This research was supported by a grant from the Citizens United for Research in Epilepsy (CURE) to SLB.

1. Materials to Construct
 <ol>
	<li>A jugular vein catheter is constructed from a piece (100 mm) of PE-50 polyethylene tubing, beveled at one end, and then filled with heparin saline (50 U heparin/mL saline).</li>
	<li>The ECG recording electrodes are constructed from two 100 mm lengths of insulated silver wire (30AWG). One end of both wires is stripped and soldered to a microconnector, and the insulation is twisted to form a third wire that is used as ground. Five-mm of insulation is stripped from the distal end of the wires and discarded.</li>
</ol>
2. Implanting Jugular Vein Catheter
<ol>
	<li>Anesthetize the animal by administration of Urethane, ip (1.2 g/kg).</li>
	<li>Routinely check (10 min intervals) depth of anesthesia by evaluating the pedal withdrawal response and/or eye reflexes. If needed, supplement anesthesia.</li>
	<li>After the animal is at the appropriate plane of anesthesia, shave the right side of the neck from the clavicle to the chin.</li>
	<li>Make a longitudinal incision (10-15 mm) in the skin above the carotid artery and open the incision.</li>
	<li>Using blunt dissection techniques, spread the underlying muscle to locate and isolate the right jugular vein.</li>
	<li>Place two pieces of surgical silk (#2) under the jugular vein, and position the sutures at the rostral and caudal most portions of the incision. Tie the rostral ligature to discontinue flow through the vein.</li>
	<li>Retract the caudal ligature to lift the vessel off the underlying tissue and make a small cut. Then insert the beveled end of the heparin-filled (50 U/ml saline) PE-50 polyethylene catheter (100 mm in length), and advance the tip approximately 8 mm to the level of the right atrium. </li>
	<li>In order to secure the catheter, tie the caudal ligature around both the vessel and catheter. Finally, close the wound with either wound clips or suture.</li>
	<li>During the implantation of the catheter, the exposed end is either clamped with a hemostat or plugged to prevent back flow of blood prior to attachment to an infusion pump.</li>
</ol>
3. Implanting CG Electrodes
<ol>
	<li>Place the animal on its back and shave the chest. </li>
	<li>For placement of the electrodes, make two longitudinal incisions (&asymp; 10 mm), one in the upper right and one in the lower left quadrants of the chest, and free the overlying skin to expose the thoracic muscles.</li>
	<li>Suture one of the exposed 5-mm tips of the silver-wire electrodes into the thoracic muscles in each of the exposed areas, and place the ground electrode subcutaneously.</li>
	<li>Close the incisions with wound clips and plug the microconnector into the recording device.</li>
	<li>Wrap the animal loosely in a towel to maintain body temperature during the procedure.</li>
</ol>
4. Recording ECG
<ol>
	<li>Recordings are made using a Powerlab Data Acquisition System and Macintosh computer or similar hardware/software (amplified, 50X; filtered, 1-1000 Hz). </li>
	<li>Following a 20-30 min equilibration period, record ECG for 20 min.</li>
</ol>
5. Inducing Ventricular Arrhythmias
<ol>
	<li>Attach the jugular vein catheter to a remote syringe placed in a programmable infusion pump.</li>
	<li>Initiate the aconitine infusion at a rate to deliver a constant dose of 5 ug/kg/min for 7 minutes. </li>
	<li>Following ventricular fibrillation or the end of the infusion period, euthanize the animal using an approved procedure. </li>
</ol>
6. Analysis of ECG 
<ol>
	<li>The QT interval represents the total duration of ventricular electrical activity. Fig. 1 shows a model ECG recording and the duration of the QT interval. Calculate the mean time between the initiation of the Q-wave and termination of the T-wave for all recorded heartbeats over a five-minute interval obtained during the 20 min recording period. </li>
	<li>To correct the QT interval for differences in heart rate (QTc), use Bazett&#x2019;s formula, QTc=QT/(RR)&frac12;, where RR is the mean interval between heartbeats. The RR interval is calculated as the duration between successive Q-waves obtained during the analyzed period.</li>
	<li>The QT dispersion (QTd) is calculated by subtracting the minimum QT from the maximum QT recorded for each animal.</li>
</ol>
7. Analysis of Heart Rate Variability
<ol><li>Software programs (i.e. LabChart 7, ADInstruments, Inc.) that analyze variability in the RR intervals obtained during the period of analysis are used for automated calculation of both temporal and/or spectral variance in heart rate. </li></ol>
8. Analysis of Susceptibility to Ventricular Arrhythmias
<ol><li>Calculate the latency from the initiation of aconitine infusion to the first premature ventricular contraction (PVC, Fig. 2, Panel 1), ventricular tachycardia (VT, Fig, 2, Panel 2) and ventricular fibrillation (VF, Fig. 2 Panel 3). </li></ol>
9. Representative Results
Fig. 3 illustrates one measure of HRV in the spectral domain (root mean squared of the standard deviation of the RR 
interval, RMSSD) in control rats and in animals that underwent SE one week prior to testing. This measure of HRV, which represents changes in the 
parasympathetic control of cardiac function 26, is significantly decreased in animals following status epilepticus. Fig. 4 shows that both 
corrected QT interval (QTc, Panel A) and QT dispersion (QTd, Panel B) are significantly increased two weeks following SE27. Taken together, these 
data predict that SE increases risk of ventricular arrhythmias. This prediction is confirmed by the findings that the periods of aconitine infusion 
necessary to induce the experimental arrhythmias (PVC, VT, VF) observed in these studies were significantly smaller in animals following SE (Fig. 5)27.
<img src="/files/ftp_upload/2726/2726fig1.jpg" alt="figure 1" /> Figure 1.Model ECG recording of a normal heartbeat showing P, QRS, and T waves. The QT interval is calculated as the time between the dashed lines.
<img src="/files/ftp_upload/2726/2726fig2.jpg" alt="figure 2" /> Figure 2. Panel 1) Premature ventricular contractions (PVCs) are isolated contractions of the ventricles (B) that occur between normal heartbeats (A). PVC recordings are characterized by the absence of P and T waves. 
Panel 2 Ventricular tachycardia (VT) is a series of multiple ventricular contractions (C) that occur with no intervening normal heartbeats (A).
Panel 3 Ventricular fibrillation (VF) is the lack of discernable rhythm and no association between QRS and P waves (D).
<img src="/files/ftp_upload/2726/2726fig3.jpg" alt="figure 3" /> Figure 3. The root mean squared of the standard deviation (RMSSD; msec) of the intervals between heartbeats observed in control rats and in animals undergoing status epilepticus (SE) 1 wk prior to testing. **p&lt;0.01 compared to Control.
<img src="/files/ftp_upload/2726/2726fig4.jpg" alt="figure 4" /> Figure 4. Mean corrected QT interval (Panel A) and QT dispersion (Panel B) calculated from ECG recordings obtained from control animals (Cont) and from rats undergoing status epilepticus (SE) two weeks prior to testing. *p&lt;0.05 compared to Cont. (Metcalf, et al., Am. J. Physiol. Heart and Circ, 2009, Am Physiol Soc, used with permission from 27).
<img src="/files/ftp_upload/2726/2726fig5.jpg" alt="figure 5" /> Figure 5. Latencies (sec) between the initiation of aconitine infusion and the 1st premature ventricular contraction (PVC), ventricular tachycardia (VT), and ventricular fibrillation (VF) in control animals (Cont) and rats undergoing status epilepticus (SE) two weeks prior to testing. *p&lt;0.05, **p&lt;0.01 compared to Cont. (Metcalf, et al., Am. J. Physiol. Heart and Circ, 2009, Am Physiol Soc, used with permission from 27).

<ol>
	<li>Chugh, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determinants+of+prolonged+QT+interval+and+their+contribution+to+sudden+death+risk+in+coronary+artery+disease:+The+Oregon+sudden+unexpected+death+study.">Determinants of prolonged QT interval and their contribution to sudden death risk in coronary artery disease: The Oregon sudden unexpected death study.</a> Circulation. 119, 663-670 (2009).</li><li>Darbar, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensitivity+and+specificity+of+QTc+dispersion+for+identification+of+risk+of+cardiac+death+in+patients+with+peripheral+vascular+disease.">Sensitivity and specificity of QTc dispersion for identification of risk of cardiac death in patients with peripheral vascular disease.</a> BMJ. 312, 874-878 (1996).</li><li>Bruyne, M. C. d. e. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=QTc+dispersion+predicts+cardiac+mortality+in+the+elderly:+The+Rotterdam+study.">QTc dispersion predicts cardiac mortality in the elderly: The Rotterdam study.</a> Circulation. 97, 467-472 (1998).</li><li>Malik, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heart+rate+variability:+standards+of+measurement,+physiological+interpretation,+and+clinical+use.">Heart rate variability: standards of measurement, physiological interpretation, and clinical use.</a> Circulation. 93, 1043-1065 (1996).</li><li>Grippo, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+susceptibility+to+ventricular+arrhythmias+in+a+rodent+model+of+experimental+depression.">Increased susceptibility to ventricular arrhythmias in a rodent model of experimental depression.</a> Am. J. Physiol. 286, H619-H626 (2004).</li><li>Li, M., Wang, J., Xie, H. H., Shen, F. M., Su, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+susceptibility+of+ventricular+arrhythmia+to+aconitine+in+conscious+hypertensive+rats.">The susceptibility of ventricular arrhythmia to aconitine in conscious hypertensive rats.</a> Acta. 28, 211-215 (2007).</li><li>Beig, M. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Voluntary+exercise+does+not+affect+stress-induced+tachycardia,+but+improves+resistance+to+cardiac+arrhythmias+in+rats.">Voluntary exercise does not affect stress-induced tachycardia, but improves resistance to cardiac arrhythmias in rats.</a> Clin. Exp. Pharm. Physiol. , (2010).</li><li>Hazari, M. S., Haykai-Coates, N., Winsett, D. W., Costa, D. L., Farraj, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+single+exposure+to+particulate+or+gaseous+ari+pollution+increases+the+risk+of+aconitine-induced+cardiac+arrhythmia+in+hypertensive+rats.">A single exposure to particulate or gaseous ari pollution increases the risk of aconitine-induced cardiac arrhythmia in hypertensive rats.</a> Toxicol. Sci. 112, 532-542 (2009).</li><li>Amran, M. S., Hashimoto, K., Homma, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+sodium-calcium+exchange+inhibitors,+KB-R7943+and+SEA0400,+on+aconitine-induced+arrhythmias+in+guinea+pigs+in+vivo,+in+vitro,+and+in+computer+simulation+studies.">Effects of sodium-calcium exchange inhibitors, KB-R7943 and SEA0400, on aconitine-induced arrhythmias in guinea pigs in vivo, in vitro, and in computer simulation studies.</a> J. Pharmacol. Exp. Ther. 310, 83-89 (2004).</li><li>Klekot, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antiarrhythmic+activity+of+a+membrane-protecting+agent+Sal'magin+in+rats+with+aconitine-induced+arrhythmias.">Antiarrhythmic activity of a membrane-protecting agent Sal'magin in rats with aconitine-induced arrhythmias.</a> Bull. Exp. Biol. Med. 142, 209-211 (2006).</li><li>Lowenstein, D. H., Alldredge, B. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Status+Epilepticus.">Status Epilepticus.</a> New England J. Med. 338, 970-976 (1998).</li><li>Walker, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Status+epilepticus:+an+evidence+based+guide.">Status epilepticus: an evidence based guide.</a> BMJ. 331, 673-677 (2005).</li><li>Shorvon, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Status epilepticus: its clinical features and treatment in children and adults. , (1994).</li><li>Boggs, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hemodynamic+monitoring+prior+to+and+at+the+time+of+death+in+status+epilepticus.">Hemodynamic monitoring prior to and at the time of death in status epilepticus.</a> Epilepsy Res. 31, 199-209 (1998).</li><li>Walton, N. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systemic+effects+of+generalized+convulsive+status+epilepticus.">Systemic effects of generalized convulsive status epilepticus.</a> Epilepsia. 34, S54-S58 (1993).</li><li>Boggs, J. G., Painter, J. A., DeLorenzo, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+electrocardiographic+changes+in+status+epilepticus.">Analysis of electrocardiographic changes in status epilepticus.</a> Epilepsy Res. 14, 87-94 (1993).</li><li>Painter, J. A., Shiel, F. O., DeLorenzo, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+pathology+findings+in+status+epilepticus.">Cardiac pathology findings in status epilepticus.</a> Epilepsia. 34, 30-30 (1993).</li><li>Lathers, C. M., Schraeder, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+pharmacology:+drugs+as+a+benefit+and/or+risk+in+sudden+unexpected+death+in+epilepsy.">Clinical pharmacology: drugs as a benefit and/or risk in sudden unexpected death in epilepsy.</a> J. Clin. Pharmacol. 42, 123-126 (2002).</li><li>Dashieff, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sudden+unexpected+death+in+epilepsy:+a+series+from+an+epilepsy+surgery+program+and+specualtion+of+the+relationship+to+sudden+cardiac+death.">Sudden unexpected death in epilepsy: a series from an epilepsy surgery program and specualtion of the relationship to sudden cardiac death.</a> J. Clin. Neurophysiol. 8, 216-222 (1991).</li><li>Tigaran, P. -. C. o. d. r. e. a., Dalager-Pedersen, S., Baandrup, S., Dam, U., M, ., Vesterby-Charles, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sudden+unexpected+death+in+epilepsy:+is+death+by+seizures+a+cardiac+event.">Sudden unexpected death in epilepsy: is death by seizures a cardiac event.</a> Am. J. Forensic Med. Pathol. 26, 99-105 (2005).</li><li>Leung, H., Kwan, P., Elger, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Finding+the+missing+link+between+ictal+bradyarrhythmia,+ictal+asystole,+and+sudden+unexpected+death+in+epilepsy.">Finding the missing link between ictal bradyarrhythmia, ictal asystole, and sudden unexpected death in epilepsy.</a> Epilepsy and Behavior. 9, 19-30 (2006).</li><li>Nei, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EEG+and+ECG+in+sudden+unexplained+death+in+epilepsy.">EEG and ECG in sudden unexplained death in epilepsy.</a> Epilepsia. 45, 338-345 (2004).</li><li>Dudek, F. E., Clark, S., Williams, P. A., Grabenstatter, H. L., Pitkanen, A., Schwartzkroin, P. A., Moshe, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Models of Seizures and Epilepsy. , 415-432 (2006).</li><li>Turski, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Limbic+seizures+produced+by+pilocarpine+in+rats:+behavioral+electroencephalographic,+and+neuropathological+study.">Limbic seizures produced by pilocarpine in rats: behavioral electroencephalographic, and neuropathological study.</a> Behav. Brain Res. 9, 315-335 (1989).</li><li>Kulkarni, S. K., George, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lithium-pilocarpine+neurotoxicity:+a+potential+model+of+status+epilepticus.">Lithium-pilocarpine neurotoxicity: a potential model of status epilepticus.</a> Methods Find. Exp. Clin. Pharamacol. 17, 551-567 (1995).</li><li>Stein, P. K., Bosner, M. S., Kleiger, R. E., Conger, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hart+rate+variability:+a+measure+of+cardiac+autonomic+tone.">Hart rate variability: a measure of cardiac autonomic tone.</a> Am. Heart J. 127, 1376-1381 (1994).</li><li>Metcalf, C. S., Poelzing, S., Little, J. G., Bealer, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Status+epilepticus+induces+cardiac+myofilament+damage+and+increased+susceptibility+to+arrhythmias+in+rat.">Status epilepticus induces cardiac myofilament damage and increased susceptibility to arrhythmias in rat.</a> Am. J. Physiol. 297, H2120-H2127 (2009).</li></ol>

We have nothing to disclose.

Two aspects of the described procedures are of critical importance. Firstly, the rate of aconitine administration to the 
heart must be equivalent across animals. This requires consistent placement of the jugular vein catheter tips relative to the heart, and careful 
adjustment of the infusion rate. The rate of aconitine delivery to the hearts must be equal in order to appropriately evaluate the onset of 
ventricular arrhythmias relative to altered susceptibility. If aconitine delivery varies, then the latency to arrhythmias may be due to
 differences in drug concentration and not alterations in cardiac function. Secondly, preparation and placement of the ECG recording electrodes 
 must yield artifact-free recordings with clearly discernable P, QRS, and T waves. While heart rate, HRV, and occurrence of ventricular arrhythmias 
 can be determined from QRS waves alone, QTc and QTd must be calculated from recordings containing clear Q-wave onset, and T-wave termination.
One obvious limitation of these techniques is that they are conducted in anesthetized animals. However, this is necessary 
for two reasons. 1) Since the ECG electrodes are implanted in skeletal muscle tissue, they are subject to artifacts produced during movement in 
conscious rats. These non-cardiac signals frequently obscure the ECG activity necessary for appropriate analysis of heart function. 2) Aconitine 
induced arrhythmias raise a potential ethical issue when evoked in conscious animals.
These procedures allow quantification of several well-accepted prognostic indicators of SCD, along with direct analysis of 
susceptibility to ventricular arrhythmias in the same animal. These techniques are valuable for determining relative cardiac risk, as well as 
efficacy of cardioprotective therapies, in seizure disorders, and any pathology associated with lethal ventricular arrhythmias that can be modeled 
in the rodent.

methods for ecg evaluation of indicators of cardiac risk, and susceptibility to aconitine-induced arrhythmias in rats following status epilepticus

Lethal cardiac arrhythmias contribute to mortality in a number of pathological conditions. Several parameters obtained from a 
non-invasive, easily obtained electrocardiogram (ECG) are established, well-validated prognostic indicators of cardiac risk in patients suffering 
from a number of cardiomyopathies. Increased heart rate, decreased heart rate variability (HRV), and increased duration and variability of cardiac 
ventricular electrical activity (QT interval) are all indicative of enhanced cardiac risk 1-4. In animal models, it is valuable to compare 
these ECG-derived variables and susceptibility to experimentally induced arrhythmias. Intravenous infusion of the arrhythmogenic agent aconitine has
 been widely used to evaluate susceptibility to arrhythmias in a range of experimental conditions, including animal models of depression 5 and 
 hypertension 6, following exercise 7 and exposure to air pollutants 8, as well as determination of the antiarrhythmic efficacy of pharmacological 
 agents 9,10. 
It should be noted that QT dispersion in humans is a measure of QT interval variation across the full set of leads from a 
standard 12-lead ECG. Consequently, the measure of QT dispersion from the 2-lead ECG in the rat described in this protocol is different than that 
calculated from human ECG records. This represents a limitation in the translation of the data obtained from rodents to human clinical medicine.
Status epilepticus (SE) is a single seizure or series of continuously recurring seizures lasting more than 30 min 
11,12 11,12, and results in mortality in 20% of cases 13. Many individuals survive the SE, but die within 30 days 14,15.
 The mechanism(s) of this delayed mortality is not fully understood. It has been suggested that lethal ventricular arrhythmias contribute to many of these 
 deaths 14-17. In addition to SE, patients experiencing spontaneously recurring seizures, i.e. epilepsy, are at risk of premature 
 sudden and unexpected death associated with epilepsy (SUDEP) 18. As with SE, the precise mechanisms mediating SUDEP are not known. 
 It has been proposed that ventricular abnormalities and resulting arrhythmias make a significant contribution 18-22.
 To investigate the mechanisms of seizure-related cardiac death, and the efficacy of cardioprotective therapies, it is 
 necessary to obtain both ECG-derived indicators of risk and evaluate susceptibility to cardiac arrhythmias in animal models of seizure disorders 
 23-25. Here we describe methods for implanting ECG electrodes in the Sprague-Dawley laboratory rat (Rattus norvegicus), following SE,
 collection and analysis of ECG recordings, and induction of arrhythmias during iv infusion of aconitine.
 These procedures can be used to directly determine the relationships between ECG-derived measures of cardiac electrical 
 activity and susceptibility to ventricular arrhythmias in rat models of seizure disorders, or any pathology associated with increased risk of sudden 
 cardiac death.

Watch this Scientific Journal Video about Methods for ECG Evaluation of Indicators of Cardiac Risk, and Susceptibility to Aconitine-induced Arrhythmias in Rats Following Status Epilepticus at JoVE.com

Methods for ECG Evaluation of Indicators of Cardiac Risk, and Susceptibility to Aconitine-induced Arrhythmias in Rats Following Status Epilepticus

Techniques for measurement of electrical activity of the heart by electrocardiogram (ECG), and analysis of cardiac risk factors and susceptibility to arrhythmias following status epilepticus (SE) in the rat are described.

Lethal cardiac arrhythmias contribute to mortality in a number of pathological conditions. Several parameters obtained from a 
non-invasive, easily ...

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17.7K Views.  University of Utah. Techniques for measurement of electrical activity of the heart by electrocardiogram (ECG), and analysis of cardiac risk factors and susceptibility to arrhythmias following status epilepticus (SE) in the rat are described. 

Video: Methods for ECG Evaluation of Indicators of Cardiac Risk, and Susceptibility to Aconitine-induced Arrhythmias in Rats Following Status Epilepticus

Gene-environment Interaction Models to Unmask Susceptibility Mechanisms in Parkinson's Disease

Lipoxygenase (LOX) activity has been implicated in neurodegenerative disorders such as Alzheimer&#39;s disease, but its effects in Parkinson&#39;s disease (PD) pathogenesis are less understood. Gene-environment interaction models have utility in unmasking the impact of specific cellular pathways in toxicity that may not be observed using a solely genetic or toxicant disease model alone.&#160;To evaluate if distinct LOX isozymes selectively contribute to PD-related neurodegeneration, transgenic (i.e. 5-LOX and 12/15-LOX deficient) mice can be challenged with a toxin that mimics cell injury and death in the disorder.&#160;Here we describe the use of a neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), which produces a nigrostriatal lesion to elucidate the distinct contributions of LOX isozymes to neurodegeneration related to PD. The use of MPTP in mouse, and nonhuman primate, is well-established to recapitulate the nigrostriatal damage in PD.&#160;The extent of MPTP-induced lesioning is measured by HPLC analysis of dopamine and its metabolites and semi-quantitative Western blot analysis of striatum for tyrosine hydroxylase (TH), the rate-limiting enzyme for the synthesis of dopamine. To assess inflammatory markers, which may demonstrate LOX isozyme-selective sensitivity, glial fibrillary acidic protein (GFAP) and Iba-1 immunohistochemistry are performed on brain sections containing substantia nigra, and GFAP Western blot analysis is performed on striatal homogenates.&#160;This experimental approach can provide novel insights into gene-environment interactions underlying nigrostriatal degeneration and PD.

Lipoxygenase (LOX) isozymes can generate products that may increase or decrease neuroinflammation and neurodegeneration.&#160;A gene-environment interaction study could identify LOX isozyme-specific effects. Using the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of nigrostriatal damage in two LOX isozyme-deficient transgenic lines allows for comparison of the contribution of LOX isozymes on dopaminergic integrity and inflammation.

Lipoxygenase (LOX) activity has been implicated in neurodegenerative disorders such as Alzheimer&#39;s disease, but its effects in Parkinson&#39;s disease ...

Ascending Aortic Constriction in Rats for Creation of Pressure Overload Cardiac Hypertrophy Model

Ascending aortic constriction is the most common and successful surgical model for creating pressure overload induced cardiac hypertrophy and heart failure. Here, we describe a detailed surgical procedure for creating pressure overload and cardiac hypertrophy in rats by constriction of the ascending aorta using a small metallic clip. After anesthesia, the trachea is intubated by inserting a cannula through a half way incision made between two cartilage rings of trachea. Then a skin incision is made at the level of the second intercostal space on the left chest wall and muscle layers are cleared to locate the ascending portion of aorta. The ascending aorta is constricted to 50&#8211;60% of its original diameter by application of a small sized titanium clip. Following aortic constriction, the second and third ribs are approximated with prolene sutures. The tracheal cannula is removed once spontaneous breathing was re-established. The animal is allowed to recover on the heating pad by gradually lowering anesthesia. The intensity of pressure overload created by constriction of the ascending aorta is determined by recording the pressure gradient using trans-thoracic two dimensional Doppler-echocardiography. Overall this protocol is useful to study the remodeling events and contractile properties of the heart during the gradual onset and progression from compensated cardiac hypertrophy to heart failure stage.

We describe a stepwise procedure for creating pressure overload and left ventricular hypertrophy in Wistar rats by constriction of the ascending aorta using a small metallic clip. This model is extensively used for studying remodeling changes during cardiac hypertrophy and for identifying and evaluating strategies for regression of such changes.

Ascending aortic constriction is the most common and successful surgical model for creating pressure overload induced cardiac hypertrophy and heart ...

Early Detection of Drug-Induced Renal Hemodynamic Dysfunction Using Sonographic Technology in Rats

The kidney normally functions to maintain hemodynamic homeostasis and is a major site of damage caused by drug toxicity. Drug-induced nephrotoxicity is estimated to contribute to 19- 25% of all clinical cases of acute kidney injury (AKI) in critically ill patients. AKI detection has historically relied on metrics such as serum creatinine (sCr) or blood urea nitrogen (BUN) which are demonstrably inadequate in full assessment of nephrotoxicity in the early phase of renal dysfunction. Currently, there is no robust diagnostic method to accurately detect hemodynamic alteration in the early phase of AKI while such alterations might actually precede the rise in serum biomarker levels. Such early detection can help clinicians make an accurate diagnosis and help in in decision making for therapeutic strategy. Rats were treated with Cisplatin to induce AKI. Nephrotoxicity was assessed for six days using high-frequency sonography, sCr measurement and upon histopathology of kidney. Hemodynamic evaluation using 2D and Color-Doppler images were used to serially study nephrotoxicity in rats, using the sonography. Our data showed successful drug-induced kidney injury in adult rats by histological examination. Color-Doppler based sonographic assessment of AKI indicated that resistive-index (RI) and pulsatile-index (PI) were increased in the treatment group; and peak-systolic velocity (mm/s), end-diastolic velocity (mm/s) and velocity-time integral (VTI, mm) were decreased in renal arteries in the same group. Importantly, these hemodynamic changes evaluated by sonography preceded the rise of sCr levels. Sonography-based indices such as RI or PI can thus be useful predictive markers of declining renal function in rodents. From our sonography-based observations in the kidneys of rats that underwent AKI, we showed that these noninvasive hemodynamic measurements may consider as an accurate, sensitive and robust method in detecting early stage kidney dysfunction. This study also underscores the importance of ethical issues associated with animal use in research.

Early stage hemodynamic dysfunction is critical to the development of kidney disease. Yet, detection methodologies are limited. Recent advances in sonography provide a noninvasive, accurate option for early detection of kidney injury. This study outlines a step-by-step, sonographic methodology for detecting kidney dysfunction using a drug-induced nephrotoxicity rat model.

The kidney normally functions to maintain hemodynamic homeostasis and is a major site of damage caused by drug toxicity. Drug-induced nephrotoxicity is ...

A Model of Cardiac Remodeling Through Constriction of the Abdominal Aorta in Rats

Heart failure is one of the leading causes of death worldwide. It is a complex clinical syndromethat includes fatigue, dyspnea, exercise intolerance, and fluid retention. Changes in myocardial structure, electrical conduction, and energy metabolism develop with heart failure, leading to contractile dysfunction, increased risk of arrhythmias, and sudden death. Hypertensive heart disease is one of the key contributing factors of cardiac remodeling associated with heart failure. The most commonly-used animal model mimicking hypertensive heart disease is created via surgical interventions, such as by narrowing the aorta. Abdominal aortic constriction is a useful experimental technique to induce a pressure overload, which leads to heart failure. The surgery can be easily performed, without the need for chest opening or mechanical ventilation. Abdominal aortic constriction-induced cardiac pathology progresses gradually, making this model relevant to clinical hypertensive heart failure. Cardiac injury and remodeling can be observed 10 weeks after the surgery. The method described here provides a simple and effective approach to produce a hypertensive heart disease animal model that is suitable for studying disease mechanisms and for testing novel therapeutics.

A rat model of abdominal aortic constriction that induces cardiac hypertrophy and remodeling is described. An efficient, highly-reproducible, and minimally-invasive method is used to provide a simple yet useful platform for research in myocardial hypertrophy and dysfunction.

Heart failure is one of the leading causes of death worldwide. It is a complex clinical syndromethat includes fatigue, dyspnea, exercise intolerance, and ...

Cardiac Magnetic Resonance for the Evaluation of Suspected Cardiac Thrombus: Conventional and Emerging Techniques

We present the conventional cardiac magnetic resonance (CMR) protocol for evaluating a suspected thrombus and highlight emerging techniques.&#160;The appearance of a mass on certain magnetic resonance (MR) sequences can help differentiate a thrombus from competing diagnoses such as a tumor. T1 and T2 signal characteristics of a thrombus are related to the evolution of hemoglobin properties. A thrombus typically does not enhance following contrast administration, which also helps differentiation from a tumor. We also highlight the emerging role of T1 mapping in the evaluation of a thrombus, which can add another level of support in diagnosis. Prior to any CMR exam, patient screening and interviews are critical to ensure safety and to optimize patient comfort. Effective communication during the exam between the technologist and the patient promotes proper breath holding technique and higher quality images. Volumetric post processing and structured reporting are helpful to ensure that the radiologist answers the ordering services&#39; question and communicates these results effectively. Optimal pre-MR safety evaluation, CMR exam execution, and post exam processing and reporting allow for delivery of high quality radiological service in the evaluation of a suspected cardiac thrombus.

The goal of this article is to describe how cardiac magnetic resonance can be used for the evaluation and diagnosis of a suspected cardiac thrombus. The method presented will describe data acquisition as well as the pre-procedure and post-procedure protocol.

We present the conventional cardiac magnetic resonance (CMR) protocol for evaluating a suspected thrombus and highlight emerging techniques.&#160;The ...

Echocardiographic Assessment of Cardiac Anatomy and Function in Adult Rats

The use of experimental animal models has become crucial in cardiovascular science. Most studies using rodent models are focused on two-dimensional imaging to study the cardiac anatomy of the left ventricle and M-mode echo to assess its dimensions. However, this could limit a comprehensive study. Herein, we describe a protocol that allows an assessment of the heart chamber size, left ventricular function (systolic and diastolic) and valvular function. A conventional medical ultrasound machine was used in this protocol and different echo views were obtained through left parasternal, apical and suprasternal windows. In the left parasternal window, the long and short axis were acquired to analyze left chamber dimensions, right ventricle and pulmonary artery dimensions, and mitral, pulmonary and aortic valve function. The apical window allows the measurement of heart chamber dimensions and evaluation of systolic and diastolic parameters. It also allows Doppler assessment with detection and quantification of heart valve disturbances (regurgitation or stenosis). Different segments and walls of the left ventricle are visualized throughout all views. Finally, the ascending aorta, aortic arch, and descending aorta can be imaged through the suprasternal window. A combination of ultrasound imaging, Doppler flow and tissue Doppler assessment have been obtained to study cardiac morphology and function. This represents an important contribution to improve the assessment of cardiac function in adult rats with impact for research using these animal models.

A non-invasive protocol for transthoracic echocardiography assessment of cardiac anatomy and function for adult rats is presented in the current study. The heart valves, all four cardiac chambers and the ascending aorta, aortic arch and descending aorta are studied in detail.

The use of experimental animal models has become crucial in cardiovascular science. Most studies using rodent models are focused on two-dimensional ...

Semi-Minimal Invasive Method to Induce Myocardial Infarction in Rats and the Assessment of Cardiac Function by an Isolated Working Heart System

Myocardial infarction (MI) remains the main contributor to morbidity and mortality worldwide. Therefore, research on this topic is mandatory. An easily and highly reproducible MI induction procedure is required to obtain further insight and better understanding of the underlying pathological changes. This procedure can also be used to evaluate the effects or potency of new and promising treatments (as drugs or interventions) in acute MI, subsequent remodeling and heart failure (HF). After intubation and pre-operative preparation of the animal, an anesthetic protocol with isoflurane was performed, and the surgical procedure was conducted&#160;quickly. Using a minimally invasive approach, the left anterior descending artery (LAD) was located and occluded by a ligature. The occlusion can be performed acutely for subsequent reperfusion (ischemia/reperfusion injury). Alternatively, the vessel can be ligated permanently to investigate the development of chronic MI, remodeling or HF. Despite common pitfalls, the drop-out rates are minimal. Various treatments such as remote ischemic conditioning can be examined for their cardioprotective potential pre-, peri- and post-operatively. The post-operative recovery was quick as the anesthesia was precisely controlled and the duration of the operation was short. Post-operative analgesia was administered for three days. The minimally invasive procedure reduces the risk of infection and inflammation. Furthermore, it facilitates rapid recovery. The &#8220;working heart&#8221; measurements were performed ex vivo and enabled precise control of preload, afterload and flow. This procedure requires specific equipment and training for adequate performance. This manuscript provides a detailed step-by-step introduction for conducting these measurements.

This article presents an efficient method to perform myocardial ischemia and subsequent chronic reperfusion in rats using a minimally invasive approach. In addition, left ventricular hemodynamic function of rats is assessed by echocardiography and isolated working heart methods.&#160;

Myocardial infarction (MI) remains the main contributor to morbidity and mortality worldwide. Therefore, research on this topic is mandatory. An easily ...

Development and Evaluation of 3D-Printed Cardiovascular Phantoms for Interventional Planning and Training

Catheter-based interventions are standard treatment options for cardiovascular pathologies. Therefore, patient-specific models could help training physicians&#39; wire-skills as well as improving planning of interventional procedures. The aim of this study was to develop a manufacturing process of patient-specific 3D-printed models for cardiovascular interventions.
To create a 3D-printed elastic phantom, different 3D-printing materials were compared to porcine biological tissues (i.e., aortic tissue) in terms of mechanical characteristics. A fitting material was selected based on comparative tensile tests and specific material thicknesses were defined. Anonymized contrast-enhanced CT-datasets were collected retrospectively. Patient-specific volumetric models were extracted from these datasets and subsequently 3D-printed. A pulsatile flow loop was constructed to simulate the intraluminal blood flow during interventions. Models&#39; suitability for clinical imaging was assessed by x-ray imaging, CT, 4D-MRI and (Doppler) ultrasonography. Contrast medium was used to enhance visibility in x-ray-based imaging. Different catheterization techniques were applied to evaluate the 3D-printed phantoms in physicians&#39; training as well as for pre-interventional therapy planning.
Printed models showed a high printing resolution (~30 &#181;m) and mechanical properties of the chosen material were comparable to physiological biomechanics. Physical and digital models showed high anatomical accuracy when compared to the underlying radiological dataset. Printed models were suitable for ultrasonic imaging as well as standard x-rays. Doppler ultrasonography and 4D-MRI displayed flow patterns and landmark characteristics (i.e., turbulence, wall shear stress) matching native data. In a catheter-based laboratory setting, patient-specific phantoms were easy to catheterize. Therapy planning and training of interventional procedures on challenging anatomies (e.g., congenital heart disease (CHD)) was possible.
Flexible patient-specific cardiovascular phantoms were 3D-printed, and the application of common clinical imaging techniques was possible. This new process is ideal as a training tool for catheter-based (electrophysiological) interventions and can be used in patient-specific therapy planning.

Here we present development of a mock circulation setup for multimodal therapy evaluation, pre-interventional planning, and physician-training on cardiovascular anatomies. With the application of patient-specific tomographic scans, this setup is ideal for therapeutic approaches, training, and education in individualized medicine.

Catheter-based interventions are standard treatment options for cardiovascular pathologies. Therefore, patient-specific models could help training ...

Analyzing Long-Term Electrocardiography Recordings to Detect Arrhythmias in Mice

Arrhythmias are common, affecting millions of patients worldwide. Current treatment strategies are associated with significant side effects and remain ineffective in many patients. To improve patient care, novel and innovative therapeutic concepts causally targeting arrhythmia mechanisms are needed. To study the complex pathophysiology of arrhythmias, suitable animal models are necessary, and mice have been proven to be ideal model species to evaluate the genetic impact on arrhythmias, to investigate fundamental molecular and cellular mechanisms, and to identify potential therapeutic targets.
Implantable telemetry devices are among the most powerful tools available to study electrophysiology in mice, allowing continuous ECG recording over a period of several months in freely moving, awake mice. However, due to the huge number of data points (&gt;1 million QRS complexes per day), analysis of telemetry data remains challenging. This article describes a step-by-step approach to analyze ECGs and to detect arrhythmias in long-term telemetry recordings using the software, Ponemah, with its analysis modules, ECG Pro and Data Insights, developed by Data Sciences International (DSI). To analyze basic ECG parameters, such as heart rate, P wave duration, PR interval, QRS interval, or QT duration, an automated attribute analysis was performed using Ponemah to identify P, Q, and T waves within individually adjusted windows around detected R waves. 
Results were then manually reviewed, allowing adjustment of individual annotations. The output from the attribute-based analysis and the pattern recognition analysis was then used by the Data Insights module to detect arrhythmias. This module allows an automatic screening for individually defined arrhythmias within the recording, followed by a manual review of suspected arrhythmia episodes. The article briefly discusses challenges in recording and detecting ECG signals, suggests strategies to improve data quality, and provides representative recordings of arrhythmias detected in mice using the approach described above.

Here we present a step-by-step protocol for a semiautomated approach to analyze murine long-term electrocardiography (ECG) data for basic ECG parameters and common arrhythmias. Data are obtained by implantable telemetry transmitters in living and awake mice and analyzed using Ponemah and its analysis modules.

Arrhythmias are common, affecting millions of patients worldwide. Current treatment strategies are associated with significant side effects and remain ...

Analysis of Raw and Processed Cyperi Rhizoma Samples Using Liquid Chromatography-Tandem Mass Spectrometry in Rats with Primary Dysmenorrhea

Cyperi rhizoma (CR) is widely used in gynecology and is a general medicine for treating women's diseases in China. Since the analgesic effect of CR is enhanced after processing with vinegar, CR processed with vinegar (CRV) is generally used clinically. However, the mechanism by which the analgesic effect is enhanced by vinegar processing is unclear. In this study, the ultra-high pressure liquid chromatographytandem mass spectrometry (UPLC-MS/MS) technique was used to examine changes in the blood levels of the exogenous constituents and metabolites between CR-treated and CRV-treated rats with dysmenorrhea. The results revealed differing levels of 15 constituents and two metabolites in the blood of these rats. Among them, the levels of (-)-myrtenol and [(1R,2S,3R,4R)-3-hydroxy-1,4,7,7-tetramethylbicyclo[2.2.1]hept-2-yl]acetic acid in the CRV group were considerably higher than in the CR group. CRV reduced the level of 2-series prostanoids and 4-series leukotrienes with proinflammatory, platelet aggregation, and vasoconstriction activities and provided analgesic effects by modulating arachidonic acid and linoleic acid metabolism and the biosynthesis of unsaturated fatty acids. This study revealed that vinegar processing enhances the analgesic effect of CR and contributes to our understanding of the mechanism of action of CRV.

Here, a comparative analysis of raw and processed Cyperi rhizoma (CR) samples is presented using ultra-high performance liquid chromatography-high-resolution tandem mass spectrometry (UPLC-MS/MS) in rats with primary dysmenorrhea. The changes in blood levels of the metabolites and the sample constituents were examined between rats treated with CR and CR processed with vinegar (CRV).

Cyperi rhizoma (CR) is widely used in gynecology and is a general medicine for treating women's diseases in China. Since the analgesic effect of CR is ...