This study was supported by Taiwan Ministry of Science and Technology grants MOST 104-2314-B-037-080-MY3 and MOST 107-2314-B-037-110 to HCL and Taiwan National Health Research Institutes grant NHRI-EX107-10724SC.

All the animal experiments described were approved by the Institutional Animal Care and Use Committee of Kaohsiung Medical University.
1. Animal Care
<ol>
	<li>To avoid difficult cannulation due to small arterial size, use rats with body weight over 200 g.</li>
	<li>Remove chow from the cage and fast rats overnight.</li>
	<li>Offer water ad libidum unless there is a special experimental design.</li>
</ol>
2. Experimental Preparation
<ol>
	<li>Obtain the following materials: forceps with teeth (Figure 1A), surgical scissors (Figure 1B), forceps with fine tips (Figure 1C, 1D), forceps with angled tips (Figure 1E), bulldog vascular clamp (Figure 1F), silk string approximately 20 cm in length (Figure 1G), micro scissors (Figure 1H), intra-arterial cannula, a sterile polyethylene (PE) tube with an internal diameter of 0.5 mm and an outer diameter of 0.9 mm, 25&#8211;30 cm in length, connected to a 26 G x 1/2&#8217;&#8217; needle (Figure 1I), two three-way stopcocks (Figure 1J) for connecting the intra-arterial cannula and the pressure transducer (Figure 1K), and 1 mL syringes filled with heparinized saline (100 IU/mL).</li>
	<li>Animal Preparation
	<ol>
		<li>Anesthetize the rat by inhalation of isoflurane (placing the rats in a 25 cm x 25 cm x 14&#8201;cm induction chamber saturated with 4% isoflurane, followed by nosecone use with 3% isoflurane). 
		NOTE: If the animal is not sufficiently anesthetized, turn up the flow rate of isoflurane.</li>
		<li>Test the pain reflex by pinching the toes.</li>
		<li>Place the rat in a supine position on a pre-cut Styrofoam board (or thick cardboard). Fix the four legs with rubber bands to immobilize the body (Figure 2A). 
		NOTE: To avoid potential noise during the ECG recording, the placement surface must not be electrically conductive.</li>
	</ol>
	</li>
	<li>Prepare instruments for BP and ECG recording. Include an analog input unit to acquire signals, a pressure transducer with a compatible hub, three bipolar needle-tipped electrocardiogram leads, and a computer with suitable software.</li>
</ol>
3. Pressure Transducer Calibration
<ol>
	<li>Before initiation of BP recording, calibrate with a standard mercury sphygmomanometer (Figure 1L).</li>
	<li>Remove the pressure cuff from the sphygmomanometer and connect the three-way stopcock on the inflation tube to the pressure transducer (Figure 1K) of the data acquisition system.</li>
	<li>Screw the air-release valve clockwise. Keep eyes on the gauge and keep pumping the inflation bulb. When the gauge shows 100 mmHg, switch the three-way stopcock to connect to the pressure transducer. Use 100 mmHg pressure for the calibration. The conversion factor for calculating BP will be determined automatically.</li>
	<li>Release the pressure by screwing the air-release valve counterclockwise until the pressure of the sphygmomanometer is back to zero.</li>
	<li>Repeat step 3.2 with pressure of 200 mmHg.</li>
	<li>Detach the pressure transducer from the mercury sphygmomanometer.</li>
	<li>Connect the pressure transducer to the three-way stopcock of the PE catheter (Figure 1I).</li>
</ol>
4. Mini-surgery for Cannulation of the Femoral Artery
<ol>
	<li>Surface landmark identification and excision of the skin (Figure 2)
	<ol>
		<li>Identify the location of inguinal crease (indentation at the junction between abdomen and thigh) (dashed line in Figure 2A).</li>
		<li>Pinch the full layer of skin at the center of the inguinal crease.&#160;The fur may be shaved or removed from the incision site using electric clippers or a depilatory cream prior to incision.</li>
		<li>Lift the skin and cut it off with the surgical scissors at an orientation approximately parallel to the ipsilateral thigh (Figure 2B). The femoral nerve and vessels are underneath the exposed subcutaneous tissue (Figure 2C).</li>
	</ol>
	</li>
	<li>Dissection of the tissue to expose the femoral artery
	<ol>
		<li>Dissect the tissue using forceps with fine tips, layer by layer. Stop the dissection at the level of femoral vessels. Make sure the dissection is not injuring the vessels underneath.</li>
		<li>Carefully use forceps (Figure 1C or 1D) to clear out the soft tissue along the femoral nerve and vessels to obtain good observation. The nerve is fiber-like in texture. The vein is dark purple and the artery is pulsatile (Figure 3A).</li>
		<li>Use forceps with angled tips (Figure 1E) to extend the exposed length of the femoral artery and vein (Figure 3B).</li>
	</ol>
	</li>
	<li>Cannulation of the femoral artery (Figure 3)
	<ol>
		<li>Use forceps to separate the femoral vein from the artery and apply a bulldog clamp at the femoral artery as cranially as possible (Figure 3C).</li>
		<li>Make a loose tie of the two silk strings, one just below the bulldog clamp and the other at the caudal terminal of the exposed femoral artery (Figure 3D).</li>
		<li>Make a small hole over the ventral side of the femoral artery (Figure 3E) using the micro scissors (Figure 1H).</li>
		<li>Insert the tip of the PE catheter through the small hole and advance the catheter cranially. 
		NOTE: Do not apply torsion while advancing the PE catheter. The torsion force may twist the femoral artery and result in luminal stenosis.</li>
		<li>Remove the bulldog clamp after the PE catheter is securely advanced into the femoral artery lumen. 
		NOTE:&#160;Keep eyes on the PE catheter while removing the bulldog clamp from the femoral artery. The observation of a backflush of blood into the PE catheter associated with observation for pulse confirms its placement in the arterial lumen.&#160;&#160;</li>
		<li>Tighten the upper silk string to secure the position of the PE catheter. 
		NOTE: Any accidental pull or dislocation of the PE catheter can result in major bleeding.</li>
		<li>Tighten the lower silk string to prevent bleeding from the caudal side of the femoral artery.</li>
	</ol>
	</li>
	<li>Confirmation of the success of femoral artery cannulation
	<ol>
		<li>Use a 1 mL syringe to inject 0.1&#8211;0.2 mL of heparinized saline into the femoral artery. The resistance on injection must be trivial. If any obvious resistance upon injection is noticed, check the whole cannulation again. 
		NOTE: Make sure the three-way stopcocks are appropriately switched before applying any negative pressure or saline injection into the femoral artery cannula. At this time point, any injection into the pressure transducer will need a re-calibration.</li>
		<li>Check if there is any oozing around the cannulation site. If not, cover the surgical site with a wet cotton ball.</li>
	</ol>
	</li>
</ol>
5. Recording of Blood Pressure
<ol>
	<li>After a smooth flush for the PE catheter, attach the PE catheter three-way stopcock to the one on the pressure transducer (Figure 4).</li>
	<li>Make sure that there are no air bubbles in the cannulation system. Also check the connection junctions of the three-way stopcock.</li>
	<li>Start the data acquisition system with sampling frequency of 1,000 Hz to record the BP. Arterial pressure waves will be demonstrated (Figure 6).</li>
	<li>Allow the whole setup to stabilize for at least 3-5 min. In cases with unsteady signals, stabilization time can be extended to 15 min.</li>
	<li>Check the cannulation site periodically to make sure there is no bleeding. 
	NOTE: When acquiring data from a pressure transducer, it is important to place the transducer at the level of the animal&#8217;s heart.</li>
</ol>
6. Surface ECG
<ol>
	<li>Check the three leads of the bipolar ECG to make sure the positive, negative, and reference platinum electrodes are intact.</li>
	<li>Insert the leads subcutaneously at the left foreleg, right foreleg, and right hindleg (Figure 5).</li>
	<li>Attach the electrode hubs to a custom-built ECG amplifier with sampling frequency of 1,000 Hz and filter frequency of 3&#8211;500 Hz. Keep ECG leads steady during recording. Motion of the ECG leads can produce unsteady baselines and artifacts.</li>
</ol>
7. Animal Euthanasia After Completing of the Experiment
<ol>
	<li>After completing of the BP and ECG recording, stop the acquisition system. Remove the electrodes. Ligate the femoral artery by tightening up the previously placed silk string right after withdrawing the PE catheter.</li>
	<li>Place the rats individually in the visible euthanasia chamber connected to compressed carbon dioxide (CO2) gas cylinders. Seal the top securely.</li>
	<li>Introduce 100% CO2 with a fill rate of about 10% to 30% of the chamber volume per minute.</li>
	<li>Keep your eyes on the rat; lack of respiration and faded eye color should appear within 2&#8722;3 min. 
	NOTE: If cessation of respiratory movement&#160;does not occur after 3 min, the system should be examined for the chamber fill rate, CO2 supply, or leaks.</li>
	<li>Maintain CO2 flow to the chamber for 1 more minute after respiration ceases.</li>
	<li>Ascertain cardiac and respiratory arrest and note fixed and dilated pupils to confirm death. 
	NOTE: If the rat is not dead but in CO2 narcosis, use a secondary method of euthanasia such as bilateral thoracotomy.</li>
</ol>

<ol>
	<li>Shiou, Y. -. L., Huang, I. C., Lin, H. -. T., Lee, H. -. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+fat+diet+aggravates+atrial+and+ventricular+remodeling+of+hypertensive+heart+disease+in+aging+rats.">High fat diet aggravates atrial and ventricular remodeling of hypertensive heart disease in aging rats.</a> Journal of the Formosan Medical Association. , (2017).</li><li>Parasuraman, S., Raveendran, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+invasive+blood+pressure+in+rats.">Measurement of invasive blood pressure in rats.</a> Journal of Pharmacology, Pharmacotherapeutics. 3 (2), 172-177 (2012).</li><li>Fink, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Does+Tail-Cuff+Plethysmography+Provide+a+Reliable+Estimate+of+Central+Blood+Pressure+in+Mice?.">Does Tail-Cuff Plethysmography Provide a Reliable Estimate of Central Blood Pressure in Mice?.</a> Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease. 6 (6), e006554 (2017).</li><li>Wilde, E., 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=Tail-Cuff+Technique+and+Its+Influence+on+Central+Blood+Pressure+in+the+Mouse.">Tail-Cuff Technique and Its Influence on Central Blood Pressure in the Mouse.</a> Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease. 6 (6), (2017).</li><li>Braga, V. A., Prabhakar, N. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Refinement+of+Telemetry+for+Measuring+Blood+Pressure+in+Conscious+Rats.">Refinement of Telemetry for Measuring Blood Pressure in Conscious Rats.</a> Journal of the American Association for Laboratory Animal Science : JAALAS. 48 (3), 268-271 (2009).</li><li>Kaese, S., Verheule, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+electrophysiology+in+mice:+a+matter+of+size.">Cardiac electrophysiology in mice: a matter of size.</a> Frontiers in Physiology. 3 (345), (2012).</li><li>Okamoto, K., Aoki, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+strain+of+spontaneously+hypertensive+rats.">Development of a strain of spontaneously hypertensive rats.</a> Japanese Circulation Journal. 27, 282-293 (1963).</li><li>Van Vliet, B. N., Chafe, L. L., Antic, V., Schnyder-Candrian, S., Montani, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+and+indirect+methods+used+to+study+arterial+blood+pressure.">Direct and indirect methods used to study arterial blood pressure.</a> Journal of Pharmacological and Toxicological Methods. 44 (2), 361-373 (2000).</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>Irvine, R. J., White, J., Chan, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+influence+of+restraint+on+blood+pressure+in+the+rat.">The influence of restraint on blood pressure in the rat.</a> Journal of Pharmacological and Toxicological Methods. 38 (3), 157-162 (1997).</li><li>Nvd Vosse, F., Stergiopulos, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pulse+Wave+Propagation+in+the+Arterial+Tree.">Pulse Wave Propagation in the Arterial Tree.</a> Annual Review of Fluid Mechanics. 43 (1), 467-499 (2011).</li><li>Cosson, E., 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=Aortic+stiffness+and+pulse+pressure+amplification+in+Wistar-Kyoto+and+spontaneously+hypertensive+rats.">Aortic stiffness and pulse pressure amplification in Wistar-Kyoto and spontaneously hypertensive rats.</a> The American Journal of Physiology: Heart and Circulatory Physiology. 292 (5), H2506-H2512 (2007).</li></ol>

The authors have nothing to disclose.

Invasive arterial cannulation allows highly accurate measurement of BP. It can be done with a PE tube without requiring an expensive catheter. Invasive BP measurement can also be performed simultaneously with a recording of the surface ECG.
The major learning curve for this method is the experimental skill required to cannulate small blood vessels. In experienced hands, the successful rate for femoral artery cannulation can approach 100%. Practice is recommended before performing real&#160;exp...

Blood pressure (BP) and electrocardiography (ECG) are basic parameters for cardiovascular physiology and medicine. Experimental animal models have been widely applied in biomedical research for various cardiovascular diseases such as hypertensive heart failure1 and procedures for ECG recording and BP measurement can be performed in experimental rats.
There are three methods for BP measurement in rats: intra-arterial cannulation (invasive)2, tail cuff plethysmography (noninvasive)3, and radio telemetry (invasive). The reliability of BP measurement by tail cuff plethysmography can be affected by animal handling during the recording. For example, the tail cuff underestimates the core BP changes that occur simultaneously during the restraint and measurement phases4. Radio telemetry is considered the best &#34;gold standard&#34; technique for monitoring BP and heart rate in awake and freely moving animals5. However, since radio telemetry hardware and software are costly, intra-arterial cannulation is also widely used as an economical alternative.
Intra-arterial cannulation requires considerable microsurgical skill but yields the real waveforms of arterial pressure. BP can be recorded through a saline-filled catheter inserted in the radial, femoral, or brachial artery. This method of direct invasive BP measurement requires pre-surgical animal preparation, anesthesia, immobilization of laboratory animals, surgical skill in tissue dissection and arterial cannulation, and proper calibration before acquiring the measurement.
Rodent surface ECG is similar to human ECG. A rat ECG has sequences of P waves, QRS complexes, T waves, and QT intervals6. The P wave, PR interval, QRS complex, and T waves reflect atrial depolarization, impulse conduction from the atrial to the AV node, ventricular depolarization, and repolarization, respectively. The QT interval is defined as the period from the initiation of the Q wave to the end-point of the T wave where it returns to the iso-electrical baseline1.
The ECG indicates the cardiac systole and diastole phases; therefore, the simultaneous recording of the surface ECG correlates with the invasive BP measurement. By using a combination of methodologies, it is possible to elucidate pathophysiological changes in a disease model or the pharmacological effects of a drug or therapy in cardiovascular medicine.
A spontaneous hypertensive rat (SHR) strain had been obtained by inbreeding of Wistar rats with high BP in Japan. The BP rises from 5 to 10 weeks of age and becomes stationary from 30 to 35 weeks of age7. Wistar-Kyoto rats (WKY) have systolic BP about 130 mmHg7 and are commonly used as normo-tensive control. We used SHR and WKY to demonstrate the result of intraarterial cannulation BP and ECG recording.

<table border="0" cellpadding="0" cellspacing="0" style="width:1101px;" width="1102">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Polyethylene tube</td>
			<td style="width:212px;">BECTON DICKINSON</td>
			<td style="width:131px;">427401</td>
			<td style="width:544px;">internal diameter of 0.5 mm, outer diameter of 0.9 mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">26G x 1/2&#34;&#34; needle</td>
			<td style="width:212px;">TERUMO</td>
			<td style="width:131px;">160426D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Adson Forceps</td>
			<td style="width:212px;">TOP Line</td>
			<td>12-540</td>
			<td style="width:544px;">12 cm (4.75&#34;) Long, Straight, 1 x 2 Teeth</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Bulldog vascular clamp</td>
			<td style="width:212px;">Teleflex</td>
			<td style="width:131px;">357581</td>
			<td>8 mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Computer&#160;</td>
			<td style="width:212px;">AUSUS</td>
			<td style="width:131px;">X453M</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Exernal analog signal recording device</td>
			<td style="width:212px;">iWorx</td>
			<td style="width:131px;">T5141538</td>
			<td>This allows the recording of up to three channels of ECG, EMG or EEG as well as GSR (skin conductance) from a single iWire input on the recording Module.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Graefe Forceps</td>
			<td>AESCULAP Surgical Instruments</td>
			<td>BD312R</td>
			<td>MICRO DRESSING FORCEPS, CURVED, SERRATED, 105 mm, 4 1/8&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Mecury sphygmomanometer</td>
			<td style="width:212px;">Spirit</td>
			<td style="width:131px;">CK-101</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Pressure transducer</td>
			<td style="width:212px;">iWorx</td>
			<td style="width:131px;">IworxBP100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Semken Forceps</td>
			<td>MEDE TECHNIK</td>
			<td>10-104</td>
			<td>100 mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Software</td>
			<td style="width:212px;">LabScribe3</td>
			<td style="width:131px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Surgical scissors</td>
			<td>HEBU</td>
			<td style="width:131px;">1714</td>
			<td>14.5 cm long</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Syringe (1 mL)</td>
			<td style="width:212px;">TERUMO</td>
			<td style="width:131px;">160426D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Three-way stopcocks</td>
			<td style="width:212px;">Cole-Parmer</td>
			<td>EW-30600-23</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Tipped forceps</td>
			<td style="width:212px;">World Precision Instruments</td>
			<td style="width:131px;">504506</td>
			<td>11 cm long, 0.1x0.06 mm Tips</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Vannas Scissors</td>
			<td style="width:212px;">World Precision Instruments</td>
			<td>500086</td>
			<td>8.5 cm long, Straight, 0.025 x 0.015 mm Tips, 7mm super fine Blades</td>
		</tr>
	</tbody>
</table>

simultaneous electrocardiography recording and invasive blood pressure measurement in rats

For studies related to cardiovascular physiology or pathophysiology, blood pressure (BP) and electrocardiography are basic observational parameters. Research focusing on cardiovascular disease models, potential cardiovascular therapeutic targets or pharmaceutical agents requires assessment of systemic arterial pressure and heart rhythm changes. In situations where radio telemetry systems are not available or affordable, the technique of femoral artery cannulation is an alternative way to obtain intra-arterial pressure waveform recordings and systemic BP measurements. This technique is economical and can be performed with standard equipment in animal facilities. However, invasive arterial pressure recording requires cannulation of small arteries, which can be a challenging surgical skill. Here, we present step-by-step protocols for femoral artery cannulation procedures. Key procedures include the calibration of the data acquisition system, tissue dissection and femoral artery cannulation, and setup of the arterial cannulation system for pressure recording. Surface electrocardiography recording procedures are also included. We also present examples of BP recordings from normotensive and hypertensive rats. This protocol allows reliable direct recordings of systemic BP with simultaneous electrocardiography.

We purchased SHR and normotensive Wistar-Kyoto WKY rats from the National Laboratory Animal Center (Taipei, Taiwan). All animals were housed in a temperature-controlled facility (20&#8722;22 &#176;C) with free access to water and standard chow on a 12 h light/dark cycle.
We used six 47-week-old rats and they were weighed before the BP and ECG measurement. The representative tracings from simultaneous recording of ECG and BP in S...

Watch this Scientific Journal Video about Simultaneous Electrocardiography Recording and Invasive Blood Pressure Measurement in Rats at JoVE.com

Simultaneous Electrocardiography Recording and Invasive Blood Pressure Measurement in Rats

Here, we describe a setup for simultaneous recording of electrocardiography and intra-arterial blood pressure (BP) in experimental rats, which can be done with standard equipment in animal facilities and can be applied to physiological or pharmacological studies to investigate pathogenic or therapeutic mechanisms in cardiovascular medicine.

For studies related to cardiovascular physiology or pathophysiology, blood pressure (BP) and electrocardiography are basic observational parameters. ...

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14.8K Views.  Kaohsiung Medical University Hospital. Here, we describe a setup for simultaneous recording of electrocardiography and intra-arterial blood pressure (BP) in experimental rats, which can be done with standard equipment in animal facilities and can be applied to physiological or pharmacological studies to investigate pathogenic or therapeutic mechanisms in cardiovascular medicine.

Video: Simultaneous Electrocardiography Recording and Invasive Blood Pressure Measurement in Rats

Epidural Intracranial Pressure Measurement in Rats Using a Fiber-optic Pressure Transducer

Elevated intracranial pressure (ICP) is a significant problem in several forms of ischemic brain injury including stroke, traumatic brain injury and cardiac arrest. This elevation may result in further neurological injury, in the form of transtentorial herniation1,2,3,4, midbrain compression, neurological deficit or increased cerebral infarct2,4. Current therapies are often inadequate to control elevated ICP in the clinical setting5,6,7 . Thus there is a need for accurate methods of ICP measurement in animal models to further our understanding of the basic mechanisms and to develop new treatments for elevated ICP.
In both the clinical and experimental setting ICP cannot be estimated without direct measurement. Several methods of ICP catheter insertion currently exist. Of these the intraventricular catheter has become the clinical 'gold standard' of ICP measurement in humans8. This method involves the partial removal of skull and the instrumentation of the catheter through brain tissue. Consequently, intraventricular catheters have an infection rate of 6-11%9. For this reason, subdural and epidural cannulations have become the preferred methods in animal models of ischemic injury. 
Various ICP measurement techniques have been adapted for animal models, and of these, fluid-filled telemetry catheters10 and solid state catheters are the most frequently used11,12,13,14,15. The fluid-filled systems are prone to developing air bubbles in the line, resulting in false ICP readings. Solid state probes avoid this problem (Figure 1). An additional problem is fitting catheters under the skull or into the ventricles without causing any brain injury that might alter the experimental outcomes. Therefore, we have developed a method that places an ICP catheter contiguous with the epidural space, but avoids the need to insert it between skull and brain. 
An optic fibre pressure catheter (420LP, SAMBA Sensors, Sweden) was used to measure ICP at the epidural location because the location of the pressure sensor (at the very tip of the catheter) was found to produce a high fidelity ICP signal in this model. There are other manufacturers of similar optic fibre technologies13 that may be used with our methodology. Alternative solid state catheters, which have the pressure sensor located at the side of the catheter tip, would not be appropriate for this model as the signal would be dampened by the presence of the monitoring screw. 
Here, we present a relatively simple and accurate method to measure ICP. This method can be used across a wide range of ICP related animal models.

A novel technique to record the pressures within the skull is described. The minimally invasive method uses a fibre-optic pressure sensing system to accurately measure intracranial pressure (ICP) in anaesthetized rats without causing significant brain trauma. The technique may be used in a wide range of experimental models.

Elevated intracranial pressure (ICP) is a significant problem in several forms of ischemic brain injury including stroke, traumatic brain injury and ...

Non-invasive Optical Measurement of Cerebral Metabolism and Hemodynamics in Infants

Perinatal brain injury remains a significant cause of infant mortality and morbidity, but there is not yet an effective bedside tool that can accurately screen for brain injury, monitor injury evolution, or assess response to therapy. The energy used by neurons is derived largely from tissue oxidative metabolism, and neural hyperactivity and cell death are reflected by corresponding changes in cerebral oxygen metabolism (CMRO2). Thus, measures of CMRO2 are reflective of neuronal viability and provide critical diagnostic information, making CMRO2 an ideal target for bedside measurement of brain health.
Brain-imaging techniques such as positron emission tomography (PET) and single-photon emission computed tomography (SPECT) yield measures of cerebral glucose and oxygen metabolism, but these techniques require the administration of radionucleotides, so they are used in only the most acute cases.
Continuous-wave near-infrared spectroscopy (CWNIRS) provides non-invasive and non-ionizing radiation measures of hemoglobin oxygen saturation (SO2) as a surrogate for cerebral oxygen consumption. However, SO2 is less than ideal as a surrogate for cerebral oxygen metabolism as it is influenced by both oxygen delivery and consumption. Furthermore, measurements of SO2 are not sensitive enough to detect brain injury hours after the insult 1,2, because oxygen consumption and delivery reach equilibrium after acute transients 3. We investigated the possibility of using more sophisticated NIRS optical methods to quantify cerebral oxygen metabolism at the bedside in healthy and brain-injured newborns. More specifically, we combined the frequency-domain NIRS (FDNIRS) measure of SO2 with the diffuse correlation spectroscopy (DCS) measure of blood flow index (CBFi) to yield an index of CMRO2 (CMRO2i) 4,5.
With the combined FDNIRS/DCS system we are able to quantify cerebral metabolism and hemodynamics. This represents an improvement over CWNIRS for detecting brain health, brain development, and response to therapy in neonates. Moreover, this method adheres to all neonatal intensive care unit (NICU) policies on infection control and institutional policies on laser safety. Future work will seek to integrate the two instruments to reduce acquisition time at the bedside and to implement real-time feedback on data quality to reduce the rate of data rejection.

We combined frequency-domain near-infrared spectroscopy measures of cerebral hemoglobin oxygenation with diffuse correlation spectroscopy measures of cerebral blood flow index to estimate an index of oxygen metabolism. We tested the utility of this measure as a bedside screening tool to evaluate the health and development of the newborn brain.

Perinatal brain injury remains a significant cause of infant mortality  and morbidity, but there is not yet an effective bedside tool that can  accurately ...

Automated Measurement of Microcirculatory Blood Flow Velocity in Pulmonary Metastases of Rats

Because the lung is a major target organ of metastatic disease, animal models to study the physiology of pulmonary metastases are of great importance. However, very few methods exist to date to investigate lung metastases in a dynamic fashion at the microcirculatory level, due to the difficulty to access the lung with a microscope. Here, an intravital microscopy method is presented to functionally image and quantify the microcirculation of superficial pulmonary metastases in rats, using a closed-chest pulmonary window and automated analysis of blood flow velocity and direction. The utility of this method is demonstrated to measure increases in blood flow velocity in response to pharmacological intervention, and to image the well-known tortuous vasculature of solid tumors. This is the first demonstration of intravital microscopy on pulmonary metastases in a closed-chest model. Because of its minimized invasiveness, as well as due to its relative ease and practicality, this technology has the potential to experience widespread use in laboratories that specialize on pulmonary tumor research.

A method is presented to measure microcirculatory blood flow velocity in pulmonary cancer metastases of the pleural surface in rats in an automated fashion, using closed-chest pulmonary intravital microscopy. This model has potential to be used as a widespread tool to perform physiologic research on pulmonary metastases in rodents.

Because the lung is a major target organ of metastatic disease, animal models to study the physiology of pulmonary metastases are of great importance. ...

Measurement of the Pressure-volume Curve in Mouse Lungs

In recent decades the mouse has become the primary animal model of a variety of lung diseases. In models of emphysema or fibrosis, the essential phenotypic changes are best assessed by measurement of the changes in lung elasticity. To best understand specific mechanisms underlying such pathologies in mice, it is essential to make functional measurements that can reflect the developing pathology. Although there are many ways to measure elasticity, the classical method is that of the total lung pressure-volume (PV) curve done over the whole range of lung volumes. This measurement has been made on adult lungs from nearly all mammalian species dating back almost 100 years, and such PV curves also played a major role in the discovery and understanding of the function of pulmonary surfactant in fetal lung development. Unfortunately, such total PV curves have not been widely reported in the mouse, despite the fact that they can provide useful information on the macroscopic effects of structural changes in the lung. Although partial PV curves measuring just the changes in lung volume are sometimes reported, without a measure of absolute volume, the nonlinear nature of the total PV curve makes these partial ones very difficult to interpret. In the present study, we describe a standardized way to measure the total PV curve. We have then tested the ability of these curves to detect changes in mouse lung structure in two common lung pathologies, emphysema and fibrosis. Results showed significant changes in several variables consistent with expected structural changes with these pathologies. This measurement of the lung PV curve in mice thus provides a straightforward means to monitor the progression of the pathophysiologic changes over time and the potential effect of therapeutic procedures.

Here we present a protocol to simply and reliably measure the lung pressure-volume curve in mice, showing that it is sufficiently sensitive to detect phenotypic parenchymal changes in two common lung pathologies, pulmonary fibrosis and emphysema. This metric provides a means to quantify the lung&#8217;s structural changes with developing pathology.

In recent decades the mouse has become the primary animal model of a variety of lung diseases. In models of emphysema or fibrosis, the essential ...

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 ...

Cavernous Nerve Stimulation and Recording of Intracavernous Pressure in a Rat

The stimulation of the cavernous nerve (CN) and measurement of intracavernous pressure (ICP) have been used extensively to test and evaluate therapies for erectile dysfunction. However, the methods used vary between laboratories, and pitfalls still exist. The goal of this study was to describe a surgical technique that would provide a reliable and reproducible model. By exposing the ischiocavernosus muscle at its point of insertion on the ischial tuberosity, the penile crus could be cannulated with minimal dissection and injury to the structures involved in erectile function. Repeated stimulation of the CN, without the need for lifting and drying, was achieved by using a 125 &#181;m bipolar silver electrode and biocompatible silicon glue to isolate the electrode-nerve complex. This method prevents neuropraxia by reducing stretching and drying the nerve and provides complete isolation of the nerve, negating electrical leakage and preventing stimulation of alternative pathways.

This study describes a simplified surgical procedure and technique for performing cavernous nerve stimulation with the isolation of the nerve-electrode complex using silicone glue and intracavernous pressure measurement.

The stimulation of the cavernous nerve (CN) and measurement of intracavernous pressure (ICP) have been used extensively to test and evaluate therapies for ...

Invasive Hemodynamic Characterization of the Portal-hypertensive Syndrome in Cirrhotic Rats

This is a detailed protocol describing invasive hemodynamic measurements in cirrhotic rats for the characterization of portal hypertensive syndrome. Portal hypertension (PHT) due to cirrhosis is responsible for the most severe complications in patients with liver disease. The full picture of the portal hypertensive syndrome is characterized by increased portal pressure (PP) due to the increased intrahepatic vascular resistance (IHVR), hyperdynamic circulation, and increased splanchnic blood flow. Progressive splanchnic arterial vasodilation and increased cardiac output with elevated heart rate (HR) but low arterial pressure characterizes the portal hypertensive syndrome.
Novel therapies are currently being developed that aim to decrease PP by either targeting IHVR or increased splanchnic blood flow &#8212; but side effects on systemic hemodynamics may occur. Thus, a detailed characterization of portal venous, splanchnic, and systemic hemodynamic parameters, including measurement of PP, portal venous blood flow (PVBF), mesenteric arterial blood flow, mean arterial pressure (MAP), and HR is needed for preclinical evaluation of the efficacy of novel treatments for PHT. Our video article provides the reader with a structured protocol for performing invasive hemodynamic measurements in cirrhotic rats. In particular, we describe the catheterization of the femoral artery and the portal vein via an ileocolic vein and the measurement of portal venous and splanchnic blood flow via perivascular Doppler-ultrasound flow probes. Representative results of different rat models of PHT are shown.

Here we describe a detailed protocol for invasive measurements of hemodynamic parameters including portal pressure, splanchnic blood flow, and systemic hemodynamics in order to characterize the portal hypertensive syndrome in rats.

This is a detailed protocol describing invasive hemodynamic measurements in cirrhotic rats for the characterization of portal hypertensive syndrome. ...

Absorbent Microbiopsy Sampling and RNA Extraction for Minimally Invasive, Simultaneous Blood and Skin Analysis

Conventional skin biopsy limits the clinical research that involves cosmetically sensitive areas or pediatric applications due to its invasiveness. Here, we describe the protocol for using&#160;an absorbent microneedle-based device, absorbent microbiopsy, for minimally invasive sampling of skin and blood mixture. Our goal is to help facilitate rapid progress in clinical research, the establishment of biomarkers for skin disease and reducing the risk for clinical research participants. In contrast to conventional skin biopsy techniques, the absorbent microbiopsy can be performed within seconds and does not require intensive training due to its simple design. In this report, we describe the use of absorbent microbiopsy, including loading and application, on a&#160;volunteer. Then, we show how to isolate RNA from the absorbed sample. Finally, we demonstrate the use of quantitative reverse transcription PCR (RT-qPCR) to quantify mRNA expression levels of both blood (CD3E and CD19) and skin (KRT14 and TYR). The methods that we describe utilize off the shelf kits and reagents. This protocol offers a minimally invasive approach for simultaneous sampling of skin and blood within the same absorbent microbiopsy matrix. We have found human ethics committees, clinicians and volunteers to be supportive of this approach to dermatological research.

In this article, we demonstrate how the absorbent microbiopsy technique is performed and how the sample can be used for RNA extraction for simple and simultaneous sampling of skin and blood in a minimally invasive manner.

Conventional skin biopsy limits the clinical research that involves cosmetically sensitive areas or pediatric applications due to its invasiveness. Here, ...

Simultaneous Flow Cytometric Characterization of Multiple Cell Types Retrieved from Mouse Brain/Spinal Cord Through Different Homogenization Methods

Recent advances in viral vector and nanomaterial sciences have opened the way for new cutting-edge approaches to investigate or manipulate the central nervous system (CNS). However, further optimization of these technologies would benefit from methods allowing rapid and streamline determination of the extent of CNS and cell-specific targeting upon administration of viral vectors or nanoparticles in the body. Here, we present a protocol that takes advantage of the high throughput and multiplexing capabilities of flow cytometry to allow a straightforward quantification of different cell subtypes isolated from mouse brain or spinal cord, namely microglia/macrophages, lymphocytes, astrocytes, oligodendrocytes, neurons and endothelial cells. We apply this approach to highlight critical differences between two tissue homogenization methods in terms of cell yield, viability and composition. This could instruct the user to choose the best method depending on the cell type(s) of interest and the specific application. This method is not suited for analysis of anatomical distribution, since the tissue is homogenized to generate a single-cell suspension. However, it allows to work with viable cells and it can be combined with cell-sorting, opening the way for several applications that could expand the repertoire of tools in the hands of the neuroscientist, ranging from establishment of primary cultures derived from pure cell populations, to gene-expression analyses and biochemical or functional assays on well-defined cell subtypes in the context of neurodegenerative diseases, upon pharmacological treatment or gene therapy.

We present a flow cytometry method to identify simultaneously different cell types retrieved from mouse brain or spinal cord. This method could be exploited to isolate or characterize pure cell populations in neurodegenerative diseases or to quantify the extent of cell targeting upon in vivo administration of viral vectors or nanoparticles.

Recent advances in viral vector and nanomaterial sciences have opened the way for new cutting-edge approaches to investigate or manipulate the central ...