We thank Katherine Clark for technical assistance. A.D.R.G. is funded in part by 5K01MH097957-03

Adult (3-4 months old) male mice on a mixed C57BL/6 background were used in this protocol. Animals were kept on a 12 hr light/dark cycle, and allowed free access to food and water. All procedures performed in this protocol were conducted according to protocols approved by the Drexel University Institutional Animal Care and Use Committee.
1. Preparing Surgical Area
<ol>
	<li>Disinfect surgical table with 70% ethanol, then cover the entire surgical bench with absorbent pads and arrange surgical instruments adjacent to stereotaxic.</li>
	<li>Set up stereotaxic equipment without manipulator arm. Arrange the heating pad on the stereotaxic and set to 37 &#176;C. Avoid overheating the animal by placing a small piece of paper towel or surgical pad between the animal and the heating pad.</li>
	<li>Using autoclaved scissors, cut small pieces of autoclaved gelfoam into a sterile Petri dish containing&#160;0.9% sterile saline solution until ready for use. 
	Note: Maintain sterile working environment by using autoclaved instruments and sterile surgical supplies. Re-sterilize surgical instruments during the procedure or in between animals by dipping into a bead sterilizer for 10-15 sec, as needed. Maintain clean gloves throughout the procedure by rubbing hands with 70% ethanol, as needed, to disinfect.</li>
</ol>
2. Prepping Mouse for Surgery
<ol>
	<li>Remove mouse from home cage and weigh (g).</li>
	<li>Place mouse into isoflurane induction chamber and set oxygen to 2 L/min and isoflurane vaporizer to 5 to induce a surgical plane of anesthesia, about 3-5 min. Monitor for slowed breathing and immobilization. Check that the mouse is fully sedated using the toe pinch reflex.</li>
	<li>When mouse is fully sedated, place in stereotaxic frame, secure the nose in the nose cone, which is attached with tubing to the isoflurane. Insert ear bars into ear canal and tighten, ensuring the head is stable.</li>
	<li>Shave the head from ear to ear, and from between the eyes to behind the ears.</li>
	<li>Sterilize the skin with alternating wipes of isopropyl alcohol and betadine iodine solution, 3 times each.</li>
	<li>Apply artificial tears to both eyes to prevent them from drying out during the surgical procedure.</li>
</ol>
3. Surgical Procedure
<ol>
	<li>Monitor the depth of anesthesia by pinching the toe or tail. The mouse is in the appropriate surgical plane when there is no response, and the respiration is slow and even.</li>
	<li>Make a parasagittal skin incision from just behind the eyes to almost between the ears in one single, firm motion using a No. 11 scalpel blade. Move skin aside and clip right side with hemostat.</li>
	<li>Clear skull of overlying membrane using the dull side of the No. 11 scalpel and cotton tipped applicators. Optionally, wipe the skull with cotton tipped applicator dipped in 0.9% saline solution. Allow to dry completely.</li>
	<li>Using a small ruler, mark the anterior border of the craniotomy at 1 mm caudal to the coronal suture, and the left edge of the craniotomy at 1 mm lateral to the sagittal suture (Figure 1), with a permanent marker. Then mark the right and caudal borders of the craniotomy at 4 mm from the sagittal and coronal sutures, respectively (Figure 1).</li>
	<li>Using a 0.5 mm drill bit, begin to make craniotomy by drilling slowly following the permanent marker outline. Be sure not to break through the skull completely. Press gently on the isolated piece of parietal bone with No. 5 forceps, areas of weakness will give way to the pressure. When the thinned bone is sufficiently weak throughout the perimeter, the bone piece is ready for removal. 
	NOTE: If the investigator experiences difficulty removing the bone in one piece, this suggests that bone was not sufficiently thinned during drilling. Consider drilling the skull further in subsequent animals to facilitate easy removal of the bone piece.</li>
	<li>Using a 10 ml syringe fitted with a 23 G needle, apply a small amount of 0.9% saline to soak the isolated bone and drilled area.</li>
	<li>Attach the manipulator arm to the stereotaxic equipment. Attach a new No. 11 scalpel blade to the probe holder with the sharp side of the blade facing rostrally. 
	NOTE: Although the rostral and caudal tissues experience the sharp and blunt edges of the blade, respectively, the mechanical damage induced by the penetrating injury is comparable throughout the extent of the lesion. We observe no appreciable differences in major features of reactive gliosis including upregulation of GFAP expression or proliferation, between rostral and caudal sections.</li>
	<li>Keeping the manipulator arm out of the way, carefully lift the isolated bone using 5/45 angled forceps. Insert the tip of the forceps into the side of the isolated bone and lift, using leverage to pull off the piece of bone left behind in one full movement. 
	NOTE: Be careful not to stab the brain or disturb the dura underneath the skull.</li>
	<li>Take a small piece of the soaked absorbable gel foam and place on the uncovered brain to prevent it from drying out and soak up any blood that might be present.</li>
	<li>Once the gel foam is in place, swing the manipulator arm into place and adjust blade to center of craniotomy over the gel foam. Remove the gel foam and lower the blade until the tip touches the dura without puncturing the dura. Mark dorsal/ventral coordinates using the vernier scale on the vertical arm of the stereotaxic.</li>
	<li>Using the manipulator arm, slowly lower the blade precisely 3 mm into the brain. This is achieved by using the vernier scale markings on the manipulator arm. Allow blade to stay in place for 5-10 sec. Move the stereotaxic arm with blade attachment rostral to caudal three times allowing the blade to reach the rostral and caudal boundaries of the craniotomy before moving to the opposite end. 
	NOTE: The dura is not removed prior to inserting the blade. In contrast to the rat dura, which is ~80 &#956;m in thickness7, the mouse dura is considerably thinner (only a few cell layers thick) and does not produce an appreciable resistance to the scalpel blade during insertion. Use a new scalpel blade for each mouse to ensure that each animal receives a consistent injury.</li>
	<li>Slowly raise the stereotaxic arm, removing the blade from the brain. After removal of the blade, immediately place another piece of gelfoam on the brain surface to soak up any excess blood or fluid.</li>
	<li>Meanwhile, remove the stereotaxic arm and dispose of the No. 11 scalpel blade. Once bleeding has stopped, remove the gelfoam.</li>
	<li>Close the wound by suturing the skin with non-absorbable suture, such as ethilon or prolene. Sutures should be removed 9-10 days following surgery.</li>
	<li>Return the mouse to its home cage, and allow the mouse to recover slowly on a heating pad and monitor for any signs of distress. Recovery from isofluorane-induced anesthesia typically occurs within 2-5 min after removal from the isofluorane. Do not leave the animal unattended until it has regained sternal recumbancy.</li>
	<li>Administer 0.5-1 ml of lactated Ringer&#8217;s solution subcutaneously to ensure hydration.</li>
</ol>
4. Post-surgical Care
<ol>
	<li>Closely monitor animals post-operatively until recovery from anesthesia before returning to the colony.
	<ol>
		<li>To reduce pain and discomfort post-operatively, administer 0.05-0.1 mg/kg buprenorphine by i.p. injection immediately following the procedure.</li>
		<li>Observe animals for 2-3 days post-operatively for severe signs of distress such as restricted movements, lack of grooming, or weight loss. Euthanize animals demonstrating any of these signs of distress and remove from the study.</li>
	</ol>
	</li>
	<li>To examine histopathology of injured tissues, euthanize animals by standard intracardial perfusion.
	<ol>
		<li>Briefly, anesthetize animals with an overdose of ketamine/xylazine, then intracardially perfused with 15-20 ml&#160;of 0.9% NaCl, or until the liver is cleared of blood, followed by 60 ml&#160;of 4% paraformaldehyde at the conclusion of the experiment.</li>
	</ol>
	</li>
	<li>Dissect brains and post-fix for 2-4 hr in 4% paraformaldehyde before transferring to 30% sucrose solution. Section brains on a cryostat at 40-60 &#181;m, and process by standard histological or immunohistochemical procedures, or as described in Garcia8,9.</li>
</ol>

<ol>
	<li>Hamby, M. E., Sofroniew, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reactive+astrocytes+as+therapeutic+targets+for+CNS+disorders.">Reactive astrocytes as therapeutic targets for CNS disorders.</a> Neurotherapeutics. 7 (4), 494-506 (2010).</li><li>Bush, T. G., 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=Leukocyte+infiltration,+neuronal+degeneration,+and+neurite+outgrowth+after+ablation+of+scar-forming,+reactive+astrocytes+in+adult+transgenic+mice.">Leukocyte infiltration, neuronal degeneration, and neurite outgrowth after ablation of scar-forming, reactive astrocytes in adult transgenic mice.</a> Neuron. 23 (2), 297-308 (1999).</li><li>Faulkner, J. 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=Reactive+astrocytes+protect+tissue+and+preserve+function+after+spinal+cord+injury.">Reactive astrocytes protect tissue and preserve function after spinal cord injury.</a> The Journal of neuroscience : the official journal of the Society for Neuroscience. 24 (9), 2143-2155 (2004).</li><li>Okada, S., 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=Conditional+ablation+of+Stat3+or+Socs3+discloses+a+dual+role+for+reactive+astrocytes+after+spinal+cord+injury.">Conditional ablation of Stat3 or Socs3 discloses a dual role for reactive astrocytes after spinal cord injury.</a> Nature Medicine. 12 (7), 829-834 (2006).</li><li>Voskuhl, R. 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=Reactive+Astrocytes+Form+Scar-Like+Perivascular+Barriers+to+Leukocytes+during+Adaptive+Immune+Inflammation+of+the+CNS.">Reactive Astrocytes Form Scar-Like Perivascular Barriers to Leukocytes during Adaptive Immune Inflammation of the CNS.</a> Journal of Neuroscience. 29 (37), 11511-11522 (2009).</li><li>Burda, J. E., Sofroniew, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reactive+Gliosis+and+the+Multicellular+Response+to+CNS+Damage+and+Disease.">Reactive Gliosis and the Multicellular Response to CNS Damage and Disease.</a> Neuron. 81 (2), 229-248 (2014).</li><li>Maikos, J. T., Elias, R. A., Shreiber, D. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+properties+of+dura+mater+from+the+rat+brain+and+spinal+cord.">Mechanical properties of dura mater from the rat brain and spinal cord.</a> Journal of neurotrauma. 25 (1), 38-51 (2008).</li><li>Garcia, A. D., Doan, N. B., Imura, T., Bush, T. G., Sofroniew, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GFAP-expressing+progenitors+are+the+principal+source+of+constitutive+neurogenesis+in+adult+mouse+forebrain.">GFAP-expressing progenitors are the principal source of constitutive neurogenesis in adult mouse forebrain.</a> Nat Neurosci. 7 (11), 1233-1241 (2004).</li><li>Garcia, A. D., Petrova, R., Eng, L., Joyner, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sonic+hedgehog+regulates+discrete+populations+of+astrocytes+in+the+adult+mouse+forebrain.">Sonic hedgehog regulates discrete populations of astrocytes in the adult mouse forebrain.</a> J Neurosci. 30 (41), 13597-13608 (2010).</li><li>Amat, J. A., Ishiguro, H., Nakamura, K., Norton, W. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenotypic+diversity+and+kinetics+of+proliferating+microglia+and+astrocytes+following+cortical+stab+wounds.">Phenotypic diversity and kinetics of proliferating microglia and astrocytes following cortical stab wounds.</a> Glia. 16 (4), 368-382 (1996).</li><li>Hozumi, I., 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=Administration+of+prosaposin+ameliorates+spatial+learning+disturbance+and+reduces+cavity+formation+following+stab+wounds+in+rat+brain.">Administration of prosaposin ameliorates spatial learning disturbance and reduces cavity formation following stab wounds in rat brain.</a> Neuroscience letters. 267 (1), 73-76 (1999).</li><li>Ness, R., David, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leptomeningeal+cells+modulate+the+neurite+growth+promoting+properties+of+astrocytes+in+vitro.">Leptomeningeal cells modulate the neurite growth promoting properties of astrocytes in vitro.</a> Glia. 19 (1), 47-57 (1997).</li><li>Byrnes, K. R., Fricke, S. T., Faden, A. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuropathological+differences+between+rats+and+mice+after+spinal+cord+injury.">Neuropathological differences between rats and mice after spinal cord injury.</a> Journal of magnetic resonance imaging : JMRI. 32 (4), 836-846 (2010).</li><li>Steward, O., 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=Genetic+approaches+to+neurotrauma+research:+opportunities+and+potential+pitfalls+of+murine+models.">Genetic approaches to neurotrauma research: opportunities and potential pitfalls of murine models.</a> Experimental neurology. 157 (1), 19-42 (1999).</li><li>Davalos, D., 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=ATP+mediates+rapid+microglial+response+to+local+brain+injury+in+vivo.">ATP mediates rapid microglial response to local brain injury in vivo.</a> Nature neuroscience. 8 (6), 752-758 (2005).</li><li>Hanisch, U. -. K., Kettenmann, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglia:+active+sensor+and+versatile+effector+cells+in+the+normal+and+pathologic+brain.">Microglia: active sensor and versatile effector cells in the normal and pathologic brain.</a> Nature neuroscience. 10 (11), 1387-1394 (2007).</li><li>Neumann, H., Kotter, M. R., Franklin, R. J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Debris+clearance+by+microglia:+an+essential+link+between+degeneration+and+regeneration.">Debris clearance by microglia: an essential link between degeneration and regeneration.</a> Brain. 132 (2), 288-295 (2008).</li><li>Nimmerjahn, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resting+Microglial+Cells+Are+Highly+Dynamic+Surveillants+of+Brain+Parenchyma+in+Vivo.">Resting Microglial Cells Are Highly Dynamic Surveillants of Brain Parenchyma in Vivo.</a> Science. 308 (5726), 1314-1318 (2005).</li><li>Horn, K. P., Busch, S. A., Hawthorne, A. L., van Rooijen, N., Silver, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Another+Barrier+to+Regeneration+in+the+CNS:+Activated+Macrophages+Induce+Extensive+Retraction+of+Dystrophic+Axons+through+Direct+Physical+Interactions.">Another Barrier to Regeneration in the CNS: Activated Macrophages Induce Extensive Retraction of Dystrophic Axons through Direct Physical Interactions.</a> Journal of Neuroscience. 28 (38), 9330-9341 (2008).</li><li>Ip, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immune+Cells+Contribute+to+Myelin+Degeneration+and+Axonopathic+Changes+in+Mice+Overexpressing+Proteolipid+Protein+in+Oligodendrocytes.">Immune Cells Contribute to Myelin Degeneration and Axonopathic Changes in Mice Overexpressing Proteolipid Protein in Oligodendrocytes.</a> Journal of Neuroscience. 26 (31), 8206-8216 (2006).</li><li>Perry, V. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contribution+of+systemic+inflammation+to+chronic+neurodegeneration.">Contribution of systemic inflammation to chronic neurodegeneration.</a> Acta neuropathologica. 120 (3), 277-286 (2010).</li><li>McKeon, R. J., Jurynec, M. J., Buck, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+chondroitin+sulfate+proteoglycans+neurocan+and+phosphacan+are+expressed+by+reactive+astrocytes+in+the+chronic+CNS+glial+scar.">The chondroitin sulfate proteoglycans neurocan and phosphacan are expressed by reactive astrocytes in the chronic CNS glial scar.</a> Journal of Neuroscience. 19 (24), 10778-10788 (1999).</li><li>Silver, J., Miller, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regeneration+beyond+the+glial+scar.">Regeneration beyond the glial scar.</a> Nature reviews. Neuroscience. 5 (2), 146-156 (2004).</li><li>Zamanian, J. 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=Genomic+analysis+of+reactive+astrogliosis.">Genomic analysis of reactive astrogliosis.</a> J Neurosci. 32 (18), 6391-6410 (2012).</li><li>Buffo, 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=Origin+and+progeny+of+reactive+gliosis:+A+source+of+multipotent+cells+in+the+injured+brain.">Origin and progeny of reactive gliosis: A source of multipotent cells in the injured brain.</a> Proceedings of the National Academy of Sciences of the United States of America. 105 (9), 3581-3586 (2008).</li><li>Sirko, S., 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=Reactive+glia+in+the+injured+brain+acquire+stem+cell+properties+in+response+to+sonic+hedgehog.+[corrected].">Reactive glia in the injured brain acquire stem cell properties in response to sonic hedgehog. [corrected].</a> Cell stem cell. 12 (4), 426-439 (2013).</li></ol>

The authors have nothing to disclose.

It is critical that the skull or underlying dura are not damaged during the drilling. Use light pressure while drilling to ensure the skull is not punctured. In addition, care should be taken while lifting the skull piece to ensure the dura is not lifted off with the bone.
The forebrain stab injury described here models a penetrating injury to the CNS. Though less clinically translatable than TBI models such as FPI or CCI, the forebrain stab lesion model serves as a useful tool for a broad ran...

A major challenge for developing successful therapies to treat the injured CNS is an incomplete understanding of the complex multicellular events that are triggered by the trauma. Reactive astrocytes are gaining increasing recognition as a promising target for novel therapies1. Though historically regarded as hostile to neural repair, reactive astrocytes are now recognized as critical components of a complex, multicellular neuroprotective response that includes attenuation of inflammatory processes and limiting secondary damage and neurodegeneration2-6. Although the neuropathological characteristics of reactive gliosis have long been well defined, the cellular and molecular mechanisms regulating reactive gliosis, and the diverse array of downstream consequences remain poorly understood. Understanding the mechanisms that drive reactive gliosis, as well as the subsequent cellular and molecular events, is an important step towards developing strategies aimed at promoting the neuroprotective properties of reactive gliosis, while attenuating the detrimental effects. 
Here we demonstrate a method to induce severe reactive astrogliosis in the forebrain of adult mice using a stab injury. In contrast to other traumatic brain injury (TBI) models, such as controlled cortical impact (CCI) or fluid percussion injury (FPI), which require specialized equipment to produce an injury, the forebrain stab requires only a stereotaxic device to stabilize the head and a No. 11 scalpel blade. Thus the forebrain stab lesion model is more broadly accessible to a wide range of laboratories that do not have access to the specialized devices necessary for creating an FPI or CCI injury. The method described here enables investigators to reliably and reproducibly trigger a robust gliosis response to investigate subsequent cellular and molecular events. Once recovered from surgery, animals that have received a forebrain stab injury can survive for prolonged periods without the need for specialized care and can be returned to the colony for acute, intermediate, or chronic studies. Though less clinically translatable than FPI or CCI models of TBI, a forebrain lesion produced by a stab injury serves as a simple yet useful experimental model to investigate basic biological mechanisms underlying reactive gliosis and other neuropathological events following trauma to the CNS.

<table><tbody><tr><td>Stereotax</td><td>Harvard Apparatus</td><td>726049</td><td></td></tr><tr><td>High speed micro drill</td><td>Harvard Apparatus</td><td>724950</td><td></td></tr><tr><td>stainless steel scalpel blade, #11</td><td>MedVet</td><td>JOR581S</td><td></td></tr><tr><td>5/45 angled forceps</td><td>Fine Science Tools</td><td>11251-35</td><td></td></tr><tr><td>Gelfoam sponge 12 cm x 7 mm</td><td>Fisher</td><td>NC9841478</td><td></td></tr><tr><td>Rb anti-GFAP</td><td>DAKO</td><td>&amp;nbsp;Z033429-2</td><td>Dilution - 1:20,000 (bright-field); 1:1,000 (fluorescence)</td></tr><tr><td>Shp anti-BrdU</td><td>Abcam</td><td>ab1893</td><td>Dilution - 1:20,000 (bright-field); 1:500 (fluorescence)</td></tr><tr><td>Biotinylated goat anti-rabbit</td><td>Vector Laboratories</td><td>BA-1000&amp;nbsp;</td><td>Dilution - 1:400 (bright-field)</td></tr><tr><td>Biotinylated rabbit anti-sheep</td><td>Vector Laboratories</td><td>BA-6000</td><td>Dilution - 1:400 (bright-field)</td></tr><tr><td>Alexafluor 488 goat anti-rabbit</td><td>Life Technologies</td><td>A-11008</td><td>Dilution - 1:400 (bright-field)</td></tr><tr><td>Alexafluor 568 donkey anti-sheep</td><td>Life Technologies</td><td>A-21099</td><td>Dilution - 1:1,000 (fluorescence)</td></tr><tr><td>DAPI Nucleic Acid Stain</td><td>Life Technologies</td><td>D3571</td><td>Dilution - 1:1,000 (fluorescence)</td></tr><tr><td>Cresyl Violet Acetate</td><td>Sigma Aldrich</td><td>C5042-10G</td><td>Dilution - 1% (bright-field)</td></tr></tbody></table>

triggering reactive gliosis in vivo by a forebrain stab injury

Following injury to the CNS, astrocytes undergo a broad range of biochemical, morphological, and molecular changes collectively referred to as reactive astrogliosis. Reactive astrocytes exert both inflammatory and protective effects that inhibit and promote, respectively, neural repair. The mechanisms underlying the diverse functional properties of reactive astrogliosis are not well understood. Achieving a greater understanding of these mechanisms is critical to developing therapeutic strategies to treat the injured CNS. Here we demonstrate a method to trigger reactive astrogliosis in the adult mouse forebrain using a forebrain stab lesion. This lesion model is simple, reliable, and requires only a stereotaxic device and a scalpel blade to produce the injury. The use of stab lesions as an injury model in the forebrain is well established and amenable to studies addressing a broad range of neuropathological outcomes, such as neuronal degeneration, neuroinflammation, and disruptions in the blood brain barrier (BBB). Thus, the forebrain stab injury model serves as a powerful tool that can be applied for a broad range of studies on the CNS response to trauma.

Because animals undergoing this procedure do not require specialized post-operative care, short or long-term time survival periods are easily incorporated into the study, depending on the need to investigate acute or chronic pathology following injury. Principal features of reactive gliosis, such as upregulation of GFAP and hypertrophy of soma, can be observed as early as 2-3 days following injury. The peak phase of proliferation for reactive astrocytes is during days 3-5 following injury10. The representative...

Watch this Scientific Journal Video about Triggering Reactive Gliosis In Vivo by a Forebrain Stab Injury at JoVE.com

Triggering Reactive Gliosis In Vivo by a Forebrain Stab Injury

This article describes a detailed protocol to produce a forebrain stab injury in adult mice. The stab injury induces severe reactive gliosis and glial scar formation which can be subsequently examined by standard immunohistochemistry methods.

Triggering Reactive Gliosis In Vivo by a Forebrain Stab Injury

Following injury to the CNS, astrocytes undergo a broad range of biochemical, morphological, and molecular changes collectively referred to as reactive ...

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10.5K Views.  Drexel University. This article describes a detailed protocol to produce a forebrain stab injury in adult mice. The stab injury induces severe reactive gliosis and glial scar formation which can be subsequently examined by standard immunohistochemistry methods. 

Video: Triggering Reactive Gliosis In Vivo by a Forebrain Stab Injury

An Injury Paradigm to Investigate Central Nervous System Repair in Drosophila

An experimental method has been developed to investigate the cellular responses to central nervous system (CNS) injury using the fruit-fly Drosophila. Understanding repair and regeneration in animals is a key question in biology. The damaged human CNS does not regenerate, and understanding how to promote the regeneration is one of main goals of medical neuroscience. The powerful genetic toolkit of Drosophila can be used to tackle the problem of CNS regeneration.
A lesion to the CNS ventral nerve cord (VNC, equivalent to the vertebrate spinal cord) is applied manually with a tungsten needle. The VNC can subsequently be filmed in time-lapse using laser scanning confocal microscopy for up to 24 hr to follow the development of the lesion over time. Alternatively, it can be cultured, then fixed and stained using immunofluorescence to visualize neuron and glial cells with confocal microscopy. Using appropriate markers, changes in cell morphology and cell state as a result of injury can be visualized. With ImageJ and purposely developed plug-ins, quantitative and statistical analyses can be carried out to measure changes in wound size over time and the effects of injury in cell proliferation and cell death. These methods allow the analysis of large sample sizes. They can be combined with the powerful genetics of Drosophila to investigate the molecular mechanisms underlying CNS regeneration and repair.

An Injury Paradigm to Investigate Central Nervous System Repair in Drosophila

An injury paradigm using the Drosophila larval ventral nerve cord to investigate central nervous system regeneration and repair is described. Stabbing followed by laser scanning confocal microscopy in time-lapse and fixed specimens, combined with quantitative analysis with purposefully developed software and genetics, are used to investigate the molecular mechanisms of CNS regeneration and repair.

An experimental method has been developed to investigate the cellular responses to central nervous system (CNS) injury using the fruit-fly Drosophila. ...

Neuroscience

Acute Brain Trauma in Mice Followed By Longitudinal Two-photon Imaging

Although acute brain trauma often results from head damage in different accidents and affects a substantial fraction of the population, there is no effective treatment for it yet. Limitations of currently used animal models impede understanding of the pathology mechanism. Multiphoton microscopy allows studying cells and tissues within intact animal brains longitudinally under physiological and pathological conditions. Here, we describe two models of acute brain injury studied by means of two-photon imaging of brain cell behavior under posttraumatic conditions. A selected brain region is injured with a sharp needle to produce a trauma of a controlled width and depth in the brain parenchyma. Our method uses stereotaxic prick with a syringe needle, which can be combined with simultaneous drug application. We propose that this method can be used as an advanced tool to study cellular mechanisms of pathophysiological consequences of acute trauma in mammalian brain in vivo. In this video, we combine acute brain injury with two preparations: cranial window and skull thinning. We also discuss advantages and limitations of both preparations for multisession imaging of brain regeneration after trauma.

Acute brain trauma is a severe injury that has no adequate treatment to date. Multiphoton microscopy allows studying longitudinally the process of acute brain trauma development and probing therapeutical strategies in rodents. Two models of acute brain trauma studied with in vivo two-photon imaging of brain are demonstrated in this protocol.

Although acute brain trauma often results from head damage in different accidents and affects a substantial fraction of the population, there is no ...

Stab Wound Injury of the Zebrafish Adult Telencephalon: A Method to Investigate Vertebrate Brain Neurogenesis and Regeneration

Adult zebrafish have an amazing capacity to regenerate their central nervous system after injury. To investigate the cellular response and the molecular mechanisms involved in zebrafish adult central nervous system (CNS) regeneration and repair, we developed a zebrafish model of adult telencephalic injury.
In this approach, we manually generate an injury by pushing an insulin syringe needle into the zebrafish adult telencephalon. At different post injury days, fish are sacrificed, their brains are dissected out and stained by immunohistochemistry and/or in situ hybridization (ISH) with appropriate markers to observe cell proliferation, gliogenesis, and neurogenesis. The contralateral unlesioned hemisphere serves as an internal control. This method combined for example with RNA deep sequencing can help to screen for new genes with a role in zebrafish adult telencephalon neurogenesis, regeneration, and repair.

To shed light on the cellular and molecular mechanisms of zebrafish adult neurogenesis and regeneration, we developed a protocol for invasive surgery causing mechanical injuries in the zebrafish adult telencephalon and subsequent monitoring of changes in the stabbed hemisphere by immunohistochemistry or in situ hybridization.

Adult zebrafish have an amazing capacity to regenerate their central nervous system after injury. To investigate the cellular response and the molecular ...

Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice

Current astrocytoma models are limited in their ability to define the roles of oncogenic mutations in specific brain cell types during disease pathogenesis and their utility for preclinical drug development. In order to design a better model system for these applications, phenotypically wild-type cortical astrocytes and neural stem cells (NSC) from conditional, genetically engineered mice (GEM) that harbor various combinations of floxed oncogenic alleles were harvested and grown in culture. Genetic recombination was induced in vitro using adenoviral Cre-mediated recombination, resulting in expression of mutated oncogenes and deletion of tumor suppressor genes. The phenotypic consequences of these mutations were defined by measuring proliferation, transformation, and drug response in vitro. Orthotopic allograft models, whereby transformed cells are stereotactically injected into the brains of immune-competent, syngeneic littermates, were developed to define the role of oncogenic mutations and cell type on tumorigenesis in vivo. Unlike most established human glioblastoma cell line xenografts, injection of transformed GEM-derived cortical astrocytes into the brains of immune-competent littermates produced astrocytomas, including the most aggressive subtype, glioblastoma, that recapitulated the histopathological hallmarks of human astrocytomas, including diffuse invasion of normal brain parenchyma. Bioluminescence imaging of orthotopic allografts from transformed astrocytes engineered to express luciferase was utilized to monitor in vivo tumor growth over time. Thus, astrocytoma models using astrocytes and NSC harvested from GEM with conditional oncogenic alleles provide an integrated system to study the genetics and cell biology of astrocytoma pathogenesis in vitro and in vivo and may be useful in preclinical drug development for these devastating diseases.

Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice

Phenotypically wild-type astrocytes and neural stem cells harvested from mice engineered with floxed, conditional oncogenic alleles and transformed via viral Cre-mediated recombination can be used to model astrocytoma pathogenesis in vitro and in vivo by orthotopic injection of transformed cells into brains of syngeneic, immune-competent littermates.

Current astrocytoma models are limited in their ability to define the roles of oncogenic mutations in specific brain cell types during disease ...

Stab Wound Injury Model of the Adult Optic Tectum Using Zebrafish and Medaka for the Comparative Analysis of Regenerative Capacity

While zebrafish have a superior capacity to regenerate their central nervous system (CNS), medaka has a lower CNS regenerative capacity. A brain injury model was developed in the adult optic tectum of zebrafish and medaka and comparative histological and molecular analyses were performed to elucidate the molecular mechanisms regulating the high regenerative capacity of this tissue across these fish species. Here a stab wound injury model is presented for the adult optic tectum using a needle and histological analyses for proliferation and differentiation of the neural stem cells (NSCs). A needle was manually inserted into the central region of the optic tectum, and then the fish were intracardially perfused, and their brains were dissected. These tissues were then cryosectioned and evaluated using immunostaining against the appropriate NSC proliferation and differentiation markers. This tectum injury model provides robust and reproducible results in both zebrafish and medaka, allowing for comparing NSC responses after injury. This method is available for small teleosts, including zebrafish, medaka, and African killifish, and enables us to compare their regenerative capacity and investigate unique molecular mechanisms.

A mechanical brain injury model in the adult zebrafish is described to investigate the molecular mechanisms regulating their high regenerative capacity. The method explains to create a stab wound injury in the optic tectum of multiple species of small fish to evaluate the regenerative responses using fluorescent immunostaining.

While zebrafish have a superior capacity to regenerate their central nervous system (CNS), medaka has a lower CNS regenerative capacity. A brain injury ...

Developmental Biology

A Drosophila In Vivo Injury Model for Studying Neuroregeneration in the Peripheral and Central Nervous System

The regrowth capacity of damaged neurons governs neuroregeneration and functional recovery after nervous system trauma. Over the past few decades, various intrinsic and extrinsic inhibitory factors involved in the restriction of axon regeneration have been identified. However, simply removing these inhibitory cues is insufficient for successful regeneration, indicating the existence of additional regulatory machinery. Drosophila melanogaster, the fruit fly, shares evolutionarily conserved genes and signaling pathways with vertebrates, including humans. Combining the powerful genetic toolbox of flies with two-photon laser axotomy/dendriotomy, we describe here the Drosophila sensory neuron &#8211; dendritic arborization (da) neuron injury model as a platform for systematically screening for novel regeneration regulators. Briefly, this paradigm includes a) the preparation of larvae, b) lesion induction to dendrite(s) or axon(s) using a two-photon laser, c) live confocal imaging post-injury and d) data analysis. Our model enables highly reproducible injury of single labeled neurons, axons, and dendrites of well-defined neuronal subtypes, in both the peripheral and central nervous system.

A Drosophila In Vivo Injury Model for Studying Neuroregeneration in the Peripheral and Central Nervous System

Here, we present a protocol using the Drosophila sensory neuron - dendritic arborization (da) neuron injury model, which combines in vivo live imaging, two-photon laser axotomy/dendriotomy, and the powerful fly genetic toolbox, as a platform for screening potential promoters and inhibitors of neuroregeneration.

The regrowth capacity of damaged neurons governs neuroregeneration and functional recovery after nervous system trauma. Over the past few decades, various ...

Stimulating and Analyzing Adult Neurogenesis in the Drosophila Central Brain

The molecular and cellular mechanisms underlying neurogenesis in response to disease or injury are not well understood. However, understanding these mechanisms is crucial for developing neural regenerative therapies. Drosophila melanogaster is a leading model for studies of neural development but historically has not been exploited to investigate adult brain regeneration. This is primarily because the adult brain exhibits very low mitotic activity. Nonetheless, penetrating traumatic brain injury (PTBI) to the adult Drosophila central brain triggers the generation of new neurons and new glia. The powerful genetic tools available in Drosophila combined with the simple but rigorous injury protocol described here now make adult Drosophila brain a robust model for neural regeneration research. Provided here are detailed instructions for (1) penetrating injuries to the adult central brain and (2) dissection, immunohistochemistry, and imaging post-injury. These protocols yield highly reproducible results and will facilitate additional studies to dissect mechanisms underlying neural regeneration.

Stimulating and Analyzing Adult Neurogenesis in the Drosophila Central Brain

This article provides detailed protocols for inflicting Penetrating Traumatic Brain Injury (PTBI) to adult Drosophila and examining the resulting neurogenesis.

The molecular and cellular mechanisms underlying neurogenesis in response to disease or injury are not well understood. However, understanding these ...

Electromagnetic Controlled Closed-Head Model of Mild Traumatic Brain Injury in Mice

Highly reproducible animal models of traumatic brain injury (TBI), with well-defined pathologies, are needed for testing therapeutic interventions and understanding the mechanisms of how a TBI alters brain function. The availability of multiple animal models of TBI is necessary to model the different aspects and severities of TBI seen in people. This manuscript describes the use of a midline closed head injury (CHI) to develop a mouse model of mild TBI. The model is considered mild because it does not produce structural brain lesions based on neuroimaging or gross neuronal loss. However, a single impact creates enough pathology that cognitive impairment is measurable at least 1 month after injury. A step-by-step protocol to induce a CHI in mice using a stereotaxically guided electromagnetic impactor is defined in the paper. The benefits of the mild midline CHI model include the reproducibility of the injury-induced changes with low mortality. The model has been temporally characterized up to 1 year after the injury for neuroimaging, neurochemical, neuropathological, and behavioral changes. The model is complementary to open skull models of controlled cortical impact using the same impactor device. Thus, labs can model both mild diffuse TBI and focal moderate-to-severe TBI with the same impactor.

The protocol describes mild traumatic brain injury in a mouse model. In particular, a step-by-step protocol to induce a mild midline closed head injury and the characterization of the animal model is fully explained.

Highly reproducible animal models of traumatic brain injury (TBI), with well-defined pathologies, are needed for testing therapeutic interventions and ...

Intravital Imaging of Fluorescent Protein Expression in Mice with a Closed-Skull Traumatic Brain Injury and Cranial Window Using a Two-Photon Microscope

The goal of this protocol is to demonstrate how to longitudinally visualize the expression and localization of a protein of interest within specific cell types of an animal's brain, upon exposure to exogenous stimuli. Here, the administration of a closed-skull traumatic brain injury (TBI) and simultaneous implantation of a cranial window for subsequent longitudinal intravital imaging in mice is shown. Mice are intracranially injected with an adeno-associated virus (AAV) expressing enhanced green fluorescent protein (EGFP) under a neuronal specific promoter. After 2 to 4 weeks, the mice are subjected to a repetitive TBI using a weight drop device over the AAV injection location. Within the same surgical session, the mice are implanted with a metal headpost and then a glass cranial window over the TBI impacting site. The expression and cellular localization of EGFP is examined using a two-photon microscope in the same brain region exposed to trauma over the course of months.

This study demonstrates delivery of a repetitive traumatic brain injury to mice and simultaneous implantation of a cranial window for subsequent intravital imaging of a neuron-expressed EGFP using two-photon microscopy.

The goal of this protocol is to demonstrate how to longitudinally visualize the expression and localization of a protein of interest within specific cell ...

Stab-Wound Mouse Model for Studying Hemorrhage and Inflammation in Traumatic Brain Injury

Traumatic brain injury (TBI) results from physical damage, often caused by accidents or sports-related incidents. The causes of TBI are diverse, including concussions, brain contusions, hematomas, and skull fractures. To replicate these different causes, various TBI mouse models have been developed using distinct protocols. Physical brain injury leads to both primary and secondary brain injuries, which exacerbate neuronal loss. Primary injury occurs immediately after the damage, often due to hemorrhage, and subsequently triggers secondary injuries, including inflammation around the lesion. Developing a TBI model suitable for assessing hemorrhage extension and inflammatory severity is therefore crucial. This protocol introduces a method for mimicking penetrating brain injury, referred to as the stab-wound TBI mouse model, to study mechanisms of hemorrhage, inflammation, and neuronal loss associated with TBI pathology. This model is created by puncturing the skull and brain with needles and is simple to execute without the need for specialized experimental equipment. Additionally, the minor injury inflicted on the mouse cerebral cortex using a needle does not affect the animal's behavior post-surgery. This feature allows researchers to study the localized effects of brain injury without concerns about broader behavioral consequences. Sample data from stab-wounded mouse cerebral cortices demonstrate the model's effectiveness in assessing blood leakage into the parenchyma, glial activation, and inflammatory cytokine production. Furthermore, this protocol facilitates the evaluation of blood coagulants and anti-inflammatory compounds, aiding in the development of therapeutic agents for TBI.

Simplified traumatic brain injury (TBI) models have facilitated the development of therapeutic approaches. This protocol outlines the creation of a stab-wound mouse cortex using needles, enabling the analysis of hemorrhage and inflammation. The stab-wound TBI mouse model offers the advantage of being performed without requiring specialized equipment.

Traumatic brain injury (TBI) results from physical damage, often caused by accidents or sports-related incidents. The causes of TBI are diverse, including ...

Triggering Reactive Gliosis In Vivo by a Forebrain Stab Injury

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