This work was supported by the German Research Foundation [FE 1929/1-1 and WA 2597/3-1]. We thank Sandra Dirschl for excellent technical assistance and Julia Rilling for veterinary advice.

<ol>
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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Baroreflex+control+of+sympathetic+outflow+in+pregnant+rats:+effects+of+captopril.">Baroreflex control of sympathetic outflow in pregnant rats: effects of captopril.</a> The American Journal of Physiology. 258 (6), 1417-1423 (1990).</li><li>Mortara, A., 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=Arterial+baroreflex+modulation+of+heart+rate+in+chronic+heart+failure:+clinical+and+hemodynamic+correlates+and+prognostic+implications.">Arterial baroreflex modulation of heart rate in chronic heart failure: clinical and hemodynamic correlates and prognostic implications.</a> Circulation. 96 (10), 3450-3458 (1997).</li><li>La Rovere, M. T., Bigger, J. T., Marcus, F. I., Mortara, A., Schwartz, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Baroreflex+sensitivity+and+heart-rate+variability+in+prediction+of+total+cardiac+mortality+after+myocardial+infarction.+ATRAMI+(Autonomic+Tone+and+Reflexes+After+Myocardial+Infarction)+Investigators.">Baroreflex sensitivity and heart-rate variability in prediction of total cardiac mortality after myocardial infarction. ATRAMI (Autonomic Tone and Reflexes After Myocardial Infarction) Investigators.</a> Lancet. 351 (9101), 478-484 (1998).</li><li>Robinson, T. G., Dawson, S. L., Eames, P. J., Panerai, R. B., Potter, 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=Cardiac+baroreceptor+sensitivity+predicts+long-term+outcome+after+acute+ischemic+stroke.">Cardiac baroreceptor sensitivity predicts long-term outcome after acute ischemic stroke.</a> Stroke. 34 (3), 705-712 (2003).</li><li>Fenske, 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=cAMP-dependent+regulation+of+HCN4+controls+the+tonic+entrainment+process+in+sinoatrial+node+pacemaker+cells.">cAMP-dependent regulation of HCN4 controls the tonic entrainment process in sinoatrial node pacemaker cells.</a> Nature Communications. 11 (1), 5555 (2020).</li><li>Fenske, 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=Comprehensive+multilevel+in+vivo+and+in+vitro+analysis+of+heart+rate+fluctuations+in+mice+by+ECG+telemetry+and+electrophysiology.">Comprehensive multilevel in vivo and in vitro analysis of heart rate fluctuations in mice by ECG telemetry and electrophysiology.</a> Nature Protocols. 11 (1), 61-86 (2016).</li><li>Thireau, J., Zhang, B. L., Poisson, D., Babuty, D. <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+in+mice:+a+theoretical+and+practical+guide.">Heart rate variability in mice: a theoretical and practical guide.</a> Experimental Physiology. 93 (1), 83-94 (2008).</li><li>Cesarovic, N., Jirkof, P., Rettich, A., Arras, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Implantation+of+radiotelemetry+transmitters+yielding+data+on+ECG,+heart+rate,+core+body+temperature+and+activity+in+free-moving+laboratory+Mice.">Implantation of radiotelemetry transmitters yielding data on ECG, heart rate, core body temperature and activity in free-moving laboratory Mice.</a> Journal of Visualized Experiments. (57), e3260 (2011).</li><li>Alam, M. A., Parks, C., Mancarella, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=long-term+blood+pressure+measurement+in+freely+moving+mice+using+telemetry.">long-term blood pressure measurement in freely moving mice using telemetry.</a> Journal of Visualized Experiments. (111), e53991 (2016).</li><li>Optical and electrophysiological approaches to examine the role of cAMP-dependent regulation of the sinoatrial pacemaker channel HCN4. Dissertation, LMU Munich Available from: <a target="_blank" href="https://edoc.ub.uni-muenchen.de/24431/1/Brox_Verena.pdf">https://edoc.ub.uni-muenchen.de/24431/1/Brox_Verena.pdf</a> (2019)</li><li>Just, A., Faulhaber, J., Ehmke, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autonomic+cardiovascular+control+in+conscious+mice.">Autonomic cardiovascular control in conscious mice.</a> American Journal of Physiology-Regulatory, Integrative and Comparative Physiology. 279 (6), 2214-2221 (2000).</li><li>Parati, G., Di Rienzo, M., Mancia, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+to+measure+baroreflex+sensitivity:+from+the+cardiovascular+laboratory+to+daily+life.">How to measure baroreflex sensitivity: from the cardiovascular laboratory to daily life.</a> Journal of Hypertension. 18 (1), 7-19 (2000).</li><li>Robbe, H. W., 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=Assessment+of+baroreceptor+reflex+sensitivity+by+means+of+spectral+analysis.">Assessment of baroreceptor reflex sensitivity by means of spectral analysis.</a> Hypertension. 10 (5), 538-543 (1987).</li><li>Pinna, G. D., Maestri, R., Raczak, G., La Rovere, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+baroreflex+sensitivity+from+the+gain+function+between+arterial+pressure+and+heart+period.">Measuring baroreflex sensitivity from the gain function between arterial pressure and heart period.</a> Clinical Science. 103 (1), 81-88 (2002).</li><li>Pinna, G. D., Maestri, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+criteria+for+estimating+baroreflex+sensitivity+using+the+transfer+function+method.">New criteria for estimating baroreflex sensitivity using the transfer function method.</a> Medical and Biological Engineering and Computing. 40 (1), 79-84 (2002).</li><li>Laude, D., Baudrie, V., Elghozi, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applicability+of+recent+methods+used+to+estimate+spontaneous+baroreflex+sensitivity+to+resting+mice.">Applicability of recent methods used to estimate spontaneous baroreflex sensitivity to resting mice.</a> American Journal of Physiology-Regulatory, Integrative and Comparative Physiology. 294 (1), 142-150 (2008).</li><li>Ma, X., Abboud, F. M., Chapleau, M. 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Significance of the method with respect to alternative methods 
In the present work, we present a detailed protocol to quantify spontaneous BRS using the sequence method. This approach utilizes spontaneous BP and reflex HR changes measured by ECG and BP telemetry. The advantage of this method is that both parameters can be recorded in conscious, freely moving, unrestrained animals without disturbing animals by walking into the room where the measurements are performed or even by physical interaction required for injection of drugs. This point is very important since it has been clearly shown that such disturbances severely interfere with HR and BP recordings. For example, the injection of drugs requires fixation of the mice, which causes a maximum stress response that increases HR up to 650-700 bpm. To circumvent these stress responses, BRS has been previously determined in anesthetized mice. However, standard anesthetics used in veterinary medicine such as ketamine/xylazine or isoflurane induce bradycardia and influence autonomic reflex responses, limiting the validity of these approaches and the interpretation of the results. To partially overcome these limitations implantable drug delivery devices, i.e., osmotic pumps, which can release drugs into the peritoneal cavity were used. However, with osmotic pumps it is not possible to apply a bolus of a defined dose of drug limiting the application of such devices. Alternatively, complex infusion catheters17 can be implanted into mice in order to administer drugs. However, these catheters are difficult to handle and require surgical skills comparable to those required for the implantation of telemetric devices, while producing less scientific outcome as compared to measurements of spontaneous BRS. Beside the technical issues associated with measuring BRS using injection of drugs, there are some limitations related to the drug action per se. Traditional approaches for determining BRS include bolus injections of vasoactive drugs. However, bolus injection of vasoconstrictors (e.g., phenylephrine) or vasodilators (e.g., sodium nitroprusside) have been considered an excessive and non-physiological stimulus for reflex HR adaption to changes in BP18. Spontaneous activity of the baroreceptor reflex can also be quantified using spectral methods. One of these methods assesses BRS in the frequency domain by calculation of the ratio between changes in HR and changes in blood pressure in a specific frequency band18,19. Other spectral methods involve the determination of the transfer function of BP and HR or the quantification of the coherence between BP and HR20,21. These methods also require telemetric acquisition of spontaneous BP and HR parameters and while they are appropriate for the determination of spontaneous BRS, they require intensive computational tools and are challenging to apply. Furthermore, all spectral methods suffer from the limitation that non-stationary signals preclude the application of spectral methods. In particular, spectral peaks induced by respiration rhythms can be reduced in human patients by asking the patient to stop breathing, while this is obviously not possible in mice. Therefore, the signal-to-noise ratio is frequently quite low in mice. Given the limitations of the methods discussed above, we favor the sequence method for determining BRS in mice. A considerable advantage of this method is the fact that it is a noninvasive technique that provides data on spontaneous BRS under real life conditions22. One further important point is that the duration of sequences analyzed using the sequence method are quite short, involving 3-5 beats. Reflex regulation of HR by the vagal nerve is very fast and well within the timeframe of these sequences. Therefore, the sequence method is well suited to evaluate the contribution of the vagal nerve to BRS. By contrast regulation by the sympathetic nervous system is much slower. In fact, during these short sequences activity of the sympathetic nervous system can be assumed to be almost constant. Therefore, the method is customized to selectively detect reflex changes of the HR driven by vagus nerve activity.
Interpretation of BRS data 
For the interpretation of BRS dysfunction or BRS data per se it is important to consider the individual functional levels which are involved in the baroreceptor reflex. On the neuronal level, afferent, central or efferent components of the reflex might be affected23. On the cardiovascular level, reduced or exaggerated responsiveness of the sinoatrial node to ANS input might be present11,24. A change on each level could lead to changes in the BRS. In order to dissect whether neuronal and/or cardiac mechanisms are responsible for observed changes in BRS, cardiac or neuron specific gene deletion, knock down or gene editing approaches could be used.
Critical steps in the protocol 
The most sophisticated and critical step in this protocol is the preparation and cannulation of the left carotid artery (Step 2.3). The tension of the caudal occlusion suture has to be sufficiently high to completely stop the blood flow before cannulation. Otherwise, even a small leakage of blood during cannulation can severely restrict visibility or even cause the mouse to bleed to death. Cannulation should be successful at the first attempt. However, upon failure of the first attempt, it is still possible to carefully retry cannulation.
The midline incision and subcutaneous tunnel from the neck to the left flank (Step 2.3) must be large enough to easily introduce the transmitter without force but must also be as small as possible to keep the transmitter in place. Otherwise, one will need to lock it into position with suture material or tissue adhesive. Since mice have a very delicate skin, necrosis of the skin can occur if the tunnel for the transmitter is too small.
If the ECG electrodes are too long to fit into the subcutaneous tunnel (Step 2.4), it is necessary to form a new tip by shortening the electrode to a proper length. The electrode must lie flat against the body over the entire length of the lead. Too long electrodes will disturb the animals and they will try to open the wound to remove the transmitter, resulting in risk of tissue irritation and wound dehiscence. Leads that are too short can of course not be extended and it may be that in this case the electrodes cannot be positioned in such a way that they correspond to Einthoven II configuration. We&#160;therefore&#160;recommend&#160;to determine the optimal length of the ECG leads on a dead mouse of the same sex, weight, and genetic background.
Mice should be given a longer recovery time after transmitter implantation if they do not have a normal circadian rhythm and this is not the phenotype of the mouse line under study (step 2.7). Another reason for disturbed circadian rhythms could be inadequate acoustic isolation of the animal facility or personnel entering the room during measurement.
ECG, BP and BRS data analysis is straight forward (Step 2.8). The most critical step is to exclude ectopic beats, sinus pauses, arrhythmic episodes or sections with low-quality signals from data analysis.

The arterial baroreceptor reflex is the major feedback control system in humans which provides a short-term - and possibly also longer term1,2 - control of arterial blood pressure (ABP). This reflex buffers perturbations in BP that occur in response to physiological or environmental triggers. It provides prompt reflex changes in heart rate, stroke volume, and total peripheral arterial resistance. The reflex originates in sensory nerve endings in the aortic arch and carotid sinuses. These nerve terminals make up the arterial baroreceptors. The somata of nerve terminals in the aortic arch are located in the nodose ganglion while those of nerve terminals in the carotid sinus are located in the petrosal ganglion. The reflex is triggered by an increase in blood pressure, which stretches and activates the baroreceptor nerve terminals (Figure 1A). Activation results in action potential volleys which are transmitted centrally via the afferent aortic depressor and carotid sinus nerves to cardiovascular brain stem nuclei such as the nucleus tractus solitarii and the dorsal nucleus of the vagal nerve. Changes in afferent nerve activity in turn modulate the autonomic efferent activity. Increased activity of baroreceptor nerves decreases sympathetic and increases parasympathetic nerve activity. Thus, the consequences of activation of baroreceptors are a reduction in heart rate, cardiac output, and vascular resistance which together counteract and buffer the increase in blood pressure3. By contrast, decreased activity of baroreceptor nerves increases sympathetic and decreases parasympathetic nerve activity, which increases heart rate, cardiac output, and vascular resistance and thus counteract the decrease in blood pressure.
Numerous studies in humans and animals have demonstrated that the baroreceptor reflex can be adjusted under physiological conditions such as exercise4, sleep5, heat stress6, or pregnancy7. Additionally, there is&#160;evidence that the baroreflex is chronically impaired in cardiovascular diseases, such as hypertension, heart failure, myocardial infarction, and stroke. In fact, baroreflex dysfunction is also utilized as a prognostic marker in several cardiovascular diseases8,9,10. Furthermore, dysfunction of the baroreflex is also present in disorders of the ANS. Given the importance of the baroreceptor reflex for health and disease states, in vivo estimation of this reflex is an important component of autonomic and cardiovascular research with certain serious clinical implications.
Genetic mouse lines are essential tools in cardiovascular research. In vivo studies of such mouse lines provide valuable insights into cardiovascular physiology and pathophysiology and in many cases serve as preclinical model systems for cardiovascular diseases. Here we provide a protocol for telemetric in vivo ECG and BP recording in conscious, unrestrained, freely moving mice and describe how baroreflex sensitivity can be determined from these recordings using the sequence method (Figure 1B). The applied method is called the sequence method, because the beat-to-beat series of systolic BP (SBP) and RR intervals are screened for short sequences of three or more beats during spontaneous increase or decrease in SBP with reflex adaption of the HR. This method is the gold-standard for baroreflex sensitivity determination since only spontaneous reflex mechanisms are investigated. The technique is superior to older techniques that involved invasive procedures such as injection of vasoactive drugs to induce BP changes.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62101/62101fig1v2.jpg" /> 
Figure 1: Schematic representation of the baroreflex and baroreflex sensitivity assessment using the sequence method. (A) Course of the baroreflex during an acute increase in blood pressure. A short-term rise in ABP is sensed by baroreceptors located in the aortic arch and carotid sinus. This information is transmitted to the central nervous system and induces a decrease in sympathetic nerve activity in parallel with an increase in parasympathetic activity. Release of acetylcholine from nerve endings located in the sinoatrial node region induces a decrease of the second messenger cAMP in sinoatrial node pacemaker cells and hence a reduction in heart rate. A short-term decrease in blood pressure has the opposite effect. (B) Schematic BP traces during an up sequence (upper left panel) and down sequence (upper right panel) of three consecutive beats. An up sequence is associated with a parallel increase in RR intervals (lower left panel) which is equivalent to a decrease in HR. A down sequence is associated with a parallel decrease in RR intervals (lower right panel) which is equivalent to an increase in HR. <a href="https://www.jove.com/files/ftp_upload/62101/62101fig1v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr >
			<td >Acepromazine maleate (Tranquisol KH) Solution Injectable 0.5 mg/mL</td>
			<td >CP-Pharma, Germany</td>
			<td >1229</td>
			<td >anesthesia</td>
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		<tr >
			<td >B.Braun Injekt-F 1 mL syringe</td>
			<td >Wolfram Droh GmbH, Germany</td>
			<td >9166017V</td>
			<td></td>
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			<td >Bepanthen eye and nose ointment</td>
			<td >Bayer AG, Germany</td>
			<td></td>
			<td></td>
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		<tr >
			<td >Blunt dissecting scissors</td>
			<td >Fine Science Tools GmbH, Germany</td>
			<td >14078-10</td>
			<td></td>
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			<td >Carprofen (Carprosol) 50 mg/mL</td>
			<td >CP-Pharma, Germany</td>
			<td >115</td>
			<td>preemptive and post-operative pain relief</td>
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			<td >Cutasept F skin desinfectant</td>
			<td >BODE Chemie GmbH, Germany</td>
			<td >9803650</td>
			<td></td>
		</tr>
		<tr >
			<td >Cotton Tipped Applicator sterile</td>
			<td >Paul Boettger GmbH &#38; Co. KG, Germany</td>
			<td>09-119-9100</td>
			<td></td>
		</tr>
		<tr >
			<td >Forceps - Micro-Blunted Tips</td>
			<td >Fine Science Tools GmbH, Germany</td>
			<td >11253-25</td>
			<td></td>
		</tr>
		<tr >
			<td >Forceps - straight</td>
			<td >Fine Science Tools GmbH, Germany</td>
			<td >11008-13</td>
			<td></td>
		</tr>
		<tr >
			<td >Gauze swabs with cut edges, 7.5x7.5 cm, cotton</td>
			<td >Paul Hartmann AG. Germany</td>
			<td >401723</td>
			<td></td>
		</tr>
		<tr >
			<td >HD&#8209;X11, Combined telemetric ECG and BP transmitters&#160;</td>
			<td >Data Sciences International, United States</td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >Homothermic blanket system with flexible probe</td>
			<td >Harvard Apparatus, United States</td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >Hot bead sterilizer</td>
			<td >Fine Science Tools GmbH, Germany</td>
			<td >18000-45</td>
			<td></td>
		</tr>
		<tr >
			<td >Ketamine 10%</td>
			<td >Ecuphar GmbH, Germany</td>
			<td >799-760</td>
			<td>anesthesia</td>
		</tr>
		<tr >
			<td >Magnet</td>
			<td >Data Sciences International, United States</td>
			<td ></td>
			<td>transmitter turn on/off</td>
		</tr>
		<tr >
			<td >Needle holder, Olsen-Hegar with suture cutter</td>
			<td >Fine Science Tools GmbH, Germany</td>
			<td >12502-12</td>
			<td></td>
		</tr>
		<tr >
			<td >Needle single use No. 17, 0.55 x 25 mm</td>
			<td >Henke-Sass Wolf GmbH, Germany</td>
			<td>4710005525</td>
			<td>24 G needle</td>
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		<tr >
			<td >Needle single use No. 20, 0.40 x 20 mm</td>
			<td >Henke-Sass Wolf GmbH, Germany</td>
			<td >4710004020</td>
			<td>27 G needle</td>
		</tr>
		<tr >
			<td >Needle-suture combination, sterile, absorbable (6-0 USP, metric 0.7, braided)</td>
			<td >Resorba Medical, Germany</td>
			<td >PA10273</td>
			<td>lead fixation</td>
		</tr>
		<tr >
			<td >Needle-suture combination, sterile, silk (5-0 USP, metric 1.5, braided)</td>
			<td >Resorba Medical, Germany</td>
			<td >4023</td>
			<td>skin closure</td>
		</tr>
		<tr >
			<td >OPMI&#160;1FR&#160;pro,&#160;Dissecting&#160;microscope</td>
			<td >Zeiss, Germany</td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >Pilca depilatory mousse</td>
			<td >Werner Schmidt Pharma GmbH, Germany</td>
			<td>6943151</td>
			<td></td>
		</tr>
		<tr >
			<td >PVP-Iodine hydrogel 10%</td>
			<td >Ratiopharm, Germany</td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >Ringer&#39;s lactate solution</td>
			<td >B. Braun Melsungen AG, Germany</td>
			<td >401-951</td>
			<td>&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;</td>
		</tr>
		<tr >
			<td >Sensitive plasters, Leukosilk</td>
			<td >BSN medical GmbH, Germany</td>
			<td >102100</td>
			<td>surgical tape</td>
		</tr>
		<tr >
			<td >Sodium chloride solution 0.9% sterile Miniplasco Connect 5 ml</td>
			<td >B. Braun Melsungen AG, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Surgibond tissue adhesive</td>
			<td >SMI, Belgium</td>
			<td >ZG2</td>
			<td></td>
		</tr>
		<tr >
			<td >Suture, sterile, silk, non-needled (5-0 USP, metric 1 braided)</td>
			<td >Resorba Medical, Germany</td>
			<td >G2105</td>
			<td>lead preparation, ligation sutures</td>
		</tr>
		<tr >
			<td >Trimmer, Wella Contura type 3HSG1</td>
			<td >Procter &#38; Gamble</td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >Vessel Cannulation Forceps</td>
			<td >Fine Science Tools GmbH, Germany</td>
			<td >18403-11</td>
			<td></td>
		</tr>
		<tr >
			<td >Xylazine (Xylariem) 2%</td>
			<td >Ecuphar GmbH, Germany</td>
			<td >797469</td>
			<td>anesthesia</td>
		</tr>
		<tr >
			<td >Data acquisition and analysis</td>
			<td >Source</td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >DSI Data Exchange Matrix</td>
			<td >Data Sciences International, United States</td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >DSI Dataquest ART 4.33</td>
			<td >Data Sciences International, United States</td>
			<td ></td>
			<td>data aquisition software</td>
		</tr>
		<tr >
			<td >DSI Ponemah</td>
			<td >Data Sciences International, United States</td>
			<td ></td>
			<td>data aquisition software</td>
		</tr>
		<tr >
			<td >DSI PhysioTel HDX-11 for mice</td>
			<td >Data Sciences International, United States</td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >DSI PhysioTel receivers RPC1</td>
			<td >Data Sciences International, United States</td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >ecgAUTO v3.3.5.11</td>
			<td >EMKA Technologies</td>
			<td ></td>
			<td>ECG and BP analysis software</td>
		</tr>
		<tr >
			<td >Microsoft Excel</td>
			<td >Microsoft Corporation, United States</td>
			<td ></td>
			<td></td>
		</tr>
	</tbody>
</table>

implantation of combined telemetric ecg and blood pressure transmitters to determine spontaneous baroreflex sensitivity in conscious mice

Blood pressure (BP) and heart rate (HR) are both controlled by the autonomic nervous system (ANS) and are closely intertwined due to reflex mechanisms. The baroreflex is a key homeostatic mechanism to counteract acute, short-term changes in arterial BP and to maintain BP in a relatively narrow physiological range. BP is sensed by baroreceptors located in the aortic arch and carotid sinus. When BP changes, signals are transmitted to the central nervous system and are then communicated to the parasympathetic and sympathetic branches of the autonomic nervous system to adjust HR. A rise in BP causes a reflex decrease in HR, a drop in BP causes a reflex increase in HR.
Baroreflex sensitivity (BRS) is the quantitative relationship between changes in arterial BP and corresponding changes in HR. Cardiovascular diseases are often associated with impaired baroreflex function. In various studies reduced BRS has been reported in e.g., heart failure, myocardial infarction, or coronary artery disease. 
Determination of BRS requires information from both BP and HR, which can be recorded simultaneously using telemetric devices. The surgical procedure is described beginning with the insertion of the pressure sensor into the left carotid artery and positioning of its tip in the aortic arch to monitor arterial pressure followed by the subcutaneous placement of the transmitter and ECG electrodes. We also describe postoperative intensive care and analgesic management. After a two-week period of post-surgery recovery long-term ECG and BP recordings are performed in conscious and unrestrained mice. Finally, we include examples of high-quality recordings and the analysis of spontaneous baroreceptor sensitivity using the sequence method.

Positive results for ECG and BP raw data 
Using this protocol high-quality ECG and BP data can be acquired (Figure 4 and Supplemental File 14), allowing not only for precise BRS analysis but also for analysis of a broad range of ECG or BP-derived parameters, e.g. ECG intervals (Figure 4B, upper panel), blood pressure parameters (Figure 4B, lower panel), heart rate and blood pressure variability, arrhythmia detection etc12,13,14,15.
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/62101/62101fig4v2.jpg" /> 
Figure 4: Telemetric ECG and BP recordings. (A) Representative, high-quality ECG trace (upper panel) and corresponding high-quality raw BP recordings (lower panel). (B) Magnification of ECG traces (upper panel). P wave, QRS complex, T wave and RR interval are indicated. Magnification of corresponding BP data (lower panel). Diastolic BP (DBP) and systolic BP (SBP) are indicated. <a href="https://www.jove.com/files/ftp_upload/62101/62101fig4v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Positive results for circadian rhythm 
A healthy mouse that has sufficiently recovered from surgery shows a physiological increase of activity, HR and BP during the activity (dark) phase (Figure 5). Many different factors can disturb this regular circadian rhythm. These include psychological stress, acoustic or electric noise and pain. For example, an acute pain condition immediately after surgery would result in an increase in heart rate with a simultaneous decrease in activity. Therefore, the circadian rhythm is an important indicator for animal health and well-being and should be routinely checked before BRS analysis.
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/62101/62101fig5v2.jpg" /> 
Figure 5: Analysis of long-term telemetry measurements to determine circadian rhythm variations. Circadian rhythm of heart rate (A), activity (B), systolic blood pressure (C) and diastolic blood pressure (D) averaged from 9 male wild-type C57BL/6J mice during 12 h light and dark cycles. Grey areas depict the activity (dark) phase and white areas depict the resting (light) phase of the animals. All parameters are physiologically elevated during the animal&#180;s activity (dark) phase. Data are represented as mean +/- SEM. <a href="https://www.jove.com/files/ftp_upload/62101/62101fig5v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Positive results for BRS analysis 
After performing the analysis as described in the protocol section 2.8 the software will detect up and down sequences, respectively. The method used is called sequence method since changes in SBP and RR intervals are examined on a beat-to-beat basis during short sequences of three or more beats with a spontaneous rise or fall in SBP (Figure 6). A continuous elevation in SBP over three heartbeats causes a reflex increase in parasympathetic activity and in consequence slows down HR, which is equivalent to longer RR intervals. The latency for the reflex HR adaption is one beat. Such a sequence is shown in Figure 6A and is defined as an up sequence. In contrast, a continuous decrease in SBP over three beats with parallel rise in HR (decrease in RR interval) is defined as a down sequence (Figure 6B). To evaluate the correlation between RR and SBP, both parameters are plotted against each other and the slope (ms/mmHg) of the linear regression line is calculated for each sequence (Figure 6A,B, lower panels). After sorting by up and down sequences the average number of&#160;sequences per 1000 beats (Figure 6C) and average gain of spontaneous BRS can be calculated for up and down sequences, respectively (Figure 6D,E). The gain of spontaneous BRS is reflected by the slope of the linear regression line calculated from the RR/SBP relation. The deviation from normal BRS values can have various causes. These include changes in ANS input or changes in the responsiveness of the sinoatrial node to autonomic nervous system input. In Figure 6 increased BRS in a mouse model for sick sinus syndrome (SSS) with exaggerated responsiveness of the sinoatrial node to vagal input is shown11.
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/62101/62101fig6v2.jpg" /> 
Figure 6: Estimation of BRS using the sequence method. (A) Representative BP trace of a wild-type C57BL/6J mouse during an up sequence of three consecutive beats (upper panel) associated with a parallel increase in RR interval (middle panel) which is equivalent to a decrease in HR. The RR intervals were plotted against the SBP (lower panel). The slope of the regression line (red line) for the up sequence depicted in the upper and middle panel (WT, black circles) was 4.10 ms/mmHg. A representative RR/SBP relationship of the sick sinus syndrome mouse model yielded an increased slope of 6.49 ms/mmHg indicating elevated BRS (SSS, grey circles). (B) Representative down sequence of a wild-type mouse with a drop in SBP (upper panel) and a subsequent decrease in RR interval (middle panel) which results in a BRS slope of 4.51 ms/mmHg (lower panel; WT, black circles). A representative RR/SBP relationship of the sick sinus syndrome mouse model (SSS, grey circles) with a slope of 7.10 ms/mmHg. The orientation of the red arrowheads indicates the direction of the sequences (up or down sequence). (C) Total amount of sequences per 1000 beats for WT and SSS mice. (D) Mean slope of the RR/SBP relationship for up sequences for WT and SSS mice. (E) Mean slope of the RR/SBP relationship for down sequences for WT and SSS mice. Statistics in (C-E) were performed from results of six male WT animals and eight male animals of the sick sinus syndrome mouse model. Boxplots show the median line, perc 25/75, and min/max value; open symbols represent the mean value. <a href="https://www.jove.com/files/ftp_upload/62101/62101fig6v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Negative result for raw data quality 
Especially during phases of higher activity signal quality might decrease (Figure 7 and Supplemental Files 15,16). This can be caused by temporary displacement or incorrect position of either the BP catheter or ECG leads or both due to motion of the animal. Also, skeletal muscle activity might be detected from the ECG leads and induce noise (Figure 7B, upper panel). With the software settings described above, these low quality beats are not detected and are therefore excluded from analysis. Nevertheless, manual inspection of the analysed raw data is mandatory.
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/62101/62101fig7v2.jpg" /> 
Figure 7: Examples of low-quality raw signals. (A) ECG signal (upper panel) is detected with good quality, but BP signal (lower panel) quality is low. (B) Qualities of ECG (upper panel) and BP (lower panel) signal are not sufficient. <a href="https://www.jove.com/files/ftp_upload/62101/62101fig7v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Negative results for BRS analysis 
The BRS analysis settings listed in protocol section 2.8.3 are in general essential for fast and correct detection of up and down sequences. The minimum correlation coefficient for the regression line is set to 0.75. Setting too low values for the minimum correlation coefficient results in false detections of sequences that do not reflect baroreflex activity but rather result from arrhythmic beats (Figure 8). For BRS analysis only episodes with stable sinus rhythm must be analysed. Ectopic beats or other arrhythmic events, e.g., sinus pauses, can be found with the HRV option of ECG and BP analysis software and must be invalidated.
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/62101/62101fig8v2.jpg" /> 
Figure 8: Sequences that do not reflect baroreflex activity. (A) ECG trace of a mouse with mild sinus dysrhythmia. (B) BP recording depicting a spontaneous increase in SBP. (C) Corresponding RR intervals indicate a decrease of HR upon the increase of BP. (D) Plot of SBP and corresponding RR intervals. The low correlation coefficient of the regression line indicates that HR reduction was not caused by activity of the baroreflex but rather by sinus dysrhythmia. (E) Raw ECG trace depicting a sinus pause. (F) Corresponding raw BP signal. The sinus pause causes a drop in diastolic blood pressure. Systolic blood pressure of the subsequent beat is almost unaffected. <a href="https://www.jove.com/files/ftp_upload/62101/62101fig8v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplemental File 1: Surgery protocol. Template for documentation of the surgical procedure and post-operative care. <a href="https://www.jove.com/files/ftp_upload/62101/SupplementalFile1.pdf" target="_blank">Please click here to download&#160;this File.</a>
Supplemental File 2: Converting Dataquest A.R.T data into IOX data for analysis in ecgAUTO software. Select animals in the subjects list (left) and Pressure and ECG in the waveforms list (right). Press OK to convert data. <a href="https://www.jove.com/files/ftp_upload/62101/SupplementalFile2.pdf" target="_blank">Please click here to download&#160;this File.</a>
Supplemental File 3: ECG settings for BRS analysis. Set parameters as listed, press ok and apply the configuration. <a href="https://www.jove.com/files/ftp_upload/62101/SupplementalFile3.pdf" target="_blank">Please click here to download&#160;this File.</a>
Supplemental File 4: BP settings for BRS analysis. Set parameters as listed, press ok and apply the configuration. Save the configuration as a configuration file to be able to load the settings easily. <a href="https://www.jove.com/files/ftp_upload/62101/SupplementalFile4.pdf" target="_blank">Please click here to download&#160;this File.</a>
Supplemental File 5: Parameters in list/to file window for &#34;sections&#34;. Choose sections to be exported under the sections&#160;&#62;&#160;txt header (selected) and press Apply!. <a href="https://www.jove.com/files/ftp_upload/62101/SupplementalFile5.pdf" target="_blank">Please click here to download&#160;this File.</a>
Supplemental file 6: Parameters in list/to file window for &#34;steps&#34;. Choose step data to be exported under the steps &#62; txt header (selected) and press Apply!. <a href="https://www.jove.com/files/ftp_upload/62101/SupplementalFile6.pdf" target="_blank">Please click here to download&#160;this File.</a>
Supplemental File 7: Parameters in list/to file window for &#34;beats&#34;. Choose values to be exported under the beats &#62; txt header (selected) and press Apply!. For BRS analysis the ticked parameters are necessary. Note the order of selection indicated by the numbers. <a href="https://www.jove.com/files/ftp_upload/62101/SupplementalFile7.pdf" target="_blank">Please click here to download&#160;this File.</a>
Supplemental File 8: TemplateBRS spreadsheet file. Spreadsheet template for automated sorting and analysis of up and down sequences. <a href="https://www.jove.com/files/ftp_upload/62101/TemplateBRS.xlsx" target="_blank">Please click here to download&#160;this File.</a>
Supplemental File 9: Copying relevant data from the Results File I. Copy the columns (Pressure)_BRS_deltaP, (Pressure)_BRS_# and (Pressure)_BRS_slope from the Results File. <a href="https://www.jove.com/files/ftp_upload/62101/SupplementalFile9.pdf" target="_blank">Please click here to download&#160;this File.</a>
Supplemental File 10: Spreadsheet template file (TemplateBRS) for data sorting and analysis I. Paste the copied data into the respective columns of the &#34;Up sequences&#34; and &#34;Down sequences&#34; spreadsheet in the TemplateBRS spreadsheet file. <a href="https://www.jove.com/files/ftp_upload/62101/SupplementalFile10.pdf" target="_blank">Please click here to download&#160;this File.</a>
Supplemental File 11: Copying relevant data from the Results File II. Copy the column (Pressure)_BRS_SBP from the Results File. <a href="https://www.jove.com/files/ftp_upload/62101/SupplementalFile11.pdf" target="_blank">Please click here to download&#160;this File.</a>
Supplemental File 12: A spreadsheet template file (TemplateBRS) for data sorting and analysis II. Paste the copied SBP data into the &#34;All sequences&#34; spreadsheet in the TemplateBRS spreadsheet file to calculate the total number of sequences. <a href="https://www.jove.com/files/ftp_upload/62101/SupplementalFile12.pdf" target="_blank">Please click here to download&#160;this File.</a>
Supplemental File 13: Filtering and analyzing the sequences. In the &#34;Up sequences&#34; spreadsheet of the TemplateBRS spreadsheet file, open the drop-down menu of the (Pressure)_BRS_# column filter and press OK without changing any parameters. This will automatically sort the data and update the calculations for sequences with 3 beats. Repeat this for the &#34;Down sequences&#34; spreadsheet. <a href="https://www.jove.com/files/ftp_upload/62101/SupplementalFile13.pdf" target="_blank">Please click here to download&#160;this File.</a>
Supplemental File 14: Screenshot of a high-quality recording detected with ECG and BP analysis software. The upper trace (ECG) shows detection of each R-peak and the lower trace (BP) shows detection of each diastolic pressure (DP) and systolic pressure (SP) peak. Areas under successfully detected peaks are marked in red. <a href="https://www.jove.com/files/ftp_upload/62101/SupplementalFile14.pdf" target="_blank">Please click here to download&#160;this File.</a>
Supplemental File 15: Screenshot of a low-quality BP recording where BP parameters are only partially detected. The upper trace (ECG) shows detection of each R-peak but the lower trace (BP) shows gaps between detected BP peaks. Detected peaks of diastolic pressure (DP) and systolic pressure (SP) are marked with red areas. <a href="https://www.jove.com/files/ftp_upload/62101/SupplementalFile15.pdf" target="_blank">Please click here to download&#160;this File.</a>
Supplemental File 16: Screenshot of a low-quality ECG and BP recording where ECG and BP parameters could not be detected. The upper trace (ECG) shows a region (purple background) where ECG parameters could not be detected. BP detection (lower trace) also failed due to low signal quality. <a href="https://www.jove.com/files/ftp_upload/62101/SupplementalFile16.pdf" target="_blank">Please click here to download&#160;this File.</a>

Watch this Scientific Journal Video about Implantation of Combined Telemetric ECG and Blood Pressure Transmitters to Determine Spontaneous Baroreflex Sensitivity in Conscious Mice at JoVE.com

Implantation of Combined Telemetric ECG and Blood Pressure Transmitters to Determine Spontaneous Baroreflex Sensitivity in Conscious Mice

The baroreflex is a heart-rate regulation mechanism by the autonomic nervous system in response to blood-pressure changes. We describe a surgical technique to implant telemetry transmitters for continuous and simultaneous measurement of electrocardiogram and blood pressure in mice. This can determine spontaneous baroreflex sensitivity, an important prognostic marker for cardiovascular disease.

Blood pressure (BP) and heart rate (HR) are both controlled by the autonomic nervous system (ANS) and are closely intertwined due to reflex mechanisms. ...

implantation-combined-telemetric-ecg-blood-pressure-transmitters-to

Research

JoVE Journal

Medicine

4.7K Views.  Ludwig-Maximilians-Universität München. The baroreflex is a heart-rate regulation mechanism by the autonomic nervous system in response to blood-pressure changes. We describe a surgical technique to implant telemetry transmitters for continuous and simultaneous measurement of electrocardiogram and blood pressure in mice. This can determine spontaneous baroreflex sensitivity, an important prognostic marker for cardiovascular disease.

Video: Implantation of Combined Telemetric ECG and Blood Pressure Transmitters to Determine Spontaneous Baroreflex Sensitivity in Conscious Mice

Hyperinsulinemic-euglycemic Clamps in Conscious, Unrestrained Mice

Type 2 diabetes is characterized by a defect in insulin action. The hyperinsulinemic-euglycemic clamp, or insulin clamp, is widely considered the "gold standard" method for assessing insulin action in vivo. During an insulin clamp, hyperinsulinemia is achieved by a constant insulin infusion. Euglycemia is maintained via a concomitant glucose infusion at a variable rate. This variable glucose infusion rate (GIR) is determined by measuring blood glucose at brief intervals throughout the experiment and adjusting the GIR accordingly. The GIR is indicative of whole-body insulin action, as mice with enhanced insulin action require a greater GIR. The insulin clamp can incorporate administration of isotopic 2[14C]deoxyglucose to assess tissue-specific glucose uptake and [3-3H]glucose to assess the ability of insulin to suppress the rate of endogenous glucose appearance (endoRa), a marker of hepatic glucose production, and to stimulate the rate of whole-body glucose disappearance (Rd).
The miniaturization of the insulin clamp for use in genetic mouse models of metabolic disease has led to significant advances in diabetes research. Methods for performing insulin clamps vary between laboratories. It is important to note that the manner in which an insulin clamp is performed can significantly affect the results obtained. We have published a comprehensive assessment of different approaches to performing insulin clamps in conscious mice1 as well as an evaluation of the metabolic response of four commonly used inbred mouse strains using various clamp techniques2. Here we present a protocol for performing insulin clamps on conscious, unrestrained mice developed by the Vanderbilt Mouse Metabolic Phenotyping Center (MMPC; URL: www.mc.vanderbilt.edu/mmpc). This includes a description of the method for implanting catheters used during the insulin clamp. The protocol employed by the Vanderbilt MMPC utilizes a unique two-catheter system3. One catheter is inserted into the jugular vein for infusions. A second catheter is inserted into the carotid artery, which allows for blood sampling without the need to restrain or handle the mouse. This technique provides a significant advantage to the most common method for obtaining blood samples during insulin clamps which is to sample from the severed tip of the tail. Unlike this latter method, sampling from an arterial catheter is not stressful to the mouse1. We also describe methods for using isotopic tracer infusions to assess tissue-specific insulin action. We also provide guidelines for the appropriate presentation of results obtained from insulin clamps.

The hyperinsulinemic-euglycemic clamp, or insulin clamp, is the gold standard for assessing insulin action in vivo. A method for performing insulin clamps in mice is described. This includes a method for arterial catheterization that permits experiments to be performed in conscious, unrestrained mice with minimal stress.

Type 2 diabetes is characterized by a defect in insulin action. The hyperinsulinemic-euglycemic clamp, or insulin clamp, is widely considered the "gold ...

Implantation of Radiotelemetry Transmitters Yielding Data on ECG, Heart Rate, Core Body Temperature and Activity in Free-moving Laboratory Mice

The laboratory mouse is the animal species of choice for most biomedical research, in both the academic sphere and the pharmaceutical industry. Mice are a manageable size and relatively easy to house. These factors, together with the availability of a wealth of spontaneous and experimentally induced mutants, make laboratory mice ideally suited to a wide variety of research areas.
In cardiovascular, pharmacological and toxicological research, accurate measurement of parameters relating to the circulatory system of laboratory animals is often required. Determination of heart rate, heart rate variability, and duration of PQ and QT intervals are based on electrocardiogram (ECG) recordings. However, obtaining reliable ECG curves as well as physiological data such as core body temperature in mice can be difficult using conventional measurement techniques, which require connecting sensors and lead wires to a restrained, tethered, or even anaesthetized animal. Data obtained in this fashion must be interpreted with caution, as it is well known that restraining and anesthesia can have a major artifactual influence on physiological parameters1, 2.
Radiotelemetry enables data to be collected from conscious and untethered animals. Measurements can be conducted even in freely moving animals, and without requiring the investigator to be in the proximity of the animal. Thus, known sources of artifacts are avoided, and accurate and reliable measurements are assured. This methodology also reduces interanimal variability, thus reducing the number of animals used, rendering this technology the most humane method of monitoring physiological parameters in laboratory animals3, 4. Constant advancements in data acquisition technology and implant miniaturization mean that it is now possible to record physiological parameters and locomotor activity continuously and in realtime over longer periods such as hours, days or even weeks3, 5.
Here, we describe a surgical technique for implantation of a commercially available telemetry transmitter used for continuous measurements of core body temperature, locomotor activity and biopotential (i.e. onelead ECG), from which heart rate, heart rate variability, and PQ and QT intervals can be established in freeroaming, untethered mice. We also present pre-operative procedures and protocols for post-operative intensive care and pain treatment that improve recovery, well-being and survival rates in implanted mice5, 6.

A surgical technique for implantation of commercially available telemetry transmitters used for continuous measurement of biopotential (one-lead ECG), heart rate, core body temperature and locomotor activity in freely moving mice is shown. Recommendations and protocols for post-operative care and pain relief, improving recovery, well being and survival rate are also presented.

The laboratory mouse is the animal species of choice for most biomedical research, in both the academic sphere and the pharmaceutical industry. Mice are a ...

Femoral Arterial and Venous Catheterization for Blood Sampling, Drug Administration and Conscious Blood Pressure and Heart Rate Measurements

In multiple fields of study, access to the circulatory system in laboratory studies is necessary. Pharmacological studies in rats using chronically implanted catheters permit a researcher to effectively and humanely administer substances, perform repeated blood sampling and assists in conscious direct measurements of blood pressure and heart rate. Once the catheter is implanted long-term sampling is possible. Patency and catheter life depends on multiple factors including the lock solution used, flushing regimen and catheter material. This video will demonstrate the methodology of femoral artery and venous catheterization of the rat. In addition the video will demonstrate the use of the femoral venous and arterial catheters for blood sampling, drug administration and use of the arterial catheter in taking measurements of blood pressure and heart rate in a conscious freely-moving rat. A tether and harness attached to a swivel system will allow the animal to be housed and have samples taken by the researcher with minimal disruption to the animal. To maintain patency of the catheter, careful daily maintenance of the catheter is required using lock solution (100 U/ml heparinized saline), machine-ground blunt tip syringe needles and the use of syringe filters to minimize potential contamination. With careful aseptic surgical techniques, proper catheter materials and careful catheter maintenance techniques, it is possible to sustain patent catheters and healthy animals for long periods of time (several weeks).

Chronic catheterization of blood vessels in the rat is often required for administration of substances, obtain blood sample over a period of time or for direct conscious blood pressure measurements. Femoral arterial catheterization of the rat and corresponding measurements of blood pressure in the conscious animal will be demonstrated.

In multiple fields of study, access to the circulatory system in laboratory studies is necessary. Pharmacological studies in rats using chronically ...

Orthotopic Implantation and Peripheral Immune Cell Monitoring in the II-45 Syngeneic Rat Mesothelioma Model

The enormous upsurge of interest in immune-based treatments for cancer such as vaccines and immune checkpoint inhibitors, and increased understanding of the role of the tumor microenvironment in treatment response, collectively point to the need for immune-competent orthotopic models for pre-clinical testing of these new therapies. This paper demonstrates how to establish an orthotopic immune-competent rat model of pleural malignant mesothelioma. Monitoring disease progression in orthotopic models is confounded by the internal location of the tumors. To longitudinally monitor disease progression and its effect on circulating immune cells in this and other rat models of cancer, a single tube flow cytometry assay requiring only 25 &#181;l whole blood is described. This provides accurate quantification of seven immune parameters: total lymphocytes, monocytes and neutrophils, as well as the T-cell subsets CD4 and CD8, B-cells and Natural Killer cells. Different subsets of these parameters are useful in different circumstances and models, with the neutrophil to lymphocyte ratio having the greatest utility for monitoring disease progression in the mesothelioma model. Analyzing circulating immune cell levels using this single tube method may also assist in monitoring the response to immune-based treatments and understanding the underlying mechanisms leading to success or failure of treatment.

The generation of an orthotopic rat model of pleural malignant mesothelioma by implantation of II-45 mesothelioma cells into the pleural cavity of immune competent rats is presented. A flow cytometric method to analyze seven immune cell subsets in these animals from a 25 &#181;l&#160;blood sample is also described.

The enormous upsurge of interest in immune-based treatments for cancer such as vaccines and immune checkpoint inhibitors, and increased understanding of ...

Long-term Blood Pressure Measurement in Freely Moving Mice Using Telemetry

During the development of new vasoactive agents, arterial blood pressure monitoring is crucial for evaluating the efficacy of the new proposed drugs. Indeed, research focusing on the discovery of new potential therapeutic targets using genetically altered mice requires a reliable, long-term assessment of the systemic arterial pressure variation. Currently, the gold standard for obtaining long-term measurements of blood pressure in ambulatory mice uses implantable radio-transmitters, which require&#160;artery cannulation. This technique eliminates the need for tethering, restraining, or anesthetizing the animals which introduce stress and artifacts during data sampling. However, arterial blood pressure monitoring in mice via catheterization can be rather challenging due to the small size of the arteries. Here we present a step-by-step guide to illustrate the crucial key passages for a successful subcutaneous implantation of radio-transmitters and carotid artery cannulation in mice. We also include examples of long-term blood pressure activity taken from freely moving mice after a period of post-surgery recovery. Following this procedure will allow reliable direct blood pressure recordings from multiple animals simultaneously.

The goal of this protocol is to assess systemic blood pressure in conscious freely moving mice using implantable radio-telemetry devices.

During the development of new vasoactive agents, arterial blood pressure monitoring is crucial for evaluating the efficacy of the new proposed drugs. ...

Novel Approach for Simultaneous Recording of Renal Sympathetic Nerve Activity and Blood Pressure with Intravenous Infusion in Conscious, Unrestrained Mice.

Renal sympathetic nerves contribute significantly to both physiological and pathophysiological phenomena. Evaluating renal sympathetic nerve activity (RSNA) is of great interest in many areas of research such as chronic kidney disease, hypertension, heart failure, diabetes and obesity. Unequivocal assessment of the role of the sympathetic nervous system is thus imperative for proper interpretation of experimental results and understanding of disease processes. RSNA has been traditionally measured in anesthetized rodents, including mice. However, mice usually exhibit very low systemic blood pressure and hemodynamic instability for several hours during anesthesia and surgery. Meaningful interpretation of RSNA is confounded by this non-physiological state, given the intimate relationship between sympathetic nervous tone and cardiovascular status. To address this limitation of traditional approaches, we developed a new method for measuring RSNA in conscious, freely-moving mice. Mice were chronically instrumented with radio-telemeters for continuous monitoring of blood pressure as well as a jugular venous infusion catheter and custom-designed bipolar electrode for direct recording of RSNA. Following a 48-72 hour recovery period, survival rate was 100% and all mice behaved normally. At this time-point, RSNA was successfully recorded in 80% of mice, with viable signals acquired up to 4 and 5 days post-surgery in 70% and 50% of mice, respectively. Physiological blood pressures were recorded in all mice (116&plusmn;2 mmHg; n=10). Recorded RSNA increased with eating and grooming, as well-established in the literature. Furthermore, RSNA was validated by ganglionic blockade and modulation of blood pressure with pharmacological agents. Herein, an effective and manageable method for clear recording of RSNA in conscious, freely-moving mice is described.

Anesthetized mice exhibit non-physiological systemic blood pressure, which precludes meaningful assessment of autonomic tone given the intimate relationship between blood pressure and the autonomic nervous system. Thus, a novel method to simultaneously record renal sympathetic nerve activity and blood pressure with intravenous infusion in conscious mice is outlined.

Renal sympathetic nerves contribute significantly to both physiological and pathophysiological phenomena. Evaluating renal sympathetic nerve activity ...

Assessment of Respiratory Function in Conscious Mice by Double-chamber Plethysmography

Air volume changes created by a conscious subject breathing spontaneously within a body box are at the basis of plethysmography, a technique used to non-invasively assess some features of the respiratory function in humans as well as in laboratory animals. The present article focuses on the application of the double-chamber plethysmography (DCP) in small animals. It provides background information on the methodology as well as a detailed step-by-step procedure to successfully assess respiratory function in conscious, spontaneously breathing animals in a non-invasive manner. The DCP can be used to monitor the respiratory function of multiple animals in parallel, as well as to identify changes induced by aerosolized substances over a chosen time period and in a repeated manner. Experiments on control and allergic mice are used herein to demonstrate the utility of the technique, explain the associated outcome parameters, as well as to discuss the related advantages and shortcomings. Overall, the DCP provides valid and theoretically sound readouts that can be trusted to evaluate the respiratory function of conscious small animals both at baseline and after challenges with aerosolized substances.

The objective of the present article is to provide a detailed description of the recommended procedures to evaluate respiratory function in conscious mice by double-chamber plethysmography.

Air volume changes created by a conscious subject breathing spontaneously within a body box are at the basis of plethysmography, a technique used to ...

Ultrasound Imaging of the Thoracic and Abdominal Aorta in Mice to Determine Aneurysm Dimensions

Contemporary high-resolution ultrasound instruments have sufficient resolution to facilitate the measurement of mouse aortas. These instruments have been widely used to measure aortic dimensions in mouse models of aortic aneurysms. Aortic aneurysms are defined as permanent dilations of the aorta, which occur most frequently in the ascending and abdominal regions. Sequential measurements of aortic dimensions by ultrasound are the principal approach for assessing the development and progression of aortic aneurysms in vivo. Although many reported studies used ultrasound imaging to measure aortic diameters as a primary endpoint, there are confounding factors, such as probe position and cardiac cycle, that may impact the accuracy of data acquisition, analysis, and interpretation. The purpose of this protocol is to provide a practical guide on the use of ultrasound to measure the aortic diameter in a reliable and reproducible manner. This protocol introduces the preparation of mice and instruments, the acquisition of appropriate ultrasound images, and data analysis.

Ultrasound imaging has become a common modality to determine the luminal dimensions of thoracic and abdominal aortic aneurysms in mice. This protocol describes the procedure to acquire reliable and reproducible two-dimensional ultrasound images of the ascending and abdominal aorta in mice.

Contemporary high-resolution ultrasound instruments have sufficient resolution to facilitate the measurement of mouse aortas. These instruments have been ...

Implantation of an Isoproterenol Mini-Pump to Induce Heart Failure in Mice

Isoproterenol (ISO), is a non-selective beta-adrenergic agonist, that is used widely to induce cardiac injury in mice. While the acute model mimics stress-induced cardiomyopathy, the chronic model, administered through an osmotic pump, mimics advanced heart failure in humans. The purpose of the described protocol is to create the chronic ISO-induced heart failure model in mice using an implanted mini-pump. This protocol has been used to induce heart failure in 100+ strains of inbred mice. Techniques on surgical pump implantation are described in detail and may be relevant to anyone interested in creating a heart failure model in mice. In addition, the weekly cardiac remodeling changes based on echocardiographic parameters for each strain and expected time to model development are presented. In summary, the method is simple and reproducible. Continuous ISO administered via the implanted mini-pump over 3 to 4 weeks is sufficient to induce cardiac remodeling. Finally, the success for ISO model creation may be assessed in vivo by serial echocardiography demonstrating hypertrophy, ventricular dilation, and dysfunction.

The chronic administration of isoproterenol via an implanted osmotic pump has been used widely to mimic advanced heart failure in mice. Here, we describe detailed methods in surgical mini-pump implantation for the continuous isoproterenol administration over 3 weeks, as well as, echocardiographic assessment for the successful model creation.

Isoproterenol (ISO), is a non-selective beta-adrenergic agonist, that is used widely to induce cardiac injury in mice. While the acute model mimics ...

Repetitive Blood Sampling from the Subclavian Vein of Conscious Rat

Rats are widely used in pharmacokinetics (PK) and toxicokinetic (TK) studies that need to collect a certain amount of blood at specific time points to detect drug exposure. The rat blood sampling method determines the quality of the plasma and further affects the precision of the test results. The subclavian vein blood collection method described in this protocol collects blood samples repeatedly in the conscious state of animals to meet the needs of PK and TK tests. The skills of restraint handling and appropriate procedure of needle incision ensure the success rate of blood collection. It is easy to operate while ensuring the quality of plasma and at the same time catering to animal welfare. However, this method requires skilled operation, and an improper one may cause animal weakness, pain, lameness, and even mortality. The current method has been used in the testing facility for a 4-week oral toxicity study in Sprague Dawley (SD) rats with TK. The maximum amount of blood collected within 24 h did not exceed 20% of the animal's total blood. The animals' body weight was more than 200 g for males and females. The data showed that the animals' bodyweight increased steadily every week, and the clinical observation was normal after repetitive sample collection.

The present protocol describes a simple and efficient method for collecting blood from the subclavian vein in rats. It enables rapid, timely, and easily identifiable sampling without anesthesia and obtains high-quality blood through repetitive sample collection.

Rats are widely used in pharmacokinetics (PK) and toxicokinetic (TK) studies that need to collect a certain amount of blood at specific time points to ...