This work was supported by Merit Review awards (BX002332 and BX000640) from the Biomedical Laboratory Research and Development Service of the Veterans Affairs Office of Research and Development, National Institutes of Health (R15HL129140), and funds from Institutional Research and Improvement account. The project is supported in part by the National Institutes of Health grant C06RR0306551.

All surgical procedures performed on animals should be carried out in accordance with Guide for the Care and Use of Laboratory Animals13 or other appropriate ethical guidelines. Protocols should be approved by the animal welfare committee at the appropriate institution before proceeding.
1. Preparing for the ECG
NOTE: Before beginning, don personal protective equipment including gloves, eyewear and a clean laboratory coat or disposable gown.
<ol>
	<li>C...

<ol>
	<li>Kochanek, K. D., Murphy, S. L., Xu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deaths:+Final+Data+for+2011.">Deaths: Final Data for 2011.</a> Natl Vital Stat Rep. 63 (3), 1-120 (2015).</li><li>Klabunde, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Cardiovascular Physiology Concepts 2edn. , 243 (2012).</li><li>Bhardwaj, R., Kandoria, A., Sharma, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myocardial+infarction+in+young+adults-risk+factors+and+pattern+of+coronary+artery+involvement.">Myocardial infarction in young adults-risk factors and pattern of coronary artery involvement.</a> Niger Med J. 55 (1), 44-47 (2014).</li><li>Xu, Z., Alloush, J., Beck, E., Weisleder, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+murine+model+of+myocardial+ischemia-reperfusion+injury+through+ligation+of+the+left+anterior+descending+artery.">A murine model of myocardial ischemia-reperfusion injury through ligation of the left anterior descending artery.</a> J Vis Exp. (86), (2014).</li><li>Fern&#225;ndez, B., 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=The+coronary+arteries+of+the+C57BL/6+mouse+strains:+implications+for+comparison+with+mutant+models.">The coronary arteries of the C57BL/6 mouse strains: implications for comparison with mutant models.</a> J Anat. 212 (1), 12-18 (2008).</li><li>Thaler, M. 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> The Only EKG Book You'll Ever Need. , (2003).</li><li>Poirier, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exercise,+heart+rate+variability,+and+longevity:+the+cocoon+mystery?.">Exercise, heart rate variability, and longevity: the cocoon mystery?.</a> Circulation. 129 (21), 2085-2087 (2014).</li><li>Boudoulas, K. D., Borer, J. S., Boudoulas, H. <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,+Life+Expectancy+and+the+Cardiovascular+System:+Therapeutic+Considerations.">Heart Rate, Life Expectancy and the Cardiovascular System: Therapeutic Considerations.</a> Cardiology. 132 (4), 199-212 (2015).</li><li>Wehrens, X. H., Kirchhoff, S., Doevendans, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+electrocardiography:+an+interval+of+thirty+years.">Mouse electrocardiography: an interval of thirty years.</a> Cardiovasc Res. 45 (1), 231-237 (2000).</li><li>Boukens, B. J., Rivaud, M. R., Rentschler, S., Coronel, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Misinterpretation+of+the+mouse+ECG:+'musing+the+waves+of+Mus+musculus.">Misinterpretation of the mouse ECG: 'musing the waves of Mus musculus.</a> J Physiol. 592 (21), 4613-4626 (2014).</li><li>O'Gara, P. T., 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=ACCF/AHA+guideline+for+the+management+of+ST-elevation+myocardial+infarction:+executive+summary:+a+report+of+the+American+College+of+Cardiology+Foundation/American+Heart+Association+Task+Force+on+Practice+Guidelines:+developed+in+collaboration+with+the+American+College+of+Emergency+Physicians+and+Society+for+Cardiovascular+Angiography+and+Interventions.">ACCF/AHA guideline for the management of ST-elevation myocardial infarction: executive summary: a report of the American College of Cardiology Foundation/American Heart Association Task Force on Practice Guidelines: developed in collaboration with the American College of Emergency Physicians and Society for Cardiovascular Angiography and Interventions.</a> Catheter Cardiovasc Interv. 82 (1), E1-E27 (2013).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Guide for the Care and Use of Laboratory Animals. , (2011).</li><li>Gao, S., Ho, D., Vatner, D. E., Vatner, S. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Echocardiography+in+Mice.">Echocardiography in Mice.</a> Curr Protoc Mouse Biol. 1, 71-83 (2011).</li><li>Jong, W. 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=Reduced+acute+myocardial+ischemia-reperfusion+injury+in+IL-6-deficient+mice+employing+a+closed-chest+model.">Reduced acute myocardial ischemia-reperfusion injury in IL-6-deficient mice employing a closed-chest model.</a> Inflamm Res. 65 (6), 489-499 (2016).</li><li>Nadtochiy, S. 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=In+vivo+cardioprotection+by+S-nitroso-2-mercaptopropionyl+glycine.">In vivo cardioprotection by S-nitroso-2-mercaptopropionyl glycine.</a> J Mol Cell Cardiol. 46 (6), 960-968 (2009).</li><li>Preda, M. B., Burlacu, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrocardiography+as+a+tool+for+validating+myocardial+ischemia-reperfusion+procedures+in+mice.">Electrocardiography as a tool for validating myocardial ischemia-reperfusion procedures in mice.</a> Comp Med. 60 (6), 443-447 (2010).</li><li>Speerschneider, T., Thomsen, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiology+and+analysis+of+the+electrocardiographic+T+wave+in+mice.">Physiology and analysis of the electrocardiographic T wave in mice.</a> Acta Physiol (Oxf. 209 (4), 262-271 (2013).</li><li>Kersten, J. R., Schmeling, T. J., Pagel, P. S., Gross, G. J., Warltier, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isoflurane+mimics+ischemic+preconditioning+via+activation+of+K(ATP)+channels:+reduction+of+myocardial+infarct+size+with+an+acute+memory+phase.">Isoflurane mimics ischemic preconditioning via activation of K(ATP) channels: reduction of myocardial infarct size with an acute memory phase.</a> Anesthesiology. 87 (2), 361-370 (1997).</li><li>Gargiulo, 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=Mice+anesthesia,+analgesia,+and+care,+Part+I:+anesthetic+considerations+in+preclinical+research.">Mice anesthesia, analgesia, and care, Part I: anesthetic considerations in preclinical research.</a> ILAR J. 53 (1), E55-E69 (2012).</li><li>Curtis, M. 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=The+Lambeth+Conventions+(II):+guidelines+for+the+study+of+animal+and+human+ventricular+and+supraventricular+arrhythmias.">The Lambeth Conventions (II): guidelines for the study of animal and human ventricular and supraventricular arrhythmias.</a> Pharmacol Ther. 139 (2), 213-248 (2013).</li></ol>

The authors have nothing to disclose.

Using ECG changes as a supplemental method for confirming myocardial ischemia and reperfusion ensures the accurate placement of the occluding ligature. Accuracy of ligature placement is critical to reducing data variability among animals. The LAD in a mouse heart is a difficult artery to visualize. Therefore, supplementing visual pallor with electrocardiographic changes will help ensure the correct placement of the ligature and resulting tissue damage.
Since the ECG pad offers a non-invasive v...

Heart disease remains the leading cause of death worldwide1,2. Not only is the left ventricle (LV) the most muscular chamber, responsible for pumping blood from the heart to the entire body3, it is a common cardiac injury site post-myocardial infarction4. Left ventricular tissue death often results in systolic heart failure. Animal models of heart disease are imperative for the advancement of biomedical cardiovascular research. The C57Bl/6 strain of mice have been a popular choice for animal models due to their quick breeding time, low cost and ease in genetic alterations. Most murine surgical models for the study of heart disease involve occlusion of the LAD branch of the left coronary artery. The LAD is sometimes called the left obtuse marginal5,6. The LAD supplies blood to the left ventricular anterior and antero-lateral walls. LAD occlusion studies are aimed at inducing anterior infarctions, sometimes extending into the inferior and lateral wall regions7.
Two models that are used frequently for myocardial infarction studies include chronic occlusion myocardial infarction and myocardial ischemia/reperfusion injury. The chronic occlusion is created by surgically suturing around and permanently blocking blood flow through the LAD. The ischemia/reperfusion injury is created much in the same way only with a transient, usually 30-60 min, ischemic period. To achieve transient ischemia, the occluding suture ties around the LAD and a small PE-10 tube which is placed parallel to the LAD on the epicardial surface of the heart, followed by a reperfusion period where the tubing and occluding suture is removed and blood is allowed to once again flow through the artery and into the myocardium. The ischemia/reperfusion surgery has been deemed to be clinically relevant due to the nature of reperfusion injury paralleling the treatment of human infarctions which includes prompt coronary angioplasty and stenting of the artery, or coronary artery bypass. Typically, during these surgeries, ischemia of the LV in a mouse heart is confirmed by visible pallor of the myocardial wall. However, by simply performing the surgeries on an electrocardiogram (ECG) pad under constant monitoring conditions, visible changes can be observed in the ECG waveform, thereby confirming ischemia and reperfusion of the mouse myocardium.
Although the murine heart is similar to the human heart in many respects, including its four-chambered structure, the hearts also have differences. One obvious difference is the average resting heart rate of adult mice is 600 - 700 beats per min (bpm) whereas that of adult humans is ~60-100 bpm8,9. Additionally, in mice the repolarization waves, J and T, often merge with the depolarization QRS-complex making a clear ST-segment difficult to discern10. To complicate the process of electrocardiographically confirming myocardial ischemia, it is the elevation of the T-wave and the ST-segment which are used as markers for the diagnosis of ischemia and myocardial infarction injury in humans, clinically referred to as ST elevation myocardial infarction or STEMI. One of the key differences between human and murine waveforms is that S-wave is immediately followed be a J-wave that transfers directly into a negative T-wave. During acute myocardial ischemia in mice the amplitude of S-wave decreases and is directly followed by an abnormal J-wave and an inverted T-wave11. The T-wave does not seem to represent a significant portion of the repolarization in mice11. Despite nomenclature and mouse vs. human differences, ECG confirmation of murine myocardial ischemia and reperfusion is still feasible and relatively simple. For the sake of simplifying waveform interpretation, the segment between the S-J-T is referred to as ST-segment herein.
STEMI guidelines published in 2013 recommend a patient door-to-balloon time of less than 90 min12.This means that the time frame from the identification of the patient&#39;s coronary artery occlusion until the artery is reopened should be less than 90 min. The beating heart is constantly working and therefore, has a high oxidative metabolism and a high level of oxygen consumption3. To provide for this, a network of capillaries is available to each myocyte3. It only takes a heart a few beats to exhaust its oxygen and nutrient supply. In a 90 min window, an ischemic heart region in a human will have been blocked from receiving between 5,400 and 9,000 heart beats worth of oxygen-rich blood. In that same 90 min window, a mouse would have 54,000 to 63,000 heart beats. Experimental time points for murine ischemia/reperfusion injury are typically between 30 and 60 min.
The importance of developing a supplemental method of confirming myocardial ischemia and reperfusion in a murine model has profound implications on the consistency and reproducibility of data in myocardial ischemia/reperfusion studies. The current practice of visually observing the heart for a change in tissue color is not adequate as a stand-alone diagnostic. Additionally, reperfusion after removal of the tubing and suture is not guaranteed. Although the artery is no longer tied off, the artery may have sustained damage during the procedure and may become impossible to reperfuse. It would be beneficial to have a record of electrocardiographic changes to confirm reperfusion rather than relying on observations of myocardial pallor and rubor (red color). Hearts that do not show the markers of ischemia/reperfusion injury can then quickly be flagged and a decision on how to proceed can be made by the investigators.
Lastly, establishing a record of ECG changes from baseline throughout the ischemic and reperfusion periods allows investigators to continue to monitor the heart after the initial surgery. Investigators currently lose sight of the heart as soon as the surgery is completed. ECG is a simple way to gain insight into changes occurring in the myocardium hours to days after the surgery. ECG recorded at time points after surgery could reveal late-developing Q-waves indicating continued or worsening tissue death. However, to effectively gage new or worsening electrocardiographic markers, a baseline ECG must be available for comparison.
This protocol will demonstrate how to prepare, obtain, and interpret the ECG to confirm ischemia and reperfusion of the mouse heart using 8 - 12 week old male C57Bl/6 mice.

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confirmation of myocardial ischemia and reperfusion injury in mice using surface pad electrocardiography

Many animal models have been established for the study of myocardial remodeling and heart failure due to its status as the number one cause of mortality worldwide. In humans, a pathologic occlusion forms in a coronary artery and reperfusion of that occluded artery is considered essential to maintain viability of the myocardium at risk. Although essential for myocardial recovery, reperfusion of the ischemic myocardium creates its own tissue injury. The physiologic response and healing of an ischemia/reperfusion injury is different from a chronic occlusion injury. Myocardial ischemia/reperfusion injury is gaining recognition as a clinically relevant model for myocardial infarction studies. For this reason, parallel animal models of ischemia/reperfusion are vital in advancing the knowledge base regarding myocardial injury. Typically, ischemia of the mouse heart after left anterior descending (LAD) coronary artery occlusion is confirmed by visible pallor of the myocardium below the occlusion (ligature). However, this offers only a subjective way of confirming correct or consistent ligature placement, as there are multiple major arteries that could cause pallor in different myocardial regions. A method of recording electrocardiographic changes to assess correct ligature placement and resultant ischemia as well as reperfusion, to supplement observed myocardial pallor, would help yield consistent infarct sizes in mouse models. In turn, this would help decrease the number of mice used. Additionally, electrocardiographic changes can continue to be recorded non-invasively in a time-dependent fashion after the surgery. This article will demonstrate a method of electrocardiographically confirming myocardial ischemia and reperfusion in real time.

A normal murine ECG is displayed in Figure 2 with alphabetic markers for electrical events P, Q, R, S, J and T. P is the initial atrial depolarization. QRS is the wave of depolarization over the ventricles. J is early repolarization and T represents heterogeneous repolarization also known as recovery11. It should be noted that many labs do not use the J-wave nomenclature and instead refer to the SJT-segment as the ST-segment10,15-17. Here, results an...

Watch this Scientific Journal Video about Confirmation of Myocardial Ischemia and Reperfusion Injury in Mice Using Surface Pad Electrocardiography at JoVE.com

Confirmation of Myocardial Ischemia and Reperfusion Injury in Mice Using Surface Pad Electrocardiography

During murine myocardial ischemia/reperfusion surgery, correct placement of the occluding ligature is typically confirmed by visible observation of myocardial pallor. Herein, a method of electrocardiographically confirming ischemia and reperfusion, to supplement observed myocardial pallor, is demonstrated in male C57Bl/6 mice.

Many animal models have been established for the study of myocardial remodeling and heart failure due to its status as the number one cause of mortality ...

The overall goal of this procedure is to use electrocardiography to confirm a successful myocardial ischemia and reperfusion in mice. This method can help increase the consistency and reproducibility of the ischemia reperfusion surgery. The main advantage of this technique is that it allows the success of the ischemia and reperfusion to be assessed during the procedure, as opposed to necessitating time-consuming post-surgical analysis.After preparing the ECG pad, place a smaller than dime-sized amount of highly conductive electrolyte gel onto each of the four metallic electrodes on the ECG pad. Then press each paw of an anesthetized supine mouse to a piece of tape, and attach the tape to an ECG electrode, taking care that each paw is in contact with the gel and the electrode. Next, configure the machine so that the ECG waveform can be visualized in real time, adjusting the resolution to account for the differences in amplitude as necessary.If the peak of the R wave or the trough of the Q wave is out of the visual frame, adjust the resolution until the entire height of the waveform can be observed. Then clear the ECG pad of tools, taking care that the mouse is still and untouched and obtain a baseline waveform of the animal's normal, healthy ECG. To perform the ischemia reperfusion, begin by removing the hair around the surgical site and disinfecting the area with betadine and alcohol solutions.Next, use a scalpel to make a vertical incision parallel to the esophagus and trachea, gently moving the lymph nodes to each side of the incision until the thin tissue covering the trachea is exposed. Using forceps, gently separate the tissue until the white cartilage rings of the trachea are visible. Then quickly insert the ventilation tubing into the mouth towards the throat.When the tip of the tubing is visible through the exposed neck area, align the tube with the start of the trachea. Gently wiggle the tube side to side while applying upward pressure until the tubing slides into the trachea, as confirmed visually through the translucent tissue. When the tube is in place, use a loop of string to hook the mouse's two front teeth and tape the string ends to the ECG pad to steady the head of the animal and to ensure the ventilation tubing does not move during surgery.Then quickly attach the ventilation tubing to the rodent ventilator and adjust the ventilation settings according to the weight of the animal. Cover the exposed trachea with a gauze soaked in warm saline to keep the tissue from drying. Then use a scalpel to make a vertical incision along the left side of the sternum.Using forceps, gently separate the fascia layer from the muscle layer, followed by careful cutting of the underlying muscles layers without damaging any visible blood vessels. Use the forceps to gently pull the third rib upwards. Then, maintaining the grip on the rib with one hand, use surgical scissors to carefully cut the intercostal tissue between the third and fourth rib.When the lungs have retracted, use the forceps to gently separate the thin layer of pericardium surrounding the heart. Then move the ribs into a position where the heart is visible between the ribs. To position the heart so that the left atrial auricle is easily visible, move the mouse's lower left paw so that it overlaps the lower right paw.Locate the left anterior descending coronary artery, or LAD, beneath the left auricle, and swiftly insert a 7-0 silk tapered suturing needle into the myocardium deeply enough to pass under the LAD, but not so deeply as to penetrate the left ventricle cavity. Hold the suturing ligature until there is about four centimeters of suturing silk left on the free end, and begin to tie a simple suture knot. Once the free end of the suturing silk has been pulled through the loops to form the knot, grasp both the free and needle ends of the suturing silk with forceps and insert an approximately one-centimeter section of PE10 tubing under the forming knot atop the epicardial surface.When the tubing is in place, return the lower left paw to its original electrode and tighten the knot so that the PE10 tubing is sutured to the heart. Release all physical contact with the mouse to allow the ECG waveform to stabilize for 10 seconds, visually checking the waveform and recording it as the time of occlusion. Then, if the ECG and myocardial color changes indicate ischemia, double-knot the suture around the PE10 tubing and cover the open chest cavity with warm saline gauze.At the end of the ischemic period, remove the gauze covering the chest cavity and visualize the heart. Cut the suturing silk atop the PE10 tubing and remove the tubing, followed by the gentle removal of the ligature from the myocardium. Release all physical contact with the mouse and allow the ECG waveform to cycle for 10 seconds, recording the waveform as the time of reperfusion.At the experimental endpoint, adjust the resolution for any changes in the amplitude, as necessary. If the T wave changes and the myocardium returns to red, indicating a successful reperfusion, suture the intercostal space with a 5-0 silk suture, while applying gentle pressure to the mouse's chest to expel any excess air that entered the cavity during surgery to close the chest cavity. Then suture the muscle layers and skin, allowing the mouse to fully recover in a constant temperature-controlled environment with monitoring.This figure follow the progression of one mouse from a baseline rating all the way through reperfusion, with the first waveform displaying a normal sinus rhythm at the recorded baseline. The second waveform displays the ECG one minute after the ligature was tied and the artery became occluded, with an elevated, hyperacute T wave peak observed, compared to the baseline, preligature waveform. The third waveform shows a complete ST segment elevation at the five-minute time point, with the S portion of the complex not reaching as deeply negatively as in the baseline waveform, before progressing into the J and T waves.At 20 minutes of ischemia, the QT interval has widened, and the ST segment is still elevated, with both sections remaining as such up to the 45-minute mark. After five minutes of reperfusion, evidence of deep, significant Q waves begin to appear, and the T wave returns to the isoelectric line. After 30 minutes, however, the negative Q waves still remain, and are likely indicative of both permanent damage and that the dying heart tissue is deflecting the electrical currents around the damaged area.Further, continuous ischemia and the progression to injury and infarction leads to an enhanced negative T wave projection that is usually permanent. While attempting this procedure, it's important to remember to obtain a baseline ECG before making any marks on the animal. ECG analysis can be applied to detect any electrical changes or abnormalities.The heart can be further processed for protein analysis to investigate proteomic changes or tissue staining to investigate proteomic or morphometric changes. After watching this video, you should have a good understanding of how to use electrocardiography to confirm successful myocardial ischemia and reperfusion in a mouse.

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Research

JoVE Journal

Medicine

13.1K Views.  East Tennessee State University. The overall goal of this procedure is to use electrocardiography to confirm a successful myocardial ischemia and reperfusion in mice. This method can help increase the consistency and reproducibility of the ischemia reperfusion surgery. The main advantage of this technique is that it allows the success of the ischemia and reperfusion to be assessed during the procedure, as opposed to necessitating time-consuming post-surgical analysis.After preparing the ECG pad, place a smaller than d...