Name: evaluation of blood-brain barrier breakdown in a mouse model of mild traumatic brain injury
Uploaded: 2024-10-18T13:05:00.000+00:00
Duration: 5 min 19 s
Description: 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 ...

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

<ol>
	<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. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Manual+restraint+and+common+compound+administration+routes+in+mice+and+rats.">Manual restraint and common compound administration routes in mice and rats.</a> J Vis Exp. 67, e2771 (2012).</li><li>Satoh, Y., 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=Molecular+hydrogen+prevents+social+deficits+and+depression-like+behaviors+induced+by+low-intensity+blast+in+mice.">Molecular hydrogen prevents social deficits and depression-like behaviors induced by low-intensity blast in mice.</a> J Neuropathol Exp Neurol. 77 (9), 827-836 (2018).</li><li>Gage, G. J., Kipke, D. R., Shain, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole+animal+perfusion+fixation+for+rodents.">Whole animal perfusion fixation for rodents.</a> J Vis Exp. 65, e3564 (2012).</li><li>Selever, J., Kong, J. Q., Arenkiel, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+approach+to+high-resolution+fluorescence+imaging+in+semi-thick+brain+slices.">A rapid approach to high-resolution fluorescence imaging in semi-thick brain slices.</a> J Vis Exp. 53, e2807 (2011).</li><li>Jiao, Y., 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=A+simple+and+sensitive+antigen+retrieval+method+for+free-floating+and+slide-mounted+tissue+sections.">A simple and sensitive antigen retrieval method for free-floating and slide-mounted tissue sections.</a> J Neurosci Methods. 93 (2), 149-162 (1999).</li><li>Nagaraja, T. N., Keenan, K. A., Fenstermacher, J. D., Knight, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+leakage+patterns+of+fluorescent+plasma+flow+markers+after+transient+focal+cerebral+ischemia+suggest+large+openings+in+blood-brain+barrier.">Acute leakage patterns of fluorescent plasma flow markers after transient focal cerebral ischemia suggest large openings in blood-brain barrier.</a> Microcirculation. 15 (1), 1-14 (2010).</li><li>Xu, Y., 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=Quantifying+blood-brain-barrier+leakage+using+a+combination+of+Evans+blue+and+high+molecular+weight+fitc-dextran.">Quantifying blood-brain-barrier leakage using a combination of Evans blue and high molecular weight fitc-dextran.</a> J Neurosci Methods. 325, 108349 (2019).</li></ol>

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 remo...

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><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&amp;nbsp;</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&amp;ndash;dextran average mol wt 40,000</td><td>Sigma-Aldrich&amp;nbsp;</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&amp;nbsp;Cover Glass (No.1)&amp;nbsp;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&lt;br/&gt; 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 m...

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

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

517 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

Controlled Cortical Impact Model of Mouse Brain Injury with Therapeutic Transplantation of Human Induced Pluripotent Stem Cell-Derived Neural Cells

Traumatic brain injury (TBI) is a leading cause of morbidity and mortality worldwide. Disease pathology due to TBI progresses from the primary mechanical insult to secondary injury processes, including apoptosis and inflammation. Animal modeling has been valuable in the search to unravel injury mechanisms and evaluate potential neuroprotective therapies. This protocol describes the controlled cortical impact (CCI) model of focal, open-head TBI. Specifically, parameters for producing a mild unilateral cortical injury are described. Behavioral consequences of CCI are analyzed using the adhesive tape removal test of bilateral sensorimotor integration. Regarding experimental therapy for TBI pathology, this protocol also illustrates a process for transplanting cultured cells into the brain. Neural cell cultures derived from human induced pluripotent stem cells (hiPSCs) were chosen for their potential to show superior functional restoration in human TBI patients. Chronic survival of hiPSCs in the host mouse brain tissue is detected using a modified DAB immunohistochemical process.

This protocol demonstrates methodologies for a mouse model of open-skull traumatic brain injury and transplantation of cultured human induced pluripotent stem cell-derived cells into the injury site. Behavioral and histologic tests of outcomes from these procedures are also described in brief.

Traumatic brain injury (TBI) is a leading cause of morbidity and mortality worldwide. Disease pathology due to TBI progresses from the primary mechanical ...

Developmental Biology

Quantification of Neurovascular Protection Following Repetitive Hypoxic Preconditioning and Transient Middle Cerebral Artery Occlusion in Mice

Experimental animal models of stroke are invaluable tools for understanding stroke pathology and developing more effective treatment strategies. A 2 week protocol for repetitive hypoxic preconditioning (RHP) induces long-term protection against central nervous system (CNS) injury in a mouse model of focal ischemic stroke. RHP consists of 9 stochastic exposures to hypoxia that vary in both duration (2 or 4 hr) and intensity (8% and 11% O2). RHP reduces infarct volumes, blood-brain barrier (BBB) disruption, and the post-stroke inflammatory response for weeks following the last exposure to hypoxia, suggesting a long-term induction of an endogenous CNS-protective phenotype. The methodology for the dual quantification of infarct volume and BBB disruption is effective in assessing neurovascular protection in mice with RHP or other putative neuroprotectants. Adult male Swiss Webster mice were preconditioned by RHP or duration-equivalent exposures to 21% O2 (i.e. room air). A 60 min transient middle cerebral artery occlusion (tMCAo) was induced 2 weeks following the last hypoxic exposure. Both the occlusion and reperfusion were confirmed by transcranial laser Doppler flowmetry. Twenty-two hr after reperfusion, Evans Blue (EB) was intravenously administered through a tail vein injection. 2 hr later, animals were sacrificed by isoflurane overdose and brain sections were stained with 2,3,5- triphenyltetrazolium chloride (TTC). Infarcts volumes were then quantified. Next, EB was extracted from the tissue over 48 hr to determine BBB disruption after tMCAo. In summary, RHP is a simple protocol that can be replicated, with minimal cost, to induce long-term endogenous neurovascular protection from stroke injury in mice, with the translational potential for other CNS-based and systemic pro-inflammatory disease states.

This protocol describes repetitive hypoxic preconditioning, or brief exposures to systemic hypoxia that reduce infarct volumes and blood-brain barrier disruption following transient middle cerebral artery occlusion in mice. It also details dual quantification of infarct volume and blood-brain barrier disruption after stroke to assess the efficacy of neurovascular protection.

Experimental animal models of stroke are invaluable tools for understanding stroke pathology and developing more effective treatment strategies. A 2 week ...

Medicine

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 ...

A Mouse Model of Single and Repetitive Mild Traumatic Brain Injury

Mild Traumatic Brain Injury (mTBI) can result in the acute loss of brain function, including a period of confusion, a loss of consciousness (LOC), focal neurological deficits and even amnesia. Athletes participating in contact sports are at high risk of exposure to large number of mTBIs. In terms of the level of injury in a sporting athlete, a mTBI is defined as a mild injury that does not cause gross pathological changes, but does cause short-term neurological deficits that are spontaneously resolved. Despite previous attempts to model mTBI in mice and rats, many have reported gross adverse effects including skull fractures, intracerebral bleeding, axonal injury and neuronal cell death. Herein, we describe our highly reproducible animal model of mTBI that reproduces clinically relevant symptoms. This model uses a custom made pneumatic impactor device to deliver a closed-head trauma. This impact is made under precise velocity and deformation parameters, creating a reliable and reproducible model to examine the mechanisms that contribute to effects of single or repetitive concussive mTBI.

Athletes absorb several hundred mild traumatic brain injuries (mTBI)/concussions every year; however, the consequence of these on the brain is poorly understood. Therefore, an animal model of single and repetitive mTBI that consistently replicates clinically relevant symptoms provides the means to advance the study of mTBI and concussion.

Mild Traumatic Brain Injury (mTBI) can result in the acute loss of brain function, including a period of confusion, a loss of consciousness (LOC), focal ...

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 ...

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

Mild traumatic brain injuries (mTBIs) are a significant public health problem. Repeated exposure to mTBI can lead to cumulative, long-lasting functional deficits. Numerous studies by our group and others have shown that mTBI stimulates cytokine expression and activates microglia, decreases cerebral blood flow and metabolism, and impairs cerebrovascular reactivity. Moreover, several works have reported an association between derangements in these neuroinflammatory and hemodynamic markers and cognitive impairments. Herein we detail methods to characterize the neuroinflammatory and hemodynamic tissue response to mTBI in mice. Specifically, we describe how to perform a weight-drop model of mTBI, how to longitudinally measure cerebral blood flow using a non-invasive optical technique called diffuse correlation spectroscopy, and how to perform a Luminex multiplexed immunoassay on brain tissue samples to quantify cytokines and immunomodulatory phospho-proteins (e.g., within the MAPK and NF&#954;B pathways) that respond to and regulate activity of microglia and other neural immune cells. Finally, we detail how to integrate these data using a multivariate systems analysis approach to understand the relationships between all of these variables. Understanding the relationships between these physiologic and molecular variables will ultimately enable us to identify mechanisms responsible for mTBI.

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.

Mild traumatic brain injuries (mTBIs) are a significant public health problem. Repeated exposure to mTBI can lead to cumulative, long-lasting functional ...

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

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

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

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

rmTBI Induction in Mice via a Closed-Head Injury Method

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

Mild traumatic brain injury is a clinically highly heterogeneous neurological disorder. Highly reproducible traumatic brain injury (TBI) animal models with well-defined pathologies are urgently needed for studying the mechanisms of neuropathology after mild TBI and testing therapeutics. Replicating the entire sequelae of TBI in animal models has proven to be a challenge. Therefore, the availability of multiple animal models of TBI is necessary to account for the diverse aspects and severities seen in TBI patients. CHI is one of the most common methods for fabricating rodent models of rmTBI. However, this method is susceptible to many factors, including the impact method used, the thickness and shape of the skull bone, animal apnea, and the type of head support and immobilization utilized. The aim of this protocol is to demonstrate a combination of the thinned-skull window and fluid percussion injury (FPI) methods to produce a precise mouse model of CHI-associated rmTBI. The primary objective of this protocol is to minimize factors that could impact the accuracy and consistency of CHI and FPI modeling, including skull bone thickness, shape, and head support. By utilizing a thinned-skull window method, potential inflammation due to craniotomy and FPI is minimized, resulting in an improved mouse model that replicates the clinical features observed in patients with mild TBI. Results from behavior and histological analysis using hematoxylin and eosin (HE) staining suggest that rmTBI can lead to a cumulative injury that produces changes in both behavior and gross morphology of the brain. Overall, the modified CHI-associated rmTBI presents a useful tool for researchers to explore the underlying mechanisms that contribute to focal and diffuse pathophysiological changes in rmTBI.

Author Spotlight: Optimizing Rodent Models for Investigating the Mechanisms and Rehabilitation in Repeated Mild 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.

Mild traumatic brain injury is a clinically highly heterogeneous neurological disorder. Highly reproducible traumatic brain injury (TBI) animal models ...

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

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

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

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

Induction of Mild Traumatic Brain Injury in a Mouse Model

<a href='https://appjove.unicartagenaproxy.elogim.com/v/64556/electromagnetic-controlled-closed-head-model-mild-traumatic-brain'>Source: Macheda, T., et. al. Electromagnetic Controlled Closed-Head Model of Mild Traumatic Brain Injury in Mice. J. Vis. Exp. (2022)</a>This video demonstrates the procedure to induce a closed-head injury in a mouse model using a stereotaxic frame. The controlled impact replicates mild traumatic brain injury, allowing researchers to study neurological effects and recovery by measuring post-injury reflexes and behavior.

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

Summary

Explore More Videos

Chapters in this video

Controlled Cortical Impact Model of Mouse Brain Injury with Therapeutic Transplantation of Human Induced Pluripotent Stem Cell-Derived Neural Cells

Quantification of Neurovascular Protection Following Repetitive Hypoxic Preconditioning and Transient Middle Cerebral Artery Occlusion in Mice

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

A Mouse Model of Single and Repetitive Mild Traumatic Brain Injury

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

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

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

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

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

Induction of Mild Traumatic Brain Injury in a Mouse Model