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 &amp;amp; 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&amp;auml;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>&lt;strong&gt;Software&lt;/strong&gt;</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>SPSS Statistics 22</td><td>IBM</td><td></td><td></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>&lt;strong&gt;Reagents&lt;/strong&gt;</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. Dit protocol beschrijft een nieuwe techniek om de drie belangrijkste parameters van ischemisch hersenletsel te meten op dezelfde set knaagdierhersenmonsters. Het gebruik van slechts één hersenmonster is zeer voordelig in termen van ethische en economische kosten.

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

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 ...

2-Vessel Occlusion/Hypotension: A Rat Model of Global Brain Ischemia

Cardiac arrest followed by resuscitation often results in dramatic brain damage caused by ischemia and subsequent reperfusion of the brain. Global brain ischemia produces damage to specific brain regions shown to be highly sensitive to ischemia 1. Hippocampal neurons have higher sensitivity to ischemic insults compared to other cell populations, and specifically, the CA1 region of the hippocampus is particularly vulnerable to ischemia/reperfusion 2.
The design of therapeutic interventions, or study of mechanisms involved in cerebral damage, requires a model that produces damage similar to the clinical condition and in a reproducible manner. Bilateral carotid vessel occlusion with hypotension (2VOH) is a model that produces reversible forebrain ischemia, emulating the cerebral events that can occur during cardiac arrest and resuscitation. We describe a model modified from Smith et al. (1984) 2, as first presented in its current form in Sanderson, et al. (2008) 3, which produces reproducible injury to selectively vulnerable brain regions 3-6. The reliability of this model is dictated by precise control of systemic blood pressure during applied hypotension, the duration of ischemia, close temperature control, a specific anesthesia regimen, and diligent post-operative care. An 8-minute ischemic insult produces cell death of CA1 hippocampal neurons that progresses over the course of 6 to 24 hr of reperfusion, while less vulnerable brain regions are spared. This progressive cell death is easily quantified after 7-14 days of reperfusion, as a near complete loss of CA1 neurons is evident at this time.
In addition to this brain injury model, we present a method for CA1 damage quantification using a simple, yet thorough, methodology. Importantly, quantification can be accomplished using a simple camera-mounted microscope, and a free ImageJ (NIH) software plugin, obviating the need for cost-prohibitive stereology software programs and a motorized microscopic stage for damage assessment.

Bilateral carotid occlusion coupled with systemic hypotension produces global brain ischemia in the rat, resulting in damage to the hippocampus with reproducible severity. Animal subjects are impaired with predictable patterns of brain damage, they recover expediently, and mortality rates are comparatively low.

Cardiac arrest followed by resuscitation often results in dramatic brain damage caused by ischemia and subsequent reperfusion of the brain. Global brain ...

Medicine

Optimized System for Cerebral Perfusion Monitoring in the Rat Stroke Model of Intraluminal Middle Cerebral Artery Occlusion

The translational potential of pre-clinical stroke research depends on the accuracy of experimental modeling. Cerebral perfusion monitoring in animal models of acute ischemic stroke allows to confirm successful arterial occlusion and exclude subarachnoid hemorrhage. Cerebral perfusion monitoring can also be used to study intracranial collateral circulation, which is emerging as a powerful determinant of stroke outcome and a possible therapeutic target. Despite a recognized role of Laser Doppler perfusion monitoring as part of the current guidelines for experimental cerebral ischemia, a number of technical difficulties exist that limit its widespread use. One of the major issues is obtaining a secure and prolonged attachment of a deep-penetration Laser Doppler probe to the animal skull. In this video, we show our optimized system for cerebral perfusion monitoring during transient middle cerebral artery occlusion by intraluminal filament in the rat. We developed in-house a simple method to obtain a custom made holder for twin-fibre (deep-penetration) Laser Doppler probes, which allow multi-site monitoring if needed. A continuous and prolonged monitoring of cerebral perfusion could easily be obtained over the intact skull.

Cerebral perfusion monitoring has been demonstrated to improve accuracy in ischemic stroke models. Technical difficulties often limit the use of this essential tool for cerebrovascular research. In this video, an optimized system is shown to obtain a single or multi-site hemodynamic monitoring during intraluminal middle cerebral artery occlusion in rats.

The translational potential of pre-clinical stroke research depends on the accuracy of experimental modeling. Cerebral perfusion monitoring in animal ...

A Middle Cerebral Artery Occlusion Technique for Inducing Post-stroke Depression in Rats

Post-stroke depression (PSD) is the most recurrent of all psychiatric complications resulting from an ischemic stroke. A greater majority (about 60%) of all ischemic stroke patients suffer from PSD, a disorder considered to be an ischemic stroke-related precursor for increased death and degradation in health. The pathophysiology of PSD is still obscure. To study the mechanism of development and occurrence of PSD further, and to find out a therapy, we attempted to develop a new protocol that requires occluding the middle cerebral artery (MCA) via the internal carotid artery (ICA) in rats. This protocol describes a model of PSD induced in rats through the middle cerebral artery occlusion (MCAO). Also used in the experiment are the Porsolt forced swim test and the sucrose preference test to confirm and evaluate the depressive mood of the rats under investigation. Rather than inserting the catheter through the external carotid artery (ECA), as stipulated for the original procedure, this MCAO technique has the monofilament passing directly through the ICA. This MCAO technique was developed a few years ago and leads to a reduction in mortality and variability. It is generally accepted that the criteria used are preferred in the selection of biological models. The data obtained with this protocol show that this model of MCAO could be a way of inducing PSD in rats and could potentially lead to the understanding of the pathophysiology and the future development of new drugs and other neuroprotective agents.

Here we present a protocol to induce post-stroke depression in rats by occluding the middle cerebral artery via the internal carotid artery. We use the Porsolt forced swim test and the sucrose preference test to confirm and evaluate induced depressive moods.

Post-stroke depression (PSD) is the most recurrent of all psychiatric complications resulting from an ischemic stroke. A greater majority (about 60%) of ...

Behavior

Stereological and Flow Cytometry Characterization of Leukocyte Subpopulations in Models of Transient or Permanent Cerebral Ischemia

Microglia activation, as well as extravasation of haematogenous macrophages and neutrophils, is believed to play a pivotal role in brain injury after stroke. These myeloid cell subpopulations can display different phenotypes and functions and need to be distinguished and characterized to study their regulation and contribution to tissue damage. This protocol provides two different methodologies for brain immune cell characterization: a precise stereological approach and a flow cytometric analysis. The stereological approach is based on the optical fractionator method, which calculates the total number of cells in an area of interest (infarcted brain) estimated by a systematic random sampling. The second characterization approach provides a simple way to isolate brain leukocyte suspensions and to characterize them by flow cytometry, allowing for the characterization of microglia, infiltrated monocytes and neutrophils of the ischemic tissue. In addition, it also details a cerebral ischemia model in mice that exclusively affects brain cortex, generating highly reproducible infarcts with a low rate of mortality, and the procedure for histological brain processing to characterize infarct volume by the Cavalieri method.

Brain myeloid cells characterization following stroke can be performed by stereology using the optical fractionator method, or by a flow cytometric analysis of brain leukocytes suspensions. Both are useful techniques to perform an accurate phenotypical distinction of the main myeloid cell subsets found in the ischemic brain.

Microglia activation, as well as extravasation of haematogenous macrophages and neutrophils, is believed to play a pivotal role in brain injury after ...

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 ...

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 ...

Laser-Induced Brain Injury in the Motor Cortex of Rats

A common technique for inducing stroke in experimental rodent models involves the transient (often denoted as MCAO-t) or permanent (designated as MCAO-p) occlusion of the middle cerebral artery (MCA) using a catheter. This generally accepted technique, however, has some limitations, thereby limiting its extensive use. Stroke induction by this method is often characterized by high variability in the localization and size of the ischemic area, periodical occurrences of hemorrhage, and high death rates. Also, the successful completion of any of the transient or permanent procedures requires expertise and often lasts for about 30 minutes. In this protocol, a laser irradiation technique is presented that can serve as an alternative method for inducing and studying brain injury in rodent models.
When compared to rats in the control and MCAO groups, the brain injury by laser induction showed reduced variability in body temperature, infarct volume, brain edema, intracranial hemorrhage, and mortality. Furthermore, the use of a laser-induced injury caused damage to the brain tissues only in the motor cortex unlike in the MCAO experiments where destruction of both the motor cortex and striatal tissues is observed.
Findings from this investigation suggest that laser irradiation could serve as an alternative and effective technique for inducing brain injury in the motor cortex. The method also shortens the time for completing the procedure and does not require expert handlers.

The protocol presented here shows a technique to create a rodent model of brain injury. The method described here uses laser irradiation and targets motor cortex.

A common technique for inducing stroke in experimental rodent models involves the transient (often denoted as MCAO-t) or permanent (designated as MCAO-p) ...

A Fibrin-Enriched and tPA-Sensitive Photothrombotic Stroke Model

An ideal thromboembolic stroke model requires certain properties, including relatively simple surgical procedures with low mortality, a consistent infarction size and location, precipitation of platelet:fibrin intermixed blood clots similar to those in patients, and an adequate sensitivity to fibrinolytic treatment. The rose bengal (RB) dye-based photothrombotic stroke model meets the first two requirements but is highly refractory to tPA-mediated lytic treatment, presumably due to its platelet-rich, but fibrin-poor clot composition. We reason that combination of RB dye (50 mg/kg) and a sub-thrombotic dose of thrombin (80 U/kg) for photoactivation aimed at the proximal branch of middle cerebral artery (MCA) may produce fibrin-enriched and tPA-sensitive clots. Indeed, the thrombin and RB (T+RB)-combined photothrombosis model triggered mixed platelet:fibrin blood clots, as shown by immunostaining and immunoblots, and maintained consistent infarct sizes and locations plus low mortality. Moreover, intravenous injection of tPA (Alteplase, 10 mg/kg) within 2 h post-photoactivation significantly decreased the infarct size in T+RB photothrombosis. Thus, the thrombin-enhanced photothrombotic stroke model may be a useful experimental model to test novel thrombolytic therapies.

Traditional photothrombotic stroke (PTS) models mainly induce dense platelet aggregates of a high resistance to tissue plasminogen activator (tPA)-lytic treatment. Here a modified murine PTS model is introduced by co-injecting thrombin and photosensitive dye for photoactivation. The thrombin-enhanced PTS model produces mixed platelet:fibrin clots and is highly sensitive to tPA-thrombolysis.

An ideal thromboembolic stroke model requires certain properties, including relatively simple surgical procedures with low mortality, a consistent ...

Early Pathological and Magnetic Resonance Detection of Cerebral Injury Using a Rat Model of Neonatal Hypoxic Ischemic Encephalopathy

Perinatal hypoxic-ischemic encephalopathy (HIE) is an acute disease that may afflict newborns, resulting in variable long- and short-term neurodevelopmental outcomes. Early diagnosis is critical to identifying infants who may benefit from intervention; however, early diagnosis relies heavily on clinical criteria. No molecular or radiological tests have shown promise in detecting early cerebral injury. Studies have shown that magnetic resonance imaging (MRI) can show changes in both blood flow/ischemia and metabolic disruption. However, they have all been used to evaluate the secondary phase of the disease (&gt;12 h) after the onset of the injury. Early diagnosis is critical to rapidly starting therapeutic hypothermia in eligible infants, which is currently recommended to be initiated within 6 h of birth. The rat model of hypoxic-ischemic injury was developed in 1981 and has been validated and used extensively to study changes in brain perfusion, cerebral injury markers, and morphology. However, it has primarily been used as a "late model", evaluating injury several days after the initial ischemic insult. The model has been known to have poor sensitivity in evaluating reliable and reproducible early cerebral changes. The objective of this study was to develop a reliable model to study early gross morphological and radiological markers of HIE using pathological staining and cerebral magnetic resonance imaging/magnetic resonance spectroscopy.

The present protocol describes a rodent model of newborn hypoxic-ischemic injury for identifying early changes in cerebral tissue by gross morphology and magnetic resonance imaging. This has benefits over existing models, which can be used to study late injury but do not allow the evaluation of reproducible early changes.

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

Samenvatting

Meer video's verkennen