rs of Stroke Biology: Radial, Relayed, and Regenerative.</a> Neurotherapeutics. 13, 348-359 (2016).</li><li>Caicco, M. J., Cooke, M. J., Wang, Y., Tuladhar, A., Morshead, C. M., Shoichet, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+hydrogel+composite+system+for+sustained+epi-cortical+delivery+of+Cyclosporin+A+to+the+brain+for+treatment+of+stroke.">A hydrogel composite system for sustained epi-cortical delivery of Cyclosporin A to the brain for treatment of stroke.</a> Journal of Controlled Release. 166 (3), 197-202 (2013).</li><li>Moshayedi, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systematic+optimization+of+an+engineered+hydrogel+allows+for+selective+control+of+human+neural+stem+cell+survival+and+differentiation+after+transplantation+in+the+stroke+brain.">Systematic optimization of an engineered hydrogel allows for selective control of human neural stem cell survival and differentiation after transplantation in the stroke brain.</a> Biomaterials. 105, 145-155 (2016).</li><li>Lu, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hemodynamic+effects+of+intraoperative+anesthetics+administration+in+photothrombotic+stroke+model:+a+study+using+laser+speckle+imaging.">Hemodynamic effects of intraoperative anesthetics administration in photothrombotic stroke model: a study using laser speckle imaging.</a> BMC Neuroscience. 18 (10), (2017).</li><li>Wiersma, A. M., Winship, I. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+Photothrombotic+Stroke+in+the+Sensorimotor+Cortex+of+Rats+and+Preparation+of+Tissue+for+Analysis+of+Stroke+Volume+and+Topographical+Cortical+Localization+of+Ischemic+Infarct.">Induction of Photothrombotic Stroke in the Sensorimotor Cortex of Rats and Preparation of Tissue for Analysis of Stroke Volume and Topographical Cortical Localization of Ischemic Infarct.</a> Bio-protocol. 8 (10), (2018).</li><li>Liang, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Region-specific+and+activity-dependent+regulation+of+SVZ+neurogenesis+and+recovery+after+stroke.">Region-specific and activity-dependent regulation of SVZ neurogenesis and recovery after stroke.</a> PNAS. 116 (27), 13621-13630 (2019).</li><li>Payne, S. L., Anandakumaran, P. N., Varga, B. V., Morshead, C. M., Nagy, A., Shoichet, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+maturation+of+human+iPSC-derived+neuroepithelial+cells+influences+transplant+survival+in+the+stroke-injured+rat+brain.">In vitro maturation of human iPSC-derived neuroepithelial cells influences transplant survival in the stroke-injured rat brain.</a> Tissue Engineering Part A. 24, 351-360 (2018).</li><li>Lee, I. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Delayed+epidural+transplantation+of+human+induced+pluripotent+stem+cell-derived+neural+progenitors+enhances+functional+recovery+after+stroke.">Delayed epidural transplantation of human induced pluripotent stem cell-derived neural progenitors enhances functional recovery after stroke.</a> Science Reports. 7, 1943 (2017).</li><li>Somaa, F. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peptide-based+scaffolds+support+human+cortical+progenitor+graft+integration+to+reduce+atrophy+and+promote+functional+repair+in+a+model+of+stroke.">Peptide-based scaffolds support human cortical progenitor graft integration to reduce atrophy and promote functional repair in a model of stroke.</a> Cell Reports. 20, 1964-1977 (2017).</li><li>Griffin, D. R., Weaver, W. M., Scumpia, P., Di Carlo, D., Segura, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accelerated+wound+healing+by+injectable+microporous+gel+scaffolds+assembled+from+annealed+building+blocks.">Accelerated wound healing by injectable microporous gel scaffolds assembled from annealed building blocks.</a> Nature Materials. 14 (7), 737-744 (2016).</li><li>Sideris, E. Y., Aaron, C. J., Carmichael, S. T., Segura, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hyaluronic+acid+particle+hydrogels+decrease+cerebral+atrophy+and+promote+pro-reparative+astrocyte/axonal+infiltration+in+the+core+after+ischemic+stroke.">Hyaluronic acid particle hydrogels decrease cerebral atrophy and promote pro-reparative astrocyte/axonal infiltration in the core after ischemic stroke.</a> bioRxiv. , (2019).</li><li>Yu, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combinated+Transplantation+of+Neural+Stem+Cells+and+Collagen+Type+I+Promote+Functional+Recovery+After+Cerebral+Ischemia+in+Rats.">Combinated Transplantation of Neural Stem Cells and Collagen Type I Promote Functional Recovery After Cerebral Ischemia in Rats.</a> The Anatomical Record. 293 (5), 911-917 (2010).</li><li>Jin, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transplantation+of+Human+Neural+Precursor+Cells+in+Matrigel+Scaffolding+Improves+Outcome+from+Focal+Cerebral+Ischemia+after+Delayed+Postischemic+Treatment+in+Rats.">Transplantation of Human Neural Precursor Cells in Matrigel Scaffolding Improves Outcome from Focal Cerebral Ischemia after Delayed Postischemic Treatment in Rats.</a> Journal of Cerebral Blood Flow &amp; Metabolism. 30 (3), 534-544 (2009).</li><li>Gelderblom, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal+and+spatial+dynamics+of+cerebral+immune+cell+accumulation+in+stroke.">Temporal and spatial dynamics of cerebral immune cell accumulation in stroke.</a> Stroke. 40 (5), 1849-1857 (2009).</li><li>Wei, L., Erinjeri, J. P., Rovainen, C. M., Woolsey, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collateral+growth+and+angiogenesis+around+cortical+stroke.">Collateral growth and angiogenesis around cortical stroke.</a> Stroke. 32 (9), 2179-2184 (2001).</li><li>Zhang, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activated+Neural+Stem+Cells+Contribute+to+Stroke-Induced+Neurogenesis+and+Neuroblast+Migration+toward+the+Infarct+Boundary+in+Adult+Rats.">Activated Neural Stem Cells Contribute to Stroke-Induced Neurogenesis and Neuroblast Migration toward the Infarct Boundary in Adult Rats.</a> Journal of Cerebral Blood Flow &amp; Metabolism. 24 (4), 441-448 (2004).</li></ol>

The authors declare that TS is a founder in Tempo Therapeutics, which seeks to commercialize MAP hydrogels.

Here we demonstrate an easily reproducible, minimally invasive, permanent stroke model and describe how to inject a biomaterial into the infarct five days after stroke. The use of the photothrombotic dye Rose Bengal and a 520 nm collimated laser connected to the stereotaxic device gives us the ability to position the stroke at the motor cortex of the mouse with enhanced precision. Five days after stroke, the location of the infarct is visible by eye at the center of irradiation, 2.0 mm medio-lateral to the bregma. Hydrog...

Stroke is the leading cause of disability and the fifth-leading cause of death in the United States1. Approximately 87% of all strokes are ischemic, while a majority of the remaining 13% are hemorrhagic2. An ischemic stroke is defined as the blockage of blood flow in an artery to the surrounding tissue. This occlusion results in oxygen deprivation and subsequent necrosis that often leads to permanent disability in surviving patients. While there has been a decrease in the mortality rate of stroke3, its prevalence is expected to increase to 3.4 million people by 20304. This increase in disabled survivors and consequent economic burden has led to a push for stroke research that focuses on mechanisms of neural repair. Following stroke there is an inflammatory period that leads to the formation of a scar that prevents the necrotic region from expanding. The region surrounding the necrotic core is termed &#8220;peri-infarct&#8221; and there is a strong evidence that the plasticity in this region, which includes increased angiogenesis, neurogenesis, and axonal sprouting, is directly linked to the observed recovery in animal models and humans5. Since there are no in vitro models that can properly replicate the complex interactions following stroke, animal models are essential for stroke research.
There are several in vivo models that can be used to produce ischemic stroke. One of the most common models used in mice is middle cerebral artery occlusion, or MCAo, through distal or proximal (via intra-arterial filament) occlusion. The proximal model, also known as filament MCAo (fMCAo), typically results in large ischemic strokes that encompass anywhere from 5% to 50% of the cerebral hemisphere, dependent upon number of factors6. In these models, a suture or filament is advanced from the internal carotid artery into the base of the middle cerebral artery (MCA) and kept in place for a specific period of time. This method for occlusion, which can be made temporary or permanent, produces an infarct that is centered in the striatum and may or may not involve overlying cortex6. The resulting stroke size is highly variable, and imaging techniques, such as laser doppler, are required to confirm the effectiveness of the procedure in each mouse. Intra-arterial or intra-luminal filament occlusion that lasts greater than 30 minutes produces strokes at the larger end of the size range. Some investigators have focused on shorter filament occlusion times, which require substantial experimental focus and lab validation7. Filament MCAo models in mice follow similar stages of cell death, ischemic progression, and formation of a peri-infarct region as seen in human stroke cases; however, the larger strokes more closely resemble the disease state of malignant cerebral infarctions, which are less common, less treatable human strokes6. Meanwhile, distal MCA occlusion requires a more involved surgery and craniectomy. In this model, the distal part of the MCA that runs along the surface of the brain is directly occluded with a suture tie or cauterization. In some variations of the technique, the carotid arteries are unilaterally or transiently-bilaterally occluded. A benefit of the distal MCAo is that it produces a cortical-based stroke that is less variable in size than the filament model. However, the distal model produces poorer behavioral output due to transection of the external carotid artery (ECA), which is also a concern with fMCAo6.
An alternative stroke model that is known for being less invasive is the photothrombotic (PT) model. The PT model results in a well-defined location of ischemia and is associated with a high survival rate8. The technique relies on a photosensitive dye injected intraperitoneally that allows for the intravascular photo-oxidation simply by irradiating the desired tissue with a light or laser9. Upon excitation, oxygen radicals are formed that cause endothelial damage, which activates platelet aggregation and clot formation in the irradiated area8,9. The tight control over stroke size and location, as well as high reproducibility of the PT model, makes it ideal for studying biomaterials. While precision is made possible using a laser and stereotaxic coordinates, there are some disadvantages that may make this model less ideal for few studies. Unlike the fMCAo model, the PT stroke model cannot be reperfused. Therefore, materials for investigating neuroprotective agents specific to damages following reperfusion or the mechanisms following reperfusion would not be useful here8. Additionally, due to the microvascular insult of the PT model, relatively small ischemic penumbra is seen. Instead, local vasogenic edema occurs, which is uncharacteristic for human stroke, making this model undesirable for preclinical drug studies focused on the peri-infarct area6,8.
The overall goal of biomaterial strategies in stroke is to either deliver bioactive agents or to act as a surrogate extracellular matrix for brain tissue growth. One strategy that we will explore using our methods is to deliver hydrogel directly into the stroke core, as opposed to the peri-infarct tissue where many current cell therapies are delivered10. The rationale for this approach is that delivery into the necrotic tissue found in the core will avoid disrupting the surrounding healthy or recovering tissue. We assume diffusion of any active agents included within the biomaterial will be able to reach the peri-infarct from the core, especially since we find that delivery of hydrogel biomaterials reduces the thickness of the glial scar11. This is important since the peri-infarct region has been shown to exhibit neuroplasticity after stroke, making it an attractive target. Furthermore, the delivery of a surrogate matrix to the stroke core can be loaded with angiogenic12 or neurogenic13 factors to guide the formation of new tissue, as well as cells for the delivery14. Cell delivery is greatly enhanced by using a matrix because it protects cells from the harsh injection forces and local environment present during delivery, as well as encourages differentiation and engraftment15.
These injectable therapeutic biomaterials have clinical relevance in stroke applications, as there are currently no medical therapies that stimulate neuronal recovery after stroke. The underlying neural circuits involved in recovery lie in brain tissue that is adjacent to the stroke core16, while the stroke core itself is devoid of viable neural tissue. We anticipate that delivering a biomaterial into the necrotic stroke core has potential to stimulate the adjacent tissue toward regenerative processes through a number of mechanisms previously mentioned, including depot release of growth factors13, stimulation of tissue in-growth and promotion of recovering brain tissue development11,12, alteration of immune responses17, and delivery of stem cell-derived therapeutics14,18. However, to effectively study the possibility of these applications, a consistent and reproducible method for inducing stroke and injecting biomaterials is needed. The PT stroke model uses techniques that offer precise control over the orientation and location of the stroke. A laser attached to the stereotaxic device guides orientations, and pumps attached to the stereotaxic devices control the injection rate of the material without the need for additional forms of imaging. Therefore, we have chosen to describe the methods for performing a PT stroke in the motor cortexes of mice and for injecting biomaterials into the stroke core. Here, we use microporous annealed particle (MAP) hydrogels as the biomaterial for injection with no added cells or growth factors. Additionally, we explain how to successfully retrieve the brain with intact biomaterial, and we discuss immunochemistry assays used to analyze the stroke outcome with and without injection of biomaterials.

<table><tbody><tr><td>10% Normal Goat Serum</td><td>VWR</td><td>100504-028</td><td>For blocking buffer</td></tr><tr><td>2-ply alcohol pre pad, Sterile, Medium</td><td>Medline</td><td>MDS090735</td><td></td></tr><tr><td>25uL Hamilton Syringe 702RN, no needle</td><td>Fishcer Scientific</td><td>14824663</td><td>Syringes used to inject biomaterials</td></tr><tr><td>25uL Positive displacement pipette</td><td>Gilson</td><td>M-25</td><td></td></tr><tr><td>2x Beam Expander, 400-650nm</td><td>Thorlabs</td><td>GBE02-A</td><td>Laser beam expander</td></tr><tr><td>Adjustable Stage Platform</td><td>Kopf Instruments</td><td>901</td><td></td></tr><tr><td>Anti-Glucose Transporter GLUT1 antibody, rabbit</td><td>Abcam</td><td>ab113435</td><td></td></tr><tr><td>Anti-Iba1 Antibody, goat</td><td>abcam</td><td>ab5076</td><td></td></tr><tr><td>BD Vacutainer Safety-Lok Blood Collection Sets. 25G, 12&quot;</td><td>Medsupply</td><td>367294</td><td>For perfusions</td></tr><tr><td>BKF12- Matte Black Aluminum Foil</td><td>Thorlabs</td><td>BKF12</td><td>To cover anything that is reflective when using laser.</td></tr><tr><td>Bone Iris Mini Scissors - 3-1/2&quot;</td><td>Sklar surgical instruments</td><td>64-2035</td><td></td></tr><tr><td>C57BL/6 Mice</td><td>Jackson Laboratory</td><td>000664</td><td>8-12 weeks of age</td></tr><tr><td>Cage Assembly Rob</td><td>Thorlabs</td><td>ER3-P4</td><td>3&quot; Long, diameter 6mm, 4 pack - for attaching laser to sterotax</td></tr><tr><td>Carbon Steel Burrs -0.5mm Diameter</td><td>Fine Science Tools</td><td>19007-05</td><td>For creating burr hole</td></tr><tr><td>Chromium(III) potassium sulfate dodecahydrate</td><td>VWR</td><td>EM1.01036.0250</td><td></td></tr><tr><td>Compact Controller for pigtailed lasers</td><td>Thorlabs</td><td>CLD1010LP</td><td></td></tr><tr><td>Cotton Swabs</td><td>VWR</td><td>89031-288</td><td></td></tr><tr><td>CP 25 pipette tips</td><td>Gilson</td><td>F148012</td><td></td></tr><tr><td>Donkey anti-goat IgG H&amp;amp;L (488)</td><td>abcam</td><td>ab150129</td><td></td></tr><tr><td>Donkey Anti-rabbit IgG H&amp;amp;L (647)</td><td>abcam</td><td>ab150075</td><td></td></tr><tr><td>Donkey Anti-rat IgG H&amp;amp;L (555)</td><td>abcam</td><td>ab150154</td><td></td></tr><tr><td>EMS DPX Mountant</td><td>Elecron Microscopy Sciences</td><td>13512</td><td>Mounting solution for slides</td></tr><tr><td>EMS Gelatin Powder Type A 300 Bloom</td><td>Electron Microscopy Sciences</td><td>16564</td><td>For gelatin coating slides</td></tr><tr><td>EMS Paraformaldehyde, Granular</td><td>VWR</td><td>100504-162</td><td>For making 45 PFA</td></tr><tr><td>ESD Worstation kit</td><td>Elmstat</td><td>WSKK5324SB</td><td>Need for setting up the laser</td></tr><tr><td>Fiber Bench Wall Plate, unthreaded</td><td>Thorlabs</td><td>HCA3</td><td>Need for connecting laser to Kopf shaft</td></tr><tr><td>FiberPort</td><td>Thorlabs</td><td>PAF-X-5-A</td><td>FC/APC&amp;amp; APC, f=4.6mm, 350-700nm, diameter 0.75mm</td></tr><tr><td>Fine Scissors - straight/sharp-blunt/10cm</td><td>Fine Science Tools</td><td>14028-10</td><td></td></tr><tr><td>GFAP Antibody, rat</td><td>Thermo Fisher Scientific</td><td>13-0300</td><td></td></tr><tr><td>Heating Plate</td><td>Kopf Instruments</td><td>HP-4M</td><td></td></tr><tr><td>ImmEdge Hydrophobic Barrier Pen</td><td>Vector Laboratories</td><td>H-4000</td><td>For staining slides</td></tr><tr><td>IMPAC 6-Integrated Multi Patient Anesthesia Center</td><td>VetEquip</td><td>901808</td><td></td></tr><tr><td>Iodine Prep Pads</td><td>Medx Supple</td><td>MED MDS093917H</td><td></td></tr><tr><td>Jewlers Forceps #5</td><td>GFS chemicals</td><td>46085</td><td></td></tr><tr><td>Laser Safety Glasses</td><td>Thorlabs</td><td>LG10B</td><td>Amber Lenses, 35% Visible light (googles versions available too)</td></tr><tr><td>M27-1084 Powerful LED Dual Goose-neck</td><td>United Scope</td><td>LED-11C</td><td></td></tr><tr><td>Medical USP Grade Oxygen</td><td>Airgas</td><td>OX USP250</td><td></td></tr><tr><td>Miltex Adson Dressing Forceps, Disecting-grade</td><td>Intefra Miltex</td><td>V96-118</td><td></td></tr><tr><td>Mini Cord/Cordless Small Animal Trimmer</td><td>Harvard aparatus</td><td>72-6110</td><td></td></tr><tr><td>Mini-pump variable flow</td><td>Thomas Scientific</td><td>70730-064</td><td>Pump for perfusions</td></tr><tr><td>Mouse Brain Matrices, Coronal Slices, 1mm</td><td>Kent Scientific</td><td>RBMA-200c</td><td>For TTC slices</td></tr><tr><td>Mouse Gas Anethesia Head Holder</td><td>Kopf Instruments</td><td>923-B</td><td></td></tr><tr><td>Nanojet Control Box</td><td>Chemyx</td><td>10050</td><td></td></tr><tr><td>Nanojet pump header</td><td>Chemxy</td><td>10051</td><td>Attach to stereotaxic device for injecting biomaterial</td></tr><tr><td>Needle RN 30G PT STY 3, 0.5 inch</td><td>Fishcer Scientific</td><td>NC9459562</td><td></td></tr><tr><td>Non-rupture ear bars 60&amp;ordm;</td><td>Kopf Instruments</td><td>922</td><td></td></tr><tr><td>PBS buffer pH 7.4</td><td>VWR</td><td>97062 338</td><td></td></tr><tr><td>Pigtaled laser 520 nm, 100mW, 5G Pin</td><td>Thorlabs</td><td>LP520-MF100</td><td></td></tr><tr><td>Positive charge glass slides</td><td>Hareta</td><td>AHS90-WH</td><td></td></tr><tr><td>Power engergy meter</td><td>Thorlabs</td><td>PM100D</td><td>Used to measure your mW laser output</td></tr><tr><td>Puralube Vet Ointment</td><td>Dr. Foster Smith</td><td>9N-76855</td><td></td></tr><tr><td>Rectal Probe Mouse</td><td>kopf Instruments</td><td>Ret-3-ISO</td><td></td></tr><tr><td>Rose Bengal Dye 95%</td><td>Sigma-Aldrich</td><td>330000-5G</td><td></td></tr><tr><td>Shaft Modified 8-32 threaded hole 1/2&quot; depth</td><td>Kopf Instruments</td><td>1770-02</td><td>For connecting laser to sterotaxic device</td></tr><tr><td>Slim photodiode power sensor</td><td>Thorlabs</td><td>S130VC</td><td>Used with power energy meter</td></tr><tr><td>SM1-Threaed 30 mm Cage Plate 0.35&quot; thick 2 Retaining</td><td>Thorlabs</td><td>CP02</td><td>For connecting laser expander</td></tr><tr><td>Sol-M U-100 Insuline syringe with 1/2 unit markings 0.5 mL</td><td>VWR</td><td>10002-726</td><td>To inject rose bengal</td></tr><tr><td>StainTray Slide Staining System</td><td>Simport Scientific</td><td>M920-2</td><td>For staining slides</td></tr><tr><td>Sterotaxic device</td><td>Kopf Instruments</td><td>940</td><td>Small Animal Stereotaxic Instrument</td></tr><tr><td>Student Adson Forceps -1x2 teeth</td><td>Fine Science Tools</td><td>91127-12</td><td></td></tr><tr><td>Student Fine Forceps - straight/broad Shanks</td><td>Fine Science Tools</td><td>91113-10</td><td></td></tr><tr><td>Temperature Controller</td><td>Kopf Instruments</td><td>TCAT-2LV</td><td></td></tr><tr><td>Tissue-Tek OCT compound</td><td>VWR</td><td>25608-930</td><td></td></tr><tr><td>Triton X-100</td><td>VWR</td><td>97063-864</td><td></td></tr><tr><td>Upper Bracket Clamp</td><td>Kopf Instruments</td><td>1770-c</td><td>For connecting laser to sterotaxic device</td></tr><tr><td>Vetbond Tissue Adhessive 3mL</td><td>Santa Cruz Biotechnology</td><td>sc-361931</td><td></td></tr><tr><td>Vogue Professional My Manicurist</td><td>Bargin Source</td><td>6400</td><td>For Burrs</td></tr><tr><td>VWR Bead Sterilizers</td><td>VWR</td><td>75999-328</td><td></td></tr><tr><td>Tissue Tek OCT compound</td><td>Sakura</td><td>4583</td><td>For tissue embeding</td></tr></tbody></table>

injection of hydrogel biomaterial scaffolds to the brain after stroke

Stroke is the leading cause of disability and the fifth-leading cause of death in the United States. Approximately 87% of all strokes are ischemic strokes and are defined as the sudden blockage of a vessel supplying blood to the brain. Within minutes of the blockage, cells begin to die and result in irreparable tissue damage. Current therapeutic treatments focus on clot removal or lysis to allow for the reperfusion and prevent more severe brain damage. Although transient brain plasticity may salvage some of the damaged tissue over time, significant fractions of patients are left with neurological deficits that will never resolve. There is a lack of therapeutic options to treat neurological deficits caused by stroke, emphasizing the need to develop new strategies to treat this growing patient population. Injectable biomaterials are currently being designed to enhance brain plasticity and improve endogenous repair through the delivery of active agents or stem cells. One method to test these approaches is to utilize a rodent stroke model, inject the biomaterial into the stroke core, and assess repair. Knowing the precise location of the stroke core is imperative for the accurate treatment after stroke, therefore, a stroke model that results in a predictable stroke location is preferable to avoid the need for imaging prior to injection. The following protocol will cover how to induce a photothrombotic stroke, how to inject a hydrogel in a controlled and precise manner, and how to extract and cryosection the brain while keeping the biomaterial intact. In addition, we will highlight how these same hydrogel materials can be used for the co-delivery of stem cells. This protocol can be generalized to the use of other injectable biomaterials into the stroke core.

The aim of this method was to demonstrate how to inject biomaterials into the brain after stroke. A photothrombotic model with rose bengal and a 520 nm laser was used for controlled orientation of the stroke lesion in both size and location. Five days after stroke the infarct could be visualized during surgery (Figure 1B) and by TTC and imaging IHC stained slides (Figure 2). An increase in laser diameter with a 2x lens lead to a visual increase in the stroke les...

Watch this Scientific Journal Video about Injection of Hydrogel Biomaterial Scaffolds to The Brain After Stroke at JoVE.com

Injection of Hydrogel Biomaterial Scaffolds to The Brain After Stroke

Stroke is a global issue with minimal treatment options and no current clinical therapy for regenerating the lost brain tissue. Here we describe methods for creating precise photothrombotic stroke in the motor cortex of rodents and subsequent injection of hydrogel biomaterials to study their effects on tissue regeneration after stroke.

Stroke is the leading cause of disability and the fifth-leading cause of death in the United States. Approximately 87% of all strokes are ischemic strokes ...

injection-of-hydrogel-biomaterial-scaffolds-to-the-brain-after-stroke

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

5.0K Views.  Duke University. Stroke is a global issue with minimal treatment options and no current clinical therapy for regenerating the lost brain tissue. Here we describe methods for creating precise photothrombotic stroke in the motor cortex of rodents and subsequent injection of hydrogel biomaterials to study their effects on tissue regeneration after stroke.

Video: Injection of Hydrogel Biomaterial Scaffolds to The Brain After Stroke

Promotion of Survival and Differentiation of Neural Stem Cells with Fibrin and Growth Factor Cocktails after Severe Spinal Cord Injury

Neural stem cells (NSCs) can self-renew and differentiate into neurons and glia. Transplanted NSCs can replace lost neurons and glia after spinal cord injury (SCI), and can form functional relays to re-connect spinal cord segments above and below a lesion. Previous studies grafting neural stem cells have been limited by incomplete graft survival within the spinal cord lesion cavity. Further, tracking of graft cell survival, differentiation, and process extension had not been optimized. Finally, in previous studies, cultured rat NSCs were typically reported to differentiate into glia when grafted to the injured spinal cord, rather than neurons, unless fate was driven to a specific cell type. To address these issues, we developed new methods to improve the survival, integration and differentiation of NSCs to sites of even severe SCI. NSCs were freshly isolated from embryonic day 14 spinal cord (E14) from a stable transgenic Fischer 344 rat line expressing green fluorescent protein (GFP) and were embedded into a fibrin matrix containing growth factors; this formulation aimed to retain grafted cells in the lesion cavity and support cell survival. NSCs in the fibrin/growth factor cocktail were implanted two weeks after thoracic level-3 (T3) complete spinal cord transections, thereby avoiding peak periods of inflammation. Resulting grafts completely filled the lesion cavity and differentiated into both neurons, which extended axons into the host spinal cord over remarkably long distances, and glia. Grafts of cultured human NSCs expressing GFP resulted in similar findings. Thus, methods are defined for improving neural stem cell grafting, survival and analysis of in vivo findings.

Fibrin matrices containing growth factors were used to retain grafted neural stem cells into sites of complete spinal cord transection. Grafted cells completely filled the lesion cavity and differentiated into multiple neural cell types, including neurons that extended axons into host spinal cord over long distances.

Neural stem cells (NSCs) can self-renew and differentiate into neurons and glia. Transplanted NSCs can replace lost neurons and glia after spinal cord ...

Neuroscience

Glioblastoma Relapse Post-Resection Model for Therapeutic Hydrogel Investigations

Tumor recurrence is an important factor indicative of a poor prognosis in glioblastoma (GBM). Many studies are attempting to identify effective therapeutic strategies to prevent the recurrence of GBM after surgery. Bioresponsive therapeutic hydrogels capable of sustaining locally released drugs are frequently used for the local treatment of GBM after surgery. However, research is limited due to the lack of a suitable GBM relapse post-resection model. Here, a GBM relapse post-resection model was developed and applied in therapeutic hydrogel investigations. This model was constructed based on the orthotopic intracranial GBM model, which is widely used in studies on GBM. Subtotal resection was performed on the orthotopic intracranial GBM model mouse to mimic the clinical treatment. The residual tumor was used to indicate the size of the tumor growth. This model is easy to build, can better mimic the situation of GBM surgical resection, and can be applied in various studies on the local treatment of GBM relapse post-resection. As a result, the GBM relapse post-resection model provides a unique GBM recurrence model for effective local treatment studies of relapse post-resection.

The present protocol establishes a glioblastoma (GBM) relapse post-resection model using microscopy to investigate the therapeutic effect of an injectable, bioresponsive hydrogel in vivo.

Tumor recurrence is an important factor indicative of a poor prognosis in glioblastoma (GBM). Many studies are attempting to identify effective ...

Cancer Research

Three-dimensional Tissue Engineered Aligned Astrocyte Networks to Recapitulate Developmental Mechanisms and Facilitate Nervous System Regeneration

Neurotrauma and neurodegenerative disease often result in lasting neurological deficits due to the limited capacity of the central nervous system (CNS) to replace lost neurons and regenerate axonal pathways. However, during nervous system development, neuronal migration and axonal extension often occur along pathways formed by other cells, referred to as &#34;living scaffolds&#34;. Seeking to emulate these mechanisms and to design a strategy that circumvents the inhibitory environment of the CNS, this manuscript presents a protocol to fabricate tissue engineered astrocyte-based &#34;living scaffolds&#34;. To create these constructs, we employed a novel biomaterial encasement scheme to induce astrocytes to self-assemble into dense three-dimensional bundles of bipolar longitudinally-aligned somata and processes. First, hollow hydrogel micro-columns were assembled, and the inner lumen was coated with collagen extracellular-matrix. Dissociated cerebral cortical astrocytes were then delivered into the lumen of the cylindrical micro-column and, at a critical inner diameter of &#60;350 &#181;m, spontaneously self-aligned and contracted to produce long fiber-like cables consisting of dense bundles of astrocyte processes and collagen fibrils measuring &#60;150 &#181;m in diameter yet extending several cm in length. These engineered living scaffolds exhibited &#62;97% cell viability and were virtually exclusively comprised of astrocytes expressing a combination of the intermediate filament proteins glial-fibrillary acidic protein (GFAP), vimentin, and nestin. These aligned astrocyte networks were found to provide a permissive substrate for neuronal attachment and aligned neurite extension. Moreover, these constructs maintain integrity and alignment when extracted from the hydrogel encasement, making them suitable for CNS implantation. These preformed constructs structurally emulate key cytoarchitectural elements of naturally occurring glial-based &#34;living scaffolds&#34; in vivo. As such, these engineered living scaffolds may serve as test-beds to study neurodevelopmental mechanisms in vitro or facilitate neuroregeneration by directing neuronal migration and/or axonal pathfinding following CNS degeneration in vivo.

We showcase the development of self-assembled, three-dimensional bundles of longitudinally aligned astrocytic somata and processes within a novel biomaterial encasement. These engineered &#34;living scaffolds&#34;, exhibiting micron-scale diameter yet extending centimeters in length, may serve as test-beds to study neurodevelopmental mechanisms or facilitate neuroregeneration by directing neuronal migration and/or axonal pathfinding.

Neurotrauma and neurodegenerative disease often result in lasting neurological deficits due to the limited capacity of the central nervous system (CNS) to ...

Bioengineering

A Hydrogel Construct and Fibrin-based Glue Approach to Deliver Therapeutics in a Murine Myocardial Infarction Model.

The murine MI model is widely recognized in the field of cardiovascular disease, and has consistently been used as a first step to test the efficacy of treatments in vivo1. The traditional, established protocol has been further fine-tuned to minimize the damage to the animal. Notably, the pectoral muscle layers are teased away rather than simply cut, and the thoracotomy is approached intercostally as opposed to breaking the ribs in a sternotomy, preserving the integrity of the ribcage. With these changes, the overall stress on the animal is decreased. 
Stem cell therapies aimed to alleviate the damage caused by MIs have shown promise over the years for their pro-angiogenic and anti-apoptotic benefits. Current approaches of delivering cells to the heart surface typically involve the injection of the cells either near the damaged site, within a coronary artery, or into the peripheral blood stream2-4. While the cells have proven to home to the damaged myocardium, functionality is limited by their poor engraftment at the site of injury, resulting in diffusion into the blood stream5. This manuscript highlights a procedure that overcomes this obstacle with the use of a cell-encapsulated hydrogel patch. The patch is fabricated prior to the surgical procedure and is placed on the injured myocardium immediately following the occlusion of the left coronary artery. To adhere the patch in place, biocompatible external fibrin glue is placed directly on top of the patch, allowing for it to dry to both the patch and the heart surface. This approach provides a novel adhesion method for the application of a delicate cell-encapsulating therapeutic construct.

This protocol aims to alleviate the limitation of poor cell engraftment for stem cell treatment of myocardial infarctions through the use of a hydrogel system and a fibrin-based glue. With this approach, cell-to-tissue contact post-infarction can be maintained, increasing the therapeutic potential of beneficial agents at the site of injury.

The murine MI model is widely recognized in the field of cardiovascular disease, and has consistently been used as a first step to test the efficacy of ...

Medicine

Implantation of Miniosmotic Pumps and Delivery of Tract Tracers to Study Brain Reorganization in Pathophysiological Conditions

Pharmacological treatment in animal models of cerebral disease imposes the problem of repeated injection protocols that may induce stress in animals and result in impermanent tissue levels of the drug. Additionally, drug delivery to the brain is delicate due to the blood brain barrier (BBB), thus significantly reducing intracerebral concentrations of selective drugs after systemic administration. Therefore, a system that allows both constant drug delivery without peak levels and circumvention of the BBB is in order to achieve sufficiently high intracerebral concentrations of drugs that are impermeable to the BBB. In this context, miniosmotic pumps represent an ideal system for constant drug delivery at a fixed known rate that eludes the problem of daily injection stress in animals and that may also be used for direct brain delivery of drugs. Here, we describe a method for miniosmotic pump implantation and post operatory care that should be given to animals in order to successfully apply this technique. We embed the aforementioned experimental paradigm in standard procedures that are used for studying neuroplasticity within the brain of C57BL6 mice. Thus, we exposed animals to 30 min brain infarct and implanted with miniosmotic pumps connected to the skull via a cannula in order to deliver a pro-plasticity drug. Behavioral testing was done during 30 days of treatment. After removal the animals received injections of anterograde tract tracers to analyze neuronal plasticity in the chronic phase of recovery. Results indicated that neuroprotection by the delivered drug was accompanied with increase in motor fibers crossing the midline of the brain at target structures. The results affirm the value of these techniques for drug administration and brain plasticity studies in modern neuroscience.

In order to study brain reorganization under pathological conditions we used miniosmotic pumps for direct protein delivery into the brain circumventing the blood brain barrier. Tract tracers are then injected to study alterations in brain connectivity under the influence of the protein.

Pharmacological treatment in animal models of cerebral disease imposes the problem of repeated injection protocols that may induce stress in animals and ...

Improved 3D Hydrogel Cultures of Primary Glial Cells for In Vitro Modelling of Neuroinflammation

In the central nervous system, numerous acute injuries and neurodegenerative disorders, as well as implanted devices or biomaterials engineered to enhance function result in the same outcome: excess inflammation leads to gliosis, cytotoxicity, and/or formation of a glial scar that collectively exacerbate injury or prevent healthy recovery. With the intent of creating a system to model glial scar formation and study inflammatory processes, we have generated a 3D cell scaffold capable of housing primary cultured glial cells: microglia that regulate the foreign body response and initiate the inflammatory event, astrocytes that respond to form a fibrous scar, and oligodendrocytes that are typically vulnerable to inflammatory injury. The present work provides a detailed step-by-step method for the fabrication, culture, and microscopic characterization of a hyaluronic acid-based 3D hydrogel scaffold with encapsulated rat brain-derived glial cells. Further, protocols for characterization of cell encapsulation and the hydrogel scaffold by confocal immunofluorescence and scanning electron microscopy are demonstrated, as well as the capacity to modify the scaffold with bioactive substrates, with incorporation of a commercial basal lamina mixture to improved cell integration.

Improved 3D Hydrogel Cultures of Primary Glial Cells for In Vitro Modelling of Neuroinflammation

Herein, we present a protocol for the 3D culture of rat brain-derived glia cells, including astrocytes, microglia, and oligodendrocytes. We demonstrate primary cell culture, methacrylated hyaluronic acid (HAMA) hydrogel synthesis, HAMAphoto-polymerization and cell encapsulation, and sample processing for confocal and scanning electron microscopic imaging.

In the central nervous system, numerous acute injuries and neurodegenerative disorders, as well as implanted devices or biomaterials engineered to enhance ...

Electrically Conductive Scaffold to Modulate and Deliver Stem Cells

Stem cell therapy has emerged as an exciting stroke therapeutic, but the optimal delivery method remains unclear. While the technique of microinjection has been used for decades to deliver stem cells in stroke models, this technique is limited by the lack of ability to manipulate the stem cells prior to injection. This paper details a method of using an electrically conductive polymer scaffold for stem cell delivery. Electrical stimulation of stem cells using a conductive polymer scaffold alters the stem cell's genes involved in cell survival, inflammatory response, and synaptic remodeling. After electrical preconditioning, the stem cells on the scaffold are transplanted intracranially in a distal middle cerebral artery occlusion rat model. This protocol describes a powerful technique to manipulate stem cells via a conductive polymer scaffold and creates a new tool to further develop stem cell-based therapy.

This protocol describes fabrication of a cell culture system to allow seeding of stem cells on a conductive polymer scaffold for in vitro electrical stimulation and subsequent in vivo implantation of the stem cell-seeded scaffold using a minimally invasive technique.

Stem cell therapy has emerged as an exciting stroke therapeutic, but the optimal delivery method remains unclear. While the technique of microinjection ...

Preparation and Characterization of Graphene-Based 3D Biohybrid Hydrogel Bioink for Peripheral Neuroengineering

Peripheral neuropathies can occur as a result of axonal damage, and occasionally due to demyelinating diseases. Peripheral nerve damage is a global problem that occurs in 1.5%-5% of emergency patients and may lead to significant job losses. Today, tissue engineering-based approaches, consisting of scaffolds, appropriate cell lines, and biosignals, have become more applicable with the development of three-dimensional (3D) bioprinting technologies. The combination of various hydrogel biomaterials with stem cells, exosomes, or bio-signaling molecules is frequently studied to overcome the existing problems in peripheral nerve regeneration. Accordingly, the production of injectable systems, such as hydrogels, or implantable conduit structures formed by various bioprinting methods has gained importance in peripheral neuro-engineering. Under normal conditions, stem cells are the regenerative cells of the body, and their number and functions do not decrease with time to protect their populations; these are not specialized cells but can differentiate upon appropriate stimulation in response to injury. The stem cell system is under the influence of its microenvironment, called the stem cell niche. In peripheral nerve injuries, especially in neurotmesis, this microenvironment cannot be fully rescued even after surgically binding severed nerve endings together. The composite biomaterials and combined cellular therapies approach increases the functionality and applicability of materials in terms of various properties such as biodegradability, biocompatibility, and processability. Accordingly, this study aims to demonstrate the preparation and use of graphene-based biohybrid hydrogel patterning and to examine the differentiation efficiency of stem cells into nerve cells, which can be an effective solution in nerve regeneration.

In this manuscript, we demonstrate the preparation of a biohybrid hydrogel bioink containing graphene for use in peripheral tissue engineering. Using this 3D biohybrid material, the neural differentiation protocol of stem cells is performed. This can be an important step in bringing similar biomaterials to the clinic.

Peripheral neuropathies can occur as a result of axonal damage, and occasionally due to demyelinating diseases. Peripheral nerve damage is a global ...

Hydrogel Arrays Enable Increased Throughput for Screening Effects of Matrix Components and Therapeutics in 3D Tumor Models

Cell-matrix interactions mediate complex physiological processes through biochemical, mechanical, and geometrical cues, influencing pathological changes and therapeutic responses. Accounting for matrix effects earlier in the drug development pipeline is expected to increase the likelihood of clinical success of novel therapeutics. Biomaterial-based strategies recapitulating specific tissue microenvironments in 3D cell culture exist but integrating these with the 2D culture methods primarily used for drug screening has been challenging. Thus, the protocol presented here details the development of methods for 3D culture within miniaturized biomaterial matrices in a multi-well plate format to facilitate integration with existing drug screening pipelines and conventional assays for cell viability. Since the matrix features critical for preserving clinically relevant phenotypes in cultured cells are expected to be highly tissue- and disease-specific, combinatorial screening of matrix parameters will be necessary to identify appropriate conditions for specific applications. The methods described here use a miniaturized culture format to assess cancer cell responses to orthogonal variation of matrix mechanics and ligand presentation. Specifically, this study demonstrates the use of this platform to investigate the effects of matrix parameters on the responses of patient-derived glioblastoma (GBM) cells to chemotherapy.

The present protocol describes an experimental platform to assess the effects of mechanical and biochemical cues on chemotherapeutic responses of patient-derived glioblastoma cells in 3D matrix-mimetic cultures using a custom-made UV illumination device facilitating high-throughput photocrosslinking of hydrogels with tunable mechanical features.

Cell-matrix interactions mediate complex physiological processes through biochemical, mechanical, and geometrical cues, influencing pathological changes ...

Development of Combinatorial Therapeutics for Spinal Cord Injury using Stem Cell Delivery

Traumatic spinal cord injury (SCI) induces permanent sensorimotor deficit below the site of injury. It affects approximately a quarter million people in the US, and it represents an immeasurable public health concern. Research has been conducted to provide effective therapy; however, SCI is still considered incurable due to the complex nature of the injury site. A variety of strategies, including drug delivery, cell transplantation, and injectable biomaterials, are investigated, but one strategy alone limits their efficacy for regeneration. As such, combinatorial therapies have recently gained attention that can target multifaceted features of the injury. It has been shown that extracellular matrices (ECM) may increase the efficacy of cell transplantation for SCI. To this end, 3D hydrogels consisting of decellularized spinal cords (dSCs) and sciatic nerves (dSNs) were developed at different ratios and characterized. Histological analysis of dSCs and dSNs confirmed the removal of cellular and nuclear components, and native tissue architectures were retained after decellularization. Afterward, composite hydrogels were created at different volumetric ratios and subjected to analyses of turbidity gelation kinetics, mechanical properties, and embedded human adipose-derived stem cell (hASC) viability. No significant differences in mechanical properties were found among the different ratios of hydrogels and decellularized spinal cord matrices. Human ASCs embedded in the gels remained viable throughout the 14-day culture. This study provides a means of generating tissue-engineered combinatorial hydrogels that present nerve-specific ECM and pro-regenerative mesenchymal stem cells. This platform can provide new insights into neuro-regenerative strategies after SCI with future investigations.

In this study, nerve-mimetic composite hydrogels were developed and characterized that can be utilized to investigate and capitalize on the pro-regenerative behavior of adipose-derived stem cells for spinal cord injury repair.

Traumatic spinal cord injury (SCI) induces permanent sensorimotor deficit below the site of injury. It affects approximately a quarter million people in ...

Injection of Hydrogel Biomaterial Scaffolds to The Brain After Stroke

Summary

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Chapters in this video

Promotion of Survival and Differentiation of Neural Stem Cells with Fibrin and Growth Factor Cocktails after Severe Spinal Cord Injury

Glioblastoma Relapse Post-Resection Model for Therapeutic Hydrogel Investigations

Three-dimensional Tissue Engineered Aligned Astrocyte Networks to Recapitulate Developmental Mechanisms and Facilitate Nervous System Regeneration

A Hydrogel Construct and Fibrin-based Glue Approach to Deliver Therapeutics in a Murine Myocardial Infarction Model.

Implantation of Miniosmotic Pumps and Delivery of Tract Tracers to Study Brain Reorganization in Pathophysiological Conditions

Improved 3D Hydrogel Cultures of Primary Glial Cells for In Vitro Modelling of Neuroinflammation

Electrically Conductive Scaffold to Modulate and Deliver Stem Cells

Preparation and Characterization of Graphene-Based 3D Biohybrid Hydrogel Bioink for Peripheral Neuroengineering

Hydrogel Arrays Enable Increased Throughput for Screening Effects of Matrix Components and Therapeutics in 3D Tumor Models

Development of Combinatorial Therapeutics for Spinal Cord Injury using Stem Cell Delivery