Name: an in vivo assessment of blood-brain barrier disruption in a rat model of ischemic stroke
Uploaded: 2018-03-11T14:00:00.000+00:00
Duration: 12 min 19 s
Description: Watch this Scientific Journal Video about An In Vivo Assessment of Blood-Brain Barrier Disruption in a Rat Model of Ischemic Stroke at JoVE.com

The authors are thankful to the Vice Chancellor for Research of the Ardabil University of Medical Sciences (Ardabil, Iran) for the financial support (grant No: 9607).

All procedures were performed in accordance with the guidelines of Ardabil University of Medical Sciences Research Council for conducting animal studies (Ethical ID number: IR.ARUMS.REC.1394.08). In this visualized study, we used adult male Sprague-Dawley rats (300-350g) obtained from Pasture Institute (Tehran, Iran).
1. Anesthesia and Flowmetry
<ol>
	<li>Induce anesthesia using 4% isoflurane and maintain it with isoflurane (1-1.5%) in a mixture of nitrous oxide (70% v/v) and oxygen (30% v/v) for the duration of the surgery.</li>
	<li>Place an anaesthetized animal in the prone posture on a feed-back controlled heating blanket and maintain the body temperature at 37&#177;0.5 &#176;C by means of a rectal probe connected to this heating unit.</li>
	<li>Apply a small amount of ophthalmic ointment on both eyes to prevent dryness.</li>
	<li>Shave the surgical region on the left side of the skull with electric clippers. Use betadine solution with a gauze pad to disinfect the skin. Rinse this region with a sterile pad containing 70% ethanol and repeat both steps for three cycles17.</li>
	<li>For analgesia, inject 0.2 mL of 0.5% bupivacaine subcutaneously into the surgery region17.</li>
	<li>Make a 1 cm long skin incision on the skull (extending from the lateral canthus of the left eye to the base of left ear) and dissect the left temporalis muscle to expose the skull. Then, create a small burr hole 5 mm lateral to and 1 mm posterior to the bregma18 to ease placement of the tip of the Doppler flowmeter pencil probe.</li>
	<li>Turn the rat from the prone to supine position, and then put the laser pencil probe into the previously drilled skull blind-hole to monitor the regional cerebral blood flow (rCBF).</li>
	<li>Record each animal&#39;s baseline rCBF and consider this as 100%. Notably, middle cerebral artery occlusion (MCAO) is started whenever a decrease in rCBF of more than 80% is detected19,20.</li>
</ol>
2. Induction of the Focal Cerebra Ischemia
<ol>
	<li>Shave the neck region and disinfect the skin with betadine solution and 70% ethanol. Inject 0.2 mL of 0.5% bupivacaine subcutaneously in surgery site for analgesia17.</li>
	<li>Make a 2 cm long surgical incision in the ventral surface of the neck to access the left common carotid artery (LCCA). Isolate the LCCA from the neighboring fascia and the vagus nerve to access bifurcation of the external carotid artery (ECA) and the internal carotid artery (ICA).</li>
	<li>Ligate permanently either the LCCA or ECA employing a 5-0 silk suture and dissect ICA free to the level of pterygopalatine artery.</li>
	<li>Loosely place a tie of 5-0 silk suture around LCCA, and then temporarily ICA clamp with a vascular micro-clip.</li>
	<li>Make a small incision on the LCCA prior to the previously placed loose tie, and then insert a 4-0 silicon-coated nylon suture into the luminal space of ICA and tighten the suture around the LCCA to secure the nylon suture and prevent blood leaking.</li>
	<li>Remove the vascular micro-clip from the ICA. Then, advance a silicon coated intraluminal filament until observing a marked decline in rCBF that indicates occlusion of the MCA origin. At the end of ischemic period (90 min), start reperfusion by withdrawing the intraluminal suture21.</li>
</ol>
3. Jugular Vein Cannulation and Evans Blue (EB) Injection
<ol>
	<li>Make a 1 cm longitudinal incision in the neck to the left side of the midline and then bluntly dissect away superficial fascia to access the external branch of the left jugular vein (LGV). Then, permanently ligate the cranial end of the LGV with 5-0 silk suture. Loosely place two ties around the vein, and then temporarily clamp the cardiac end of the LGV with a vascular micro-clamp.</li>
	<li>Make a small incision on the LGV between two sutures, and then insert heparinized serum filled catheter into the luminal space of the LGV and advance it approximately 10 mm. Afterward, tighten the ligatures around the vein to secure the catheter and prevent bleeding. Inject small amounts of serum to avoid collapsing the vein.</li>
	<li>At the beginning of the reperfusion period, slowly inject EB dye (1 mL/kg of 2% EB solution in saline) over a 5 min period. Subsequently, wash the cannula by injection of 0.5 mL normal saline and withdraw the injection cannula from the vein12,22.</li>
	<li>Finally, suture the neck and head incisions and inject 0.05 mg/kg buprenorphine intraperitoneally (IP) and repeat the injection every 6-8 h during reperfusion period for post-operative analgesia17.</li>
	<li>Inject 5 mL of pre-warmed saline(IP) to provide hydration in the recovery cage17.</li>
	<li>Place the animal in a special recovery cage equipped with temperature and air conditioning control and monitor the recovery of the animal from anesthesia.</li>
</ol>
4. Assessment of the Blood Brain Barrier Permeability
<ol>
	<li>After 24 h of reperfusion, deeply anesthetize the animal with sodium thiopental. Then, open the thoracic cavity by making a small hole beneath the sternum.</li>
	<li>Make a small opening in the right atrium and inject 250 mL of pre-warmed 0.9% saline (37 &#730;C) at a pressure of 110 mmHg through the left ventricle for 15 min to wash EB away from the circulation until the normal saline exits from the atrium becomes colorless23.</li>
	<li>Immediately remove the brain from the skull and place it in the brain matrix. Dissect the olfactory bulb and cerebellum, and then using a clean razor blade, separate the right hemisphere of the brain from the left (Lesioned) along the midline.</li>
	<li>Weigh each hemisphere and homogenize them separately in 2.5 mL of phosphate buffered saline, and mix this with 2.5 mL of trichloroacetic acid (60%) for 2 min using a vortex machine.</li>
	<li>Centrifuge brain samples for 30 min at 1,322 x g and allow the samples to cool down in a 4 &#176;C refrigerator for 10 min.</li>
	<li>Use the supernatants to estimate the EB absorbance by a spectrophotometer at 610 nm23.</li>
	<li>Calculate EB concentrations against a standard curve and express results as &#181;g/g of the brain tissue.</li>
</ol>
5. Production of the EB Standard Curve
<ol>
	<li>Prepare 10 sample solutions of EB with concentrations of 1-10 &#181;g of EB in 5 mL of phosphate buffered saline.</li>
	<li>Measure the absorbance values of each sample at 610 nm using a spectrophotometer.</li>
	<li>Make a scatter chart using a spreadsheet and present EB concentrations on the X axis and the absorbance values on the Y axis (Figure 1).</li>
	<li>Define a linear trend-line for the curve with its equation. Then, use it to obtain the EB concentration of samples.</li>
	<li>Report the final results of EB extravasation as &#181;g/g of the brain tissue weight.</li>
</ol>
6. Sham Operation
<ol>
	<li>Perform the same surgical process with rats in sham-operated group (including making an incision in the neck region and EB injection) but exclude the MCAO.</li>
</ol>

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The authors have nothing to disclose.

Thus far, various methods such as autoradiography and detection of the radioactive tracers24,25, immunofluorescence microscopy26,27, and EB extravasation technique20,23 have been used to evaluate the blood-brain barrier damage. EB dye is strongly able to bind to the serum albumin and is used as a tracer for detecting vascular leakage and quantifyi...

Vasogenic brain edema due to blood-brain barrier (BBB) disruption remains an important complication of the ischemic stroke and a major determinant of the survival rate in the stroke patients1,2. The blood-brain barrier (BBB), which is formed by brain capillary endothelial cells (BCECs) and composed of distinct neurovascular components (e.g., tight junctions among BCECs, pericytes, astroglial, and neuronal cells3), provides a specialized and dynamic interface between the central nervous system (CNS) and peripheral blood-circulation4,5. Insults such as ischemia-reperfusion injuries could disrupt the functional integrity of the BBB and lead to subsequent penetration of circulating leukocytes into the brain parenchyma that ultimately trigger cerebral inflammation and primary brain injuries6,7. Animal models are needed for the exact detection of the dysfunction of BBB following occurrence of a stroke. Such models are of great importance for studying underlying pathophysiological mechanisms and introducing new neuroprotective strategies. In vitro cell culture-based models of the BBB have been highly developed and used for molecular study of the BBB physiopathology8,9,10. Nevertheless, in vivo animal models, which produce ischemic damage of the BBB similar to human clinical conditions, are also very worthwhile in this regard. Quantitative detection of the extravasation of Evans blue (EB) is a well-accepted and sensitive technique that has been used for assessment of the BBB integrity and function in neurodegenerative diseases, including ischemic stroke11,12,13,14. This method is cost-effective, feasible, reproducible, and completely applicable in any experimental laboratory. Its implementation does not require advanced equipment, such as radioactive tracers15 or magnetic resonance imaging (MRI)16, that are prerequisites for other methods. In this article, we comprehensively demonstrate basic technical processes of BBB assessment using EB extravasation in rat models of ischemic stroke.

<table><tbody><tr><td>Isoflurane</td><td>Piramal</td><td>AWN 34041100</td><td>20 - 25 &amp;deg;C</td></tr><tr><td>2,3,5-Triphenyltetrazolium chloride (TTC)</td><td>Molekula</td><td>31216368</td><td>4 years</td></tr><tr><td>Sprague&amp;ndash;Dawley rats&amp;nbsp;</td><td>Pasture Institute (Tehran, Iran)</td><td></td><td>300-350g</td></tr><tr><td>Evans Blue&amp;nbsp;</td><td>Sigma-Aldrich&amp;nbsp;</td><td>314-13-6</td><td></td></tr><tr><td>Trichloroacetic acid&amp;nbsp;</td><td>Sigma-Aldrich&amp;nbsp;</td><td>76-03-9</td><td>2 years</td></tr><tr><td>Bupivacaine HCl (0.5%)</td><td>Delpharm Tours</td><td></td><td>below&amp;nbsp; 25 &amp;deg;C</td></tr><tr><td>Bupernorphine</td><td>Exir (Iran)</td><td></td><td></td></tr><tr><td>Sodium Carbonate</td><td>Sigma-Aldrich&amp;nbsp;</td><td>497-19-8</td><td></td></tr><tr><td>Sodium chloride&amp;nbsp;</td><td>Sigma-Aldrich&amp;nbsp;</td><td>7647-14-5</td><td></td></tr><tr><td>Di- Sodium hydrogen phosphate</td><td>EMD Millipore&amp;nbsp;</td><td>231-448-7</td><td></td></tr><tr><td>Potassium chloride</td><td>Sigma-Aldrich&amp;nbsp;</td><td>&amp;nbsp;7447-40-7</td><td></td></tr><tr><td>Ethanol&amp;nbsp;</td><td>Sigma-Aldrich&amp;nbsp;</td><td>64-17-5</td><td></td></tr><tr><td>silicone(Xantopren)</td><td>Heraeus</td><td>EN ISO 4823</td><td></td></tr><tr><td>Activator universal plus</td><td>Heraeus</td><td>66037445</td><td></td></tr><tr><td>Micro-Dissecting forceps</td><td>Stoelting</td><td>52100-41</td><td></td></tr><tr><td>Spring Scisors</td><td>Stoelting</td><td>52130-00</td><td></td></tr><tr><td>Operating&amp;nbsp; Scissors</td><td>Roboz</td><td>52140-70</td><td></td></tr><tr><td>Brain matrix&amp;nbsp;</td><td>Stoelting</td><td>51390</td><td></td></tr><tr><td>Anesthesia Machine for Small Animals |</td><td>&amp;nbsp;Kent Scientific</td><td>SS-01</td><td></td></tr><tr><td>Power Lab system</td><td>AD Instruments</td><td>ML880</td><td></td></tr><tr><td>Laser Doppler flowmeter</td><td>AD Instruments</td><td>ML191</td><td></td></tr><tr><td>Heating feed back system</td><td>Harvard Appratus</td><td>72-7560</td><td></td></tr><tr><td>Vascular micro clamp</td><td>FineScience Tools</td><td>18055-03</td><td></td></tr><tr><td>Silk 5-0 suture thread</td><td>Ethicon</td><td>682G</td><td></td></tr><tr><td>Ethilon 4-0 suture thread&amp;nbsp;</td><td>Ethicon</td><td>EH6740G</td><td></td></tr></tbody></table>

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.

There was no significant difference in EB levels in the right hemisphere versus the left hemisphere of the sham-operated rats (1.06 &#177; 0.1 &#181;g/g and 1.1 &#177; 0.09 &#181;g/g, respectively). As shown in Figures 2A-2B, induction of transient ischemia (90 min ischemia/ 24 h reperfusion) caused a significant difference in EB levels (10.41 &#177; 0.84 &#181;g/g, p &#60;0.001) in the left hemisphere of ischemic rats, as compared to the respective hemisphere in...

Watch this Scientific Journal Video about An In Vivo Assessment of Blood-Brain Barrier Disruption in a Rat Model of Ischemic Stroke at JoVE.com

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.

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 ...

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Research

JoVE Journal

Neuroscience

11.3K Views.  Ardabil University of Medical Sciences. 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.

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

Focal Cerebral Ischemia Model by Endovascular Suture Occlusion of the Middle Cerebral Artery in the Rat

Stroke is the leading cause of disability and the third leading cause of death in adults worldwide1. In human stroke, there exists a highly variable clinical state; in the development of animal models of focal ischemia, however, achieving reproducibility of experimentally induced infarct volume is essential. The rat is a widely used animal model for stroke due to its relatively low animal husbandry costs and to the similarity of its cranial circulation to that of humans2,3. In humans, the middle cerebral artery (MCA) is most commonly affected in stroke syndromes and multiple methods of MCA occlusion (MCAO) have been described to mimic this clinical syndrome in animal models. Because recanalization commonly occurs following an acute stroke in the human, reperfusion after a period of occlusion has been included in many of these models. In this video, we demonstrate the transient endovascular suture MCAO model in the spontaneously hypertensive rat (SHR). A filament with a silicon tip coating is placed intraluminally at the MCA origin for 60 minutes, followed by reperfusion. Note that the optimal occlusion period may vary in other rat strains, such as Wistar or Sprague-Dawley. Several behavioral indicators of stroke in the rat are shown. Focal ischemia is confirmed using T2-weighted magnetic resonance images and by staining brain sections with 2,3,5-triphenyltetrazolium chloride (TTC) 24 hours after MCAO.

Surgical induction of ischemic brain damage in the rat is a widely used model for stroke research. Here we demonstrate the induction of focal cerebral ischemia by occlusion of the middle cerebral artery. Visualization of the resulting infarct by histological staining and magnetic resonance imaging is also shown.

Stroke is the leading cause of disability and the third leading cause of death in adults worldwide1. In human stroke, there exists a highly variable ...

Rat Model of Blood-brain Barrier Disruption to Allow Targeted Neurovascular Therapeutics

Endothelial cells with tight junctions along with the basement membrane and astrocyte end feet surround cerebral blood vessels to form the blood-brain barrier1. The barrier selectively excludes molecules from crossing between the blood and the brain based upon their size and charge. This function can impede the delivery of therapeutics for neurological disorders. A number of chemotherapeutic drugs, for example, will not effectively cross the blood-brain barrier to reach tumor cells2. Thus, improving the delivery of drugs across the blood-brain barrier is an area of interest.
The most prevalent methods for enhancing the delivery of drugs to the brain are direct cerebral infusion and blood-brain barrier disruption3. Direct intracerebral infusion guarantees that therapies reach the brain; however, this method has a limited ability to disperse the drug4. Blood-brain barrier disruption (BBBD) allows drugs to flow directly from the circulatory system into the brain and thus more effectively reach dispersed tumor cells. Three methods of barrier disruption include osmotic barrier disruption, pharmacological barrier disruption, and focused ultrasound with microbubbles. Osmotic disruption, pioneered by Neuwelt, uses a hypertonic solution of 25% mannitol that dehydrates the cells of the blood-brain barrier causing them to shrink and disrupt their tight junctions. Barrier disruption can also be accomplished pharmacologically with vasoactive compounds such as histamine5 and bradykinin6. This method, however, is selective primarily for the brain-tumor barrier7. Additionally, RMP-7, an analog of the peptide bradykinin, was found to be inferior when compared head-to-head with osmotic BBBD with 25% mannitol8. Another method, focused ultrasound (FUS) in conjunction with microbubble ultrasound contrast agents, has also been shown to reversibly open the blood-brain barrier9. In comparison to FUS, though, 25% mannitol has a longer history of safety in human patients that makes it a proven tool for translational research10-12.
In order to accomplish BBBD, mannitol must be delivered at a high rate directly into the brain's arterial circulation. In humans, an endovascular catheter is guided to the brain where rapid, direct flow can be accomplished. This protocol models human BBBD as closely as possible. Following a cut-down to the bifurcation of the common carotid artery, a catheter is inserted retrograde into the ECA and used to deliver mannitol directly into the internal carotid artery (ICA) circulation. Propofol and N2O anesthesia are used for their ability to maximize the effectiveness of barrier disruption13. If executed properly, this procedure has the ability to safely, effectively, and reversibly open the blood-brain barrier and improve the delivery of drugs that do not ordinarily reach the brain 8,13,14.

Blood-brain barrier disruption aids the delivery of certain drugs to the brain. Mannitol delivered intra-arterially shrinks cells surrounding blood vessels in order to physically disrupt the barrier.

Endothelial cells with tight junctions along with the basement membrane and astrocyte end feet surround cerebral blood vessels to form the blood-brain ...

Medicine

Setting-up an In Vitro Model of Rat Blood-brain Barrier (BBB): A Focus on BBB Impermeability and Receptor-mediated Transport

The blood brain barrier (BBB) specifically regulates molecular and cellular flux between the blood and the nervous tissue. Our aim was to develop and characterize a highly reproducible rat syngeneic in vitro model of the BBB using co-cultures of primary rat brain endothelial cells (RBEC) and astrocytes to study receptors involved in transcytosis across the endothelial cell monolayer. Astrocytes were isolated by mechanical dissection following trypsin digestion and were frozen for later co-culture. RBEC were isolated from 5-week-old rat cortices. The brains were cleaned of meninges and white matter, and mechanically dissociated following enzymatic digestion. Thereafter, the tissue homogenate was centrifuged in bovine serum albumin to separate vessel fragments from nervous tissue. The vessel fragments underwent a second enzymatic digestion to free endothelial cells from their extracellular matrix. The remaining contaminating cells such as pericytes were further eliminated by plating the microvessel fragments in puromycin-containing medium. They were then passaged onto filters for co-culture with astrocytes grown on the bottom of the wells. RBEC expressed high levels of tight junction (TJ) proteins such as occludin, claudin-5 and ZO-1 with a typical localization at the cell borders. The transendothelial electrical resistance (TEER) of brain endothelial monolayers, indicating the tightness of TJs reached 300 ohm&#183;cm2 on average. The endothelial permeability coefficients (Pe) for lucifer yellow (LY) was highly reproducible with an average of 0.26 &#177;&#160;0.11 x 10-3 cm/min. Brain endothelial cells organized in monolayers expressed the efflux transporter P-glycoprotein (P-gp), showed a polarized transport of rhodamine 123, a ligand for P-gp, and showed specific transport of transferrin-Cy3 and DiILDL across the endothelial cell monolayer. In conclusion, we provide a protocol for setting up an in vitro BBB model that is highly reproducible due to the quality assurance methods, and that is suitable for research on BBB transporters and receptors.

Setting-up an In Vitro Model of Rat Blood-brain Barrier BBB: A Focus on BBB Impermeability and Receptor-mediated Transport

The aim of the present study was to validate the reproducibility of an in vitro BBB model involving a rat syngeneic co-culture of endothelial cells and astrocytes. The endothelial cell monolayer presented high TEER and low LY permeability. Expression of specific TJ proteins, functional responses to inflammation and functionality of transporters and receptors were assessed.

The blood brain barrier (BBB) specifically regulates molecular and cellular flux between the blood and the nervous tissue. Our aim was to develop and ...

A Triple Primary Cell Culture Model of the Human Blood-Brain Barrier for Studying Ischemic Stroke In Vitro

Ischemic stroke is a major cause of death and disability worldwide with limited therapeutic options. The neuropathology of ischemic stroke is characterized by an interruption in blood supply to the brain leading to cell death and cognitive dysfunction. During and after ischemic stroke, blood-brain barrier (BBB) dysfunction facilitates injury progression and contributes to poor patient recovery. Current BBB models primarily include endothelial monocultures and double co-cultures with either astrocytes or pericytes.
Such models lack the ability to fully imitate a dynamic brain microenvironment, which is essential for cell-to-cell communication. Additionally, commonly used BBB models often contain immortalized human endothelial cells or animal-derived (rodent, porcine, or bovine) cell cultures that pose translational limitations. This paper describes a novel well-insert-based BBB model containing only primary human cells (brain microvascular endothelial cells, astrocytes, and brain vascular pericytes) enabling the investigation of ischemic brain injury in vitro. 
The effects of oxygen-glucose deprivation (OGD) on barrier integrity were assessed by passive permeability, transendothelial electrical resistance (TEER) measurements,and direct visualization of hypoxic cells. The presented protocol offers a distinct advantage inmimicking the intercellular environment of the BBB in vivo, serving as a more realistic in vitro BBB model for developing new therapeutic strategies in the setting of ischemic brain injury.

A Triple Primary Cell Culture Model of the Human Blood-Brain Barrier for Studying Ischemic Stroke In Vitro

Here, we describe the method for establishing a triple cell culture model of the blood-brain barrier based on primary human brain microvascular endothelial cells, astrocytes, and pericytes. This multicellular model is suitable for studies of neurovascular unit dysfunction during ischemic stroke in vitro or for the screening of drug candidates.

Ischemic stroke is a major cause of death and disability worldwide with limited therapeutic options. The neuropathology of ischemic stroke is ...

Biochemistry

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 ...

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 ...

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 ...

Real-Time Monitoring and Modulation of Blood Pressure in a Rabbit Model of Ischemic Stroke

Control of blood pressure, in terms of both absolute values and its variability, affects outcomes in ischemic stroke patients. However, it remains challenging to identify the mechanisms that lead to poor outcomes or evaluate measures by which these effects can be mitigated because of the prohibitive limitations inherent to human data. In such cases, animal models can be utilized to conduct rigorous and reproducible evaluations of diseases. Here we report refinement of a previously described model of ischemic stroke in rabbits that is augmented with continuous blood pressure recording to assess the impacts of modulation on blood pressure. Under general anesthesia, femoral arteries are exposed through surgical cutdowns to place arterial sheaths bilaterally. Under fluoroscopic visualization and roadmap guidance, a microcatheter is advanced into an artery of the posterior circulation of the brain. An angiogram is performed by injecting the contralateral vertebral artery to confirm occlusion of the target artery. With the occlusive catheter remaining in position for a fixed duration, blood pressure is continuously recorded to allow for tight titration of blood pressure manipulations, whether through mechanical or pharmacological means. At the completion of the occlusion interval, the microcatheter is removed, and the animal is maintained under general anesthesia for a prescribed length of reperfusion. For acute studies, the animal is then euthanized and decapitated. The brain is harvested and processed to measure the infarct volume under light microscopy and further assessed with various histopathological stains or spatial transcriptomic analysis. This protocol provides a reproducible model that can be utilized for more thorough preclinical studies on the effects of blood pressure parameters during ischemic stroke. It also facilitates effective preclinical evaluation of novel neuroprotective interventions that might improve care for ischemic stroke patients.

Continuous arterial blood pressure recording allows the investigation of impacts of various hemodynamic parameters. This report demonstrates the application of continuous arterial blood pressure monitoring in a large animal model of ischemic stroke for determination of stroke pathophysiology, impact of different hemodynamic factors, and the assessment of novel treatment approaches.

Control of blood pressure, in terms of both absolute values and its variability, affects outcomes in ischemic stroke patients. However, it remains ...

Immunofluorescence Staining to Study Cell Death in Rat MCAO Stroke Models

Evaluating Cell Death Signaling by Immunofluorescence in a Rat Model of Ischemic Stroke

Stroke is a leading cause of death and disability worldwide. Most cases of stroke are ischemic and result from the occlusion of the middle cerebral artery (MCA). Current pharmacological approaches for the treatment of ischemic stroke are limited; therefore, novel therapies providing effective neuroprotection against ischemic injury following stroke are urgently needed. In the brain tissue following ischemic stroke, the area of the ischemic penumbra is salvageable but at risk of progressing to irreversible damage. The penumbral area surrounding the infarcted core is a conceptual target for neuroprotection. Since the blood flow is partially maintained in the penumbral area, neuronal and non-neuronal cells in this area transiently survive after stroke, and these cells that are still viable may be rescued by appropriate medical interventions. Understanding the pathophysiology of the penumbra is important in the development of neuroprotective therapies because the cell death pathways activated in the ischemic penumbra may indicate therapeutic targets, such as RUNX1 and cathepsins. These protein targets functioning as mediators of programmed cell death can be further exploited in translational research. With moderate size and similarity to the human brain, the in vivo rat model of MCA occlusion (MCAO) mimics the human ischemic stroke and offers an applicable tool to investigate the penumbral pathology, examine the cell death signaling, and evaluate the effects of potential targets in the context of MCAO. Here, we describe how to induce MCAO in rats and how to perform immunofluorescence staining for the detection of cell death signaling in the rat brain following MCAO.

Rat in vivo models are indispensable tools to investigate the pathological mechanisms and therapeutic targets for ischemic stroke. This work will describe performing immunofluorescence staining in infarcted brain slices following middle cerebral artery occlusion (MCAO) in rats.

Stroke is a leading cause of death and disability worldwide. Most cases of stroke are ischemic and result from the occlusion of the middle cerebral artery ...

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

Summary

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Chapters in this video

Focal Cerebral Ischemia Model by Endovascular Suture Occlusion of the Middle Cerebral Artery in the Rat

Rat Model of Blood-brain Barrier Disruption to Allow Targeted Neurovascular Therapeutics

Setting-up an In Vitro Model of Rat Blood-brain Barrier (BBB): A Focus on BBB Impermeability and Receptor-mediated Transport

A Triple Primary Cell Culture Model of the Human Blood-Brain Barrier for Studying Ischemic Stroke In Vitro

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

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

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

Real-Time Monitoring and Modulation of Blood Pressure in a Rabbit Model of Ischemic Stroke

Evaluating Cell Death Signaling by Immunofluorescence in a Rat Model of Ischemic Stroke