We thank Maryna Kuscheriava, Maksym Kryvonosov, Daryna Yakumenko and Evgenia Goncharyk of the Department of Physiology, Faculty of Biology, Ecology, and Medicine, Oles Honchar, Dnipro University, Dnipro, Ukraine for their support and helpful contributions to our discussions. The data obtained are part of Ruslan Kuts&#8217;s PhD thesis.

The following procedures were conducted according to the recommendations of the Declaration of Helsinki and Tokyo and the Guidelines for the Use of Experimental Animals of the European Community. The experiments were also approved by the Animal Care Committee at the Ben-Gurion University of the Negev.
1. Preparing rats for the experimental procedure
<ol>
	<li>Select adult male Sprague-Dawley rats without overt pathology, each weighing between 300 and 350 g.</li>
	<li>Maintain all rats at room temperature at 22 &#176;C, with 12 hours of light and dark cycles before experiment.</li>
	<li>Ensure that food and water are available ad libitum.</li>
	<li>Perform all procedures between 6:00 a.m. and 2:00 p.m.</li>
</ol>
2. Preparing rats for surgery
<ol>
	<li>Anesthetize the rats for 30 min with isoflurane (4% for induction and 2% for maintenance) and 24% oxygen (1.5 L/min).
	<ol>
		<li>Test the level of anesthesia in the rats by ensuring they do not have a pedal withdrawal reflex.</li>
	</ol>
	</li>
	<li>Insert the 24-gauge catheter into the tail vein. 
	NOTE: Tail warming for vasodilation is not performed.
	<ol>
		<li>Place the rats on the table in a supine position. Use medical tape to affix all four of the rats&#8217; limbs.</li>
	</ol>
	</li>
	<li>Place the probe for temperature measurement into the rat rectum before surgery.</li>
	<li>During the procedure, maintain a heating plate to support a 37 &#176;C core body temperature.</li>
	<li>Add ointment in both of the rat&#8217;s eyes for protection.</li>
	<li>Shave the surgical area and disinfect with three applications of 10% povidone-iodine followed by 70% isopropyl alcohol.</li>
</ol>
3. Right side middle cerebral artery occlusion
NOTE: MCAO is performed by a modified technique, as previously described16,22,23, with the use of instruments described by McGarry et al.24 and Ulu&#231; et al.25.
<ol>
	<li>Dissect the skin and superficial fascia at the ventral midline of neck with surgical tweezers and scissors with curved blades.</li>
	<li>Identify the muscle triangle, consisting of the ICA, external carotid artery (ECA) and common carotid artery (CCA).</li>
	<li>Carefully separate the right CCA and ICA from the vagus nerve with microforceps for vascular surgery.</li>
	<li>Expose the right CCA and the ICA. Block the blood flow coming from the CCA to the ICA using either micro-clips or special tourniquets for vascular surgery. Make an incision (approximately 1 mm) on the ICA using microscissors for vascular surgery.</li>
	<li>Insert a monofilament catheter (4-0 nylon) directly through the ICA, about 18.5-19 mm from the bifurcation point of the right CCA into the circle of Willis until reaching a mild resistance, to occlude the MCA26.</li>
	<li>Ligate around the ICA above the bifurcation of CCA.</li>
	<li>For the sham-operated control group, perform an insertion of nylon thread instead of steps 3.5 and 3.616,22.</li>
	<li>Administer 5 mL of 0.9% sodium chloride by intraperitoneal injection.</li>
	<li>Close the wound by suture and take the rat to a recovery area. 
	NOTE: A few minutes after the end of anesthesia, the rat will wake up and move independently around the cage.</li>
	<li>At 23 h after MCAO, inject 2% Evans blue in saline (4 mL/kg)23,26 into the tail vein for both operated groups via a cannula27. 
	NOTE: This is used as a blood-brain permeability tracer. Allow to circulate for 60 minutes.</li>
</ol>
4. Determination of infarct zone
<ol>
	<li>Measure IZ at 24 h after MCAO as described previously9,15,18,19,26. 
	NOTE: Rats that lost more than 20% of their weight or developed seizures or hemiplegia are excluded from the experiment.</li>
	<li>Euthanize the rat by replacing the inspired gas mixture with 20% oxygen and 80% carbon dioxide until the rat ceases to breathe spontaneously.</li>
	<li>Open the chest with a 5-6 cm lateral incision through the abdominal wall under the rib cage using scissors and surgical forceps.</li>
	<li>Perform a diaphragmatic incision along the entire length of the rib cage with scissors and surgical forceps.</li>
	<li>Carefully displacing the lungs, cut through the rib cage up to the collarbone on the right and left sides28.</li>
	<li>Perfuse with 200 mL of normal saline through the left ventricle of the heart.</li>
	<li>Puncture or incise the right atrium of the heart with scissors.</li>
	<li>Perform decapitation using a guillotine and collect brain tissue.</li>
	<li>Using iris scissors, cut from the foramen magnum to the distal edge of the posterior skull surface on both sides.</li>
	<li>Separate the olfactory bulbs, nervous connections along the ventral surface and dorsal surface of the skull from the brain.</li>
	<li>Remove the brain from the head.</li>
	<li>Produce 6 brain slices by creating 2 mm thick horizontal sections with a .009&#34; stainless steel, uncoated, single edge razor blade.</li>
	<li>Incubate for 30 min at 37 &#176;C in 0.05% TTC.</li>
	<li>Place the brain tissue on the microscope slides and perform optical scanning of these 6 brain-slices with a resolution of 1600x1600 dpi (see Supplement 1 for example).</li>
	<li>Add a blue filter with a photo editor (e.g., Adobe Photoshop CS2) using the Channel Mixer function (Image &#62; Adjustments &#62; Channel Mixer) and save the image as a JPEG file format. 
	NOTE: After applying the blue filter, the image will appear greyscale.</li>
	<li>Open the saved image in ImageJ 1.37v29,30. 
	NOTE: This computer program uses a threshold function to isolate and calculate the pixels that are either black or white (see Figure 1).</li>
	<li>For each of the 6 brain slices of the image, select and save each hemisphere (right injured ipsilateral and left uninjured contralateral) as a separate image file using the &#8220;polygon selection&#8221; tool from the main menu.</li>
	<li>Set the cut-off for determining IZ by using an auto threshold function from the main menu of the ImageJ software by selecting Image &#62; Adjust &#62; Threshold, and measure the number of pixels in each hemisphere of a single brain set. 
	NOTE: Macros may be used for this step in ImageJ software (see Supplement 2 for the code). The cut off is a critical parameter for determining which pixels to convert to white and which to convert to black depending on the shade of gray (see Supplement 3 and Supplement 4 as examples). ImageJ then compares white and black pixels to determine IZ. Based on the staining protocol and scanner settings, we used a constant cut-off value of 0.220.</li>
	<li>Perform measurement of IZ correcting for tissue swelling using the Ratios of Ipsilateral and Contralateral Cerebral Hemispheres (RICH) method13,23 (see example in Supplement 5). 
	<img alt="Equation 1" src="/files/ftp_upload/61309/61309equ01.jpg" /> 
	NOTE: Infarct size is assessed as a percentage of the contralateral hemisphere.</li>
</ol>
5. Determination of brain edema31
NOTE: Use ImageJ 1.37v for measurement of BE32,33.
<ol>
	<li>Measure BE 24 h after MCAO. For calculation of BE, use the data from left and right hemisphere volume (in units).</li>
	<li>Perform optical scanning with a resolution of 1600x1600 dpi (see Supplement 1 for example).</li>
	<li>Select brain hemispheres and set the cut-off for determining BE with ImageJ 1.37v, as described above in sections 4.17-4.19.</li>
	<li>Express the BE area as a percentage of the standard areas of the unaffected contralateral hemisphere, calculated by the RICH method using following equation (see example in Supplement 5)23,34. 
	<img alt="Equation 2" src="/files/ftp_upload/61309/61309equ02v2.jpg" /> 
	NOTE: Extent of BE is assessed as a percentage of the contralateral hemisphere.</li>
</ol>
6. Determination of BBB disruption
<ol>
	<li>Measure BBB disruption 24 h after MCAO.</li>
	<li>Divide right and left hemispheres into six slices and put each one into a microcentrifuge tube.</li>
	<li>Homogenize each slice of the brain tissue in trichloroacetic acid, based on the calculation of 1 g of brain tissue in 4 mL of 50% trichloroacetic acid.</li>
	<li>Centrifuge at 10,000 x g for 20 min.</li>
	<li>Dilute supernatant liquid 1:3 with 96% ethanol.</li>
	<li>Perform luminescence spectrophotometry by utilizing spectrophotometry software, installing the plate, and performing a sample reading using the following parameters: Fluorescence intensity excitation wavelength of 620 nm (bandwidth 10 nm) and an emission wavelength of 680 nm (bandwidth 10 nm)23,35 ; Mod top; Number Flesh 25; Manual 100; Shaking 1 sec, 1 mm. 
	NOTE: Use an excitation wavelength of 620 nm (bandwidth 10 nm) and an emission wavelength of 680 nm (bandwidth 10 nm).23,35</li>
</ol>

The authors have nothing to disclose.

The principal goal of the present protocol was to demonstrate consistent measurements of three main parameters of ischemic injury: IZ, BE and BBB permeability. Previous studies in this field have demonstrated the possibility of performing one or two of these parameters together in the same sample. Besides the cost reduction that this three-part method offers, it also provides a more desirable bioethical model that limits the number of animals that must be operated on and subsequently euthanized. As in all histological te...

One of the most common causes of morbidity and mortality worldwide is stroke. Globally, ischemic stroke represents 68% of all stroke cases, while in the United States ischemic stroke accounts for 87% of stroke cases1,2. It is estimated that the economic burden of stroke reaches $34 billion in the United States2 and &#8364;45 billion in the European Union3. Animal models of stroke are necessary to study its pathophysiology, develop new methods for evaluation, and propose new therapeutic options4.
Ischemic stroke occurs with occlusion of a major cerebral artery, usually the middle cerebral artery or one of its branches5. Thus, models of ischemic stroke have historically involved middle cerebral artery occlusion (MCAO)6,7,8,9,10,11,12. Following MCAO, neurological injury is most commonly assessed by measuring infarct zone (IZ) using a 2,3,5-triphenyltetrazolium chloride (TTC) staining method13, brain edema (BE) using drying or calculating hemispheric volumes14,15,16, and blood brain barrier (BBB) permeability by a spectrometry technique using Evans blue staining17,18,19.
The traditional MCAO method uses separate sets of brains for each of the three brain measurements. For a large sample size, this results in a significant number of euthanized animals, with added ethical and financial considerations. An alternative method to alleviate these costs would involve measurements of all three parameters in a single set of post-MCAO rodent brains.
Previous attempts have been made to measure combinations of parameters in the same brain sample. Simultaneous immunofluorescent staining methods20 as well as other molecular and biochemical analyses21 have been described after TTC staining in the same brain sample. We have previously calculated brain hemisphere volumes to assess brain edema and performed TTC staining to calculate infarct zone in the same brain set15.
In the present protocol, we present a modified MCAO technique that measures ischemic brain injury through determining IZ, BE, and BBB permeability in the same set of rodent brains. IZ is measured by TTC staining, BE is determined by calculating hemispheric volume, and BBB permeability is obtained by spectrometry methods19. In this protocol, we used a modified MCAO model, based on direct insertion and fixation of the monofilament catheter into the internal carotid artery (ICA) and further blocking of blood flow to the middle cerebral artery (MCA)22. This modified method shows a decreased rate of mortality and morbidity compared to the traditional MCAO method16,22.
This new approach provides a financially-sound and ethical model for measuring neurological injury after MCAO. This assessment of the main parameters of ischemic brain injury will help to comprehensively investigate its pathophysiology.

<table>
	<tbody>
		<tr>
			<td>2 mL Syringe</td>
			<td>Braun</td>
			<td>4606027V</td>
			<td></td>
		</tr>
		<tr>
			<td>2% chlorhexidine in 70% alcohol solution</td>
			<td>Sigma-Aldrich</td>
			<td>500 cc</td>
			<td>Provides general antisepsis of the skin in the operatory field</td>
		</tr>
		<tr>
			<td>27 G Needle with Syringe</td>
			<td>Braun</td>
			<td>305620</td>
			<td></td>
		</tr>
		<tr>
			<td>3-0 Silk sutures</td>
			<td>Henry Schein</td>
			<td>1007842</td>
			<td></td>
		</tr>
		<tr>
			<td>4-0 Nylon suture</td>
			<td>4-00</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Brain &#38; Tissue Matrices</td>
			<td>Sigma-Aldrich</td>
			<td>15013</td>
			<td></td>
		</tr>
		<tr>
			<td>Cannula Venflon 22 G</td>
			<td>KD-FIX</td>
			<td>183603985447</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge Sigma 2-16P</td>
			<td>Sigma-Aldrich</td>
			<td>Sigma 2-16P</td>
			<td></td>
		</tr>
		<tr>
			<td>Compact Analytical Balances</td>
			<td>Sigma-Aldrich</td>
			<td>HR-AZ/HR-A</td>
			<td></td>
		</tr>
		<tr>
			<td>Digital weighing scale</td>
			<td>Sigma-Aldrich</td>
			<td>Rs 4,000</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting scissors</td>
			<td>Sigma-Aldrich</td>
			<td>Z265969</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf pipette</td>
			<td>Sigma-Aldrich</td>
			<td>Z683884</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf tube</td>
			<td>Sigma-Aldrich</td>
			<td>EP0030119460</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescence detector</td>
			<td>Tecan, M&#228;nnedorf Switzerland</td>
			<td>Model: Infinite 200 PRO multimode reader</td>
			<td>Optional.</td>
		</tr>
		<tr>
			<td>Fluorescence detector</td>
			<td>Molecular Devices LLC</td>
			<td>VWR cat. # 10822 512 SpectraMax Paradigm Multi Mode Microplate Reader Base Instrument</td>
			<td>Optional.</td>
		</tr>
		<tr>
			<td>Gauze sponges</td>
			<td>Fisher</td>
			<td>22-362-178</td>
			<td></td>
		</tr>
		<tr>
			<td>Heater with thermometer</td>
			<td>Heatingpad-1</td>
			<td>Model: HEATINGPAD-1/2</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemostatic microclips</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Horizon-XL</td>
			<td>Mennen Medical Ltd</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Infusion cuff</td>
			<td>ABN</td>
			<td>IC-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro forceps</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Micro scissors</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Multiset</td>
			<td>Teva Medical</td>
			<td>998702</td>
			<td></td>
		</tr>
		<tr>
			<td>Olympus BX 40 microscope</td>
			<td>Olympus</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Operating forceps</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Operating scissors</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Optical scanner</td>
			<td>Canon</td>
			<td>Cano Scan 4200F</td>
			<td>Resolution 3200 x 6400 dpi</td>
		</tr>
		<tr>
			<td>Petri dishes</td>
			<td>Sigma-Aldrich</td>
			<td>P5606</td>
			<td></td>
		</tr>
		<tr>
			<td>Purina Chow</td>
			<td>Purina</td>
			<td>5001</td>
			<td>Rodent laboratory chow given to rats, mice and hamster is a life-cycle nutrition that has been used in biomedical research for over 5 decades. Provided to rats ad libitum in this experiment.</td>
		</tr>
		<tr>
			<td>Rat cages</td>
			<td>Techniplast</td>
			<td>2000P</td>
			<td>Conventional housing for rodents. Cages were used for housing rats throughout the experiment</td>
		</tr>
		<tr>
			<td>Scalpel blades #11</td>
			<td>Sigma-Aldrich</td>
			<td>S2771</td>
			<td></td>
		</tr>
		<tr>
			<td>Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Adobe Photoshop CS2 for Windows</td>
			<td>Adobe</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ImageJ 1.37v</td>
			<td>NIH</td>
			<td></td>
			<td>The source code is freely available. The author, Wayne Rasband (wayne@codon.nih.gov), is at the Research Services Branch, National Institute of Mental Health, Bethesda, Maryland, USA</td>
		</tr>
		<tr>
			<td>Office 365 ProPlus</td>
			<td>Microsoft</td>
			<td>-</td>
			<td>Microsoft Office Excel</td>
		</tr>
		<tr>
			<td>Windows 10</td>
			<td>Microsoft</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>2,3,5-Triphenyltetrazolium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>298-96-4</td>
			<td></td>
		</tr>
		<tr>
			<td>50% trichloroacetic acid</td>
			<td>Sigma-Aldrich</td>
			<td>76-03-9</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol 96 %</td>
			<td>Romical</td>
			<td></td>
			<td>Flammable liquid</td>
		</tr>
		<tr>
			<td>Evans blue 2%</td>
			<td>Sigma-Aldrich</td>
			<td>314-13-6</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane, USP 100%</td>
			<td>Piramamal Critical Care, Inc</td>
			<td>NDC 66794-017</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Infarct zone measurement
An independent-sample t-test indicated that 19 rats that underwent permanent MCAO demonstrated a significant increase in brain infarct volume compared to the 16 sham-operated rats (MCAO=7.49% &#177; 3.57 vs. Sham = 0.31% &#177; 1.9, t(28.49) = 7.56, p &#60; 0.01 (see Figure 2A)). The data is expressed as a mean percentage of the contralateral hemisphere &#177; SD.
Brain...

Watch this Scientific Journal Video about Measuring Post-Stroke Cerebral Edema, Infarct Zone and Blood-Brain Barrier Breakdown in a Single Set of Rodent Brain Samples at JoVE.com

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

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 ...

measuring-post-stroke-cerebral-edema-infarct-zone-blood-brain-barrier

Research

JoVE Journal

Neuroscience

7.8K Views.  Ben-Gurion University of the Negev. 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.

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

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 ...

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 Blood-brain Barrier Permeability Assay in Mice Using Fluorescently Labeled Tracers

Blood-brain barrier (BBB) is a specialized barrier that protects the brain microenvironment from toxins and pathogens in the circulation and maintains brain homeostasis. The principal sites of the barrier are endothelial cells of the brain capillaries whose barrier function results from tight intercellular junctions and efflux transporters expressed on the plasma membrane. This function is regulated by pericytes and astrocytes that together form the neurovascular unit (NVU). Several neurological diseases such as stroke, Alzheimer&#39;s disease (AD), brain tumors are associated with an impaired BBB function. Assessment of the BBB permeability is therefore crucial in evaluating the severity of the neurological disease and the success of the treatment strategies employed.
We present here a simple yet robust permeability assay that have been successfully applied to several mouse models both, genetic and experimental. The method is highly quantitative and objective in comparison to the tracer fluorescence analysis by microscopy that is commonly applied. In this method, mice are injected intraperitoneally with a mix of aqueous inert fluorescent tracers followed by anesthetizing the mice. Cardiac perfusion of the animals is performed prior to harvesting brain, kidneys or other organs. Organs are homogenized and centrifuged followed by fluorescence measurement from the supernatant. Blood drawn from the cardiac puncture just before perfusion serves for normalization purpose to the vascular compartment. The tissue fluorescence is normalized to the wet weight and serum fluorescence to obtain a quantitative tracer permeability index. For additional confirmation, the contralateral hemi-brain preserved for immunohistochemistry can be utilized for tracer fluorescence visualization purposes.

An In Vivo Blood-brain Barrier Permeability Assay in Mice Using Fluorescently Labeled Tracers

Here we present a mouse brain vascular permeability assay using intraperitoneal injection of fluorescent tracers followed by perfusion that is applicable to animal models of blood-brain barrier dysfunction. One hemi-brain is used for assessing permeability quantitatively and the other for tracer visualization/immunostaining. The procedure takes 5 - 6 h for 10 mice.

Blood-brain barrier (BBB) is a specialized barrier that protects the brain microenvironment from toxins and pathogens in the circulation and maintains ...

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 ...

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 ...

Focused Ultrasound Induced Blood-Brain Barrier Opening for Targeting Brain Structures and Evaluating Chemogenetic Neuromodulation

Acoustically Targeted Chemogenetics (ATAC) allows for the noninvasive control of specific neural circuits. ATAC achieves such control through a combination of focused ultrasound (FUS) induced blood-brain barrier opening (FUS-BBBO), gene delivery with adeno-associated viral (AAV) vectors, and activation of cellular signaling with engineered, chemogenetic, protein receptors and their cognate ligands. With ATAC, it is possible to transduce both large and small brain regions with millimeter precision using a single noninvasive ultrasound application. This transduction can later allow for a long-term, noninvasive, device-free neuromodulation in freely moving animals using a drug. Since FUS-BBBO, AAVs, and chemogenetics have been used in multiple animals, ATAC should also be scalable for the use in other animal species. This paper expands upon a previously published protocol and outlines how to optimize the gene delivery with FUS-BBBO to small brain regions with MRI-guidance but without a need for a complicated MRI-compatible FUS device. The protocol, also, describes the design of mouse targeting and restraint components that can be 3D-printed by any lab and can be easily modified for different species or custom equipment. To aid reproducibility, the protocol describes in detail how the microbubbles, AAVs, and venipuncture were used in ATAC development. Finally, an example data is shown to guide the preliminary investigations of studies utilizing ATAC.

This protocol delineates steps necessary for the gene delivery through focused ultrasound blood brain barrier (BBB) opening, evaluation of the resulting gene expression, and measurement of neuromodulation activity of chemogenetic receptors through histological tests.

Acoustically Targeted Chemogenetics (ATAC) allows for the noninvasive control of specific neural circuits. ATAC achieves such control through a ...

Transendothelial Resistance Measurement to Assess Cell Barrier Integrity

Barrier Functional Integrity Recording on bEnd.3 Vascular Endothelial Cells via Transendothelial Electrical Resistance Detection

The blood-brain barrier (BBB) is a dynamic physiological structure composed of microvascular endothelial cells, astrocytes, and pericytes. By coordinating the interaction between restricted transit of harmful substances, nutrient absorption, and metabolite clearance in the brain, the BBB is essential in preserving central nervous system homeostasis. Building in vitro models of the BBB is a valuable tool for exploring the pathophysiology of neurological disorders and creating pharmacological treatments. This study describes a procedure for creating an in vitro monolayer BBB cell model by seeding bEnd.3 cells into the upper chamber of a 24-well plate. To assess the integrity of cell barrier function, the conventional epithelial cell voltmeter was used to record the transmembrane electrical resistance of normal cells and CoCl2-induced hypoxic cells in real-time. We anticipate that the above experiments will provide effective ideas for the creation of in vitro models of BBB and drugs to treat disorders of central nervous system diseases.

Author Spotlight: Applications of TEER Detection to Assess Cell Barrier Integrity 

This protocol describes a dependable and efficient in vitro model of the brain blood barrier. The method uses mouse cerebral vascular endothelial cells bEnd.3 and measures transmembrane electrical resistance.

The blood-brain barrier (BBB) is a dynamic physiological structure composed of microvascular endothelial cells, astrocytes, and pericytes. By coordinating ...

A 3D Blood-Brain Barrier Model Derived from Human Pluripotent Stem-Cells

Reconstruction of the Blood-Brain Barrier In Vitro to Model and Therapeutically Target Neurological Disease

The blood-brain barrier (BBB) is a key physiological component of the central nervous system (CNS), maintaining nutrients, clearing waste, and protecting the brain from pathogens. The inherent barrier properties of the BBB pose a challenge for therapeutic drug delivery into the CNS to treat neurological diseases. Impaired BBB function has been related to neurological disease. Cerebral amyloid angiopathy (CAA), the deposition of amyloid in the cerebral vasculature leading to a compromised BBB, is a co-morbidity in most cases of Alzheimer&#39;s disease (AD), suggesting that BBB dysfunction or breakdown may be involved in neurodegeneration. Due to limited access to human BBB tissue, the mechanisms that contribute to proper BBB function and BBB degeneration remain unknown. To address these limitations, we have developed a human pluripotent stem cell-derived BBB (iBBB) by incorporating endothelial cells, pericytes, and astrocytes in a 3D matrix. The iBBB self-assembles to recapitulate the anatomy and cellular interactions present in the BBB. Seeding iBBBs with amyloid captures key aspects of CAA. Additionally, the iBBB offers a flexible platform to modulate genetic and environmental factors implicated in cerebrovascular disease and neurodegeneration, to investigate how genetics and lifestyle affect disease risk. Finally, the iBBB can be used for drug screening and medicinal chemistry studies to optimize therapeutic&#160;delivery to the CNS. In this protocol, we describe the differentiation of the three types of cells (endothelial cells, pericytes, and astrocytes) arising from human pluripotent stem cells, how to assemble the differentiated cells into the iBBB, and how to model CAA in vitro using exogenous amyloid. This model overcomes the challenge of studying live human brain tissue with a system that has both biological fidelity and experimental flexibility, and enables the interrogation of the human BBB and its role in neurodegeneration.

Author Spotlight: A Personalized Approach Towards Investigating Alzheimer's Disease Using an In Vitro Blood-Brain Barrier Model 

The blood-brain barrier (BBB) has a crucial role in sustaining a stable and healthy brain environment. BBB dysfunction is associated with many neurological diseases. We have developed a 3D, stem-cell-derived model of the BBB to investigate cerebrovascular pathology, BBB integrity, and how the BBB is altered by genetics and disease.

The blood-brain barrier (BBB) is a key physiological component of the central nervous system (CNS), maintaining nutrients, clearing waste, and protecting ...