Name: experimental models to study the neuroprotection of acidic postconditioning against cerebral ischemia
Uploaded: 2017-07-31T12:30:00.000+00:00
Duration: 10 min 13 s
Description: Watch this Scientific Journal Video about Experimental Models to Study the Neuroprotection of Acidic Postconditioning Against Cerebral Ischemia at JoVE.com

This work was funded by the National Natural Science Foundation of China (81573406, 81373393, 81273506, 81221003, 81473186 and 81402907), Zhejiang Provincial Natural Science Foundation (LR15H310001) and the Program for Zhejiang Leading Team of S&amp;T Innovation Team (2011R50014).

All experiments were approved by and conducted in accordance with the ethical guidelines of the Zhejiang University Animal Experimentation Committee and were in complete compliance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Efforts were made to minimize any pain or discomfort, and the minimum number of animals was used.
1. OGD of Corticostriatal Slices
<ol>
	<li>Solution preparation:
	<ol>
		<li>Prepare 1,000 mL r-ACSF (124 mmol/L, NaCl, 5 mmol/L KCl, 1.25 mmol/L KH2PO4, 2 mmol/L MgSO4, 26 mmol/L NaHCO3, 2 mmol/L CaCl2, 10 mmol/L glucose, final pH 7.4). Bubble with 5% CO2 and 95% O2 throughout the experiment.</li>
		<li>Prepare 200 mL glucose-free ACSF (gf-ACSF) as above but excluding glucose, bubbled with 5% CO2 and 95% N2. Bubble the gases for 30 min before applying to brain slices to reduce the oxygen content in the solution. Continue bubbling until the OGD procedure is finished.</li>
		<li>Prepare acidic ACSF (a-ACSF) by equilibrating 200 mL r-ACSF with 20% CO2 and 80% O2. Bubble the gases for 30 min before applying to brain slices to reduce the pH of the solution to pH 6.8, and continue bubbling until APC procedure is finished.</li>
		<li>Place three tissue holders into r-ACSF, gf-ACSF, a-ACSF, respectively.</li>
	</ol>
	</li>
	<li>Brain slice preparation: 
	Note: 8-week-old, male C57Bl/6J mice were used in this study. The animals were housed under controlled temperature (22 &#177; 2 &#176;C), with a 12 h light-dark cycle period and access to pelleted food and water.
	<ol>
		<li>Sacrifice a mouse with an isoflurane overdose in an induction chamber. Decapitate the mouse. Dissect out the brain using small scissors and forceps. Remove the brain with a thin spatula and drop it carefully into a glass beaker containing ice-cold r-ACSF equilibrated with 5% CO2 and 95% O2. 
		NOTE: These steps should be done as quickly but meticulously as possible.</li>
		<li>Keep the brain in the ice-cold r-ACSF for 5 min. Adjust the gas pressure to avoid movements of the brain.</li>
		<li>Place cryoprecipitate glue on the vibratome plate in two strips. Put a piece of 3% agarose upon the glue strip away from the blade to support the brain.</li>
		<li>Invert a 10-cm Petri dish on ice and then put a filter paper on the dish. Moisten the filter paper with drops of ice-cold r-ACSF.</li>
		<li>Transfer the brain to the filter paper with forceps. Cut off the frontal pole and the cerebellum with blade and forceps. 
		NOTE: The transverse sections should be as smooth as possible and vertical to sagittal plane.</li>
		<li>Place the remaining brain tissue vertically upon the other strip of glue and keep the brain leaning against the agarose. Add ice-cold r-ACSF to the cutting reservoir to submerge the brain and add ice to the ice holder area. Keep the r-ACSF in the reservoir bubbled with 5% CO2 and 95% O2.</li>
		<li>Raise the reservoir and adjust the position of the razor to place the razor as close to the brain as possible and just above the brain.</li>
		<li>Choose &#34;CONT&#34; mode to slice the brain sections continuously. Set the cutting thickness to 400 &#181;m, vibrating frequency to 6 - 8, and speed to 3 - 4. Press the &#34;start/stop&#34; button to start automatic cutting.</li>
		<li>Cut off the tip of a 3 mL Pasteur pipette to make an opening matching the size of the brain slices. 
		NOTE: The opening should be large enough to avoid additional damage to the brain slices.</li>
		<li>Draw out five brain slices one by one with the tip-cutPasteur pipette and place them into the tissue holder in ice-cold r-ACSF bubbled with 5% CO2 and 95% O2. Do not collect the first slice produced. Adjust the gas pressure to avoid movements of the brain slices. 
		NOTE: The brain slices should not cover each other.</li>
		<li>Place brain slices with r-ACSF at room temperature for 30 min and then at 37 &#176;C in a water bath for another 10 min to recover synaptic functions.</li>
	</ol>
	</li>
	<li>OGD and APC:
	<ol>
		<li>Place gf-ACSF and a-ACSF into a 37 &#176;C water bath and bubble the buffers with the corresponding gases as mentioned above in 1.1.2 and 1.1.3 for 30 min.</li>
		<li>Leave one slice in r-ACSF as a control. Transfer the other four brain slices into the tissue holder with preheated gf-ACSF carefully and incubate for 15 min. Transfer the slices back into the tissue holder in r-ACSF to achieve reperfusion.</li>
		<li>To study the time window of APC, transfer one slice directly from gf-ACSF to a-ACSF. Transfer the other three slices to r-ACSF at first, and then transfer two of them to the tissue holder in a-ACSF 5 and 15 min after reperfusion respectively.</li>
		<li>Incubate the two slices in a-ACSF for 3 min and then transfer them back to r-ACSF. Incubate the three slices in r-ACSF for another 1 h. Set the remaining slices in r-ACSF as the OGD group.</li>
		<li>For a dose response study, transfer all four slices to the tissue holder in r-ACSF at first after OGD and incubate for 5 min. Then leave one slice in r-ACSF as the OGD group and transfer the other three slices to a-ACSF. Incubate the slices in a-ACSF for 1, 3 and 5 min respectively and then transfer them back to r-ACSF. Incubate the three slices in r-ACSF for another 1 h.</li>
	</ol>
	</li>
	<li>Slice viability determination:
	<ol>
		<li>Prepare 0.25% 2,3,5-triphenyltetrazolium hydrochloride (TTC) normal saline solution. Add 1.25 g TTC powder to 500 mL normal saline solution. 
		NOTE: After the powder completely dissolves, transfer the solution into a 24-well plate (500 &#181;L per well) covered in foil and store it at 4 &#176;C. TTC and tissue stained with TTC are light sensitive.</li>
		<li>Transfer the slices into a 24-well plate (one slice per well) containing TTC solution and stretch out the slice in the solution. Incubate the slices in TTC solution at 37 &#176;C in a shallow water bath for 30 min.</li>
		<li>Transfer the slices into clean 1.5 mL centrifuge tubes covered in foil and measure the dry weight of the slices.</li>
		<li>Add ethanol/dimethyl sulfoxide (1:1) into the centrifuge tubes (v:w = 10:1) to extract formazan. Incubate the slices in ethanol/dimethyl sulfoxide at room temperature away from light for 24 h.</li>
		<li>Add all ethanol/dimethyl sulfoxide in the tubes into a 96-well plate and measure the absorbance at 490 nm by a plate reader.</li>
		<li>Normalize the absorbance to the dry weight of the slice and express viability as the percentage of control slice.</li>
	</ol>
	</li>
</ol>
2. MCAO
<ol>
	<li>Preparation:
	<ol>
		<li>Disinfect the workbench, the surface of the Laser Doppler Flowmetry (LDF) instrument and its accessories with 70% ethyl alcohol.</li>
		<li>Prepare autoclaved instruments: two scissors, 10 cm; two forceps, 10 cm; one ophthalmic forceps, 11 cm; one micro ophthalmic scissors, 9 cm; one microvessel clamp, 1.8 cm; oneneedle drive,r 12.5 cm; several surgical sutures (&#9651; 1/2 4 &#215; 10); cotton swabs.</li>
		<li>Prepare a syringe (without needle) with saline solution to maintain a hydrated operation area.</li>
		<li>Prepare the anesthesia gas (100% O2&#160;+&#160;isoflurane).</li>
	</ol>
	</li>
	<li>Detection of brain blood flow:
	<ol>
		<li>Set up the LDF instrument.</li>
		<li>Inject analgesics intraperitoneally into the mouse: metamizol 200 mg/kg, carprofen 4 mg/kg and buprenorphin 0.1 mg/kg.</li>
		<li>Place the mouse into an induction chamber with 4% isoflurane in oxygen to anesthetize it until spontaneous movement of body and vibrissae stops.</li>
		<li>Place the mouse in a prone position with its nose fitted into the nose cone and maintain isoflurane at 1.5% for the subsequent procedure.</li>
		<li>Apply dexpanthenol eye ointment on both eyes.</li>
		<li>Disinfect the head with 70% ethyl alcohol.</li>
		<li>Make a right paramedian skin incision between eyes and ears using a scalpel to expose the bregma.</li>
		<li>Rub the skull with sterile cotton.</li>
		<li>Apply one to two drops of surgical glue to the surface of the skull.</li>
		<li>Place the tip of fiber optic probe 5&#160;mm caudal to the bregma and 6 mm lateral to the midline.</li>
		<li>Start recording the blood flow on the computer software. Wait until a stable blood flow baseline occurs and then apply a 20 &#181;L catalyst upon the glue to fix the probe. 
		NOTE: An ideal baseline should be more than 500 flux.</li>
		<li>If the baseline is less than 200 flux, adjust the position finely to get a suitable baseline. Keep the probe attached to the skull for the duration of the experiment.</li>
		<li>Disinfect the head with iodophor after the fiber optic probe attached.</li>
	</ol>
	</li>
	<li>MCAO:
	<ol>
		<li>Place and fix the anesthetized mouse (with LDF probe attached to its skull) in a supine position and maintain its body temperature using a heat lamp throughout the surgery. Disinfect the neck with 70% ethyl alcohol.</li>
		<li>Make a paramedian skin incision in the neck using a scalpel; blunt dissect the soft tissues with forceps to expose vessels. Add one drop of saline to the exposed tissues to keep themhydrated.</li>
		<li>Dissect the common carotid artery from surrounding tissue and the vagus nerve using ophthalmic forceps. Be careful not to damage the vagus nerve.&#160;Disinfect skin of neck again with iodophor after the common carotid artery exposed.</li>
		<li>Place a microvessel clamp at distal end of the common carotid artery and then tie a dead knot with 6-0 silk sutures at the proximal end. Place the clamp as proximal as possible for subsequent operations. Ensure that the length of the vessel between the clamp and dead knot is as long as possible for subsequent operations.</li>
		<li>Make a temporary suture proximal to the clamp by tying a loose knot. Ensure that there is enough space between two knots for monofilament insertion.</li>
		<li>Make a smalllongitudinal incision between the two knots with micro ophthalmic scissors. Ensure the incision is as close to the dead knot as possible for subsequent operations. Be careful not to cut off the vessel.</li>
		<li>Insert a 12-mm tip-blunted monofilamentthrough the incision to enter the artery lumen and advance it a few mm. Tighten the loose knot around the tip of the monofilament and then remove the clamp.</li>
		<li>Advance the filament into the internal carotid artery (10 mm to occlude the middle cerebral artery) with ophthalmic forceps until the LDF software shows a sharp decline (&#62;80% reduction from baseline blood flow) in blood flow. Record the start time of occlusion. 
		NOTE: LDF instrument setup is described in section 2.2.</li>
		<li>Occlude the middle cerebral artery for 1 h. Cut off the LDF probe and put the mice in a 30 &#176;C incubator for the duration of occlusion.</li>
	</ol>
	</li>
	<li>Reperfusion and APC:
	<ol>
		<li>Re-anesthetize the animals with isoflurane as described above 55 min after the beginning time of occlusion. Place and fix mice in a supine position. Open the neck incision and re-expose the common carotid artery.</li>
		<li>Gently pull out the filament with ophthalmic forceps after occlusion period to achieve reperfusion and turn the temporary suture into a permanent one by tightening the knot.</li>
		<li>For acidosis treatment, change the gas inhaled by the nose cone to the mix gases containing 20% CO2, 20% O2 and 60% N2 for 5 min at 5, 50, or 100 min after reperfusion. Close the incision with an interrupted surgical suture.</li>
		<li>Place mice in a 30 &#176;C heat cage until mice recover consciousness and then return the mice to clean, individual cages. Provide mice with a Petri dish of water and moistened food. Monitor the mice closely after the surgery for excessive pain and death.</li>
	</ol>
	</li>
	<li>Brain Infarct volume measurement:
	<ol>
		<li>Measure brain infarct volume by usingTTC staining 24 h after reperfusion.</li>
		<li>Prepare 0.25% TTC solution as mentioned in step 1.4.1 prior to the designated time of sacrifice. Transfer the solution into a 24-well plate (1 mL per well) covered in foil and store it at 4 &#176;C. 
		NOTE: TTC and tissue stained with TTC are light sensitive.</li>
		<li>At 24 h after reperfusion, sacrifice the animal with an isoflurane overdose in an induction chamber. Decapitate the mice. Dissect out the brains using small scissors and forceps. Examine blooding points on the brains to exclude the mice that underwent subarachnoid hemorrhage at the Circle of Willis.</li>
		<li>Place the brain on a clean glass slide on a -20 &#176;C ice pack. Place the brain and the glass slide in a -20 &#176;C fridge for 5 min to make the brain easier to slice.</li>
		<li>Take out the brain and the glass slide from -20 &#176;C and put them back on the -20 &#176;C ice pack. Dissect the frontal pole and the cerebellum with blade and forceps.</li>
		<li>Slice the brain sections horizontally to a 1-mm thickness with a blade to produce 5 slices. Transfer the slices into the 24-well plate containing TTC solution (1 slice per well) with forceps and stretch out the slices in the solution. Incubate the slices in TTC solution at 37 &#176;C in a shallow water bath for 30 min.</li>
		<li>Aspirate the TTC solution. Add 10% formaldehyde (1 mL per well) and incubate at room temperature for 30 min. Place the slices in the order in which they were cut upon a cellophane sheet and scan the segments into the photographic plate imager.</li>
		<li>Analyze the infarct size as a percentage of the whole brain slice using ImageJ analysis software8based on visual identification; see Figure 2 (left).</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

Here we present two experimental models to study the neuroprotection of APC against cerebral ischemia. In brain slices, APC is achieved by incubating mice corticostriatal slices in acidic buffer bubbled with 20% CO2 after reperfusion onset, while in the MCAO model, APC is achieved by inhaling 20% CO2 to mice after reperfusion. Both models reflect the neuroprotection of APC against cerebral ischemia. The protection was comparable with that achieved by ischemic postconditioning but with a wider time window. In the MCAO model, the time window could be as long as 50 min after reperfusion compared to 10 min for ischemic postconditioning15. In addition, the procedure of APC is easier and safer.
In the corticostriatal slices model, gentle and rapid operations are crucial to warrant the viability of brain slices to the maximum extent. For the MCAO performance, the cerebral blood flow monitoring is required, and the sharp decline of blood flow must be observed to ensure the occlusion of middle cerebral artery. After the brains were dissected out for TTC staining, careful examination of the brains is necessary for exclusion of subarachnoid hemorrhage.
The extension and the duration of acidosis are critical to achieve the neuroprotection of APC. Prolonged duration of acidosis treatment is not beneficial in corticostriatal slices model (Figure 1A). In addition, our previous study showed that both 10% and 20% CO2 inhalation, rather than 30% CO2 inhalation confer neuroprotection on mice8. These suggest mild acidosis is crucial for the neuroprotection of APC, and thus the concentration of CO2 and the duration of APC are indispensable for acidosis neuroprotection.
In addition to TTC staining, many techniques can be combined to satisfy diverse research needs. For instance, extra-cellular electric recording can be added to the final step to observe how ischemia and acidosis affect neural potential16. The perfusion solution of brain slice can also be collected to measure the concentration change of amino acid neurotransmitters after APC by HPLC17. Slices of a certain brain regions can be prepared to study the responses of a particular area to ischemia and APC. Overall, many alternatives can be introduced to illuminate the impacts of ischemia and acidosis.

Stroke is one of the leading causes of mortality and disability worldwide. Great efforts have been made to find effective treatments for stroke in the last decades, however, the achievement is quite unsatisfactory. Postconditioning is a process manipulated by subtoxic stresses following an ischemic episode. Postconditioning, including ischemic, hypoxic, low-glucose and remote ischemic postconditioning, trigger endogenous adaptive mechanisms, and have been proven to be promising therapies against cerebral ischemia1,2,3,4. However, ischemic postconditioning may introduce additional injury. Limb remote ischemic postconditioning usually needs several cycles of 5 - 20 min occlusion and reperfusion on the ipsilateral or bilateral hind limbs5,6,7. Therefore, these postconditioning manipulations are dangerous or impractical in clinical practice. To overcome these disadvantages, we have developed APC as a therapy for focal cerebral ischemia in mice8. Induced simply by inhaling 20% CO2, APC significantly reduces ischemic brain injury in a more feasible and safer way. Recently we have proved that APC extends the reperfusion window, highlighting the significance of APC for stroke therapy9.
Here we present two experimental models to study the neuroprotection of APC against cerebral ischemia. The first one is the oxygen-glucose deprivation (OGD) model in mice corticostriatal slices. Rapid preparation and transfer of the brain slices into an artificial environment, usually artificial cerebrospinal fluid (ASCF), can maintain cell viability and neuronal circuitry, which makes it possible to study brain function in vitro10,11. OGD in ASCF mimics cerebral ischemia and induces ischemic injury12,13,14. After OGD, the brain slices are refreshed in regular ASCF (r-ASCF) to provide reperfusion and then treated with APC using acidic ASCF bubbled with 20% CO2. The corticostriatal slice maintains the intact histological characterization compared with primary cultured cells.
To study brain function in vivo, the mouse middle cerebral artery occlusion (MCAO) model is employed. The middle cerebral artery is blocked by inserting a flame-blunted monofilament via the common carotid artery. As one of the most widely used stroke models, the MCAO model shows clinical relevance and the application of a monofilament makes it easier to achieve reperfusion. Simply by inhaling normoxic mixed gas containing 20% CO2 after the onset of reperfusion, APC showed significant protective effects against cerebral ischemia indicated by reduced brain infarct volumes.

<table><tbody><tr><td>Sodium chloride</td><td>Sigma</td><td>S5886</td><td></td></tr><tr><td>Potassium chloride</td><td>Sigma</td><td>P5405</td><td></td></tr><tr><td>Potassium phosphate monobasic</td><td>Sigma</td><td>P9791</td><td></td></tr><tr><td>Magnesium sulfate</td><td>Sigma</td><td>M2643</td><td></td></tr><tr><td>Sodium bicarbonate</td><td>Sigma</td><td>S5761</td><td></td></tr><tr><td>Calcium chloride dihydrate</td><td>Sigma</td><td>C5080</td><td></td></tr><tr><td>D-(+)-Glucose</td><td>Sigma</td><td>G7021</td><td></td></tr><tr><td>Vibratome</td><td>Leica</td><td>VT1000 S</td><td></td></tr><tr><td>2,3,5-triphenyltetrazolium hydrochloride</td><td>Sigma</td><td>T8877</td><td></td></tr><tr><td>Absolute Ethanol</td><td>Aladdin Industrial Corporation</td><td>E111993</td><td></td></tr><tr><td>Dimethyl sulfoxide</td><td>Sigma</td><td>D8418</td><td></td></tr><tr><td>Laser Doppler Flowmetry</td><td>Moor Instruments Ltd</td><td>Model Moor VMS-LDF2</td><td></td></tr><tr><td>Diethyl ether anhydrous</td><td>Sinopharm Chemical Reagent Corporation</td><td>80059618</td><td></td></tr><tr><td>Trichloroacetaldehycle hydrate</td><td>Sinopharm Chemical Reagent Corporation</td><td>30037517</td><td></td></tr><tr><td>10% Formalin</td><td>Aladdin Industrial Corporation</td><td>F111936</td><td></td></tr><tr><td>24-well plates</td><td>Jet Biofil</td><td>TCP-010-024</td><td></td></tr></tbody></table>

experimental models to study the neuroprotection of acidic postconditioning against cerebral ischemia

Stroke is one of the leading causes of mortality and disability worldwide, with limited therapeutic approaches. As an endogenous strategy for neuroprotection, postconditioning treatments have proven to be promising therapies against cerebral ischemia. However, complicated procedures and potential safety issues limit their clinical application. To overcome these disadvantages, we have developed acidic postconditioning (APC) as a therapy for experimental focal cerebral ischemia. APC refers to the mild acidosis treatment by inhaling CO2 during reperfusion following ischemia. Here we present two models to execute APC in vitro and in vivo, respectively. The oxygen-glucose deprivation (OGD) treatment of mice and the corticostriatal occlusion and middle cerebral artery occlusion (MCAO) of mice were employed to mimic cerebral ischemia. APC can be simply achieved by transferring brain slices to acidic buffer bubbled with 20% CO2, or by mice inhaling 20% CO2. APC showed significant protective effects against cerebral ischemia, as reflected by tissue viability and brain infarct volume.

In the corticostriatal slice model described above, corticostriatal slice viability was quantified by TTC assay at 1 h after reperfusion. TTC conversion was calculated by normalizing the absorption at 490 nm to the control slice. According to TTC conversion, APC protected against OGD-induced reperfusion injury in an onset time and duration-dependent manner. In detail, both 1 and 3 min of acidosis treatment significantly improved viability at 5 min after 15 min OGD, whereas 5 min did not (OGD: 0.609 &#177; 0.029, 5/1: 0.758 &#177; 0.034, 5/3: 0.821 &#177; 0.041, 5/5: 0.672 &#177; 0.053, data reported as mean &#177; SEM) (Figure 1A). The neuroprotection by acidosis treatment remained protective within 5 min after reperfusion, whereas 15 min did not (OGD: 0.584 &#177; 0.044, 0/3: 0.762 &#177; 0.036, 5/3: 0.833 &#177; 0.062, 15/3: 0.627 &#177; 0.038) (Figure 1B).
In the MCAO model, 5 min of acidosis treatment initiated at 5 min after the onset of reperfusion rescued cell death caused by ischemic insult, reflected by smaller infarct volumes (MCAO: 33.4 &#177; 4.4%, 5/5: 16.6 &#177; 2.7%, 50/5: 19.5 &#177; 2.1%, 100/5: 37 &#177; 2.1%). The neuroprotection was still robust, even when the onset time was delayed to 50 min after reperfusion. However, acidosis treatment initiated at 100 min did not block the ischemic injuries (Figure 2).
All data were collected and analyzed in a blinded fashion. Data are presented as mean &#177; SEM. In the corticostriatal slice model, each group has 6-8 samples. In the MCAO model, each group has 9 - 10 samples. One-way analysis of variance with least significant difference was applied for multiple comparisons.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55931/55931fig1.jpg" /> 
Figure 1. APC Protects Against OGD-induced Reperfusion Injury in Corticostriatal Slices. Corticostriatal slices were treated with acidosis (pH 6.8) for indicated durations (A) and indicated recovery periods (B) after OGD. The cell viability was assessed by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay at 24 h after reperfusion, and corticostriatal slice viability was quantified by TTC assay at 1 h after reperfusion. Values show mean &#177; SEM. n= 6 - 8 for each group; *p&#60; 0.05 and **p&#60; 0.01 by one-way analysis of variance; R, reperfusion. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55931/55931fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/55931/55931fig2.jpg" /> 
Figure 2. APC Protects Against MCAO-induced Injury in Mice. Animals were subjected to 60 min MCAO and treated by inhaling 20% CO2 for 5 min at 5, 50, or 100 min after reperfusion. Infarct volume was quantified by 2,3,5-triphenyltetrazolium hydrochloride staining at 24 h after reperfusion (indicated by black dotted line on the left panel). Values show mean &#177; SEM. n = 8 - 10 for each group; *p&#60; 0.05 and **p&#60; 0.01 by one-way analysis of variance; R, reperfusion. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55931/55931fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Experimental Models to Study the Neuroprotection of Acidic Postconditioning Against Cerebral Ischemia at JoVE.com

Experimental Models to Study the Neuroprotection of Acidic Postconditioning Against Cerebral Ischemia

Acidic postconditioning protects against cerebral ischemia. Here we present two models to execute APC. They are achieved respectively by transferring corticostriatal slices to acidic buffer after oxygen-glucose deprivation in vitro and by inhaling 20% CO2 after middle cerebral artery occlusion in vivo.

Stroke is one of the leading causes of mortality and disability worldwide, with limited therapeutic approaches. As an endogenous strategy for ...

experimental-models-to-study-neuroprotection-acidic-postconditioning

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Neuroscience

7.4K Views.  Zhejiang University. Acidic postconditioning protects against cerebral ischemia. Here we present two models to execute APC. They are achieved respectively by transferring corticostriatal slices to acidic buffer after oxygen-glucose deprivation in vitro and by inhaling 20% CO2 after middle cerebral artery occlusion in vivo.

Video: Experimental Models to Study the Neuroprotection of Acidic Postconditioning Against Cerebral Ischemia

Strategies for Study of Neuroprotection from Cold-preconditioning

Neurological injury is a frequent cause of morbidity and mortality from general anesthesia and related surgical procedures that could be alleviated by development of effective, easy to administer and safe preconditioning treatments. We seek to define the neural immune signaling responsible for cold-preconditioning as means to identify novel targets for therapeutics development to protect brain before injury onset. Low-level pro-inflammatory mediator signaling changes over time are essential for cold-preconditioning neuroprotection. This signaling is consistent with the basic tenets of physiological conditioning hormesis, which require that irritative stimuli reach a threshold magnitude with sufficient time for adaptation to the stimuli for protection to become evident.
Accordingly, delineation of the immune signaling involved in cold-preconditioning neuroprotection requires that biological systems and experimental manipulations plus technical capacities are highly reproducible and sensitive. Our approach is to use hippocampal slice cultures as an in vitro model that closely reflects their in vivo counterparts with multi-synaptic neural networks influenced by mature and quiescent macroglia / microglia. This glial state is particularly important for microglia since they are the principal source of cytokines, which are operative in the femtomolar range. Also, slice cultures can be maintained in vitro for several weeks, which is sufficient time to evoke activating stimuli and assess adaptive responses. Finally, environmental conditions can be accurately controlled using slice cultures so that cytokine signaling of cold-preconditioning can be measured, mimicked, and modulated to dissect the critical node aspects. Cytokine signaling system analyses require the use of sensitive and reproducible multiplexed techniques. We use quantitative PCR for TNF-&alpha; to screen for microglial activation followed by quantitative real-time qPCR array screening to assess tissue-wide cytokine changes. The latter is a most sensitive and reproducible means to measure multiple cytokine system signaling changes simultaneously. Significant changes are confirmed with targeted qPCR and then protein detection. We probe for tissue-based cytokine protein changes using multiplexed microsphere flow cytometric assays using Luminex technology. Cell-specific cytokine production is determined with double-label immunohistochemistry. Taken together, this brain tissue preparation and style of use, coupled to the suggested investigative strategies, may be an optimal approach for identifying potential targets for the development of novel therapeutics that could mimic the advantages of cold-preconditioning.

We seek to define the neural immune signaling responsible for cold-preconditioning as means to identify novel targets for therapeutics development to protect brain before injury onset. We present strategies for such work that require biological systems, experimental manipulations plus technical capacities that are highly reproducible and sensitive.

Neurological injury is a frequent cause of morbidity and mortality from general anesthesia and related surgical procedures that could be alleviated by ...

The Application Of Permanent Middle Cerebral Artery Ligation in the Mouse

Focal cerebral ischemia is among the most common type of stroke seen in patients. Due to the clinical significance there has been a prolonged effort to develop suitable animal models to study the events that unfold during ischemic insult. These techniques include transient or permanent, focal or global ischemia models using many different animal models, with the most common being rodents.
The permanent MCA ligation method which is also referred as pMCAo in the literature is used extensively as a focal ischemia model in rodents 1-6. This method was originally described for rats by Tamura et al. in 1981 7. In this protocol a craniotomy was used to access the MCA and the proximal regions were occluded by electrocoagulation. The infarcts involve mostly cortical and sometimes striatal regions depending on the location of the occlusion. This technique is now well established and used in many laboratories 8-13. Early use of this technique led to the definition and description of &#x201C;infarct core&#x201D; and &#x201C;penumbra&#x201D; 14-16, and it is often used to evaluate potential neuroprotective compounds 10, 12, 13, 17. Although the initial studies were performed in rats, permanent MCA ligation has been used successfully in mice with slight modifications 18-20 .
This model yields reproducible infarcts and increased post-survival rates. Approximately 80% of the ischemic strokes in humans happen in the MCA area 21 and thus this is a very relevant model for stroke studies. Currently, there is a paucity of effective treatments available to stroke patients, and thus there is a need for good models to test potential pharmacological compounds and evaluate physiological outcomes. This method can also be used for studying intracellular hypoxia response mechanisms in vivo. 
Here, we present the MCA ligation surgery in a C57/BL6 mouse. We describe the pre-surgical preparation, MCA ligation surgery and 2,3,5 Triphenyltetrazolium chloride (TTC) staining for quantification of infarct volumes.

Middle cerebral artery (MCA) ligation is a technique to study focal cerebral ischemia in animal models. In this method, the middle cerebral artery is exposed by craniotomy and ligated by cauterization. This method gives highly reproducible infarct volumes and increased post-operative survival rates compared to other methods available.

Focal cerebral ischemia is among the most common type of stroke seen in patients. Due to the clinical significance there has been a prolonged effort to ...

Medicine

Bilateral Common Carotid Artery Occlusion as an Adequate Preconditioning Stimulus to Induce Early Ischemic Tolerance to Focal Cerebral Ischemia

There is accumulating evidence, that ischemic preconditioning - a non-damaging ischemic challenge to the brain - confers a transient protection to a subsequent damaging ischemic insult. We have established bilateral common carotid artery occlusion as a preconditioning stimulus to induce early ischemic tolerance to transient focal cerebral ischemia in C57Bl6/J mice. In this video, we will demonstrate the methodology used for this study.

There is accumulating evidence, that ischemic preconditioning (PC) &#x2013; a non-damaging ischemic challenge to the brain - confers a transient protection to a subsequent damaging ischemic insult. We established bilateral common carotid artery occlusion (BCCAO) as a preconditioning stimulus to induce early ischemic tolerance (IT) to transient focal cerebral ischemia (induced by middle cerebral artery occlusion, MCAO) in C57Bl6/J mice.

There is accumulating evidence, that ischemic preconditioning - a non-damaging ischemic challenge to the brain - confers a transient protection to a ...

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 ...

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

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As such, primary or &#39;neuronal-like&#39; cell culture systems are commonly utilized. While&#160;these systems are relatively easy to work with, and are useful model systems in which various functional outcomes (such as cell death) can be readily quantified, the examined outcomes and pathways in cultured immature neurons (such as excitotoxicity-mediated cell death pathways) are not necessarily the same as those observed in mature brain, or in intact tissue. Therefore, there is the need to develop models in which cellular mechanisms in mature neural tissue can be examined. We have developed an in vitro technique that can be used to investigate a variety of molecular pathways in intact nervous tissue. The technique described herein utilizes rat cortical tissue, but this technique can be adapted to use tissue from a variety of species (such as mouse, rabbit, guinea pig, and chicken) or brain regions (for example, hippocampus, striatum, etc.). Additionally, a variety of stimulations/treatments can be used (for example, excitotoxic, administration of inhibitors, etc.). In conclusion, the brain slice model described herein can be used to examine a variety of molecular mechanisms involved in excitotoxicity-mediated brain injury.

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

We have developed a brain slice model which can be used to examine molecular mechanisms involved in excitotoxicity-mediated brain injury.&#160;This technique generates viable mature brain tissue and reduces animal numbers required for experimentation, whilst keeping the neuronal circuitry, cellular interactions, and postsynaptic compartments partly intact.

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As ...

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 ...

Lateral Chronic Cranial Window Preparation Enables In Vivo Observation Following Distal Middle Cerebral Artery Occlusion in Mice

Focal cerebral ischemia (i.e., ischemic stroke) may cause major brain injury, leading to a severe loss of neuronal function and consequently to a host of motor and cognitive disabilities. Its high prevalence poses a serious health burden, as stroke is among the principal causes of long-term disability and death worldwide1. Recovery of neuronal function is, in most cases, only partial. So far, treatment options are very limited, in particular due to the narrow time window for thrombolysis2,3. Determining methods to accelerate recovery from stroke remains a prime medical goal; however, this has been hampered by insufficient mechanistic insights into the recovery process. Experimental stroke researchers frequently employ rodent models of focal cerebral ischemia. Beyond the acute phase, stroke research is increasingly focused on the sub-acute and chronic phase following cerebral ischemia. Most stroke researchers apply permanent or transient occlusion of the MCA in mice or rats. In patients, occlusions of the MCA are among the most frequent causes of ischemic stroke4. Besides proximal occlusion of the MCA using the filament model, surgical occlusion of the distal MCA is probably the most frequently used model in experimental stroke research5. Occlusion of a distal (to the branching of the lenticulo-striate arteries) MCA branch typically spares the striatum and primarily affects the neocortex. Vessel occlusion can be permanent or transient. High reproducibility of lesion volume and very low mortality rates with respect to the long-term outcome are the main advantages of this model. Here, we demonstrate how to perform a chronic cranial window (CW) preparation lateral to the sagittal sinus, and afterwards how to surgically induce a distal stroke underneath the window using a craniotomy approach. This approach can be applied for sequential imaging of acute and chronic changes following ischemia via epi-illuminating, confocal laser scanning, and two-photon intravital microscopy.

Lateral Chronic Cranial Window Preparation Enables In Vivo Observation Following Distal Middle Cerebral Artery Occlusion in Mice

Surgical occlusion of a distal middle cerebral artery branch (MCAo) is a frequently used model in experimental stroke research. This manuscript describes the basic technique of permanent MCAo, combined with the insertion of a lateral cranial window, which offers the opportunity for longitudinal intravital microscopy in mice.

Focal cerebral ischemia (i.e., ischemic stroke) may cause major brain injury, leading to a severe loss of neuronal function and consequently to a host of ...

A Preclinical Model to Assess Brain Recovery After Acute Stroke in Rats

The purpose of this study was to establish and validate an animal brain ischemia model in the recovery and sequela stages. A middle cerebral artery occlusion/reperfusion (MCAO/R) model in male Sprague-Dawley rats was chosen. By changing the rat&#39;s weight (260&#8722;330 g), the thread bolt type (2636/2838/3040/3043) and the brain infarct time (2-3 h), a higher Longa&#39;s score, a larger infarct volume and a greater model success ratio were screened using the Longa&#39;s score and TTC staining. The optimum model condition (300 g, 3040 thread bolt, 3 h brain infarct time) was acquired and used in a 1-90 day observation period after reperfusion via assessment of sensorimotor functions and infarct volume. At these conditions, the bilateral asymmetry test had a significant difference from 1 to 90 days, and the grid-walking test had a significant difference from 1 to 60 days; both differences could be a suitable sensorimotor functional test. Thus, the most appropriate condition of a novel rat model in the recovery and sequela stages of brain ischemia was found: 300 g rats that underwent MCAO with a 3040 thread bolt for a 3 h brain infarct and then reperfused. The appropriate sensorimotor functional tests were a bilateral asymmetry test and a grid-walking test.

The&#160;purpose of this study is to establish and validate an animal model for research in the recovery and sequela stages of brain ischemia by testing brain infarction and sensorimotor function after middle cerebral artery occlusion/reperfusion (MCAO/R) after 1-90 days in rats.

The purpose of this study was to establish and validate an animal brain ischemia model in the recovery and sequela stages. A middle cerebral artery ...

Modified MCAO/R Model with Preserved ECA for Enhanced Reperfusion and Stability

A Modified Model Preparation for Middle Cerebral Artery Occlusion Reperfusion

The middle cerebral artery occlusion reperfusion (MCAO/R) model is crucial for understanding the pathological mechanisms of stroke and for drug development.However, among the commonly used modeling methods, the Koizumi method often faces scrutiny due to its ligation of the common carotid artery (CCA) and its inability to achieve adequate reperfusion. Similarly, the Longa method has been criticized for disconnecting and ligating the external carotid artery (ECA). This study aims to introduce a modified model preparation method that preserves the integrity of the ECA, involves inserting a monofilament nylon suture through the CCA, repairing the ligated CCA incision, and maintaining reperfusion from the CCA. Reperfusion of blood flow was confirmed using laser speckle flow imaging. Evaluation methods such as the Longa scale, Modified Neurological Severity Score, triphenyltetrazolium chloride (TTC) staining, and immunofluorescence labeling of neurons demonstrated that this approach could induce stable ischemic nerve damage. This modified MCAO/R model protocol is simple and stable, providing valuable guidance for practitioners in the field of cerebral ischemia.

Author Spotlight: A Novel Approach to Cerebral Ischemia Modeling &#8211; Enhancing Reperfusion and Simplifying Procedure

This protocol describes the process of preparing for middle cerebral artery occlusion reperfusion via the common carotid artery.

Experimental Models to Study the Neuroprotection of Acidic Postconditioning Against Cerebral Ischemia

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Chapters in this video

Strategies for Study of Neuroprotection from Cold-preconditioning

The Application Of Permanent Middle Cerebral Artery Ligation in the Mouse

Bilateral Common Carotid Artery Occlusion as an Adequate Preconditioning Stimulus to Induce Early Ischemic Tolerance to Focal Cerebral Ischemia

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

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

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

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

Lateral Chronic Cranial Window Preparation Enables In Vivo Observation Following Distal Middle Cerebral Artery Occlusion in Mice

A Preclinical Model to Assess Brain Recovery After Acute Stroke in Rats

A Modified Model Preparation for Middle Cerebral Artery Occlusion Reperfusion