We thank Mayumi Watanabe for the cryosectioning technique. This work was supported by an Advanced Research on Military Medicine grant from the Ministry of Defense, Japan.

All experiments were conducted in accordance with the ethical guidelines for animal experiments established by the National Defense Medical College (Tokorozawa, Japan). The study protocol was approved by the Committee for Animal Research at the National Defense Medical College (approval no. 23011-1). Male C57BL/6J mice aged 8 weeks and weighing 19-23 g were used in this study. As illustrated in Figure 1, a single Evans blue injection and FITC-dextran perfusion were performed at various time points in relation to a single BSW exposure. Details of the reagents and equipment used are listed in the Table of Materials.
1. Anesthesia
NOTE: This is a modified protocol that increases the amount of medetomidine by 2.5 times compared with the original one12. The dosages for medetomidine hydrochloride, midazolam, and butorphanol were 0.75 mg/kg, 4 mg/kg, and 5 mg/kg, respectively.
<ol>
	<li>Prepare a mixture of medetomidine hydrochloride (75 &#181;g/mL), midazolam (400 &#181;g/mL), and butorphanol (500 &#181;g/mL) in physiological saline.</li>
	<li>Administer the mixture intraperitoneally to anesthetize the mouse (10 &#181;L/g)13.</li>
	<li>After approximately 5-10 min, verify sufficient anesthesia by observing the lack of tail pinch and pedal withdrawal reflexes.</li>
	<li>Keep the mouse warm until it recovers from the effects of anesthesia.</li>
</ol>
2. BSW exposure
NOTE: An in-house shock tube was used in this study14.
<ol>
	<li>Anesthetize the mouse as described in step 1.</li>
	<li>Position the mouse 5 cm away from the exit end of the shock tube, ensuring its body axis is parallel to but not aligned with the tube&#39;s axis.</li>
	<li>Deliver a single BSW exposure with a peak overpressure of 25 kPa to the head. 
	NOTE: Keep the mouse warm until it recovers from the effects of anesthesia. Regularly monitor the condition of the treated mouse. If signs of distress, such as difficulty feeding or drinking, hunching, or prolonged abnormal appearance without signs of recovery, are observed, euthanize the mouse and terminate the experiment early. Avoid administering analgesics, as they may potentially affect the glial response.</li>
</ol>
3. Evans blue injection into the tail vein
NOTE: Evans blue solution should be administered intravenously without anesthesia. In the presence of anesthesia, the dye often does not penetrate the body sufficiently, likely due to decreased blood pressure and body temperature13. Evans blue was administered at a dose of 100 mg/kg.
<ol>
	<li>Prepare the Evans blue solution (4% w/v in saline) in a microtube, then vortex and store it in the dark before use.</li>
	<li>Inject the solution into the tail vein (2.5 &#181;L/g)13.</li>
</ol>
4. Transcardial perfusion with FITC-dextran solution and fixation
<ol>
	<li>Add heparin to phosphate-buffered saline (PBS) to obtain a concentration of 1 U/mL, then add FITC-dextran to the heparinized PBS to achieve a concentration of 3 mg/mL.</li>
	<li>Completely dissolve the FITC-dextran powder before use. To do this, gently shake the solution for 30 min or more. Before use, carefully check for any undissolved FITC-dextran powder. If any remains, continue shaking until it is completely dissolved.</li>
	<li>Anesthetize the mouse as described in step 1.</li>
	<li>Proceed with the standard perfusion fixation protocol using a peristaltic pump15,16. First, perfuse with heparinized PBS containing FITC-dextran for 2 min at a rate of 4.0 mL/min. Then, perfuse with 10% formalin neutral buffer solution or 4% paraformaldehyde-PBS for 2 min at a rate of 4.0 mL/min, followed by 8 min at a rate of 3.5 mL/min.</li>
	<li>Carefully remove the brain using surgical scissors and tweezers15,16. After dissection, post-fix the brain overnight in the same fixative (i.e., 10% formalin neutral buffer solution or 4% paraformaldehyde-PBS) at 4 &#176;C.</li>
	<li>On the following day, replace the fixative with PBS.</li>
</ol>
5. Brain tissue processing
NOTE: Even if perfusion fixation is complete, Evans blue and FITC-dextran may disperse into the buffer. Therefore, the procedures leading up to step 6.8 should be completed within a week after perfusion fixation. Additionally, avoid exposing the sample to light.
<ol>
	<li>Prepare a 24-well plate with 500 &#181;L of 20% glycerol in phosphate buffer (PB; pH 7.4) in each well.</li>
	<li>Using a brain slicer, cut the brain coronally into 12 slices, each 1 mm thick.</li>
	<li>Transfer each slice into the corresponding well of the 24-well plate and store it at 4 &#176;C for at least 2 h.</li>
	<li>Replace the solution with 50% glycerol-PB and store it at 4 &#176;C for at least 2 h.</li>
	<li>Finally, replace the solution with 100% glycerol. For immediate microscopic observation, allow the slices to stand for at least 2 h at room temperature or store them at 4 &#176;C. The slices will now be translucent and ready for microscopic observation.</li>
</ol>
6. Fluorescence measurement of dye extravasation
NOTE: Fluorescence labeling efficiency varies from mouse to mouse. Therefore, fluorescence intensity needs to be normalized. Fluorescence values are expressed relative to those within the gigantocellular reticular nucleus (GRN) because this region is minimally affected by BSW treatment.
<ol>
	<li>Add 500 &#181;L of glycerol to the bottom of a 35-mm glass-bottom dish.</li>
	<li>Transfer the slice to the glycerol solution and cover its surface with a coverglass.</li>
	<li>Remove excess glycerol from the edge of the coverglass.</li>
	<li>Measure the fluorescence intensity of the slice under a fluorescence microscope.</li>
	<li>After microscopic measurement, return the slice to the well.</li>
	<li>Repeat the measurement for all 12 slices.</li>
	<li>Add 500 &#181;L of PB to each well and store the 24-well plate at 4 &#176;C overnight; on the following day, the slices will be saturated with 50% glycerol-PB.</li>
	<li>Replace the solution with 30% glycerol-PB and store it at 4 &#176;C for at least 2 h.</li>
	<li>Perform the procedures in step 7 within a few weeks.</li>
</ol>
7. Cryosectioning and immunohistochemistry
<ol>
	<li>Prepare a 24-well plate by adding 1000 &#181;L of a mixture of equal volumes of tissue freezing medium and 30% glycerol-PB to well 1. Then, add 1000 &#181;L of tissue freezing medium to well 2.</li>
	<li>Transfer the slice to well 1 and shake the plate at 4 &#176;C for 1 h.</li>
	<li>Transfer the slice to well 2 and shake the plate at 4 &#176;C for 1 h.</li>
	<li>Quickly freeze the slice with isopentane using dry ice, taking care not to bend the slice. Store the slices at -80 &#176;C until cryosectioning.</li>
	<li>Cryosection the slice to a thickness of 6 &#181;m. After drying on the microscope slide, store the sections at -80 &#176;C.</li>
	<li>For antigen retrieval17, immerse the sections in 10 mM sodium citrate (pH 6.0), heat to 100 &#176;C once, and then maintain at 98 &#176;C for 1 h.</li>
	<li>Wash the sections extensively in distilled water. The sections are now ready for immunohistochemical staining.</li>
</ol>

Fluorescent Dye-Based Visualization of Blood-Brain Barrier Breakdown in Mice

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	<li>Sweeney, M. D., Zhao, Z., Montagne, A., Nelson, A. R., Zlokovic, B. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood-brain+barrier:+From+physiology+to+disease+and+back.">Blood-brain barrier: From physiology to disease and back.</a> Physiol Rev. 99 (1), 21-78 (2019).</li><li>Kabu, 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=Blast-associated+shock+waves+result+in+increased+brain+vascular+leakage+and+elevated+ros+levels+in+a+rat+model+of+traumatic+brain+injury.">Blast-associated shock waves result in increased brain vascular leakage and elevated ros levels in a rat model of traumatic brain injury.</a> PLoS One. 10 (5), e0127971 (2015).</li><li>Kuriakose, M., Rama Rao, K. V., Younger, D., Chandra, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal+and+spatial+effects+of+blast+overpressure+on+blood-brain+barrier+permeability+in+traumatic+brain+injury.">Temporal and spatial effects of blast overpressure on blood-brain barrier permeability in traumatic brain injury.</a> Sci Rep. 8 (1), 8681 (2018).</li><li>Yeoh, S., Bell, E. D., Monson, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distribution+of+blood-brain+barrier+disruption+in+primary+blast+injury.">Distribution of blood-brain barrier disruption in primary blast injury.</a> Ann Biomed Eng. 41 (10), 2206-2214 (2013).</li><li>Readnower, R. 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=Increase+in+blood-brain+barrier+permeability,+oxidative+stress,+and+activated+microglia+in+a+rat+model+of+blast-induced+traumatic+brain+injury.">Increase in blood-brain barrier permeability, oxidative stress, and activated microglia in a rat model of blast-induced traumatic brain injury.</a> J Neurosci Res. 88 (16), 3530-3539 (2010).</li><li>Shetty, A. K., Mishra, V., Kodali, M., Hattiangady, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood-brain+barrier+dysfunction+and+delayed+neurological+deficits+in+mild+traumatic+brain+injury+induced+by+blast+shock+waves.">Blood-brain barrier dysfunction and delayed neurological deficits in mild traumatic brain injury induced by blast shock waves.</a> Front Cell Neurosci. 8, 232 (2014).</li><li>Nishii, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evans+blue+and+fluorescein+isothiocyanate-dextran+double+labeling+reveals+the+precise+sequence+of+vascular+leakage+and+glial+responses+after+exposure+to+mild-level+blast-associated+shock+waves.">Evans blue and fluorescein isothiocyanate-dextran double labeling reveals the precise sequence of vascular leakage and glial responses after exposure to mild-level blast-associated shock waves.</a> J Neurotrauma. 40 (11-12), 1228-1242 (2023).</li><li>Hoffmann, 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=High+and+low+molecular+weight+fluorescein+isothiocyanate+(FITC)-dextrans+to+assess+blood-brain+barrier+disruption:+Technical+considerations.">High and low molecular weight fluorescein isothiocyanate (FITC)-dextrans to assess blood-brain barrier disruption: Technical considerations.</a> Transl Stroke Res. 2 (1), 106-111 (2011).</li><li>Saunders, N. R., Dziegielewska, K. M., M&#248;llg&#229;rd, K., Habgood, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Markers+for+blood-brain+barrier+integrity:+How+appropriate+is+Evans+blue+in+the+twenty-first+century+and+what+are+the+alternatives.">Markers for blood-brain barrier integrity: How appropriate is Evans blue in the twenty-first century and what are the alternatives.</a> Front Neurosci. 9, 385 (2015).</li><li>Manaenko, A., Chen, H., Kammer, J., Zhang, J. H., Tang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+Evans+blue+injection+routes:+Intravenous+versus+intraperitoneal,+for+measurement+of+blood-brain+barrier+in+a+mice+hemorrhage+model.">Comparison Evans blue injection routes: Intravenous versus intraperitoneal, for measurement of blood-brain barrier in a mice hemorrhage model.</a> J Neurosci Methods. 195 (2), 206-210 (2011).</li><li>Yen, L. F., Wei, V. C., Kuo, E. Y., Lai, T. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+patterns+of+cerebral+extravasation+by+Evans+blue+and+sodium+fluorescein+in+rats.">Distinct patterns of cerebral extravasation by Evans blue and sodium fluorescein in rats.</a> PLoS One. 8 (7), e68595 (2013).</li><li>Kawai, S., Takagi, Y., Kaneko, S., Kurosawa, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+three+types+of+mixed+anesthetic+agents+alternate+to+ketamine+in+mice.">Effect of three types of mixed anesthetic agents alternate to ketamine in mice.</a> Exp Anim. 60 (5), 481-487 (2011).</li><li>Machholz, E., Mulder, G., Ruiz, C., Corning, B. F., Pritchett-Corning, K. 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The authors have nothing to disclose.

A novel double-labeling technique using Evans blue and FITC-dextran was used to accurately visualize the precise spatiotemporal distribution of BBB breakdown in a single brain. In the low-intensity BSW model, noticeable variations in the extent, location, and degree of dye extravasation were observed in examined brains (Figure 2 and Figure 3). Between-dye mismatches revealed that BBB breakdown commenced approximately 3 h after BSW exposure, with substantial remodeling occurring by day 1 and continuing through day 7 (Figure 4). In severe cases, active microglia were recruited to the sites of BBB breakdown, and clusters of reactive astrocytes were closely linked to these areas (Figure 5). By performing immunohistochemistry on the same slices used for dye extravasation analysis, a precise correlation was established between glial reactions and dye extravasation. For more detailed data and analysis, please refer to Nishii et al.7.
Because FITC-dextran was perfused at a constant concentration for an identical duration immediately before perfusion fixation, the distribution and intensity of the dye likely accurately represent the extent of BBB breakdown at the time of perfusion fixation. However, it has been demonstrated in a previous report that the fluorescence of FITC-dextran tends to be weak in a low-intensity BSW model7. Labeling with Evans blue for a longer duration was advantageous, as it could detect more subtle changes in BBB breakdown (Figure 2). However, owing to the longer duration of labeling, caution may be required when interpreting fluorescent images of Evans blue. The blood concentration of Evans blue was the highest immediately after injection and gradually decreased over a few hours (Figure 1)11. It is speculated that the intensity of labeling would correlate with the severity and duration of BBB breakdown as well as the blood concentration of Evans blue; however, it would decrease exponentially once repaired. All of these factors vary from time to time and from one experiment to another. Therefore, a conclusion should not be drawn from a single image of dye extravasation, and statistical considerations are always necessary.
Depending on the purpose of the experiment, it might be interesting to change the molecular weight as well as the combination of dyes. In this study, a combination of Evans Blue and FITC-dextran was used. After binding with serum albumin, Evans blue behaved as a 69-kDa tracer, and the 40-kDa FITC-dextran used in this study showed a similar extent of BBB breakdown as Evans blue. The spatiotemporal distribution of BBB breakdown was systematically investigated using a combination of dyes that emit different fluorescence7. In a previous study that utilized a combination of Evans blue and sodium fluorescein (376 Da), sodium fluorescein, owing to its smaller molecular weight, could label a broader range of BBB breakdown after BSW exposure3. In other studies, combinations of Evans blue and higher-molecular-weight FITC-dextran were used to compare the extent of BBB breakdown18,19. All previous reports administered Evans blue and FITC-dextran almost simultaneously. The unique aspect of the protocol presented here is that the two dyes were introduced at different time points, allowing for the examination of the temporal progression of BBB breakdown.
The cumulative nature of Evans blue may prove beneficial in different BBB breakdown models. Because it records the history of BBB breakdown, an understanding of the onset and progression of the disease may be facilitated. Researchers should be aware of the positive and negative characteristics of dyes and utilize them to the fullest extent9. This protocol has a broad range of applications for various BBB breakdown models.

Blood-brain barrier (BBB) breakdown and dysfunction are caused by systemic inflammation, infections, autoimmune diseases, injuries, and neurodegenerative diseases1. In mild traumatic brain injury (mTBI) resulting from exposure to blast-induced shock waves (BSWs), a significant correlation has been observed between the intensity of BSWs and the amount of fluorescent dye leakage due to BBB breakdown2,3,4. One notable feature of BBB breakdown in mTBI is that it begins immediately or within a few hours after exposure to BSWs and is usually a transient process that lasts for about a week before delayed, chronic neurological disorders emerge3,5,6,7. Although the details remain unclear, BBB breakdown is part of the long-lasting pathological cascade and may also serve as a prognostic factor in mTBI6. Therefore, understanding the spatial and temporal distributions of BBB breakdown in the brain is important.
Fluorescent dyes are used to determine the extent of BBB breakdown3,8. Because the blood concentration of the dyes and the magnitude and extent of BBB breakdown change over time, caution is required when interpreting images of dye extravasation. For instance, the absence of dye extravasation does not necessarily indicate the absence of BBB breakdown. No BBB breakdown may be detected before or after an increase in the dye&#39;s blood concentration. Even if the dye successfully accumulated where BBB breakdown occurred, it may have been lost over time after the breakdown ceased. In general, water-soluble and biologically inert substances are quickly excreted in the urine9. Therefore, to determine whether BBB breakdown occurs at a specific time, the most reliable results are obtained when a fluorescent dye is administered into the bloodstream for a short period immediately before fixing the animal. Commercially available fluorescein isothiocyanate (FITC)-dextran with a specific molecular weight should be used in this manner.
Evans blue is a widely recognized blue azo dye with a strong affinity for serum albumin. The dye exhibits red fluorescence when excited by green light in biological systems2. Due to its inert nature, the Evans blue-serum albumin complex remains in the blood for up to 2 h, making it a useful 69 kDa tracer for labeling regions with a compromised BBB for at least this duration10,11. Therefore, it is important to consider potential uncertainties surrounding the pharmacokinetics and toxicity of Evans blue9. However, a recent study showed that Evans blue continues to accumulate in areas where the BBB is absent or disrupted7. This feature enabled Evans Blue to record the history of the BBB breakdown, while FITC-dextran was used to record the BBB breakdown at a specific time point after the BSW exposure. Although Evans blue can be administered intravenously or intraperitoneally10, intravenous administration is preferred for time-sensitive experiments. This study aimed to demonstrate the use of Evans blue and FITC-dextran to detect the spatiotemporal distribution of BBB breakdown following exposure to BSW.
Second, the study presented a technique for freezing brain slices after observing the fluorescence of BBB breakdown and preparing thinner slices suitable for immunohistochemical procedures. The use of glycerol as a mounting medium and cryoprotectant simplifies the immunohistochemistry process. By comparing images of BBB breakdown with those from immunohistochemistry, the spatiotemporal distribution of BBB breakdown can be correlated with the tissue response of the same sample.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10% Formalin Neutral Buffer Solution</td>
			<td>FUJIFILM Wako Chemicals</td>
			<td>062-01661</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti GFAP, Rabbit</td>
			<td>DAKO-Agilent</td>
			<td>IR524</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti Iba1, Rabbit</td>
			<td>FUJIFILM Wako Chemicals</td>
			<td>019-19741</td>
			<td></td>
		</tr>
		<tr>
			<td>Chicken anti-Mouse IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488</td>
			<td>Thermo Fisher Scientific</td>
			<td>A-21200</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryo Mount</td>
			<td>Muto Pure Chemicals</td>
			<td>33351</td>
			<td>tissue freezing medium</td>
		</tr>
		<tr>
			<td>Domitor</td>
			<td>Orion Corporation</td>
			<td></td>
			<td>medetomidine</td>
		</tr>
		<tr>
			<td>Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 546</td>
			<td>Thermo Fisher Scientific</td>
			<td>A10040</td>
			<td></td>
		</tr>
		<tr>
			<td>Evans Blue</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>E2129</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon 24-well Polystyrene Clear Flat Bottom Not Treated Cell Culture Plate, with Lid, Individually Wrapped, Sterile, 50/Case</td>
			<td>CORNING</td>
			<td>351147</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescein isothiocyanate&#8211;dextran average mol wt 40,000</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>FD40S</td>
			<td>FITC-dextran</td>
		</tr>
		<tr>
			<td>Glass Base Dish 27mm (No.1 Glass)</td>
			<td>AGC TECHNO GLASS</td>
			<td>3910-035</td>
			<td>35 mm glass bottom dish</td>
		</tr>
		<tr>
			<td>IX83 Inverted Microscope</td>
			<td>OLYMPUS</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MAS Hydrophilic Adhesion Microscope Slides</td>
			<td>Matsunami Glass</td>
			<td>MAS-04</td>
			<td></td>
		</tr>
		<tr>
			<td>Matsunami&#160;Cover Glass (No.1)&#160;18 x 18mm</td>
			<td>Matsunami Glass</td>
			<td>C018181</td>
			<td></td>
		</tr>
		<tr>
			<td>Midazolam Injection 10mg [SANDOZ]</td>
			<td>Sandoz</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde EMPROVE ESSENTIAL DAC</td>
			<td>Merck Millipore</td>
			<td>1.04005.1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Peristaltic Pump</td>
			<td>ATTO</td>
			<td>SJ-1211 II-H</td>
			<td></td>
		</tr>
		<tr>
			<td>RODENT BRAIN MATRIX 
			Adult Mouse, 30 g, Coronal</td>
			<td>ASI INSTRUMENTS</td>
			<td>RBM-2000C</td>
			<td>brain slicer</td>
		</tr>
		<tr>
			<td>Vetorphale</td>
			<td>Meiji Animal Health</td>
			<td>VETLI5</td>
			<td>butorphanol</td>
		</tr>
	</tbody>
</table>

evaluation of blood-brain barrier breakdown in a mouse model of mild traumatic brain injury

Fluorescent dyes are used to determine the extent of dye extravasation that occurs due to blood-brain barrier (BBB) breakdown. Labeling with these dyes is a complex process influenced by several factors, such as the concentration of dyes in the blood, permeability of brain vessels, duration of dye extravasation, and reduction in dye concentration in the tissue due to degradation and diffusion. In a mild traumatic brain injury model, exposure to blast-induced shock waves (BSWs) triggers BBB breakdown within a limited time window. To determine the precise sequence of BBB breakdown, Evans blue, and fluorescein isothiocyanate-dextran were injected intravascularly and intracardially into mice at various time points relative to BSW exposure. The distribution of dye fluorescence in brain slices was then recorded. Differences in the distribution and intensity between the two dyes revealed the spatiotemporal sequence of BBB breakdown. Immunostaining of the brain slices showed that astrocytic and microglial responses correlated with the sites of BBB breakdown. This protocol has broad potential for application in studies involving different BBB breakdown models.

Figure 1A shows the time course of dye injection in relation to the onset of BSW, with a peak overpressure of 25 kPa. In the &#39;Post&#39; protocol, Evans blue solution was administered intravascularly 2 h before FITC-dextran perfusion, which was conducted 6 h, 1 day, 3 days, and 7 days after BSW exposure. In the &#39;Pre&#39; protocol, Evans blue solution was injected immediately before BSW exposure. In the &#39;Post&#39; protocol, the concentration of Evans blue is expected to reach its maximum approximately 2 h before perfusion fixation, whereas in the &#39;Pre&#39; protocol, it is expected to reach its maximum around the time of BSW exposure (Figure 1B). The concentration of FITC-dextran in the brain-blood vessels was maintained constant for 2 min during perfusion.
Evans blue labeling showed that BBB breakdown started within 6 h and continued until 7 days after BSW exposure (Figure 2A). Notably, BBB breakdown did not occur immediately after BSW exposure, as there was no Evans blue dye extravasation at 3 h in the &#39;Pre&#39; protocol (Figure 2B). Surprisingly, long-term labeling with Evans blue indicated its cumulative nature in the &#39;Pre&#39; protocol (Figure 2B)7.
The use of two distinct dyes, each introduced at intervals of 2 h or more, allowed the examination of temporal changes in BBB integrity by comparing their distribution. Figure 3 shows fluorescence images of the slices that exhibited a significant difference in the distribution of Evans blue and FITC-dextran.
Numerous fluorescence hotspots of various sizes were observed. Although some hotspots were bright enough to rank among the top 0.1% of pixels, many were only slightly brighter than the surrounding tissues. The discrepancy between the dyes suggests that changes occurred within this interval. Hotspots displaying only Evans blue fluorescence indicated ongoing BBB breakdown at the time of Evans blue injection, which was later repaired by the FITC-dextran injection. In contrast, hotspots showing only FITC-dextran fluorescence suggested that BBB disruption occurred between the two dye injections. Lastly, hotspots exhibiting fluorescence from both dyes indicated continuous BBB breakdown during the entire injection period.
Figure 4A presents the number of hotspots positive for Evans blue (including both Evans blue-only and Evans blue/FITC-dextran double-positive hotspots), FITC-dextran (including both FITC-dextran-only and Evans blue/FITC-dextran double-positive hotspots), or both dyes (only Evans blue/FITC-dextran double-positive hotspots). In the &#39;Pre&#39; protocol at 3 h, numerous FITC-dextran-only hotspots were observed. At 6 h and 1 day in the &#39;Post&#39; protocol, both Evans blue-only and FITC-dextran-only hotspots were detected. However, at 3 days and 7 days in the &#39;Post&#39; protocol, the hotspots were predominantly positive for both dyes. These findings suggest that BBB breakdown occurred at 3 h, was remodeled by 1 day, and persisted until 7 days after BSW exposure (Figure 4B). Importantly, the extent of BBB breakdown tended to decrease by 7 days, as previously mentioned.
Following the completion of the dye extravasation experiments, immunohistochemical staining of the slices was carried out7. The immunohistochemical image scans were consistently compared with those of the dye extravasations, confirming that the carry-over effect of the fluorescent substances was practically negligible7. As shown in Figure 5, clusters of reactive astrocytes were closely associated with sites of BBB breakdown. Further observations revealed that activated and amoeboid microglia were present alongside reactive astrocytes 3 days after BSW exposure.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66653/66653fig01.jpg" /> 
Figure 1: Experimental design. (A) Time course of the experiment. Evans blue solution was intravenously injected after (&#39;Post&#39;&#160;protocol) or immediately before (&#39;Pre&#39;&#160;protocol) BSW exposure, whereas FITC-dextran was transcardially perfused for 2 min before fixation. (B) Expected concentrations of Evans blue and FITC-dextran in the blood vessels in the &#39;Post&#39;&#160;and &#39;Pre&#39;&#160;protocols, respectively. BSW, blast-induced shock waves; FITC, fluorescein isothiocyanate. This figure is adapted from Nishii et al.7. <a href="https://www.jove.com/files/ftp_upload/66653/66653fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/66653/66653fig02.jpg" /> 
Figure 2: Comparison of fluorescence intensity in brain slices. The fluorescence intensity of the brightest 0.1% of pixels in the brain was assessed across all 12 slices and compared between the &#39;Post&#39;&#160;(A) and &#39;Pre&#39;&#160;(B) conditions. The variability and significance of the differences are indicated. xxx denotes p &#60; 0.001; xxxx denotes p &#60; 0.0001 (p values from F-test). *p &#60; 0.05; ****p &#60; 0.0001 (analysis of variance with Dunnett&#39;s multiple comparisons post hoc test). This figure is adapted from Nishii et al.7. <a href="https://www.jove.com/files/ftp_upload/66653/66653fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/66653/66653fig03.jpg" /> 
Figure 3: BBB breakdown observed at various time points using double labeling with Evans blue and FITC-dextran. Evans blue (magenta), FITC-dextran (green), and merged images are shown. The dye injection protocols are presented on the left side. Some hotspots showed predominant fluorescence of Evans blue (arrow) or FITC-dextran (arrowheads). Asterisks indicate the choroid plexus and related vessels. BBB, blood-brain barrier; FITC, fluorescein isothiocyanate; CVO, circumventricular organs of the third ventricle. Scale bar: 1 mm. This figure is adapted from Nishii et al.7. <a href="https://www.jove.com/files/ftp_upload/66653/66653fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/66653/66653fig04.jpg" /> 
Figure 4: Quantification of the fluorescence hotspots. (A) Numbers of the hotspots positive for Evans blue (magenta), FITC-dextran (green), and both dyes (gray). They were collected from the images obtained at 3 h in the &#39;Pre&#39;&#160;protocol as well as from the images obtained at 6 h, 1 day, 3 days, and 7 days in the &#39;Post&#39;&#160;protocol. From 3 h to 1 day, a significant portion of the fluorescence hotspots showed a between-dye mismatch, whereas, from 3 days to 7 days, such mismatch was hardly observed. (B) Scheme summarizing the time course of BBB breakdown. The vertical axis conceptually represents the intensity of dye leakage due to BBB breakdown. The zigzag pattern from 3 h to 1 day indicates that the location and intensity of dye leakage are unstable during this period. FITC, fluorescein isothiocyanate; BBB, blood-brain barrier; BSW, blast-induced shock waves. This figure is adapted from Nishii et al.7. <a href="https://www.jove.com/files/ftp_upload/66653/66653fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/66653/66653fig05.jpg" /> 
Figure 5: Expression of GFAP and Iba1 as markers for astrocytes and microglia, respectively. Each column of the figure contains fluorescence images of dye extravasation with Evans blue or FITC-dextran, along with GFAP or Iba1 immunohistochemical images. The color bar indicates normalized fluorescence intensities of Evans blue and FITC-dextran using those within GRN. The dye injection protocols are presented on the left side. The slice of 1 day in the &#39;Pre&#39;&#160;protocol shows fluorescence of both Evans blue and FITC-dextran, whereas the slice of 3 days in the &#39;Pre&#39;&#160;protocol shows only fluorescence of Evans blue (arrows). The regions denoted by the arrows in the dye extravasation images align with the immunohistochemical images. Arrowheads indicate clusters of reactive astrocytes. The polyclonal GFAP antibody labeled astrocytes in and around the white matter as well as in nervous tissue adjacent to the pia mater. As a result, astrocytes within and close to the dcw are labeled in this figure. The magnified images corresponding to the frame or arrow in the Iba1 column are depicted in the insets. Scale bars: 1 mm and 200 &#181;m for the dye extravasation and immunohistochemical images, respectively. GFAP, glial fibrillary acidic protein; Iba1, ionized calcium-binding adapter molecule 1; FITC, fluorescein isothiocyanate; BSW, blast-induced shock waves; dcw, deep cerebral white matter. This figure is adapted from Nishii et al.7. <a href="https://www.jove.com/files/ftp_upload/66653/66653fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Evaluation of Blood-Brain Barrier Breakdown in a Mouse Model of Mild Traumatic Brain Injury

A method was developed to visualize dye extravasation due to blood-brain barrier (BBB) breakdown by administering two fluorescent dyes to mice at different time points. The use of glycerol as a cryoprotectant facilitated immunohistochemistry on the same sample.

Fluorescent dyes are used to determine the extent of dye extravasation that occurs due to blood-brain barrier (BBB) breakdown. Labeling with these dyes is ...

evaluation-blood-brain-barrier-breakdown-mouse-model-mild-traumatic

Nanyang Technological University

Research

JoVE Journal

Neuroscience

445 Views.  National Defense Medical College. A method was developed to visualize dye extravasation due to blood-brain barrier (BBB) breakdown by administering two fluorescent dyes to mice at different time points. The use of glycerol as a cryoprotectant facilitated immunohistochemistry on the same sample.

Video: Evaluation of Blood-Brain Barrier Breakdown in a Mouse Model of Mild Traumatic Brain Injury

Lateral Fluid Percussion: Model of Traumatic Brain Injury in Mice

Traumatic brain injury (TBI) research has attained renewed momentum due to the increasing awareness of head injuries, which result in morbidity and mortality. Based on the nature of primary injury following TBI, complex and heterogeneous secondary consequences result, which are followed by regenerative processes 1,2. Primary injury can be induced by a direct contusion to the brain from skull fracture or from shearing and stretching of tissue causing displacement of brain due to movement 3,4. The resulting hematomas and lacerations cause a vascular response 3,5, and the morphological and functional damage of the white matter leads to diffuse axonal injury 6-8. Additional secondary changes commonly seen in the brain are edema and increased intracranial pressure 9. Following TBI there are microscopic alterations in biochemical and physiological pathways involving the release of excitotoxic neurotransmitters, immune mediators and oxygen radicals 10-12, which ultimately result in long-term neurological disabilities 13,14. Thus choosing appropriate animal models of TBI that present similar cellular and molecular events in human and rodent TBI is critical for studying the mechanisms underlying injury and repair.
Various experimental models of TBI have been developed to reproduce aspects of TBI observed in humans, among them three specific models are widely adapted for rodents: fluid percussion, cortical impact and weight drop/impact acceleration 1. The fluid percussion device produces an injury through a craniectomy by applying a brief fluid pressure pulse on to the intact dura. The pulse is created by a pendulum striking the piston of a reservoir of fluid. The percussion produces brief displacement and deformation of neural tissue 1,15. Conversely, cortical impact injury delivers mechanical energy to the intact dura via a rigid impactor under pneumatic pressure 16,17. The weight drop/impact model is characterized by the fall of a rod with a specific mass on the closed skull 18. Among the TBI models, LFP is the most established and commonly used model to evaluate mixed focal and diffuse brain injury 19. It is reproducible and is standardized to allow for the manipulation of injury parameters. LFP recapitulates injuries observed in humans, thus rendering it clinically relevant, and allows for exploration of novel therapeutics for clinical translation 20.
We describe the detailed protocol to perform LFP procedure in mice. The injury inflicted is mild to moderate, with brain regions such as cortex, hippocampus and corpus callosum being most vulnerable. Hippocampal and motor learning tasks are explored following LFP.

Lateral fluid percussion (LFP), an established model of traumatic brain injury in mice, is demonstrated. LFP fulfills three major criteria for animal models: validity, reliability and clinical relevance. The procedure, consisting of surgical craniotomy, fixation of hub followed by induction of injury, resulting in focal and diffuse injuries, is described.

Traumatic brain injury (TBI) research has attained renewed momentum due to the increasing awareness of head injuries, which result in morbidity and ...

Functional Neuroimaging Using Ultrasonic Blood-brain Barrier Disruption and Manganese-enhanced MRI

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains technically challenging. One approach, Activation-Induced Manganese-enhanced MRI (AIM MRI), has been used successfully to map neuronal activity in rodents 1-5. In AIM MRI, Mn2+ acts a calcium analog and accumulates in depolarized neurons 6,7. Because Mn2+ shortens the T1 tissue property, regions of elevated neuronal activity will enhance in MRI. Furthermore, Mn2+ clears slowly from the activated regions; therefore, stimulation can be performed outside the magnet prior to imaging, enabling greater experimental flexibility. However, because Mn2+ does not readily cross the blood-brain barrier (BBB), the need to open the BBB has limited the use of AIM MRI, especially in mice.
One tool for opening the BBB is ultrasound. Though potentially damaging, if ultrasound is administered in combination with gas-filled microbubbles (i.e., ultrasound contrast agents), the acoustic pressure required for BBB opening is considerably lower. This combination of ultrasound and microbubbles can be used to reliably open the BBB without causing tissue damage 8-11.
Here, a method is presented for performing AIM MRI by using microbubbles and ultrasound to open the BBB. After an intravenous injection of perflutren microbubbles, an unfocused pulsed ultrasound beam is applied to the shaved mouse head for 3 minutes. For simplicity, we refer to this technique of BBB Opening with Microbubbles and UltraSound as BOMUS 12. Using BOMUS to open the BBB throughout both cerebral hemispheres, manganese is administered to the whole mouse brain. After experimental stimulation of the lightly sedated mice, AIM MRI is used to map the neuronal response.
To demonstrate this approach, herein BOMUS and AIM MRI are used to map unilateral mechanical stimulation of the vibrissae in lightly sedated mice 13. Because BOMUS can open the BBB throughout both hemispheres, the unstimulated side of the brain is used to control for nonspecific background stimulation. The resultant 3D activation map agrees well with published representations of the vibrissae regions of the barrel field cortex 14. The ultrasonic opening of the BBB is fast, noninvasive, and reversible; and thus this approach is suitable for high-throughput and/or longitudinal studies in awake mice.

A technique is described for broadly opening the blood-brain barrier in the mouse using microbubbles and ultrasound. Using this technique, manganese can be administered to the mouse brain. Because manganese is an MRI contrast agent that accumulates in depolarized neurons, this approach enables imaging of neuronal activity.

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains ...

Investigations on Alterations of Hippocampal Circuit Function Following Mild Traumatic Brain Injury

Traumatic Brain Injury (TBI) afflicts more than 1.7 million people in the United States each year and even mild TBI can lead to persistent neurological impairments 1. Two pervasive and disabling symptoms experienced by TBI survivors, memory deficits and a reduction in seizure threshold, are thought to be mediated by TBI-induced hippocampal dysfunction 2,3. In order to demonstrate how altered hippocampal circuit function adversely affects behavior after TBI in mice, we employ lateral fluid percussion injury, a commonly used animal model of TBI that recreates many features of human TBI including neuronal cell loss, gliosis, and ionic perturbation 4-6.
	Here we demonstrate a combinatorial method for investigating TBI-induced hippocampal dysfunction. Our approach incorporates multiple ex vivo physiological techniques together with animal behavior and biochemical analysis, in order to analyze post-TBI changes in the hippocampus. We begin with the experimental injury paradigm along with behavioral analysis to assess cognitive disability following TBI. Next, we feature three distinct ex vivo recording techniques: extracellular field potential recording, visualized whole-cell patch-clamping, and voltage sensitive dye recording. Finally, we demonstrate a method for regionally dissecting subregions of the hippocampus that can be useful for detailed analysis of neurochemical and metabolic alterations post-TBI.
	These methods have been used to examine the alterations in hippocampal circuitry following TBI and to probe the opposing changes in network circuit function that occur in the dentate gyrus and CA1 subregions of the hippocampus (see Figure 1). The ability to analyze the post-TBI changes in each subregion is essential to understanding the underlying mechanisms contributing to TBI-induced behavioral and cognitive deficits. 
	The multi-faceted system outlined here allows investigators to push past characterization of phenomenology induced by a disease state (in this case TBI) and determine the mechanisms responsible for the observed pathology associated with TBI.

A multi-faceted approach to investigating functional changes to hippocampal circuitry is explained. Electrophysiological techniques are described along with the injury protocol, behavioral testing and regional dissection method. The combination of these techniques can be applied in similar fashion for other brain regions and scientific questions.

Traumatic Brain Injury (TBI) afflicts more than 1.7 million people in the United States each year and even mild TBI can lead to persistent neurological ...

Assessment of Blood-brain Barrier Permeability by Intravenous Infusion of FITC-labeled Albumin in a Mouse Model of Neurodegenerative Disease

Disruption of blood-brain barrier (BBB) integrity is a common feature for different neurological and neurodegenerative diseases. Although the interplay between perturbed BBB homeostasis and the pathogenesis of brain disorders needs further investigation, the development and validation of a reliable procedure to accurately detect BBB alterations may be crucial and represent a useful tool for potentially predicting disease progression and developing targeted therapeutic strategies.
Here, we present an easy and efficient procedure for evaluating BBB leakage in a neurodegenerative condition like that occurring in a preclinical mouse model of Huntington disease, in which defects in the permeability of BBB are clearly detectable precociously in the disease. Specifically, the high molecular weight fluorescein isothiocyanate labelled (FITC)-albumin, which is able to cross the BBB only when the latter is impaired, is acutely infused into a mouse jugular vein and its distribution in the vascular or parenchymal districts is then determined by fluorescence microscopy.
Accumulation of green fluorescent-albumin in the brain parenchyma functions as an index of aberrant BBB permeability and, when quantitated by using Image J processing software, is reported as Green Fluorescence Intensity.

In this study, we present an easy and efficient procedure for evaluating the disruption of the blood-brain barrier under neurodegenerative conditions. To achieve our objective, we infused high molecular weight fluorescein isothiocyanate labelled (FITC)-albumin into mouse jugular vein and evaluated its leakage into the brain parenchyma by fluorescence microscopy.

Disruption of blood-brain barrier (BBB) integrity is a common feature for different neurological and neurodegenerative diseases. Although the interplay ...

An In Vivo Assessment of Blood-Brain Barrier Disruption in a Rat Model of Ischemic Stroke

Ischemic stroke leads to vasogenic cerebral edema and subsequent primary brain injury, which is mediated through destruction of the blood-brain barrier (BBB). Rats with induced ischemic stroke were established and used as in vivo models to investigate the functional integrity of the BBB. Spectrophotometric detection of Evans blue (EB) in the brain samples with ischemic injury could provide reliable justification for the research and development of novel therapeutic modalities. This method generates reproducible results, and is applicable in any laboratory without a need for special equipment. Here, we present a visualized and technical guideline on the detection of the extravasation of EB following induction of ischemic stroke in rats.

An In Vivo Assessment of Blood-Brain Barrier Disruption in a Rat Model of Ischemic Stroke

The overall goal of this procedure is to provide a highly reproducible technique for in vivo assessment of the blood-brain barrier disruption in rat models of ischemic stroke.

Ischemic stroke leads to vasogenic cerebral edema and subsequent primary brain injury, which is mediated through destruction of the blood-brain barrier ...

A Novel In Vitro Model of Blast Traumatic Brain Injury

Traumatic brain injury is a leading cause of death and disability in military and civilian populations. Blast traumatic brain injury results from the detonation of explosive devices, however, the mechanisms that underlie the brain damage resulting from blast overpressure exposure are not entirely understood and are believed to be unique to this type of brain injury. Preclinical models are crucial tools that contribute to better understand blast-induced brain injury. A novel in vitro blast TBI model was developed using an open-ended shock tube to simulate real-life open-field blast waves modelled by the Friedlander waveform. C57BL/6N mouse organotypic hippocampal slice cultures were exposed to single shock waves and the development of injury was characterized up to 72 h using propidium iodide, a well-established fluorescent marker of cell damage that only penetrates cells with compromised cellular membranes. Propidium iodide fluorescence was significantly higher in the slices exposed to a blast wave when compared with sham slices throughout the duration of the protocol. The brain tissue injury is very reproducible and proportional to the peak overpressure of the shock wave applied.

A Novel In Vitro Model of Blast Traumatic Brain Injury

This paper describes a novel model of primary blast traumatic brain injury. A compressed-air driven shock tube is used to expose in vitro mouse hippocampal slice cultures to a single shock wave. This is a simple and rapid protocol generating a reproducible brain tissue injury with a high throughput.

Traumatic brain injury is a leading cause of death and disability in military and civilian populations. Blast traumatic brain injury results from the ...

Effects of Blast-induced Neurotrauma on Pressurized Rodent Middle Cerebral Arteries

Though there have been studies on the histopathological and behavioral effects of blast exposure, fewer have been dedicated to blast&#39;s cerebral vascular effects. Impact (i.e., non-blast) traumatic brain injury (TBI) is known to decrease pressure autoregulation in the cerebral vasculature in both humans and experimental animals. The hypothesis that blast-induced traumatic brain injury (bTBI), like impact TBI, results in impaired cerebral vascular reactivity was tested by measuring myogenic dilatory responses to reduced intravascular pressure in rodent middle cerebral arterial (MCA) segments from rats subjected to mild bTBI using an Advanced Blast Simulator (ABS) shock tube. Adult, male Sprague-Dawley rats were anesthetized, intubated, ventilated and prepared for Sham bTBI (identical manipulation and anesthesia except for blast injury) or mild bTBI. Rats were randomly assigned to receive Sham bTBI or mild bTBI followed by sacrifice 30 or 60 min post-injury. Immediately after bTBI, righting reflex (RR) suppression times were assessed, euthanasia at the time points post-injury was completed, the brain was harvested and the individual MCA segments were collected, mounted and pressurized. As the intraluminal pressure perfused through the arterial segments was reduced in 20 mmHg increments from 100 to 20 mmHg, MCA diameters were measured and recorded. With decreasing intraluminal pressure, MCA diameters steadily increased significantly above baseline in the Sham bTBI groups while MCA dilator responses were significantly reduced (p &#60; 0.05) in both bTBI groups as evidenced by the impaired, smaller MCA diameters recorded for the bTBI groups. In addition, RR suppression in the bTBI groups was significantly (p &#60; 0.05) higher than in the Sham bTBI groups. MCA&#39;s collected from the Sham bTBI groups exhibited typical vasodilatory properties to decreases in intraluminal pressure while MCA&#39;s collected following bTBI exhibited significantly impaired myogenic vasodilatory responses to reduced pressure that persisted for at least 60 min after bTBI.

Here, we present a protocol to describe methods for ex vivo vascular reactivity determination following a primary blast traumatic brain injury (bTBI) using isolated, pressurized, rodent middle cerebral arterial (MCA) segments. bTBI induction is accomplished using a shock tube, also known as an Advanced Blast Simulator (ABS) device.

Though there have been studies on the histopathological and behavioral effects of blast exposure, fewer have been dedicated to blast&#39;s cerebral ...

Advanced Diffusion Imaging in The Hippocampus of Rats with Mild Traumatic Brain Injury

Mild traumatic brain injury (mTBI) is the most common type of acquired brain injury. Since patients with traumatic brain injury show a tremendous variability and heterogeneity (age, gender, type of trauma, other possible pathologies, etc.), animal models play a key role in unraveling factors that are limitations in clinical research. They provide a standardized and controlled setting to investigate the biological mechanisms of injury and repair following TBI. However, not all animal models mimic the diffuse and subtle nature of mTBI effectively. For example, the commonly used controlled cortical impact (CCI) and lateral fluid percussion injury (LFPI) models make use of a craniotomy to expose the brain and induce widespread focal trauma, which are not commonly seen in mTBI. Therefore, these experimental models are not valid to mimic mTBI. Thus, an appropriate model should be used to investigate mTBI. The Marmarou weight drop model for rats induces similar microstructural alterations and cognitive impairments as seen in patients who sustain mild trauma; therefore, this model was selected for this protocol. Conventional computed tomography and magnetic resonance imaging (MRI) scans commonly show no damage following a mild injury, because mTBI induces often only subtle and diffuse injuries. With diffusion weighted MRI, it is possible to investigate microstructural properties of brain tissue, which can provide more insight into the microscopic alterations following mild trauma. Therefore, the goal of this study is to obtain quantitative information of a selected region-of-interest (i.e., hippocampus) to follow up disease progression after obtaining a mild and diffuse brain injury.

The overall goal of this procedure is to obtain quantitative microstructural information of the hippocampus in a rat with mild traumatic brain injury. This is done using an advanced diffusion-weighted magnetic resonance imaging protocol and region-of-interest based analysis of parametric diffusion maps.

Mild traumatic brain injury (mTBI) is the most common type of acquired brain injury. Since patients with traumatic brain injury show a tremendous ...

Low-intensity Blast Wave Model for Preclinical Assessment of Closed-head Mild Traumatic Brain Injury in Rodents

Traumatic brain injury (TBI) is a large-scale public health problem. Mild TBI is the most prevalent form of neurotrauma and accounts for a large number of medical visits in the United States. There are currently no FDA-approved treatments available for TBI. The increased incidence of military-related, blast-induced TBI further accentuates the urgent need for effective TBI treatments. Therefore, new preclinical TBI animal models that recapitulate aspects of human blast-related TBI will greatly advance the research efforts into the neurobiological and pathophysiological processes underlying mild to moderate TBI as well as the development of novel therapeutic strategies for TBI.
Here we present a reliable, reproducible model for the investigation of the molecular, cellular, and behavioral effects of mild to moderate blast-induced TBI. We describe a step-by-step protocol for closed-head, blast-induced mild TBI in rodents using a bench-top setup consisting of a gas-driven shock tube equipped with piezoelectric pressure sensors to ensure consistent test conditions. The benefits of the setup that we have established are its relative low-cost, ease of installation, ease of use and high-throughput capacity. Further advantages of this non-invasive TBI model include the scalability of the blast peak overpressure and the generation of controlled reproducible outcomes. The reproducibility and relevance of this TBI model has been evaluated in a number of downstream applications, including neurobiological, neuropathological, neurophysiological and behavioral analyses, supporting the use of this model for the characterization of processes underlying the etiology of mild to moderate TBI.

We present here a protocol of a blast wave model for rodents to investigate neurobiological and pathophysiological effects of mild to moderate traumatic brain injury. We established a gas-driven, bench-top setup equipped with pressure sensors allowing for reliable and reproducible generation of blast-induced mild to moderate traumatic brain injury.

Traumatic brain injury (TBI) is a large-scale public health problem. Mild TBI is the most prevalent form of neurotrauma and accounts for a large number of ...

Measuring Post-Stroke Cerebral Edema, Infarct Zone and Blood-Brain Barrier Breakdown in a Single Set of Rodent Brain Samples

One of the most common causes of morbidity and mortality worldwide is ischemic stroke. Historically, an animal model used to stimulate ischemic stroke involves middle cerebral artery occlusion (MCAO). Infarct zone, brain edema and blood-brain barrier (BBB) breakdown are measured as parameters that reflect the extent of brain injury after MCAO. A significant limitation to this method is that these measurements are normally obtained in different rat brain samples, leading to ethical and financial burdens due to the large number of rats that need to be euthanized for an appropriate sample size. Here we present a method to accurately assess brain injury following MCAO by measuring infarct zone, brain edema and BBB permeability in the same set of rat brains. This novel technique provides a more efficient way to evaluate the pathophysiology of stroke.

This protocol describes a novel technique of measuring the three most important parameters of ischemic brain injury on the same set of rodent brain samples. Using only one brain sample is highly advantageous in terms of ethical and economic costs.

One of the most common causes of morbidity and mortality worldwide is ischemic stroke. Historically, an animal model used to stimulate ischemic stroke ...