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>

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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. 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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 range of studies aimed at investigating various biochemical, cellular, or molecular events triggered by a discrete CNS insult. Although cognitive deficits have been reported in rats, 3 weeks after a bilateral stab injury11, it should be noted that the unilateral stab lesion model, as described here, is best suited for studies addressing the cellular response to injury. Fundamental aspects of various neuropathological processes, such as reactive gliosis and scar formation can be readily observed and studied. In contrast to FPI or CCI which produce diffuse and widespread neuropathology, the localized glial activation and pathology, facilitate intra-animal comparisons, between the injured and uninjured hemispheres. In addition, the infiltration of meningeal cells into the CNS at the lesion site presents an opportunity to investigate the interactions between these cells and local reactive astrocytes. Indeed, such interactions are critical in the formation of scar tissue, and have been shown to negatively regulate the permissive properties of reactive astrocytes to axonal regeneration12.
Here we use standard immunohistochemical procedures to demonstrate some of the principal features of reactive astrogliosis such as hypertrophy of cell bodies and processes, increased GFAP expression, and injury-induced proliferation. Note that although cavities are not observed in mice at 1-2 weeks following this procedure, studies using rats report cavity formation, 3 weeks following the injury11. Differences in neuropathology between mice and rats are also observed in spinal cord injuries. Whereas rats that undergo a spinal cord injury exhibit cysts or cavity formation at the lesion site, such cavities do not form in mice13,14.
The procedure is simple, reliable, easily reproducible, and requires minimal equipment. It can be easily modified to perform rat or mouse studies. In particular, use of this model with various transgenic mouse lines or in pharmacological studies can provide novel insight into mechanisms regulating the CNS response to injury. Lesion size and severity can be modified by adjusting the depth of the blade and travel distance of the stereotaxic arm holding the blade to produce discrete punctures rather than longitudinal mechanical lesions. Thus, the forebrain stab injury model can serve as an excellent experimental platform with which to study specific aspects of various neuropathological responses to injury.
Injury to the CNS triggers a complex response that is dynamic and multicellular6. In addition to reactive astrocytes, microglia mobilize quickly to phagocytose debris and initiate both pro and anti-inflammatory signaling cascades15-18. Activated microglia and invading macrophages produce cytokines and chemokines, creating a hostile environment for normal cell function and survival19-21. Extracellular matrix (ECM) molecules, such as chondroitin sulfate proteoglycan (CSPG), are produced from a variety of cell types, including reactive astrocytes and fibroblasts, and create a hostile environment for the regeneration and structural reorganization of surviving neurons22,23. Here we demonstrate some of the principal features of reactive astrogliosis that occur following a forebrain stab injury. Upregulation of intermediate filaments such as GFAP, astrocyte proliferation, and glial scar formation, followed by subsequent tissue remodeling are readily assessed using standard immunohistochemistry procedures.
It should be noted that while some features of reactive gliosis are emphasized here, reactive gliosis is highly context dependent, with varying features and gene expression profiles that emerge depending on the specific trigger24. Nevertheless, a number of fundamental properties regarding the CNS response to injury and attempts at repair can be modeled and studied in forebrain stab lesioned tissue. Indeed, it has been shown that targeted ablation of proliferating reactive astrocytes following a forebrain stab injury leads to increased leukocyte infiltration into the CNS and increased neuronal degeneration, demonstrating the neuroprotective properties of reactive astrocytes2. More recently, reactive astrocytes isolated following a penetrating stab lesion, but not a non-invasive injury, demonstrate neural stem cell potential in vitro25,26. Thus the forebrain stab is a powerful injury model for studying a broad range of biochemical, cellular, and molecular events triggered by injury to the CNS. The ease and simplicity of this injury model will facilitate further studies that can lead to novel insights into the CNS response to injury and repair mechanisms.

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 border="0" cellpadding="0" cellspacing="0">
	<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 12cmx7mm</td>
			<td>Fisher</td>
			<td>NC9841478</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibodies and other reagents</td>
			<td>Manufacturer</td>
			<td>Catalog Number</td>
			<td>Dilution (brightfield) 
			Dilution (fluorescence)</td>
		</tr>
		<tr>
			<td>Rb anti-GFAP</td>
			<td>DAKO</td>
			<td>&#160;Z033429-2</td>
			<td>1:20k 
			1:1k</td>
		</tr>
		<tr>
			<td>Shp anti-BrdU</td>
			<td>Abcam</td>
			<td>ab1893</td>
			<td>1:2k 
			1:500</td>
		</tr>
		<tr>
			<td>Biotinylated goat anti-rabbit</td>
			<td>Vector Laboratories</td>
			<td>BA-1000&#160;</td>
			<td>1:400</td>
		</tr>
		<tr>
			<td>Biotinylated rabbit anti-sheep</td>
			<td>Vector Laboratories</td>
			<td>BA-6000</td>
			<td>1:400</td>
		</tr>
		<tr>
			<td>Alexafluor 488 goat anti-rabbit</td>
			<td>Life Technologies</td>
			<td>A-11008</td>
			<td>1:400</td>
		</tr>
		<tr>
			<td>Alexafluor 568 donkey anti-sheep</td>
			<td>Life Technologies</td>
			<td>A-21099</td>
			<td>1:1000</td>
		</tr>
		<tr>
			<td>DAPI Nucleic Acid Stain</td>
			<td>Life Technologies</td>
			<td>D3571</td>
			<td>1:1000</td>
		</tr>
		<tr>
			<td>Cresyl Violet Acetate</td>
			<td>Sigma Aldrich</td>
			<td>C5042-10G</td>
			<td>1%</td>
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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 results shown below are from animals that received a stab wound lesion 7 days earlier.
The general morphology and cytoarchitecture of the forebrain following a forebrain stab injury can be visualized by Nissl staining (Figure 2). Although the blade track is most prominent throughout the center of the lesion, the disrupted cortical cytoarchitecture reveals the rostral and caudal extent of the damaged tissue. Reactive astrocytes can be observed by immunohistochemistry for GFAP (Figure 3). Note that many cortical astrocytes do not exhibit immunohistochemically detectable levels of GFAP in the absence of injury. However, GFAP expression is dramatically upregulated in the hemisphere ipsilateral to the injury while remaining at relatively low levels in the contralateral hemisphere (Figure 3), suggesting that reactive astrocytes are restricted to the ipsilateral hemisphere. Note that while the cortical tissue ipsilateral to the lesion demonstrates marked upregulation of GFAP, other astrocytic markers, such as S100&#946; are constitutively expressed in the absence of an injury (Figure 4), and maintain similar expression levels following injury (Figure 4). In addition to increased GFAP expression, reactive astrocytes undergo cellular hypertrophy. Cell bodies and processes become enlarged and show intense staining for GFAP (Figure 3).
The proliferation of reactive astrocytes can be observed by administering the thymidine analog, 5-bromo-2&#39;-deoxyuridine&#160;(BrdU), or by immunostaining for the proliferative markers Ki67 or PCNA. We routinely administer 200 mg/kg BrdU, i.p., to animals during days 3-5 following injury, the peak of reactive gliosis10 (Figure 3). However the precise dosage and timing of BrdU should be independently considered for each study, bearing in mind that BrdU will permanently label cells undergoing proliferation, as well as their progeny, at the time of administration, but that cells that enter the cell cycle before BrdU starts, or after BrdU administration is complete, will not be marked. In Figure 4, we show extensive co-localization between GFAP and BrdU at 1 week post injury, indicating that many reactive astrocytes proliferated during the time course of BrdU administration. Note that proliferating reactive astrocytes are predominantly localized adjacent to the lesion core, whereas reactive astrocytes localized distal from the lesion core are largely non-proliferative (Figure 4).
<img alt="Figure 1" src="/files/ftp_upload/52825/52825fig1.jpg" /> 
Figure 1: Schematic of the mouse skull, depicting the area of the craniotomy. Blue lines depict the initial markings identifying the boundaries of the area to be drilled. The top and left marks are measured at 1 mm below or lateral to the coronal or sagittal sutures, respectively. The bottom and right marks are measured at 4 mm from the coronal and sagittal sutures, respectively. The craniotomy is performed by drilling a circle within the marked boundaries (dotted line), creating a craniotomy that is roughly 3 mm in diameter (dashed line). Schematic is not to scale.
<img alt="Figure 2" src="/files/ftp_upload/52825/52825fig2.jpg" /> 
Figure 2: Nissl staining throughout the rostral-caudal extent of the lesion volume. (A-C) Coronal slices of injured brains 1 week post injury, showing the hemisphere ipsilateral to the lesion. Insets depict zoomed in images of the boxed regions. The blade track is most prominent in the center of the lesion, ~2.5 mm from Bregma (arrow in B). Note the disrupted cortical cytoarchitecture (insets) in sections anterior (A) and posterior (C) to the lesion center. Scale bar 500 &#956;m, inset, 250 &#956;m. <a href="https://www.jove.com/files/ftp_upload/52825/52825fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/52825/52825fig3.jpg" /> 
Figure 3: Brightfield immunohistochemistry for GFAP 1 week following a forebrain stab injury. (A-B)&#160;Low magnification images of GFAP staining in the contralateral (A) and ipsilateral (B) hemispheres of the same tissue section from an injured animal. The lesion site is shown in (B), and the corresponding region in the uninjured contralateral hemisphere is shown in (A). Scale bar, 100 &#956;m. (C-D) High magnification images of normal (C) and reactive (D) astrocytes from the contralateral and ipsilateral hemispheres, respectively. Note the dramatic hypertrophy of the cell body and processes of reactive astrocyte in (D), compared to (C). Scale bar, 10 &#956;m.
<img alt="Figure 4" src="/files/ftp_upload/52825/52825fig4.jpg" /> 
Figure 4: Reactive astrocytes proliferate following a forebrain stab injury. (A-B) Immunofluorescent staining for BrdU (red) and GFAP (green) in uninjured, control (A) and injured (B) brains, 1 week following stab injury. (C-D) Immunofluroescent staining for BrdU (red) and the astrocytic marker S100&#946; (green) in the uninjured (C) and injured (D) hemispheres, 1 week following stab injury. Animals received BrdU over days 3-5 post injury. Note that many astrocytes are proliferating at the lesion site in the injured cortex (B and D, insets, arrowheads), whereas astrocytes in the uninjured cortex are not proliferating (A and C, insets). Counterstaining with DAPI (blue). Scale bars, 100 &#956;m, inset, 25 &#956;m.

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 ...

triggering-reactive-gliosis-in-vivo-by-a-forebrain-stab-injury

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10.4K 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 in vivo Rodent Model of Contraction-induced Injury and Non-invasive Monitoring of Recovery

Muscle strains are one of the most common complaints treated by physicians. A muscle injury is typically diagnosed from the patient history and physical exam alone, however the clinical presentation can vary greatly depending on the extent of injury, the patient's pain tolerance, etc. In patients with muscle injury or muscle disease, assessment of muscle damage is typically limited to clinical signs, such as tenderness, strength, range of motion, and more recently, imaging studies. Biological markers, such as serum creatine kinase levels, are typically elevated with muscle injury, but their levels do not always correlate with the loss of force production. This is even true of histological findings from animals, which provide a "direct measure" of damage, but do not account for all the loss of function. Some have argued that the most comprehensive measure of the overall health of the muscle in contractile force. Because muscle injury is a random event that occurs under a variety of biomechanical conditions, it is difficult to study. Here, we describe an in vivo animal model to measure torque and to produce a reliable muscle injury. We also describe our model for measurement of force from an isolated muscle in situ. Furthermore, we describe our small animal MRI procedure.

An in vivo Rodent Model of Contraction-induced Injury and Non-invasive Monitoring of Recovery

An in vivo animal model of injury is described. The method takes advantage of the subcutaneous position of the fibular nerve. Velocity, timing of muscle activation, and arc of motion are all pre-determined and synchronized using commercial software. Post injury changes are monitored in vivo using MR imaging/spectroscopy.

Muscle strains are one of the most  common complaints treated by physicians. A muscle injury is typically diagnosed from the patient history and  physical ...

Implantation of a Carotid Cuff for Triggering Shear-stress Induced Atherosclerosis in Mice

It is widely accepted that alterations in vascular shear stress trigger the expression of inflammatory genes in endothelial cells and thereby induce atherosclerosis (reviewed in 1 and 2). The role of shear stress has been extensively studied in vitro investigating the influence of flow dynamics on cultured endothelial cells 1,3,4 and in vivo in larger animals and humans 1,5,6,7,8. However, highly reproducible small animal models allowing systematic investigation of the influence of shear stress on plaque development are rare. Recently, Nam et al. 9 introduced a mouse model in which the ligation of branches of the carotid artery creates a region of low and oscillatory flow. Although this model causes endothelial dysfunction and rapid formation of atherosclerotic lesions in hyperlipidemic mice, it cannot be excluded that the observed inflammatory response is, at least in part, a consequence of endothelial and/or vessel damage due to ligation.
In order to avoid such limitations, a shear stress modifying cuff has been developed based upon calculated fluid dynamics, whose cone shaped inner lumen was selected to create defined regions of low, high and oscillatory shear stress within the common carotid artery 10. By applying this model in Apolipoprotein E (ApoE) knockout mice fed a high cholesterol western type diet, vascular lesions develop upstream and downstream from the cuff. Their phenotype is correlated with the regional flow dynamics 11 as confirmed by in vivo Magnetic Resonance Imaging (MRI) 12: Low and laminar shear stress upstream of the cuff causes the formation of extensive plaques of a more vulnerable phenotype, whereas oscillatory shear stress downstream of the cuff induces stable atherosclerotic lesions 11. In those regions of high shear stress and high laminar flow within the cuff, typically no atherosclerotic plaques are observed.
 
In conclusion, the shear stress-modifying cuff procedure is a reliable surgical approach to produce phenotypically different atherosclerotic lesions in ApoE-deficient mice.

The constricting cuff presented in this article is designed to induce atherosclerosis in the murine common carotid artery. Due to the conical shape of its inner lumen the implanted cuff generates well-defined regions of low, high and oscillatory shear stress triggering the development of atherosclerotic lesions of different inflammatory phenotypes.

It is widely accepted that alterations in vascular shear stress trigger the expression of inflammatory genes in endothelial cells and thereby induce ...

In vivo 19F MRI for Cell Tracking

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. Here, we describe a general protocol for cell labeling, imaging, and image processing. The technique is applicable to various cell types and animal models, although here we focus on a typical mouse model for tracking murine immune cells. The most important issues for cell labeling are described, as these are relevant to all models. Similarly, key imaging parameters are listed, although the details will vary depending on the MRI system and the individual setup. Finally, we include an image processing protocol for quantification. Variations for this, and other parts of the protocol, are assessed in the Discussion section. Based on the detailed procedure described here, the user will need to adapt the protocol for each specific cell type, cell label, animal model, and imaging setup. Note that the protocol can also be adapted for human use, as long as clinical restrictions are met.

In vivo 19F MRI for Cell Tracking

We describe a general protocol for in vivo cell tracking using MRI in a mouse model with ex vivo labeled cells. A typical protocol for cell labeling, image acquisition processing and quantification is included.

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. ...

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

A complex integration of molecular and electrical signals is needed to transform a quiescent uterus into a contractile organ at the end of pregnancy. Despite the discovery of key regulators of uterine contractility, this process is still not fully understood. Transgenic mice provide an ideal model in which to study parturition. Previously, the only method to study uterine contractility in the mouse was ex vivo isometric tension recordings, which are suboptimal for several reasons. The uterus must be removed from its physiological environment, a limited time course of investigation is possible, and the mice must be sacrificed. The recent development of radiometric telemetry has allowed for longitudinal, real-time measurements of in vivo intrauterine pressure in mice. Here, the implantation of an intrauterine telemeter to measure pressure changes in the mouse uterus from mid-pregnancy until delivery is described. By comparing differences in pressures between wild type and transgenic mice, the physiological impact of a gene of interest can be elucidated. This technique should expedite the development of therapeutics used to treat myometrial disorders during pregnancy, including preterm labor.

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

This manuscript provides a protocol for implanting telemeters in the mouse for the purpose of measuring intrauterine pressures during pregnancy.

A complex integration of molecular and electrical signals is needed to transform a quiescent uterus into a contractile organ at the end of pregnancy. ...

Detecting Anastasis In Vivo by CaspaseTracker Biosensor

Anastasis (Greek for &#8220;rising to life&#8221;) is a recently discovered cell recovery phenomenon whereby dying cells can reverse late-stage cell death processes that are generally assumed to be intrinsically irreversible. Promoting anastasis could in principle rescue or preserve injured cells that are difficult to replace such as cardiomyocytes or neurons, thereby facilitating tissue recovery. Conversely, suppressing anastasis in cancer cells, undergoing apoptosis after anti-cancer therapies, may ensure cancer cell death and reduce the chances of recurrence. However, these studies have been hampered by the lack of tools for tracking the fate of cells that undergo anastasis in live animals. The challenge is to identify the cells that have reversed the cell death process despite their morphologically normal appearance after recovery. To overcome this difficulty, we have developed Drosophila and mammalian CaspaseTracker biosensor systems that can identify and permanently track the anastatic cells in vitro or in vivo. Here, we present in vivo protocols for the generation and use of the CaspaseTracker dual biosensor system to detect and track anastasis in Drosophila melanogaster after transient exposure to cell death stimuli. While conventional biosensors and protocols can label cells actively undergoing apoptotic cell death, the CaspaseTracker biosensor can permanently label cells that have recovered after caspase activation - a hallmark of late-stage apoptosis, and simultaneously identify active apoptotic processes. This biosensor can also track the recovery of the cells that attempted other forms of cell death that directly or indirectly involved caspase activity. Therefore, this protocol enables us to continuously track the fate of these cells and their progeny, facilitating future studies of the biological functions, molecular mechanisms, physiological and pathological consequences, and therapeutic implications of anastasis. We also discuss the appropriate controls to distinguish cells that undergo anastasis from those that display non-apoptotic caspase activity in vivo.

Detecting Anastasis In Vivo by CaspaseTracker Biosensor

Anastasis is technically challenging to detect in vivo because the cells that have reversed the cell death process can be morphologically indistinguishable from normal healthy cells. Here we describe protocols for detecting and tracking cells that undergo anastasis in live animals by using our newly developed in vivo CaspaseTracker biosensor system.

Anastasis (Greek for &#8220;rising to life&#8221;) is a recently discovered cell recovery phenomenon whereby dying cells can reverse late-stage cell death ...

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic patient tumors in different assays1. Human tumor xenografts represent the gold standard with respect to tumor biology, drug discovery, and metastasis research 2-4. Tumor xenografts can be derived from different types of material like tumor cell lines, tumor tissue from primary patient tumors4 or serially transplanted tumors. When propagated in vivo, xenografted tissue is infiltrated and vascularized by cells of mouse origin. Multiple factors such as the tumor entity, the origin of xenografted material, growth rate and region of transplantation influence the composition and the amount of mouse cells present in tumor xenografts. However, even when these factors are kept constant, the degree of mouse cell contamination is highly variable.
Contaminating mouse cells significantly impair downstream analyses of human tumor xenografts. As mouse fibroblasts show high plating efficacies and proliferation rates, they tend to overgrow cultures of human tumor cells, especially slowly proliferating subpopulations. Mouse cell derived DNA, mRNA, and protein components can bias downstream gene expression analysis, next-generation sequencing, as well as proteome analysis 5. To overcome these limitations, we have developed a fast and easy method to isolate untouched human tumor cells from xenografted tumor tissue. This procedure is based on the comprehensive depletion of cells of mouse origin by combining automated tissue dissociation with the benchtop tissue dissociator and magnetic cell sorting. Here, we demonstrate that human target cells can be can be obtained with purities higher than 96% within less than 20 min independent of the tumor type.

Human tumor xenografts are vascularized and infiltrated by cells of mouse origin during the growth phase in vivo. To circumvent the bias caused by these contaminating cells during downstream analysis, we developed a method allowing for comprehensive depletion of all mouse cells by magnetic cell sorting.

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...

Open Tracheostomy Gastric Acid Aspiration Murine Model of Acute Lung Injury Results in Maximal Acute Nonlethal Lung Injury

Acid pneumonitis is a major cause of sterile acute lung injury (ALI) in humans. Acid pneumonitis spans the clinical spectrum from asymptomatic to acute respiratory distress syndrome (ARDS), characterized by neutrophilic alveolitis, and injury to both alveolar epithelium and vascular endothelium. Clinically, ARDS is defined by acute onset of hypoxemia, bilateral patchy pulmonary infiltrates and non-cardiogenic pulmonary edema. Human studies have provided us with valuable information about the physiological and inflammatory changes in the lung caused by ARDS, which has led to various hypotheses about the underling mechanisms. Unfortunately, difficulties determining the etiology of ARDS, as well as a wide range of pathophysiology have resulted in a lack of critical information that could be useful in developing therapeutic strategies.
Translational animal models are valuable when their pathogenesis and pathophysiology accurately reproduce a concept proven in both in vitro and clinical settings. Although large animal models (e.g., sheep) share characteristics of the anatomy of human trachea-bronchial tree, murine models provide a host of other advantages including: low cost; short reproductive cycle lending itself to greater data acquisition; a well understood immunologic system; and a well characterized genome leading to the availability of a variety of gene deletion and transgenic strains. A robust model of low pH induced ARDS requires a murine ALI that targets mainly the alveolar epithelium, secondarily the vascular endothelium, as well as the small airways leading to the alveoli. Furthermore, a reproducible injury with wide differences between different injurious and non-injurious insults is important.
The murine gastric acid aspiration model presented here using hydrochloric acid employs an open tracheostomy and recreates a pathogenic scenario that reproduces the low pH pneumonitis injury in humans. Additionally, this model can be used to examine interaction of a low pH insult with other pulmonary injurious entities (e.g., food particles, pathogenic bacteria).

This protocol induces acute lung injury in a mouse that has close fidelity to the pathogenesis of acid pneumonitis observed in humans. We generate a maximal acute nonlethal low pH lung injury and account for differences in rodent-human anatomic respiratory structure using an open tracheostomy coupled with circumferential pressure release.

Acid pneumonitis is a major cause of sterile acute lung injury (ALI) in humans. Acid pneumonitis spans the clinical spectrum from asymptomatic to acute ...

Reproducible Arterial Denudation Injury by Infrarenal Abdominal Aortic Clamping in a Murine Model

Percutaneous vascular interventions uniformly result in arterial denudation injuries that subsequently lead to thrombosis and restenosis. These complications can be attributed to impairments in re-endothelialization within the wound margins. Yet, the cellular and molecular mechanisms of re-endothelialization remain to be defined. While several animal models to study re-endothelialization after arterial denudation are available, few are performed in the mouse because of surgical limitations. This undermines the opportunity to exploit transgenic mouse lines and investigate the contribution of specific genes to the process of re-endothelialization. Here, we present a step-by-step protocol for creating a highly reproducible murine model of arterial denudation injury in the infrarenal abdominal aorta using external vascular clamping. Immunocytochemical staining of injured aortas for fibrinogen and &#946;-catenin demonstrate the exposure of a pro-thrombotic surface and the border of intact endothelium, respectively. The method presented here has the advantages of speed, excellent overall survival rate, and relative technical ease, creating a uniquely practical tool for imposing arterial denudation injury in transgenic mouse models. Using this method, investigators may elucidate the mechanisms of re-endothelialization under normal or pathological conditions.

Understanding the cellular and molecular mechanisms of re-endothelialization following arterial denudation injury is of paramount importance in preventing thrombosis and restenosis of arteries. Here we describe a protocol for reproducible arterial denudation injury of the infrarenal abdominal aorta. The procedure was developed to investigate the underlying mechanisms that regulate endothelial regeneration using mouse models.

Percutaneous vascular interventions uniformly result in arterial denudation injuries that subsequently lead to thrombosis and restenosis. These ...

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 ...

Inducing Acute Lung Injury in Mice by Direct Intratracheal Lipopolysaccharide Instillation

Airway administration of lipopolysaccharide (LPS) is a common way to study pulmonary inflammation and acute lung injury (ALI) in small animal models. Various approaches have been described, such as the inhalation of aerosolized LPS as well as nasal or intratracheal instillation. The presented protocol describes a detailed step-by-step procedure to induce ALI in mice by direct intratracheal LPS instillation and perform FACS analysis of blood samples, bronchoalveolar lavage (BAL) fluid, and lung tissue. After intraperitoneal sedation, the trachea is exposed and LPS is administered via a 22 G venous catheter. A robust and reproducible inflammatory reaction with leukocyte invasion, upregulation of proinflammatory cytokines, and disruption of the alveolo-capillary barrier is induced within hours to days, depending on the LPS dosage used. Collection of blood samples, BAL fluid, and lung harvesting, as well as the processing for FACS analysis, are described in detail in the protocol. Although the use of the sterile LPS is not suitable to study pharmacologic interventions in infectious diseases, the described approach offers minimal invasiveness, simple handling, and good reproducibility to answer mechanistic immunological questions. Furthermore, dose titration as well as the use of alternative LPS preparations or mouse strains allow modulation of the clinical effects, which can exhibit different degrees of ALI severity or early vs. late onset of disease symptoms.

Presented here is a step-by-step procedure to induce acute lung injury in mice by direct intratracheal lipopolysaccharide instillation and to perform FACS analysis of blood samples, bronchoalveolar lavage fluid, and lung tissue. Minimal invasiveness, simple handling, good reproducibility, and titration of disease severity are advantages of this approach.

Airway administration of lipopolysaccharide (LPS) is a common way to study pulmonary inflammation and acute lung injury (ALI) in small animal models. ...

Triggering Reactive Gliosis In Vivo by a Forebrain Stab Injury

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An in vivo Rodent Model of Contraction-induced Injury and Non-invasive Monitoring of Recovery

Implantation of a Carotid Cuff for Triggering Shear-stress Induced Atherosclerosis in Mice

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Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

Detecting Anastasis In Vivo by CaspaseTracker Biosensor

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Open Tracheostomy Gastric Acid Aspiration Murine Model of Acute Lung Injury Results in Maximal Acute Nonlethal Lung Injury

Reproducible Arterial Denudation Injury by Infrarenal Abdominal Aortic Clamping in a Murine Model

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

Inducing Acute Lung Injury in Mice by Direct Intratracheal Lipopolysaccharide Instillation