Name: injection of hydrogel biomaterial scaffolds to the brain after stroke
Uploaded: 2020-10-01T13:00:00
Description: Watch this Scientific Journal Video about Injection of Hydrogel Biomaterial Scaffolds to The Brain After Stroke at JoVE.com

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 prematu...

<ol>
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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. 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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&#34;</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&#34;</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&#34; 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&#38;L (488)</td>
			<td>abcam</td>
			<td>ab150129</td>
			<td></td>
		</tr>
		<tr>
			<td>Donkey Anti-rabbit IgG H&#38;L (647)</td>
			<td>abcam</td>
			<td>ab150075</td>
			<td></td>
		</tr>
		<tr>
			<td>Donkey Anti-rat IgG H&#38;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&#38; 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&#186;</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&#34; 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&#34; 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 ...

Research

JoVE Journal

Biology

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Zebrafish Brain Ventricle Injection

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The Gateway to the Brain: Dissecting the Primate Eye

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Intraperitoneal Injection into Adult Zebrafish

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Mapping the After-effects of Theta Burst Stimulation on the Human Auditory Cortex with Functional Imaging

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Intraductal Injection for Localized Drug Delivery to the Mouse Mammary Gland

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Reconstitution of a Transmembrane Protein, the Voltage-gated Ion Channel, KvAP, into Giant Unilamellar Vesicles for Microscopy and Patch Clamp Studies

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Sampling Blood from the Lateral Tail Vein of the Rat

Blood samples are commonly obtained in many experimental contexts to measure targets of interest, including hormones, immune factors, growth factors, ...

A High Output Method to Isolate Cerebral Pericytes from Mouse

In recent years, cerebral pericytes have become the focus of extensive research in vascular biology and pathology. The importance of pericytes in blood ...