Name: Stab-Wound Mouse Model for Studying Hemorrhage and Inflammation in Traumatic Brain Injury (Video) | JoVE Journal
Uploaded: 2025-02-21T14:00:00.000+00:00
Duration: 4 min 3 s
Description: Learn a simple needle-based stab-wound mouse model for studying hemorrhage and inflammation in traumatic brain injury. This reproducible protocol enables analysis of neuronal and glial communication during TBI repair.

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5 minutes

We thank Ayana Hamano, Minori Yamashita, Misaki Endo, Hirono Kobayashi, and Nito Nakahira for helping with the immunohistochemistry and real-time qPCR. This work was supported by the JSPS KAKENHI 19K16122, Takeda Science Foundation, Astellas Foundation for Research on Metabolic Disorders, The Mitsubishi Foundation, Brain Science Foundation, and The Uehara Memorial Foundation to K.H.

All animal care protocols were approved by the Institutional Animal Care and Use Committee of Ochanomizu University, Japan, and were performed in accordance with the guidelines established by the Ministry of Education, Science, and Culture in Japan. Six-week-old adult C57BL/6J female mice (20-25 g) were used. All mice were provided ad libitum access to food and water in a clean environment. Details of the reagents and equipment used are listed in the Table of Materials.
1. Stab-wound surgery to the cerebral cortex
<ol>
	<li>Preparation of reagents 
	NOTE: The anesthetic protocol is similar to that previously described15. If the animal facility recommends a specific anesthetic, please follow their instructions. It has been previously confirmed that pentobarbital sodium is effective for stab-wound surgery in the cerebral cortex4,13,16,17.
	<ol>
		<li>Combination anesthetic MMB: Dissolve 1.875 mL of 1 mg/mL medetomidine, 2.0 mL of 5 mg/mL midazolam, and 2.5 mL of 5 mg/mL butorphanol tartrate in 18.625 mL of phosphate-buffered saline (PBS) to create a total volume of 25 mL. This mixture will have a concentration of 75 mg/L medetomidine, 400 mg/L midazolam, and 500 mg/L butorphanol tartrate mixture. Store at 4 &#176;C for up to 8 weeks prior to use.</li>
		<li>Anesthesia antagonist: Dissolve 150 &#956;L of 5 mg/mL atipamezole hydrochloride in 9.85 mL of PBS to create a total volume of 10 mL, with a concentration of 75 mg/L atipamezole hydrochloride. Store at 4&#160;&#176;C, protected from light, for up to 8 weeks prior to use.</li>
	</ol>
	</li>
	<li>Surgery procedure
	<ol>
		<li>Mouse anesthesia: Temporarily anesthetize the mice using an isoflurane inhalation solution-soaked paper until they fall asleep. Then, inject 10&#160;&#956;L/g combination anesthetic MMB intraperitoneal using a 1-mL syringe with a 27 G x 3/4&#34; needle. After 3-5 min, confirm the anesthesia effectiveness by performing a toe pinch and checking the righting reflex. 
		NOTE: Ensure to pay close attention not to give an overdose of anesthesia when using isoflurane inhalation solution.</li>
		<li>Place the mouse on its ventral side into a paper towel. After the transfer, reconfirm the mouse is still anesthetized with a toe pinch.</li>
		<li>Shave the fur on the skin of the occipital region. Sterilize the fur on the back of the mouse&#39;s head with 70% ethanol using a cotton swab. Grasp the occipital skin with blunt forceps and make a 1.0-1.5 mm-wide incision to expose the occipital bone without damaging the skull or any organ. Gently and slowly open the incision to observe the boundary between the cerebral cortex and cerebellum through the skull (Figure 1B). 
		NOTE: Ensure no damage occurs to the skull or brain for the no wound mouse.</li>
		<li>Mouse craniotomy: Create a small hole in the right hemisphere of the occipital bone using a 19 G x 1&#8226;1/2&#34; needle. Gently rotate the needle to make an insertion point for stab-wound needling, taking care not to damage the brain parenchyma. The hole should be positioned on the midpoint of right hemisphere interaural line between lambda and edge (Figure 1B). This hole serves as a guide for the insertion of the stab-wound needle in the subsequent step.</li>
		<li>Stab-wound: Insert a 27 G x 3/4&#34; needle from the insertion point and stab the cerebral cortex along the rostrocaudal axis (Figure 1B,C). The needle is inserted until it contacts the front of the skull, then gently withdrawn. The appropriate depth is one where the needle can be seen through the surface of the cerebral cortex. The left side of the cerebral cortex is used as the undamaged control.</li>
		<li>Suture the skin incision using a nylon 3-0 suture with a 1/2 round needle.</li>
		<li>Administer 10&#160;&#956;L/g of the anesthesia antagonist intraperitoneally as the combination anesthetic MMB. The mouse should be able to walk within 8-16 h after the anesthesia antagonist injection.</li>
	</ol>
	</li>
</ol>
2. Assessment of hemorrhage and recovery from BBB breakdown
<ol>
	<li>Immunoenzymatic staining of a stab-wounded brain section for mouse IgG leakage 
	NOTE: Here, slide-mounted frozen brain sections were used. Free-floating frozen brain sections are also suitable for this protocol.
	<ol>
		<li>Mouse anesthesia: Induce anesthesia by placing the mouse near an isoflurane inhalation solution-soaked paper until it falls asleep. Then, immediately administer 100 &#956;L/kg combination anesthetic MMB intraperitoneally. Confirm the effect of anesthesia with a toe pinch and righting reflex.</li>
		<li>Place the mouse on its back and pin both arms and feet onto the dissection board.</li>
		<li>Mouse thoracotomy: Grasp the abdominal skin using blunt forceps and make a 1.0-1.5 cm incision through the skin and peritoneum to expose the ribcage. Carefully open the ribcage with scissors while avoiding damage to the heart to expose it for cardiac perfusion.</li>
		<li>Cardiac perfusion fixation: Prepare a peristaltic perfusion pump and attach a tube with a 19 G x 1&#8226;1/2&#34; needle at one end. Fill the tube with PBS by turning on the pump before use. Then, while the pump is running, insert the needle tip into the left ventricle and secure it with forceps. Make a 0.5-1.0 mm incision in the right atrium. Perfuse the mouse18 at a rate of 4-6 mL/min for 5 min with at least 20 mL of PBS to remove the blood.</li>
		<li>Stop the pump and transfer the tube to a solution of 4% paraformaldehyde (PFA) in PBS. Restart the pump at a rate of 4-6 mL/min for 5 min with at least 20 mL of 4% PFA in PBS for fixation18. 
		NOTE: If the pump is not available, it is also possible to use a 50 mL syringe with a 19 G x 1&#8226;1/2&#34; needle for perfusion.</li>
		<li>Brain dissection: Place the mouse on its ventral side on the dissection board and expose the skull by making an incision through the midline of the skin from the occipital area to the nose. Carefully expose the brain completely by removing the skull with scissors, taking care not to damage the brain. Lift the brain with curved forceps and transfer it to a 15-mL tube filled with 10 mL of 4% PFA in PBS.</li>
		<li>Incubate the dissected brain overnight in the 4% PFA in PBS at 4&#160;&#176;C for complete fixation. Following this, incubate the brain overnight in 10 mL of 15% sucrose in PBS and transfer it to 10 mL of 30% sucrose in PBS for an additional day on a seesaw shaker. This gradual sucrose replacement helps to prevent tissue damage from ice when the brain is later stored in a freezer.</li>
		<li>Brain embedding: Embed the brain coronally in an embedding medium within a tissue embedding mold and then freeze using 2-methylbutane with dry ice19.</li>
		<li>Frozen brain sections: Using a cryostat, mount serial coronal sections (20 &#956;m-thick) of the embedded brain on a positive-charged slide glass. The brain sections are air-dried completely at room temperature overnight and stored in a -80&#160;&#176;C freezer.</li>
		<li>Blocking buffer preparation: Mix 10% newborn calf serum, 30 mg/mL bovine albumin, 10 mg/mL glycine, and 0.4% Triton X-100 in TBS to create the blocking buffer. Store at 4&#160;&#176;C for up to 2 weeks prior to use.</li>
		<li>Mouse IgG staining: After the stab-wounded cerebral cortex sections are air-dried at room temperature for 1 h, post-fix them with 500 &#956;L of 4% PFA per slide for 30 min. Then, wash the section with PBS (500 &#956;L/slide) for 5 min, followed by two washes with Tris-buffered saline (TBS, 500 &#956;L/slide) for 5 min each. 
		NOTE: Perform all procedures in a moisturizing box to prevent the buffers from evaporating.</li>
		<li>Quench endogenous enzyme activity in sections with 10% methanol and 3% hydrogen peroxide in TBS for 5 min, followed by two washes with Tris-buffered saline (TBS, 500 &#956;L/slide) for 5 min each.</li>
		<li>Perform blocking using a blocking buffer (500 &#956;L/slide) for 1 h at room temperature to prevent non-specific binding of the antibody. Then, incubate the sections with a biotin-conjugated mouse IgG antibody (1:300 dilution with the blocking buffer, 300 &#956;L/slide) for 1 h at room temperature.</li>
		<li>While incubating with the mouse IgG antibody, prepare the mixture of 1 &#956;L of Reagent A and 1 &#956;L of Reagent B from the avidin/biotin-based peroxidase system kit in 300 &#956;L of the blocking buffer. Incubate this mixture for 30 min at room temperature before use. After the mouse IgG antibody incubation is complete, wash the sections with TBS for 5 min, three times. Then, incubate the sections with the prepared mixture of Reagent A and B (300 &#956;L/slide) for 1 h at room temperature.</li>
		<li>Wash the sections with 0.1 M Tris-HCl pH 8.0 for 5 min, three times, over the course of 1 h. Then, develop color using 0.05% 3,3&#39;-diaminobenzidine (DAB) in 0.05% hydrogen peroxide-added 0.1 M Tris-HCl pH 8.0 (500 &#956;L/slide) for 5 min to 1 h (Figure 2A). Observe under a microscope for color change before stopping the reaction.</li>
		<li>Wash the sections with 0.1 M Tris-HCl pH 8.0 for 5 min three times, and air dry the sections for 15 min. Thereafter, dehydrate the sections by immersing them in 95% ethanol for 2 min twice, followed by 100% ethanol for 2 min, twice. Clear the sections in xylene for 5 min twice. Coverslip the sections using a water-insoluble mounting medium and allow them to dry.</li>
		<li>Analysis of mouse IgG staining intensity: Capture images of the sections using a microscope with a 20x objective.</li>
		<li>Using ImageJ software, quantify the staining intensity by measuring the mean gray values in five randomly selected fields (8 &#215; 8 &#956;m2 for each) from immunoenzymatically stained regions in each section. Normalize the staining intensity using the mean gray value from each contralateral; there is no wounded region as a background correction.&#160;Calculate the average of the normalized mean gray values from the five fields to determine the mean gray value of mouse IgG staining for each section.</li>
	</ol>
	</li>
	<li>Assessment of Evans blue leakage into the brain parenchyma 
	NOTE: Evans blue protocol is similar to the method previously described11. Fluorescein isothiocyanate (FITC)-labeled dextran also works for recovery from BBB breakdown10.
	<ol>
		<li>Evans blue injection: Administer 2% Evans blue in PBS (3 mL/kg) to the stab-wounded mouse through the tail vein 1 h before dissection.</li>
		<li>Fresh brain extraction: 1 h after Evans blue administration, perfuse the mouse with PBS as described in steps 2.1.1-2.1.4. Then, dissect and slice the fresh brain coronally into 1 mm-thick sections using a brain slicer. Select four slices and cut out brain pieces (1 mm2 square, each) that include the stab-wound region or intact regions in the no-wound cerebral cortex, according to the graph paper (Figure 1D).</li>
		<li>Evans blue quantification: Collect the four brain pieces in a 1.5 mL tube and homogenize them with 200 &#956;L of trichloroacetic acid using a tissue grinder. Centrifuge the homogenate at 10,000 x g at 4&#160;&#176;C for 20 min. Then, collect the supernatant with a pipette and dilute it with 600 &#956;L of ethanol. Prepare a standard curve of Evans blue, ranging from 0-1.0 ng/mL, to quantify its concentration. Measure the fluorescence intensity at 680 nm with an excitation wavelength of 620 nm using a plate reader.</li>
	</ol>
	</li>
</ol>
3. Assessment of inflammation level in the brain after stab-wound
<ol>
	<li>Immunoenzymatic staining of stab-wounded brain section for glial cells
	<ol>
		<li>Preparation of the brain sections: Make the brain sections-mounted slide glasses as described in steps 2.1.1-2.1.9.</li>
		<li>Glial staining: After air-drying the stab-wounded cerebral cortex sections for 1 h, fix the sections again with 4% PFA (500 &#956;L/slide) for 30 min. Then, wash the sections with PBS (500 &#956;L/slide) for 5 min, followed by two washes with TBS (500 &#956;L/slide) for 5 min each.</li>
		<li>Quench endogenous enzyme activity in sections with 10% methanol and 3% hydrogen peroxide in TBS for 5 min, followed by two washes with Tris-buffered saline (TBS, 500 &#956;L/slide) for 5 min each.</li>
		<li>Perform blocking using a blocking buffer (500 &#956;L/slide) for 1 h at room temperature to prevent non-specific binding of the antibody.</li>
		<li>Incubate the sections overnight with the primary antibody (300 &#956;L/slide). Use the following dilutions for the primary antibody: 1:3,000 dilution for Iba1, and 1:1,000 dilution for GFAP, diluted in the blocking buffer.</li>
		<li>Wash the section three times with TBS for 5 min each. Then, incubate with the biotin-conjugated secondary antibody (1:300 dilution with the blocking buffer, 300 &#956;L/slide) for 1 h at room temperature.</li>
		<li>While incubating with the secondary antibody, prepare the mixture of 1 &#956;L of Reagent A and 1 &#956;L of Reagent B from the avidin/biotin-based peroxidase system kit in 300 &#956;L of the blocking buffer. Then incubate the mixture at room temperature for 30 min before use.</li>
		<li>After the secondary antibody incubation is complete, wash the sections three times with TBS for 5 min each, and incubate the sections with the prepared mixture of Reagent A and B (300 &#956;L/slide) for 1 h at room temperature.</li>
		<li>Wash the sections with 0.1 M Tris-HCl pH 8.0 for 5 min, three times, over the course of 1 h. Then, develop color using 0.05% DAB in 0.05% hydrogen peroxide-added 0.1 M Tris-HCl pH 8.0 (500 &#956;L/slide) for 30 min to 3 h. Observe under a microscope for color change before stopping the reaction.</li>
		<li>Wash the sections with 0.1 M Tris-HCl pH 8.0 for 5 min three times, and air dry the sections for 15 min. After that, dehydrate the sections by immersing them in 95% ethanol for 2 min twice, followed by 100% ethanol for 2 min, twice. Clear the sections in xylene for 5 min twice. Coverslip the sections using a water-insoluble mounting medium and allow them to dry. Capture the section images using the microscope with an objective of 20x.</li>
	</ol>
	</li>
	<li>Immunofluorescent staining of stab-wounded brain section for glial cells
	<ol>
		<li>Preparation of the brain sections: Make the brain sections-mounted slide glasses as described in steps 2.1.1-2.1.9.</li>
		<li>Glial staining: After air-drying the stab-wounded cerebral cortex sections for 1 h, fix the sections again with 4% PFA (500 &#956;L/slide) for 30 min. Then, wash the sections with PBS (500 &#956;L/slide) for 5 min, followed by two washes with TBS (500 &#956;L/slide) for 5 min each. Perform blocking using a blocking buffer (500 &#956;L/slide) for 1 h at room temperature to prevent non-specific binding of the antibody.</li>
		<li>Incubate the sections overnight with the primary antibody (300 &#956;L/slide). Use the following dilutions for the primary antibody: 1:3,000 dilution for Iba1, and 1:1,000 dilution for GFAP, diluted in blocking buffer.</li>
		<li>Wash the section three times with TBS for 5 min each. Then, incubate with the fluorescent-conjugated secondary antibody (1:300 dilution with the blocking buffer, 300 &#956;L/slide) for 1 h at room temperature.</li>
		<li>Wash the sections three times with TBS for 5 min each and incubate the sections with 0.4 &#956;g/mL DAPI solution in TBS (500 &#956;L/slide) for 5 min at room temperature.</li>
		<li>Wash the sections with TBS (500 &#956;L/slide) for 5 min, and with distilled water (500 &#956;L/slide) for 5 min. After air drying the sections for 15 min, coverslip them with a water-based mounting medium and allow them to dry. Capture the section images using a confocal microscope with a 20x objective.</li>
	</ol>
	</li>
	<li>Real-time qPCR for glial cell markers and inflammatory cytokines 
	NOTE: The primer sequences16,20,21,22 used in the presented protocol are listed in Table 1.
	<ol>
		<li>Fresh brain tissue preparation: Perfuse the mouse with PBS as described in steps 2.1.1-2.1.4. Then, extract and slice the fresh brain coronally into 1 mm-thick sections using a brain slicer. Select four slices and cut out brain pieces (1 mm2 square, each) that include the stab-wound region or intact regions on the contralateral side of the cerebral cortex, according to the graph paper. Determine the region to cut out, which includes the lesion at the center (Figure 1D).</li>
		<li>RNA extraction: Collect the four brain pieces in a 1.5 mL tube and homogenize them with 1 mL of TRIzol reagent by pipetting. Incubate the mixture for 5 min at room temperature.&#160;Then, add 200 &#956;L of chloroform, vortex well, and incubate for an additional 2 min at room temperature. Centrifuge the mixture at 12,000 x g for 15 min at 4&#160;&#176;C. Carefully transfer the upper, clear layer into a new 1.5 mL tube, avoiding any white precipitates, which contain DNA and proteins.</li>
		<li>Add 500 &#956;L of isopropanol to the collected upper layer, mix well, and incubate for 10 min at room temperature. After centrifugation at 12,000 x g for 10 min at 4&#160;&#176;C, remove the supernatant and add 1 mL of 70% ethanol to wash the RNA pellet.&#160;Centrifuge at 7,500 x g for 5 min at 4&#160;&#176;C, remove the supernatant completely, and air dry the pellet for 10 min. Dissolve the RNA pellet in 15 &#956;L of RNase-free water and denature the RNA for 10 min at 55&#176;C.</li>
		<li>Measure the RNA concentration using a microvolume spectrophotometer. Store the RNA at -80&#160;&#176;C immediately.</li>
		<li>cDNA synthesis: For cDNA synthesis, use a commercially available DNA synthesis kit. Prepare a total of 10 &#956;L of Reaction buffer in a 1.5 &#956;L tube for each sample, which includes 2 &#956;L of 5 x RT Buffer, 0.5 &#956;L of RT Enzyme Mix, 0.5 &#956;L of Primer Mix, 1 &#956;g of RNA, and nuclease-free water. Incubate at 37&#160;&#176;C for 15 min for the reverse transcription reaction and at 98&#176;C for 5 min for the enzyme inactivation reaction. Then, add 90 &#956;L of nuclease-free water into the buffer and mix well. Store the cDNA solution at -20&#176;C.</li>
		<li>Quantification of RNA expression: For real-time qPCR analysis, use a commercially available qPCR master mix kit. Prepare the Mix solution for each sample by combining 5 &#956;L of KOD SYBR qPCR Mix, 0.4 &#956;L of 5 &#956;M forward primer, 0.4 &#956;L of 5 &#956;M reverse primer, and 0.2 &#956;L of 50 x ROX in 2 &#956;L of nuclease-free water.</li>
		<li>Make a mixture of 2 &#956;L of cDNA solution and 8 &#956;L of Mix solution in an 8-strip tube, mix well, and spin down.</li>
		<li>Initiate the PCR reaction with the following cycling conditions: 98&#160;&#176;C for 2 min, followed by 40 cycles of 98&#176;C for 10 s, 60&#176;C for 10 s, and 68&#176;C for 30 s. Perform the Melting/Dissociation Curve Analysis step using a real-time qPCR machine.</li>
		<li>mRNA expression analysis: Analyze mRNA expression level using the 2-Delta-Delta-Ct method, which is performed by normalization with a housekeeping gene, Gapdh, then with that of the contralateral side23.</li>
	</ol>
	</li>
</ol>

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<li>Wei, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Microglia+activation%3A+One+of+the+checkpoints+in+the+CNS+inflammation+caused+by+angiostrongylus+cantonensis+infection+in+rodent+model%5BTitle%5D)+AND+%22Parasitol+Res%22%5BJournal%5D)">Microglia activation: One of the checkpoints in the CNS inflammation caused by angiostrongylus cantonensis infection in rodent model.</a> Parasitol Res. 114 (9), 3247-3254 (2015).</li>
<li>Livak, K. J., Schmittgen, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Analysis+of+relative+gene+expression+data+using+real-time+quantitative+pcr+and+the+2%28-delta+delta+c%28t%29%29+method%5BTitle%5D)+AND+%22Methods%22%5BJournal%5D)">Analysis of relative gene expression data using real-time quantitative pcr and the 2(-delta delta c(t)) method.</a> Methods. 25 (4), 402-408 (2001).</li>
<li>Hazy, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Divergent+age-dependent+peripheral+immune+transcriptomic+profile+following+traumatic+brain+injury%5BTitle%5D)+AND+%22Sci+Rep%22%5BJournal%5D)">Divergent age-dependent peripheral immune transcriptomic profile following traumatic brain injury.</a> Sci Rep. 9 (1), 8564(2019).</li>
<li>Xia, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Osthole+confers+neuroprotection+against+cortical+stab-wound+injury+and+attenuates+secondary+brain+injury%5BTitle%5D)+AND+%22J+Neuroinflammation%22%5BJournal%5D)">Osthole confers neuroprotection against cortical stab-wound injury and attenuates secondary brain injury.</a> J Neuroinflammation. 12, 155(2015).</li>
<li>Cieri, M. B., Villarreal, A., Gomez-Cuautle, D. D., Mailing, I., Ramos, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Progression+of+reactive+gliosis+and+astroglial+phenotypic+changes+following+stab-wound-induced+traumatic+brain+injury+in+mice%5BTitle%5D)+AND+%22J+Neurochem%22%5BJournal%5D)">Progression of reactive gliosis and astroglial phenotypic changes following stab-wound-induced traumatic brain injury in mice.</a> J Neurochem. 167 (2), 183-203 (2023).</li>
<li>Barreda-Manso, M. A., Yanguas-Casas, N., Nieto-Sampedro, M., Romero-Ramirez, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neuroprotection+and+blood-brain+barrier+restoration+by+salubrinal+after+a+cortical+stab+injury%5BTitle%5D)+AND+%22J+Cell+Physiol%22%5BJournal%5D)">Neuroprotection and blood-brain barrier restoration by salubrinal after a cortical stab injury.</a> J Cell Physiol. 232 (6), 1501-1510 (2017).</li>
<li>Rapp, P. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Patient+characterization+protocols+for+psychophysiological+studies+of+traumatic+brain+injury+and+post-TBI+psychiatric+disorders%5BTitle%5D)+AND+%22Front+Neurol%22%5BJournal%5D)">Patient characterization protocols for psychophysiological studies of traumatic brain injury and post-TBI psychiatric disorders.</a> Front Neurol. 4, 91(2013).</li>
<li>Brody, D. L., Benetatos, J., Bennett, R. E., Klemenhagen, K. C., Mac Donald, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+pathophysiology+of+repetitive+concussive+traumatic+brain+injury+in+experimental+models%3B+new+developments+and+open+questions%5BTitle%5D)+AND+%22Mol+Cell+Neurosci%22%5BJournal%5D)">The pathophysiology of repetitive concussive traumatic brain injury in experimental models; new developments and open questions.</a> Mol Cell Neurosci. 66 (Pt B), 91-98 (2015).</li>
<li>Meehan, W. P., Zhang, J., Mannix, R., Whalen, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Increasing+recovery+time+between+injuries+improves+cognitive+outcome+after+repetitive+mild+concussive+brain+injuries+in+mice%5BTitle%5D)+AND+%22Neurosurgery%22%5BJournal%5D)">Increasing recovery time between injuries improves cognitive outcome after repetitive mild concussive brain injuries in mice.</a> Neurosurgery. 71 (4), 885-891 (2012).</li>
<li>Chen, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+novel+simple+traumatic+brain+injury+mouse+model%5BTitle%5D)+AND+%22Chin+Neurosurg+J%22%5BJournal%5D)">A novel simple traumatic brain injury mouse model.</a> Chin Neurosurg J. 8 (1), 8(2022).</li>
<li>Machado, C. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Weight-drop+model+as+a+valuable+tool+to+study+potential+neurobiological+processes+underlying+behavioral+and+cognitive+changes+secondary+to+mild+traumatic+brain+injury%5BTitle%5D)+AND+%22J+Neuroimmunol%22%5BJournal%5D)">Weight-drop model as a valuable tool to study potential neurobiological processes underlying behavioral and cognitive changes secondary to mild traumatic brain injury.</a> J Neuroimmunol. 385, 578242(2023).</li>
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</ol>

The authors have nothing to disclose.

Here, a protocol for creating a TBI mouse model using needles was introduced. This protocol allows for a quantitative assessment of recovery from the breakdown of the BBB and inflammation after brain injury using histological and molecular biological approaches. Alternative protocols, such as the repetitive concussive TBI model and weight-drop TBI model, can also be used to analyze BBB breakdown and inflammation. These models replicate TBI pathology under controlled conditions, including specific parameters for impact st...

Traumatic brain injury (TBI) is caused by physical damage, often resulting from accidents, including traffic accidents and fall accidents. TBI is classified into two types: penetrating brain injury, which occurs when a sharp object perforates the skull as well as the brain, and closed brain injury, which is caused by violent shaking of the brain inside without a break in the skull1.
The causes of TBI are very diverse, including concussions, brain contusions, hematomas, and skull fractures; therefore, TBI mouse models have been developed using various protocols to replicate these different causes. For example, a repetitive concussive TBI model involves brain shaking, where mice are stuck several times using an electromagnetically controlled rubber impactor2. Additionally, in the weight-drop TBI model, a strong external force is exerted on the head by a standardized weight-drop device, causing focal blunt injury with an intact skull3. Furthermore, the stab-wound TBI model is prepared by puncturing the skull and brain using a needle4 (Figure 1A). Since several TBI models have been developed, it is important to choose a model based on the specific pathology that needs to be observed.
Brain injury caused by physical damage leads to primary and secondary brain injuries, which further exacerbate neuronal loss. Primary injury occurs immediately after the damage, resulting from the breakdown of the blood-brain barrier (BBB), hemorrhage, and hematoma. Therefore, minimizing hemorrhage and hematoma expansion is crucial, as these factors can exacerbate the severity of TBI symptoms. Secondary injury is triggered by intraparenchymal blood components, which subsequently lead to inflammation around the lesion5. The prognosis after brain injury depends on the inflammatory dynamics; therefore, it is crucial to rapidly mitigate both primary and secondary injuries for a favorable prognosis6,7,8.
The BBB is composed of pericytes, tight junctions between endothelial cells, and the endfeet of astrocytes, which work together to restrict the leakage of substances from the blood vessels in healthy brains9. In the presented stab-wound system, the BBB is physically disrupted. Common methods for evaluating the BBB integrity include staining for immunoglobulin G (IgG) and assessing the leakage of fluorescence tracers, such as Evans blue and dextran10,11. IgG staining labels blood components that leak from the lesion site and deposit in the brain. As the BBB recovers, leakage of blood components into the brain decreases, and these deposits are gradually degraded. Therefore, IgG staining is used to assess the extent of BBB recovery after brain injury. Additionally, the level of leakage of intravenously administered tracer into the brain parenchyma reflects the recovery of BBB. This method provides a clearer evaluation of the BBB dynamics, as tracer leakage directly indicates the transition of blood components from the bloodstream to the brain parenchyma. Furthermore, minimizing the hemorrhage leads to a milder primary injury, which is supported by prompt blood coagulation and timely fibrinolysis. Therefore, quantifying the expression of blood coagulation and fibrinolysis regulators is an effective way to analyze this process. Regarding the molecular mechanism underlying coagulation, hemorrhage after brain injury is stopped by fibrin formation. Subsequently, the fibrin-rich thrombus is degraded by tissue plasminogen activator (tPA) and urokinase plasminogen activator (uPA). In the stab-wound TBI mouse model, fibrin formation peaks on 1 day after the injury and reduces thereafter10. Thus, the recovery level of the BBB can be predicted by quantifying blood components and tracer extravasation into the brain parenchyma, as well as the expression of blood coagulation factors.
Quantification methods for inflammation in the secondary injury process include glial activation and inflammatory cytokine expression. Prolonged inflammation is mainly induced by excessive microglia and astrocyte accumulation around the lesion site. For example, in a stab-wound TBI model, stab-wounds stimulate the reactivation of glial cells around the lesion to remove the cell debris and blood components. This glial reactivation typically peaks 3 days after the stab-wound12,13. In addition to their phagocytosis function, reactivated glial cells secrete excessive inflammatory cytokines, resulting in neuronal loss around the lesion14. It has been reported that the attenuation of glial inflammation contributes to a favorable prognosis after brain injury12,14. Determining the level of inflammation is useful for evaluating the severity and prognosis. Therefore, it is essential to develop a TBI model suitable for assessing hemorrhage extension and inflammatory severity. This study introduces a stab-wound mouse model that mimics penetrating brain injury, with the aim of studying the mechanisms of hemorrhage, inflammation, and neuronal loss in TBI pathology.

<table><tbody><tr><td>19 G x 1&amp;bull;1/2&quot; needle</td><td>TERUMO</td><td>NN-1938R&amp;nbsp;</td><td></td></tr><tr><td>27 G x 3/4&quot; needle</td><td>TERUMO</td><td>NN-2719S</td><td></td></tr><tr><td>anti-GFAP antibody</td><td>Sigma-Aldrich</td><td>G9269</td><td></td></tr><tr><td>anti-Iba1 antibody</td><td>Wako</td><td>019-19741</td><td></td></tr><tr><td>Atipamezole Hydrochloride</td><td>Nippon Zenyaku Kogyo</td><td></td><td>Product name: Antisedan</td></tr><tr><td>Biotin-conjugated mouse IgG antibody</td><td>Vector Laboratories</td><td>BA-9200</td><td></td></tr><tr><td>Biotin-conjugated rabbit IgG antibody</td><td>Vector Laboratories</td><td>BA-1000</td><td></td></tr><tr><td>Bovine albumin</td><td>Nacalai tesque</td><td>01860-07</td><td></td></tr><tr><td>Brain Slicer</td><td>Visikol</td><td>BSLM-2</td><td></td></tr><tr><td>Butorphanol Tartrate</td><td>Meiji Animal Health</td><td></td><td>Product name: Vetorphale 5 mg</td></tr><tr><td>Confocal microscope</td><td>Zeiss</td><td>LSM700</td><td></td></tr><tr><td>Cryostat&amp;nbsp;</td><td>Leica</td><td>CM1520</td><td></td></tr><tr><td>DAB</td><td>Sigma-Aldrich</td><td>D5637-1G</td><td></td></tr><tr><td>DAPI</td><td>Roche</td><td>10236276001</td><td></td></tr><tr><td>Evans blue</td><td>Wako</td><td>056-04061</td><td></td></tr><tr><td>Fluorescent-conjugated rabbit IgG antibody</td><td>Invitrogen</td><td>A-21206</td><td></td></tr><tr><td>Fluoromount-G</td><td>Invitrogen</td><td>4958-02</td><td>Water-based mounting medium</td></tr><tr><td>Isoflurane Inhalation Solution</td><td>Viatris</td><td>v002139</td><td></td></tr><tr><td>KOD SYBR qPCR Mix</td><td>TOYOBO</td><td>QKD-201</td><td>qPCR master mix kit</td></tr><tr><td>Medetomidine</td><td>Nippon Zenyaku Kogyo</td><td></td><td>Product name: Domitor</td></tr><tr><td>Microscope</td><td>Olympus</td><td>FSX100</td><td></td></tr><tr><td>Microvolume spectrophotometer</td><td>ThermoFisher Scientific</td><td>NanoDrop One</td><td></td></tr><tr><td>Midazolam 10 mg/2 mL</td><td>Sandoz</td><td>1124401A1060</td><td></td></tr><tr><td>MOUNT QUICK</td><td>Daido Sangyo</td><td>DM01</td><td>Water insoluble mounting medium</td></tr><tr><td>Newborn calf serum</td><td>Gibco</td><td>16010159</td><td></td></tr><tr><td>O.C.T. compound</td><td>Sakura Finetek Japan</td><td>45833</td><td>Embedding medium</td></tr><tr><td>Peel-A-Way, Truncated 22 mm Square Top</td><td>Ted Pella</td><td>27118</td><td>Tissue embedding mold</td></tr><tr><td>Peristaltic perfusion pump</td><td>ATTO</td><td>SJ-1211</td><td></td></tr><tr><td>Plate reader</td><td>Fisher Scientific</td><td>Cytation 3</td><td></td></tr><tr><td>Real-time qPCR machine</td><td>ThermoFisher Scientific</td><td>StepOne Plus</td><td></td></tr><tr><td>ReverTra Ace qPCR RT Kit</td><td>TOYOBO</td><td>FSQ-101</td><td>cDNA synthesis kit</td></tr><tr><td>Superfrost Plus Slide Glass</td><td>Fisher Scientific</td><td>12-550-15</td><td>Positive-charged slide glass</td></tr><tr><td>Suture with needle&amp;nbsp;</td><td>Alfresa</td><td>HT2003NA75-KF2</td><td></td></tr><tr><td>TRIzol Reagent</td><td>Invitrogen</td><td>15596026</td><td></td></tr><tr><td>VECTASTAIN ABC Standard Kit</td><td>Vector Laboratories</td><td>PK-4000</td><td>Avidin/biotin-based peroxidase system kit</td></tr></tbody></table>

stab-wound mouse model for studying hemorrhage and inflammation in traumatic brain injury

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.

To analyze recovery from BBB breakdown, hemorrhage level in the stab-wounded cerebral cortices was assessed by measuring the extravasation level of serum IgG at 1, 3, 5, and 7 days after brain injury. The mouse IgG staining images revealed blood leakage and deposition in the cerebral cortices following brain injury. This was reduced after more than 7 days, as the BBB recovered, and the IgG protein degraded (Figure 2B). IgG extravasation levels were quantified by measuring the intensity of Ig...

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 ...

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The autologous blood injection model of intracerebral hemorrhage in mice described in this protocol uses the double injection technique to minimize risk of blood reflux up the needle track, no anticoagulants in the pumping system, and eliminates all dead space and expandable tubing in the system.

Autologous Blood Injection to Model Spontaneous Intracerebral Hemorrhage in Mice

Clinically relevant animal models of intracerebral hemorrhage (ICH) are needed to extend our knowledge of hemorrhagic stroke and to examine novel therapeutic strategies. In this study, we describe and evaluate two ICH models that implement unilateral injections of either autologous whole blood or bacterial collagenase into the basal ganglia (corpus striatum) of mice.

Modeling Intracerebral Hemorrhage in Mice: Injection of Autologous Blood or Bacterial Collagenase 

This video demonstrates the procedure for inducing an open-skull traumatic brain injury in a mouse by making a midline skull incision, drilling through the skull and removing the bone flap to expose the brain cortex, and applying high pressure to induce trauma. The wound is then sutured to prepare the mouse model for subsequent experiments.

Generating a Traumatic Brain Injury Mouse Model Using an Impactor Probe

The video demonstrates a method to generate a mouse model of traumatic brain injury (TBI) using a lateral fluid percussion device (LFPI). Upon anesthetizing the mouse, the brain is exposed by creating a window on the skull. A saline-filled cannula is attached to the window and is then connected to the LFPI. The pendulum of the device is released to create a pressure pulse that inflicts a traumatic brain injury.

Generating a Mouse Model of Traumatic Brain Injury Using a Lateral Fluid Percussion Device

This video demonstrates the procedure for inducing traumatic brain injury (TBI) in a mouse model. An anesthetized mouse with an exposed skull is taken to create an opening in the skull, and then a controlled cortical injury is induced using an impact device. The injury damages the brain tissue, causing impaired cognitive function and motor coordination.

Generating a Mouse Model of Traumatic Brain Injury Using an Impactor Device

A standardized mouse model of subarachnoid hemorrhage by intraluminal Circle of Willis perforation is described. Vessel perforation and subarachnoid bleeding are monitored by intracranial pressure monitoring. In addition various vital parameters are recorded and controlled to maintain physiologic conditions.

A Murine Model of Subarachnoid Hemorrhage

Preclinical models of intracerebral hemorrhage are utilized to mimic certain aspects of clinical disease. Thus, mechanisms of injury and potential therapeutic strategies may be explored. In this protocol, two models of intracerebral hemorrhage are described, intrastriatal (basal ganglia) injections of autologous blood or collagenase.

Intrastriatal Injection of Autologous Blood or Clostridial Collagenase as Murine Models of Intracerebral Hemorrhage

Intraparenchymal hemorrhage and neuroinflammation accompanied by cerebral contusion can trigger severe secondary brain injury. This protocol details a mouse controlled cortical impact (CCI) model allowing researchers to study hemorrhage contusion and post-traumatic immune responses and explore potential therapeutics.

A Preclinical Controlled Cortical Impact Model for Traumatic Hemorrhage Contusion and Neuroinflammation

This protocol presents methods to characterize the neuroinflammatory and hemodynamic response to mild traumatic brain injury and to integrate these data as part of a multivariate systems analysis using partial least squares regression.

Systems Analysis of the Neuroinflammatory and Hemodynamic Response to Traumatic Brain Injury

This protocol presents a modified mouse model of repetitive mild traumatic brain injury (rmTBI) induced via a closed-head injury (CHI) method. The approach features a thinned-skull window and fluid percussion to reduce the inflammation commonly caused by meninges exposure, along with improved reproducibility and accuracy in modeling rmTBI in rodents.

Modified Mouse Model of Repetitive Mild Traumatic Brain Injury Incorporating Thinned-Skull Window and Fluid Percussion

Learn a simple needle-based stab-wound mouse model for studying hemorrhage and inflammation in traumatic brain injury. This reproducible protocol enables analysis of neuronal and glial communication during TBI repair.

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

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