This work was supported by a grant from the Natural Sciences and Engineering Research Council of Canada. Kim Gilbert holds a studentship from Fonds de la recherche du Qu&#233;bec - Sant&#233;.

Animals are handled in compliance with regulation of the local Animal Care Committee, in accordance with the Guidelines of the Canadian Council on Animal Care. Rats are housed individually under constant conditions (temperature of 21-22 &#186;C and humidity of 40-50%), including a 12 hr dark-light cycle, beginning at 08:00 am. Chow pellets and tap water are available ad libitum throughout the study. An acclimatisation period of 3 days after delivery by the supplier is allowed before the experiment. Male Sprague-Dawley rats weighing between 300-800 g have been routinely used with this protocol.
1. MI Surgery
<ol>
	<li>Induce anesthesia with intraperitoneal injections of ketamine 60 mg/kg and xylazine 10 mg/kg. Confirm proper anesthetization by absence of reflex when the paws are pinched.</li>
	<li>Shave surgical site. Intubate rat with endotracheal tubing: catheter (16G 1.77 inches) and place animal in lateral decubitus position. Place the animals on a heating pad to maintain temperature around 37 &#176;C. Connect the endotracheal tubing to isoflurane 2%.</li>
	<li>Prepare surgical site with chlorhexidine gluconate and isopropyl alcohol. During surgery, use sterile gloves and sterilized instruments. Apply ophthalmic ointment to both eyes to prevent dryness while under anesthesia. Put sterile field on animal and the instruments on another sterile field next to the animal.</li>
	<li>Incise skin with scalpel blade #10. Peel off muscle tissue with a hemostatic clamp. Place the animal in ventral decubitus position. Open thoracic wall with a hemostatic clamp and position chest retractor. Open pericardium with a hemostatic clamp.</li>
	<li>To produce coronary occlusion, loop a 360 mm-long 4-0 silk suture around the descending coronary artery, passing through the contiguous myocardial tissue. Insert both ends of the silk suture into a 14G, 1.25 cm long plastic tube.</li>
	<li>Pull both ends of the silk suture and push the tube down against the artery to occlude it, for 40 min. Secure occlusion by clamping the plastic tube with an hemostatic clamp.</li>
	<li>After 40 min, release the occlusion by releasing the hemostatic clamp, followed by removing the flexible tubing and silk suture. 
	Note: Sham control rats undergo the same surgical procedure minus the occlusion of the coronary artery.</li>
	<li>Before closing the thoracic wall, place a flexible catheter (18G, 4.5 cm) through the thoracic cavity to draw air out of the thorax with a 5-ml syringe to prevent pneumothorax.</li>
	<li>Close thorax cavity with 2-0 suture, stitch the muscle with 4-0 silk suture and the skin with 3-0 silk suture.</li>
	<li>Before closing the last skin point, again draw air out of the thorax with 5-ml syringe through the catheter placed previously in the thorax cavity.</li>
	<li>Suture last skin point. Stop isoflurane ventilation.</li>
	<li>Monitor rat awakening. Do not leave an animal unattended until it has regained sufficient consciousness to maintain sternal recumbency.</li>
	<li>Treat rat every 8 hr for 24 hr with an analgesic (buprenorphine, 0.05 mg/kg i.p.) and with an antibiotic (a single dose of duplocillin, 0.2 mg/kg i.p.).</li>
	<li>After the surgery, house rats individually until time of sacrifice, i.e., 3 days post-MI.</li>
	<li>Infarct size, mainly necrosis, is expressed as the proportion of infarcted tissue in the area at risk of the left ventricle. This can measured using triphenyltetrazolium coloration11.</li>
</ol>
2. Amygdala Isolation
<ol>
	<li>Place the animal head first in a cone-shaped bag, with its nose protruding out of the narrow end aperture. Secure rat to avoid any movement. Position its head appropriately on the guillotine, between the scissor blades. Decapitate animal.</li>
	<li>Place head of the rat in a dish kept on crushed ice. Perform all procedures for tissue isolation on crushed ice (4 &#176;C).</li>
	<li>Cut open skull with scissors, carefully detach bone sheaths with bone rongeurs by pulling up and avoiding mutilating the tissue underneath.</li>
	<li>Place the flat blade of a spatula between the bottom of the skull and the posterior ventral surface of the brain and carefully detach brain from skull by gently pushing the blade forward along the bottom of the skull until the brain can be slowly lifted up out of the skull. Discard the skull.</li>
	<li>Place brain on its dorsal surface. Identify hypothalamus (structure in front of the cerebellum) and cut the brain coronally in front of its anterior end and behind its posterior end; discard the anterior and posterior parts of the brain if not needed for other uses.</li>
	<li>Visualize the amygdalas as small spheres underneath the temporal lobes, bilaterally, just next to the hypothalamus.</li>
	<li>Flip the brain flat on its frontal end, dorsal surface away from the experimenter.</li>
	<li>Start with left or right side of the brain. Separate the hemispheres and then the cortex from the contiguous amygdala. Cut way about 8 mm of this loose cortex to allow isolation of the amygdala. Cut away amygdala with scalpel.</li>
	<li>Isolate the basolateral part of the amygdala from its centromedial part by cutting along the dark suture that runs across it, almost half and half. This structure is not always easily identifiable.</li>
	<li>Put amygdala subparts in separate identified vials and keep on crushed ice.</li>
	<li>Repeat dissection procedure with the other hemisphere.</li>
	<li>Immerse vials in liquid nitrogen for 1 min. Keep vial in -80 &#176;C freezer until needed.</li>
</ol>
3. Caspase-3 Activity
<ol>
	<li>Prepare lysis buffer with 0.32 M of sucrose, 1% Triton-X-100, 10 mM of Tris-HCl, pH 8, 5 mM of ethylenediaminetetraacetic acid, 2 mM of dithiothreitol, 1 mM of phenylmethylsulfonyl fluoride, 10 &#181;g/&#181;l of leupeptin, 10 &#181;g/&#181;l of pepstatin A and 10 &#181;g/&#181;l of aprotinin.</li>
	<li>Add 150 &#181;l of lysis buffer to each sample (5-10 mg). Keep on ice. Sonicate each sample on ice for 5 sec at maximal intensity (40 W). Incubate tissue in lysis buffer on ice for 30 min. Vortex each sample for 5 min.</li>
	<li>Perform 3 freeze/thaw cycles by placing samples alternately in liquid nitrogen and on a thermostatically-controlled heating plate set at 37 &#176;C. Centrifuge tissue at 13,000 g (4 &#176;C) for 10 min. Carefully remove supernatant and keep in tube on ice.</li>
	<li>Prepare standard protein concentrations between 0 and 10 &#181;g/ml with bovine serum albumin. Prepare blanks with buffer only.</li>
	<li>Add 200 &#181;l of Bradford reagent to each standard. Read standard at 595 nm. For protein levels, add 5 &#181;l of sample supernatant to 795 &#181;l of distilled water and 200 &#181;l of Bradford reagent. Read each sample at 595 nm and quantify protein with standard curve.</li>
	<li>Prepare reaction buffer with 50 mM Tris-HCl, pH 7, 5 mM of MgCl2, 1 mM of ethylene glycol tetraacetic acid, 0.1% of 3-cholamidopropyl)dimethylammonio]-1-propanesulfonate and 1 mM of dithiothreitol.</li>
	<li>Add 25 &#181;g of protein to reaction buffer and caspase-3 substrates to obtain positive and negative reactive samples. For negative samples, add reaction buffer, 25 &#181;g of protein, 0.4 &#181;l of Ac-DEVD-CHO 800 &#181;M and 0.8 &#181;l of Ac-DEVD-AMC 10 mM. For positive samples, add 25 &#181;g of protein to reaction buffer and 0.8 &#181;l of Ac-DEVD-AMC 10 mM. Process all negative and positive samples in triplicate. Complete the final volume of each sample up to 200 &#181;l by adding reaction buffer.</li>
	<li>Produce negative controls by adding 188.7 &#181;l of reaction buffer to 0.5 &#181;l of Ac-DEVD-CHO 800 &#181;M, 0.8 &#181;l Ac-DEVD-AMC 10 mM and 10 &#181;l of lysis buffer. For positive controls, add 189.2 &#181;l of reaction buffer, 0.8 &#181;l of Ac-DEVD-AMC 10 mM and 10 &#181;l of lysis buffer.</li>
	<li>Incubate samples and controls in the dark for 3 hr at 37oC.</li>
	<li>Stop reaction with 600 &#181;l glycine 0.4 M and NaOH 0.4 M (pH 10) in each sample and control.</li>
	<li>Quantify fluorescence by spectrofluorometry at excitation wavelength of 365 nm and emission wavelength of 465 nm. Add 2 ml of distilled water to reaction in glass cuvette. Read controls and samples for 10 sec with 1 point at each sec.</li>
	<li>Quantify specific activity in each sample: ((Fluorescence of negative samples minus fluorescence of positive samples) x volume of 2.8 ml) divided by (time of incubation 3 hr x quantity of protein 25 mg).</li>
</ol>

<ol>
	<li>Denault, J. B., Salvesen, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caspases:+keys+in+the+ignition+of+cell+death.">Caspases: keys in the ignition of cell death.</a> Chemical Reviews. 102, 4489-4500 (2002).</li><li>Boucher, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Post-ischemic+cardioprotection+by+A2A+adenosine+receptors:+dependent+of+phosphatidylinositol+3-kinase+pathway.">Post-ischemic cardioprotection by A2A adenosine receptors: dependent of phosphatidylinositol 3-kinase pathway.</a> Journal of Cardiovascular Pharmacology. 43, 416-422 (2004).</li><li>Kaloustian, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Celecoxib+after+the+onset+of+reperfusion+reduces+apoptosis+in+the+amygdala.">Celecoxib after the onset of reperfusion reduces apoptosis in the amygdala.</a> Apoptosis. 12, 1945-1951 (2007).</li><li>Kaloustian, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apoptosis+time+course+in+the+limbic+system+after+myocardial+infarction+in+the+rat.">Apoptosis time course in the limbic system after myocardial infarction in the rat.</a> Brain Research. 1216, 87-91 (2008).</li><li>Rousseau, G., Bah, T. M., Godbout, R., Lakshmanadoss, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+19,+Post-Myocardial+Infarction+Depression.">Chapter 19, Post-Myocardial Infarction Depression.</a> Novel Strategies in Ischemic Heart Disease . , 333-362 (2012).</li><li>Wann, B. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apoptosis+detected+in+the+amygdala+following+myocardial+infarction+in+the+rat.">Apoptosis detected in the amygdala following myocardial infarction in the rat.</a> Biological Psychiatry. 59, 430-433 (2006).</li><li>Arseneault-Breard, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combination+of+Lactobacillus+helveticus+R0052+and+Bifidobacterium+longum+R0175+reduces+post-myocardial+infarction+depression+symptoms+and+restores+intestinal+permeability+in+a+rat+model.">Combination of Lactobacillus helveticus R0052 and Bifidobacterium longum R0175 reduces post-myocardial infarction depression symptoms and restores intestinal permeability in a rat model.</a> British Journal of Nutrition. 107, 1793-1799 (2012).</li><li>Bah, T. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Escitalopram+reduces+circulating+pro-inflammatory+cytokines+and+improves+depressive+behavior+without+affecting+sleep+in+a+rat+model+of+post-cardiac+infarct+depression.">Escitalopram reduces circulating pro-inflammatory cytokines and improves depressive behavior without affecting sleep in a rat model of post-cardiac infarct depression.</a> Behavioural Brain Research. 225, 243-251 (2011).</li><li>Bah, T. M., Kaloustian, S., Rousseau, G., Godbout, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pretreatment+with+pentoxifylline+has+antidepressant-like+effects+in+a+rat+model+of+acute+myocardial+infarction.">Pretreatment with pentoxifylline has antidepressant-like effects in a rat model of acute myocardial infarction.</a> Behavioural Pharmacology. 22, 779-784 (2011).</li><li>Gilbert, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Attenuation+of+post-myocardial+infarction+depression+in+rats+by+n-3+fatty+acids+or+probiotics+starting+after+the+onset+of+reperfusion.">Attenuation of post-myocardial infarction depression in rats by n-3 fatty acids or probiotics starting after the onset of reperfusion.</a> British Journal of Nutrition. 109, 50-56 (2013).</li><li>Gilbert, K., Bernier, J., Godbout, R., Rousseau, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resolvin+D1,+a+metabolite+of+omega-3+polyunsaturated+fatty+acid,+decreases+post-myocardial+infarct+depression.">Resolvin D1, a metabolite of omega-3 polyunsaturated fatty acid, decreases post-myocardial infarct depression.</a> Marine Drugs. 12, 5396-5407 (2014).</li><li>Wann, B. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioural+signs+of+depression+and+apoptosis+in+the+limbic+system+following+myocardial+infarction:+effects+of+sertraline.">Behavioural signs of depression and apoptosis in the limbic system following myocardial infarction: effects of sertraline.</a> Journal of Psychopharmacology. 23, 451-459 (2009).</li><li>Gorr, T. A., Vogel, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Western+blotting+re-visited:+critical+perusal+of+under-appreciated+technical+issues.">Western blotting re-visited: critical perusal of under-appreciated technical issues.</a> Proteomics: Clinical Applications. , (2015).</li><li>McKernan, D. P., Dinan, T. G., Cryan, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term='Killing+the+blues':+a+role+for+cellular+suicide+(apoptosis)+in+depression+and+the+antidepressant+response.">'Killing the blues': a role for cellular suicide (apoptosis) in depression and the antidepressant response.</a> Progress in Neurobiology. 88, 246-263 (2009).</li><li>Green, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apoptotic+pathways:+paper+wraps+stone+blunts+scissors.">Apoptotic pathways: paper wraps stone blunts scissors.</a> Cell. 102, 1-4 (2000).</li></ol>

The authors have nothing to disclose.

Our experience with this protocol over the last 10 years led to the identification of critical methodological issues. First, rapid brain dissection in a Petri dish on crushed ice is important to maintain preparation quality. Second, it must be realized that tissue sonication is shorter for the cerebral amygdala compared to other types of tissues, such as the heart (10 sec) or kidneys (20 sec). Third, great care should be taken to carefully mix the substrate before adding it to the samples. We encountered variable signals when the mixing sequence was inadequate. Fourth, no bubbles should be present in the quartz cell when reading the samples. Otherwise, the signal could be altered.
We made a change in the last few years to increase reproducibility of the results: increasing substrate volume to avoid volume less than 1 &#181;l. Still, small variations may occur between successive assays, and at least 3 control samples should be used to control for variations. Fluorescence measures of these controls are averaged and the mean is set at 100%. Fluorescence measures in experimental samples are transformed in percent of the control samples.
Another limitation of the technique is that only a portion of tissue is used and, therefore, caspase-3 activity could be underestimated. Indeed, the time window of apoptosis is short in any given cell and could be missed even though it occurs in neighboring areas at the time of sampling1,4,15. The reverse is also possible: apoptosis could be particularly intense in portions of tissue, resulting in overestimation of caspase-3 activity. We recommend the use of remaining tissues (amygdala) for complementary techniques, such as TUNEL assays or Bax/Bcl2 ratio (see Introduction).
Spectrofluorometry has at least two advantages over Western blotting. First, it directly generates quantitative data. Second, it measures enzymatic activity itself rather than protein or RNA expression, which may be unrelated, and it is known that protein expression may be increased without any change in enzymatic activity. Moreover, spectrofluorometry can be performed on other tissues or with different animal species and can be adjusted for other caspase subtypes, with different substrates, so that safe comparisons can be made.
In conclusion, this document demonstrates the use of spectrofluorometry to measure caspase-3 activity in the amygdala, providing evidence that apoptosis occurs in this region of the limbic system 3 days after MI.

Caspases or Cysteine-dependent aspartate-directed proteases are a family of enzymes that are involved in various homeostasis processess1. Caspases can be broadly classified according to their roles in apoptosis or inflammation. Caspase-1, -4 , -5 and -12 are involved in inflammation whereas those engaged in apoptosis can be sub-classified as initiator caspases (caspase-8 and -9) and executioner caspases (caspase-3, -6 and -7).
Caspases play significant roles in many pathologies, mainly in disorders where apoptosis can be excessive, as observed after myocardial infarction (MI) or cerebral ischemia.
In the rat model of post-MI syndrome used in our lab, it is established that caspase-3 is activated not only in the myocardium2 but also in the limbic system which is implicated in the control of mood and emotions3-6. It is also seen that caspase-3 is activated in different regions of the limbic system, such as the amygdala and hippocampus, and this activation peaks around 3 days after ischemic insult4. Interestingly, caspase-3 activity correlates with post-MI behavioral impairments, and its attenuation through pharmacological and nutritional interventions reduces these injuries, suggesting a possible link between caspase-3 and post-MI depression7-12.
Caspase-3 activation can be measured by various techniques. Western blotting ascertains the enzymatic properties of the caspases and can detect active enzymes as well as their pro-enzyme forms. Western blotting, however, is semi-quantitative, and small variations can be lost through weak signal-to-noise ratios13. A better technique is based on the cleavage of a substrate (DEVD-AMC) by caspase-3 that releases a fluorogenic compound and can be measured by spectrofluorometry. Caspase-3 activation is a reliable marker of apoptosis. Measurement of apoptosis can be unstable because the time window of occurrence is short, and neighboring cells could phagocytize apoptotic bodies or cells14. Apoptosis can be further confirmed by other techniques, such as Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, that detects DNA fragmentation resulting from apoptotic signaling cascades6, or the ratio of pro-/anti-apoptotic proteins, such as Bax/Bcl26.

<table><tbody><tr><td>Spectroflurometer</td><td>Photon technology Instrument (now Horiba)</td><td>Ratiomaster M-40</td><td>with DeltaRAM&amp;nbsp; Random Access Monochromator&amp;nbsp;</td></tr><tr><td>Respirator</td><td>Harvard apparatus</td><td>683</td><td>small animal ventilation</td></tr><tr><td>Glass cuvette</td><td>NSG precision cells</td><td>3G10</td><td>Optical Glass (340-2,500 nm) 10 mm path</td></tr><tr><td>Sonicator</td><td>Cole Parmer</td><td>4710&amp;nbsp; series</td><td></td></tr><tr><td>Spectrometer</td><td>Varian</td><td>Cary 50 Bio</td><td></td></tr></tbody></table>

caspase-3 activity in the rat amygdala measured by spectrofluorometry after myocardial infarction

Myocardial infarction (MI) has dramatic mid- and long-term consequences at the physiological and behavioral levels, but the mechanisms involved are still unclear. Our laboratory has developed a rat model of post-MI syndrome that displays impaired cardiac functions, neuronal loss in the limbic system, cognitive deficits and behavioral signs of depression. At the neuronal level, caspase-3 activation mediates post-MI apoptosis in different limbic regions, such as the amygdala &#8211; peaking at 3 days post-MI. Cognitive and behavioral impairments appear 2-3 weeks post-MI and these correlate statistically with measures of caspase-3 activity. The protocol described here is used to induce MI, collect amygdala tissue and measure caspase-3 activity using spectrofluorometry. To induce MI, the descending coronary artery is occluded for 40 min. The protocol for evaluation of caspase-3 activation starts 3 days after MI: the rats are sacrificed and the amygdala isolated rapidly from the brain. Samples are quickly frozen in liquid nitrogen and kept at -80 &#176;C until actual analysis. The technique performed to assess caspase-3 activation is based on cleavage of a substrate (DEVD-AMC) by caspase-3, which releases a fluorogenic compound that can be measured by spectrofluorometry. The methodology is quantitative and reproducible but the equipment required is expensive and the procedure for quantifying the samples is time-consuming. This technique can be applied to other tissues, such as the heart and kidneys. DEVD-AMC can be replaced by other substrates to measure the activity of other caspases.

The amygdala was isolated in 8 sham (control) and 8 MI rats three days after the induction of the ischemia/reperfusion protocol. Caspase-3 activity was measured in this tissue using a fluorochrome that can be cleaved by active caspase-3 and detected by spectrofluorometry. Positive and negative measurements (see steps 3.11, 3.12 and 3.16) were performed in triplicate and measured during 10 sec, with 1 point at each sec. Differences between positive and negative controls were calculated and the average of the differences for all sham tissue performed during this experiment was set at 100%. Results from MI rats were scaled according to this reference and Figure 1 illustrates the final results: it indicates a significantly higher activity in MI rats compared to sham (p &#60;0.05).
<img alt="Figure 1" src="/files/ftp_upload/53207/53207fig1.jpg" /> 
Figure 1.&#160;Caspase-3 activity in the amygdala of MI rats, expressed as percentage of mean activity in sham controls, set to 100% (8 rats per group). *Indicates significant between-group difference (p &#60;0.05). The data are expressed as mean &#177; SEM.

Watch this Scientific Journal Video about Caspase-3 Activity in the Rat Amygdala Measured by Spectrofluorometry After Myocardial Infarction at JoVE.com

Caspase-3 Activity in the Rat Amygdala Measured by Spectrofluorometry After Myocardial Infarction

Myocardial infarction (MI) is not only followed by impaired cardiac function but also by apoptosis in the amygdala, a brain region involved in the behavioral consequences of MI. This protocol describes how to induce MI, collect amygdala tissue and measure caspase-3 activity, a marker of apoptosis, therein.

Myocardial infarction (MI) has dramatic mid- and long-term consequences at the physiological and behavioral levels, but the mechanisms involved are still ...

caspase-3-activity-rat-amygdala-measured-spectrofluorometry-after

Research

JoVE Journal

Neuroscience

11.7K Views.  Hospital du Sacre-Coeur de Montreal. Myocardial infarction (MI) is not only followed by impaired cardiac function but also by apoptosis in the amygdala, a brain region involved in the behavioral consequences of MI. This protocol describes how to induce MI, collect amygdala tissue and measure caspase-3 activity, a marker of apoptosis, therein.

Video: Caspase-3 Activity in the Rat Amygdala Measured by Spectrofluorometry After Myocardial Infarction

Acute Myocardial Infarction in Rats

With heart failure leading the cause of death in the USA (Hunt), biomedical research is fundamental to advance medical treatments for cardiovascular diseases. Animal models that mimic human cardiac disease, such as myocardial infarction (MI) and ischemia-reperfusion (IR) that induces heart failure as well as pressure-overload (transverse aortic constriction) that induces cardiac hypertrophy and heart failure (Goldman and Tarnavski), are useful models to study cardiovascular disease. In particular, myocardial ischemia (MI) is a leading cause for cardiovascular morbidity and mortality despite controlling certain risk factors such as arteriosclerosis and treatments via surgical intervention (Thygesen). Furthermore, an acute loss of the myocardium following myocardial ischemia (MI) results in increased loading conditions that induces ventricular remodeling of the infarcted border zone and the remote non-infarcted myocardium. Myocyte apoptosis, necrosis and the resultant increased hemodynamic load activate multiple biochemical intracellular signaling that initiates LV dilatation, hypertrophy, ventricular shape distortion, and collagen scar formation. This pathological remodeling and failure to normalize the increased wall stresses results in progressive dilatation, recruitment of the border zone myocardium into the scar, and eventually deterioration in myocardial contractile function (i.e. heart failure). The progression of LV dysfunction and heart failure in rats is similar to that observed in patients who sustain a large myocardial infarction, survive and subsequently develops heart failure (Goldman). The acute myocardial infarction (AMI) model in rats has been used to mimic human cardiovascular disease; specifically used to study cardiac signaling mechanisms associated with heart failure as well as to assess the contribution of therapeutic strategies for the treatment of heart failure. The method described in this report is the rat model of acute myocardial infarction (AMI). This model is also referred to as an acute ischemic cardiomyopathy or ischemia followed by reperfusion (IR); which is induced by an acute 30-minute period of ischemia by ligation of the left anterior descending artery (LAD) followed by reperfusion of the tissue by releasing the LAD ligation (Vasilyev and McConnell). This protocol will focus on assessment of the infarct size and the area-at-risk (AAR) by Evan's blue dye and triphenyl tetrazolium chloride (TTC) following 4-hours of reperfusion; additional comments toward the evaluation of cardiac function and remodeling by modifying the duration of reperfusion, is also presented. Overall, this AMI rat animal model is useful for studying the consequence of a myocardial infarction on cardiac pathophysiological and physiological function.

The rat model of acute myocardial infarction (AMI) is useful to study the consequence of a MI on cardiac pathophysiological and physiological function.

With heart failure leading the cause of death in the USA (Hunt), biomedical research is fundamental to advance medical treatments for cardiovascular ...

Medicine

Measuring Caspase Activity Using a Fluorometric Assay or Flow Cytometry

The activation of cysteine proteases, known as caspases, remains an important process in multiple forms of cell death. Caspases are critical initiators and executioners of apoptosis, the most studied form of programmed cell death. Apoptosis occurs during developmental processes and is a necessary event in tissue homeostasis. Pyroptosis is another form of cell death that utilizes caspases and is a critical process in activating the immune system through the activation of the inflammasome, which results in the release of members of the interleukin-1 (IL-1) family. To assess caspase activity, target substrates can be assessed. However, sensitivity can be an issue when examining single cells or low-level activity. We demonstrate how a fluorogenic substrate can be used with a population-based assay or single-cell assay by flow cytometry. With proper controls, different amino acid sequences can be used to identify which caspases are active. Using these assays, the simultaneous loss of the inhibitors of apoptosis proteins upon tumor necrosis factor (TNF) stimulation has been identified, which primarily induces apoptosis in macrophages rather than other forms of cell death.

The present protocol describes two methods to measure caspase activity through a fluorogenic substrate using flow cytometry or a spectrofluorometer.

The activation of cysteine proteases, known as caspases, remains an important process in multiple forms of cell death. Caspases are critical initiators ...

Biochemistry

Histological Quantification of Chronic Myocardial Infarct in Rats

Myocardial infarction is defined as cardiomyocyte death due to prolonged ischemia; an inflammatory response and scar formation (fibrosis) follow the ischemic injury. Following the initial acute phase, chronic remodeling of the left ventricle (LV) modifies the structure and function of the heart. Permanent coronary ligation in small animals has been widely used as a reference model for a chronic model of MI. Thinning of the infarcted wall progressively develops to transmural fibrosis. Histological assessment of infarct size is commonly performed; nevertheless, a standardization of the methods for quantification is missing. Indeed, important methodological aspects, such as the number of sections analyzed and the sampling and quantification methods, are usually not described and therefore preclude comparison across investigations. Too often, quantification is performed on a single section obtained at the level of the papillary muscles. Because novel strategies aimed at reducing infarct expansion and remodeling are under investigation, there is an important need for the standardization of accurate heart sampling protocols. We describe an accurate method to quantify the infarct size using a systematic sampling of harvested rat heart and image analyses of trichromatic stained histological sections obtained from base to apex. We also provide evidence that calculating the expansion index (EI) allowed for infarct size assessment, taking into account changes of the left ventricle throughout the remodeling.

The post-mortem assessment of myocardial infarction (MI) in rodents is based on quantification of the infarct on stained heart sections. We describe an accurate method to quantify the infarct size using systematic sampling of harvested rat hearts from base to apex and image analyses of trichrome-stained histological sections.

Myocardial infarction is defined as cardiomyocyte death due to prolonged ischemia; an inflammatory response and scar formation (fibrosis) follow the ...

Murine Myocardial Infarction Model using Permanent Ligation of Left Anterior Descending Coronary Artery

Myocardial infarction (MI) and acute coronary diseases are among the most prominent causes of death in population with western lifestyle. The murine models of MI with permanent ligation of left-anterior descending (LAD) coronary artery closely mimics MI in humans. Murine models benefit from the extensive genetic engineering available nowadays. Here we propose a reproducible murine surgical model of myocardial infarction by permanent LAD coronary ligation. Our technique comprises anesthesia with ketamine/xylazine that can be rapidly reversed by administration of an antagonist, intubation without tracheotomy for mechanical-assisted ventilation, ventilation with application of extrinsic positive end-expiratory pressure (PEEP) to avoid alveolar collapse, a thoracotomy method limiting to the minimum surgical lesions made to skeletal muscles, and lung inflation without thoracentesis. This method is sparsely invasive, reproducible and reduces post-surgery mortality and complications.

Herein we describe a surgical procedure showing how to achieve permanent ligation of the left-anterior descending coronary artery in mice. This model is of high relevance to investigate the pathophysiology of myocardial infarction and the concomitant biological processes.

Myocardial infarction (MI) and acute coronary diseases are among the most prominent causes of death in population with western lifestyle. The murine ...

A New Model of Heart Failure Post-Myocardial Infarction in the Rat

Ligation of the left anterior descending (LAD) coronary artery has been widely used to establish the rat model of heart failure (HF) post myocardial infarction (MI). However, the disadvantages of this model include high mortality rate after ligation and larger variations both in the infarct size and the degree of impaired cardiac function. In addition, a ventilator or exteriorization of the heart is indispensable for the previous models, which complicates the procedure during the ligation. In this study, we developed a reliable and reproducible model without the ventilator or exteriorization of the heart by ligating the LAD coronary artery. Four weeks after the procedure, we found that the serum concentrations of CK-MB, NT-proBNP, and Renin, which were used to assist diagnoses of MI and HF, were significantly higher in the MI group compared to the sham group. In contrast, the value of left ventricle ejection fraction (LVEF) in the MI group was obviously less than in the sham group. Furthermore, the infarct size and cardiac fibrosis area were individually confirmed and quantitatively analyzed by TTC staining and Masson's trichrome staining. Smaller variations were found in either infarct size or fibrosis area in the MI group, which helped to develop a reliable and reproducible model of HF post-MI. This new model of HF post-MI in the rat is vital for studying the potential mechanisms of MI and HF. This new method can be used to develop the new drug for treatment of MI and HF in rats by using pharmacological strategies.

We successfully developed a reliable and reproducible model of heart failure post-myocardial infarction in the rat without ventilation or exteriorization of the heart. This simplifies the procedure and benefits the further studies about the potential mechanisms behind the heart failure.

Ligation of the left anterior descending (LAD) coronary artery has been widely used to establish the rat model of heart failure (HF) post myocardial ...

An Intact Pericardium Ischemic Rodent Model

This protocol has shown that the pericardium and its contents play an essential anti-fibrotic role in the ischemic rodent model (coronary ligation to induce myocardial injury). The majority of pre-clinical myocardial infarction models require the disruption of pericardial integrity with loss of the homeostatic cellular milieu. However, recently a methodology has been developed by us to induce myocardial infarction, which minimizes pericardial damage and retains the heart&#39;s resident immune cell population. An improved cardiac functional recovery in mice with an intact pericardial space following coronary ligation has been observed. This method provides an opportunity to study inflammatory responses in the pericardial space following myocardial infarction. Further development of the labeling techniques can be combined with this model to understand the fate and function of pericardial immune cells in regulating the inflammatory mechanisms that drive remodeling in the heart, including fibrosis.

This protocol outlines the steps for inducing myocardial infarction in mice while preserving the pericardium and its contents.

This protocol has shown that the pericardium and its contents play an essential anti-fibrotic role in the ischemic rodent model (coronary ligation to ...

A Fluorescence Assay for Evaluating the Permeability of a Cardiac Microvascular Endothelial Barrier in a Rat Model of Ischemia/reperfusion

Revascularization therapies for culprit arteries, regardless of percutaneous coronary intervention and coronary artery bypass grafting, are considered the best strategy for improving the clinical prognosis of patients with acute coronary syndrome (ACS). Nonetheless, myocardial reperfusion following effective revascularization can trigger significant cardiomyocyte death and coronary endothelial collapse, known as myocardial ischemia/reperfusion injury (MIRI). Usually, endothelial cells and their intercellular tight junctions cooperatively maintain the microvascular endothelial barrier and its relatively low permeability but fail in reperfusion areas. 
Microvascular endothelial hyperpermeability induced by ischemia/reperfusion (IR) contributes to myocardial edema, increased infiltration of pro-inflammatory cells, and aggravated intramyocardial hemorrhage, which may worsen the prognosis of ACS. The tracer used in this study-70,000 Da FITC-dextran, a branched glucose molecule labeled by fluorescein isothiocyanate (FITC)-appears too large to infiltrate the cardiac microvascular endothelium in normal conditions. However, it is capable of infiltrating a broken barrier after MIRI. Thus, the higher the endothelial permeability is, the more FITC-dextran accumulates in the extravascular intercellular space. Thus, the intensity of fluorescence from FITC can indicate the permeability of the microvascular endothelial barrier. This protocol takes advantage of FITC-dextran to evaluate the cardiac microvascular endothelial barrier functionally, which is detected by an automated quantitative pathology imaging system.

Here, we describe a method to functionally assess the endothelial barrier of cardiac microvessels after ischemia/reperfusion injury via the measurement of the mean fluorescence intensity of extravasated 70,000 Da FITC-dextran in comparison with Evans Blue.

Revascularization therapies for culprit arteries, regardless of percutaneous coronary intervention and coronary artery bypass grafting, are considered the ...

An Experimental Model of Myocardial Infarction for Studying Cardiac Repair and Remodeling in Knockout Mice

Cardiovascular disease is the most prevalent cause of death in Western countries, with acute myocardial infarction (MI) being the most prevalent form. This paper describes a protocol for studying the role of galectin 3 (Gal-3) in the temporal evolution of cardiac healing and remodeling in an experimental animal model of MI. 
The procedures described include an experimental model of MI with a permanent coronary ligature in male C57BL/6J (control) and Gal-3 knockout (KO) mice, an echocardiography procedure to study cardiac remodeling and systolic function in vivo, a histological evaluation of interstitial myocardial fibrosis with picrosirius red-stained and rhodamine-conjugated lectin-stained sections for studying myocyte hypertrophy by the cross-sectional area (MCSA), and the quantification of infarct size and cardiac remodeling (scar thinning, septum thickness, and expansion index) by planimetry in slices stained with Masson's trichrome and triphenyl tetrazolium chloride. Gal-3 KO mice with MI showed disrupted cardiac remodeling and an increase in the scar thinning ratio and the expansion index. At the onset of MI, myocardial function and cardiac remodeling were also severely affected. At 4 weeks post MI, the natural evolution of fibrosis in infarcted Gal-3 KO mice was also affected. 
In summary, the experimental model of MI is a suitable model for studying the temporal evolution of cardiac repair and remodeling in mice with the genetic deletion of Gal-3 and other animal models. The lack of Gal-3 affects the dynamics of cardiac repair and disrupts the evolution of cardiac remodeling and function after MI.

Here, we describe an experimental model of myocardial infarction, an echocardiography procedure to study cardiac remodeling and function, and procedures for quantifying fibrosis and hypertrophy in picrosirius red-stained and rhodamine-stained sections, as well as the infarct size and expansion index in slices stained with Masson's trichrome.

Cardiovascular disease is the most prevalent cause of death in Western countries, with acute myocardial infarction (MI) being the most prevalent form. ...

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 ...

Differential Genes Analysis and Validation of Disulfideptosis in Rat Myocardial Ischemia-Reperfusion Injury Model

Myocardial ischemia-reperfusion injury (MIRI) is an additional injury that occurs during the process of restoring heart tissue blood flow after ischemia-induced injury. MIRI seriously affects the efficacy and short-term and long-term prognosis of reperfusion after myocardial infarction. At present, the mechanism of MIRI is not fully clear. Disulfideptosis is a novel mode of cell death, and the relationship between MIRI and disulfideptosis-related genes (DRGs) expression is still unclear. Firstly, this study explores the differentially expressed genes associated with disulfideptosis in MIRI through bioinformatics analysis. Secondly, by constructing a rat model of MIRI, DRGs were further detected. This study identified 12 related genes, including Myh9, SLC7A11, SLC3A2, Myh7b, ACTB, FLNB, Actn1, Actn4, Flnc, Dbn1 and Pdlim1. Myocardial tissue of rats with MIRI shows obvious pathological damage and apoptosis events. The results of immunohistochemistry indicated that MIRI stimulation increased the expression of GLUT1 protein in myocardial tissue but restricted the expression of F-actin protein. In addition, significant differences in the expression of three proteins were validated using external datasets and MIRI rat models. This study demonstrated that DRGs had significant predictive value in MIRI, providing new prospects for exploring biomarkers and potential therapeutic targets of MIRI.

The protocol describes the identification of several novel disulfideptosis-related differential genes associated with myocardial ischemia-reperfusion injury by bioinformatics analysis and experimental validation.