This work was supported by NHMRC Program Grant [ID 1074386], a Leducq Transatlantic Network of Excellence in Cardiovascular Research grant [RMG], and a grant from the RT Hall Trust [RMG &#38; SEI].

All animal experiments described were approved by the Garvan/St Vincent&#39;s Hospital Animal Ethics Committee in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes and the ARRIVE guidelines, and all experiments were performed by an experienced small animal surgeon (JW) with guidance from a pediatric anesthetist (JJS).
1. Preparation of instruments
<ol>
	<li>On the day of surgery, set up specialized equipment for intubation of 10-day-old pups (Figure 1A,B). This includes a warming lamp, intubation platform, fiber-optic light, small forceps, a laryngoscope fashioned out of a piece of 0.02 mm diameter copper wire (60 mm in length with the end of the wire fashioned into a 3 mm diameter circle at an angle of 175&#176;; Figure 1B), and a 24-gauge plastic intravenous (i.v.) cannula, which is used as an endotracheal tube.</li>
	<li>Ensure that the cannula consists of a 19 mm long plastic tubing (0.7 mm OD) attached to a 21 mm plastic female luer lock adaptor (Figure 1B). Stiffen the tubing of the cannula by inserting a piece of copper wire via the luer lock adaptor. Use a cannula with a total volume of 130 &#956;L for a mouse with tidal volume of ~8 &#956;L/g13,14.</li>
</ol>
2. Anesthesia of 10-day-old mice
<ol>
	<li>On the day of surgery, remove the dam from a cage of 10-day-old C57BL/6J pups and place the cage on a warming pad (37 &#176;C).</li>
	<li>Anesthetize pups by 10 &#956;L per g body weight intraperitoneal injection using a 0.5 mL insulin syringe and 29 G needle with ketamine/xylazine/atropine in the ratios detailed in Table 1 for different weight groupings.</li>
	<li>Immediately after injection, place the pup into a warmed (37 &#176;C) plexiglass chamber that has been pre-oxygenated with 100% oxygen.</li>
</ol>
3. Intubation of 10-day-old mice
<ol>
	<li>After 3-4 min of oxygenation, transfer the pup to a platform for intubation essentially as for adult mice. Perform this with the pup in the supine position (Figure 1C) or suspended at a 45&#176; angle15. Maintain temperature with a warming lamp.
	<ol>
		<li>Before intubation, assess the depth of anesthesia by the paw pinch reflex. For optimal intubation, the paw pinch reflex must still be present but markedly reduced from that of a conscious animal.</li>
	</ol>
	</li>
	<li>After securing the anesthetized pup supine to an intubation platform (Figure 1C), hold the tongue with small forceps and use a laryngoscope fashioned out of a piece of copper wire (Figure 1B) to expose the glottis and vocal cords. Aid visualization of the vocal cords by trans-illumination with a flexible fiber-optic light (Figure 1D).</li>
	<li>Using a stiffened cannula, tilt the cannula so that the luer lock end is slightly lower (~10&#176;) than the tip, and as soon as the vocal cords separate, insert the cannula and advance it until the luer lock adaptor is just outside the mouth. Remove the wire immediately after intubation. 
	NOTE: No resistance during intubation is expected in mice of this age unless the cannula is advanced too far, and resistance is felt from the carina.
	<ol>
		<li>Assess the depth of anesthesia after intubation by the ability of the animal to breathe spontaneously. Confirm successful tracheal intubation of spontaneously breathing pups by briefly blocking the intubation catheter to check that this prevents chest movement.</li>
	</ol>
	</li>
	<li>Immediately transfer the intubated pup to a warming pad (37 &#176;C) and connect the endotracheal cannula to a ventilator delivering 100% oxygen at a flow rate of 1 L/min with 30 &#956;L/stroke, 40 &#956;L/stroke, or 50 &#956;L/stroke for 3.15-4.59 g, 4.50-5.49 g, or 5.50-8.10 g pups, respectively, and 150 strokes/min.</li>
	<li>Perform these procedures rapidly, within &#60;15 s to minimize re-breathing.</li>
</ol>
4. Myocardial infarction surgery of 10-day-old mice
<ol>
	<li>To induce a surgical plane of anesthesia for surgery, switch the gas flowing into the ventilator from 100% oxygen to 4.5% isoflurane in oxygen (the isoflurane concentration being determined by a vaporizer) for 4-5min.
	<ol>
		<li>After switching to isoflurane, confirm tracheal intubation again by checking that the frequency of chest wall movement equals that of the ventilator. Loss of spontaneous breathing followed by absence of a tail or paw pinch reflex indicates that a surgical plane of anesthesia has been reached (after 4-5 min).</li>
	</ol>
	</li>
	<li>Maintain anesthesia with 2% isoflurane in oxygen.</li>
	<li>Perform myocardial infarction surgery under a surgical microscope (10x and 16x objective) as described in16.
	<ol>
		<li>Disinfect the skin in a circular motion several times with alternating rounds of an iodine-based or chlorhexidine-based scrub and 70% alcohol. Using fine scissors, make a horizontal skin incision between the third and fourth rib (fourth intercostal space) in the left lateral wall of the chest. Using fine forceps, open the thorax by blunt dissection of the intercostal space and use a retractor to keep the space open.</li>
		<li>Induce a myocardial infarction by ligation of the left coronary artery just distal to the left atrial appendage with 9-0 polypropylene monofilament suture. After the ~10 min infarction surgery, close the skin with 7-0 prolene and disinfect the incision with betadine. Clean the pup of blood with 70% ethanol or saline. 
		NOTE: Ligation of the left coronary artery in preadolescent pups is essentially bloodless, as it is with adult ligations.</li>
		<li>Administer one after the other with a 0.5 mL insulin syringe and 29 G needle: atipamezole (1-5 mg/kg, 10 &#956;L, intraperitoneal) for rapid recovery from sedation, analgesia (buprenorphine, 0.075 mg/kg, 10 &#956;L, subcutaneous), and saline (50 &#956;L, intraperitoneal).</li>
	</ol>
	</li>
	<li>Allow animals to recover by discontinuing the isoflurane. Ensure spontaneous breathing resumes within a few minutes thereafter.
	<ol>
		<li>Return the pup to the warmed pre-oxygenated chamber and monitor continuously during recovery until the righting reflex is regained, at which point extubate the pup.</li>
		<li>Gently rub the pup with home cage bedding, keep the pup warm, check that breathing is regular, and that the pup is capable of spontaneous movement. This will reduce post-surgery cannibalism by the dam.</li>
	</ol>
	</li>
	<li>Return the dam to the cage when all pups have fully recovered from anesthesia. 
	NOTE: The overall time taken for preparation, anesthesia, intubation, surgery, and recovery of one pup can range from 40-60 min.</li>
	<li>House dam and pups overnight in a cage placed half on/half off a 37 &#176;C warming pad.</li>
</ol>
5. Post-surgery assessment of infarct size
<ol>
	<li>On the 3rd day post-surgery, anesthetize pups by placing them in a plexiglass chamber pre-equilibrated with 4.5% isoflurane in oxygen at 1 mL/min flow rate.</li>
	<li>Once a surgical plane of anesthesia has been reached (after 4-5 min), assessed by the paw pinch reflex, remove the pup from the chamber and secure in supine position on a warming pad by taping the tail.</li>
	<li>Place a thread over the incisors and tape into position to keep the head extended and place the head into a nose cone connected to a ventilator delivering 4.5% isoflurane in oxygen at 200 &#181;L/stroke, 150 strokes/min. Maintain a surgical plane of anesthesia with 2% isoflurane in oxygen.</li>
	<li>Disinfect the skin with 70% ethanol. Using fine scissors, make a 1 cm incision in the skin over the right common carotid artery along the trachea and cannulate the exposed vessel using a single lumen polyethylene tube (OD 0.61 mm, ID 0.28 mm) to administer 0.2 mL of heparinized saline (200 U) for 1 min to prevent blood clotting.</li>
	<li>Increase isoflurane to 4.5% in oxygen for 1 min before rapidly administering 0.2 mL of 3.3 M KCl within 2 s to arrest the heart in diastole.</li>
	<li>Dissect the right jugular vein via the same incision and transect it. Perfuse the heart with 0.2 mL of phosphate-buffered saline (PBS), and then perfuse with 0.1mL of 0.2% Alcian Blue to stain the non-infarcted remote myocardium. Check successful perfusion, evidenced by the washing out of blood, PBS, and then Alcian Blue via the jugular vein.</li>
	<li>Open the thorax and excise the heart by dissecting the surrounding connective tissue and vessels to release the heart. Rinse the heart in PBS, remove the atria if desired, and photograph the heart with a camera mounted on a surgical microscope using a 10x objective.</li>
</ol>

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R., 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=Regulation+of+neonatal+and+adult+mammalian+heart+regeneration+by+the+miR-15+family.">Regulation of neonatal and adult mammalian heart regeneration by the miR-15 family.</a> Proceedings of the National Academy of Sciences of the United States of America. 110 (1), 187-192 (2013).</li><li>Naqvi, N., 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=A+proliferative+burst+during+preadolescence+establishes+the+final+cardiomyocyte+number.">A proliferative burst during preadolescence establishes the final cardiomyocyte number.</a> Cell. 157 (4), 795-807 (2014).</li><li>Bogush, N., 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=DUSP5+expression+in+left+ventricular+cardiomyocytes+of+young+hearts+regulates+thyroid+hormone+(T3)-induced+proliferative+ERK1/2+signaling.">DUSP5 expression in left ventricular cardiomyocytes of young hearts regulates thyroid hormone (T3)-induced proliferative ERK1/2 signaling.</a> Scientific Reports. 10 (1), 21918 (2020).</li><li>Flecknell, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Laboratory Animal Anaesthesia. 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The authors have no conflicts of interest to disclose.

Currently, there are no well-documented methods for anesthesia and intubation of 10-day-old mice for cardiothoracic surgery. To this end, we have titrated ketamine/xylazine/atropine dosage regimens to body weight, whereby doses of 20/4/0.12 mg/kg, 30/4/0.12 mg/kg, and 50/6/0.18 mg/kg facilitated intubation of pups with low (3.15-4.49 g), mid (4.50-5.49 g), and high (5.50-8.10 g) body weight, respectively. Survival post-intubation correlated with body weight (59%, 70%, and 80% for low-, mid-, and high-weight groups, respe...

Murine models are invaluable tools in preclinical cardiothoracic research, in particular because of the ease with which genetically-engineered mouse lines can be generated, and also the ease with which the mice can be surgically manipulated to provide pathological disease models to allow, for example, the study of myocardial regeneration after cardiac injury1. In this regard, it is of interest that, unlike adult mice in which cardiomyocytes have withdrawn from the cell cycle, 0-2-day-old neonatal mouse hearts repair with minimal scarring after apical resection or induction of myocardial infarction2,3,4.&#160;In contrast, 7-day-old neonatal hearts regenerate incompletely with a higher incidence of scarring2,3. Since cardiomyocytes in the apex of the left ventricle retain proliferative capacity for up to 2 weeks after birth, mechanistic studies of regeneration after cardiac injury in 0-14-day-old mice may be informative for identifying therapeutic targets for regeneration of the injured adult heart5.
The development of mouse models of cardiac injury involves surgical manipulation under anesthesia. This requires that the thorax be opened to access the heart, which generally mandates intubation and mechanical ventilation. Mouse strain, body weight, and age influence sensitivity to anesthetics6. Adult mice can be anesthetized with a wide range of agents, a common regimen for intubation being ketamine/xylazine/atropine at 100/13/0.5 mg/kg6,7. Neonatal mice (0-7 days old) lack a centralized pain reflex, and can be effectively immobilized on ice and subjected to surgery without intubation6,8,9. Preadolescent (8-14-day-old) mouse pups cannot be anesthetized with hypothermia9,10; they require intubation for cardiothoracic surgery. There are no previous studies on cardiothoracic surgery in preadolescent mice less than 14 days old. In our experience, intubation of isoflurane-anesthetized preadolescent mice under 14 days of age is difficult. The recommended injectable anesthetic regimen reported for mice older than 7 days is 50-150 mg/kg ketamine and 5-10 mg/kg xylazine10. Preadolescent mice are still developing neurologically and their responses to drugs and drug metabolism are very different from adult animals6. This poses increased risk of fluid, electrolyte, and acid-base imbalance, as well as hypoglycemia and hypothermia due to not only their high metabolic rate, which rapidly depletes their limited energy stores, but also due to their thermoregulatory immaturity6,11,12. Thus, there is little information on anesthetic regimens that both facilitate intubation and maximize survival of preadolescent mice.
Here we empirically titrated dosage regimens of ketamine/xylazine/atropine in 10-day-old C57BL/6J mouse pups ranging in weight from 3-8 g to achieve a plane of anesthesia sufficient to allow endotracheal intubation for subsequent cardiothoracic surgery, while minimizing animal mortality. We also refined animal handling practices to reduce mortality from intubation, surgery, and post-surgical maternal cannibalism.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Atipamezole (Antisedan)</td>
			<td>Provet (NSW) Pty Ltd</td>
			<td>ATIP I</td>
			<td></td>
		</tr>
		<tr>
			<td>Atropine 600 mcg/mL</td>
			<td>Clifford Hallam Healthcare Pty Ptd</td>
			<td>1957699 PFIZER-0143386</td>
			<td></td>
		</tr>
		<tr>
			<td>Betadine</td>
			<td>Livingstone International</td>
			<td>BU0520</td>
			<td></td>
		</tr>
		<tr>
			<td>Buprenorphine (Temgesic)</td>
			<td>Provet (NSW) Pty Ltd</td>
			<td>TEMG I</td>
			<td></td>
		</tr>
		<tr>
			<td>Fiber-optic light</td>
			<td>Leica&#160;</td>
			<td>3011350</td>
			<td>CLS 150X</td>
		</tr>
		<tr>
			<td>GraphPad Prism</td>
			<td>GraphPad Software, LLC</td>
			<td>Version 9.1.2</td>
			<td></td>
		</tr>
		<tr>
			<td>Intubation platform&#160;</td>
			<td>-</td>
			<td>-</td>
			<td>Any sturdy box (e.g. plastic tip box) with approximate dimensions 12 (L) x 8.5 x (W) x 7.5 cm (H)</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Provet (NSW) Pty Ltd</td>
			<td>ISOF 07</td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamine 100 mg/mL</td>
			<td>Provet (NSW) Pty Ltd</td>
			<td>KETAI1</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic intravenous cannula 24-gauge Polywin Safety&#160;</td>
			<td>BD Insyte&#160;</td>
			<td>CE0086</td>
			<td>19 mm length of plastic tubing (0.7 mm outer diameter) attached to a 21mm plastic female luer lock adaptor; total volume of annula 130 &#956;L</td>
		</tr>
		<tr>
			<td>Single lumen polyethylene tube</td>
			<td>Critchley Electrical Products Pty Ltd Auburn NSW</td>
			<td></td>
			<td>Outer diameter 0.61 mm, inner diameter 0.28 mm</td>
		</tr>
		<tr>
			<td>Small forceps</td>
			<td>F.S.T.</td>
			<td>NO 11051-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical microscope (camera optional)</td>
			<td>Leica&#160;</td>
			<td>M651 (Leica IC80 HD camera)</td>
			<td>10x and 16x objective</td>
		</tr>
		<tr>
			<td>Suture 7-0 prolene</td>
			<td>Ethicon</td>
			<td>8708H</td>
			<td></td>
		</tr>
		<tr>
			<td>Suture 9-0 polypropylene monofilament</td>
			<td>Ethicon</td>
			<td>2813</td>
			<td></td>
		</tr>
		<tr>
			<td>V-1 Tabletop with Active Scavenging isoflurane anesthesia systm</td>
			<td>VetEquip</td>
			<td>901820</td>
			<td></td>
		</tr>
		<tr>
			<td>Vented 2-Liter plexiglass induction chamber</td>
			<td>VetQuip Pty Ltd</td>
			<td>942102</td>
			<td>25 cm (L) x 13 cm (W) x 11 cm (H)</td>
		</tr>
		<tr>
			<td>Warming lamp</td>
			<td>Brilant Lighting</td>
			<td>99223</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>Provet (NSW) Pty Ltd</td>
			<td>XYLA Z 2</td>
			<td></td>
		</tr>
	</tbody>
</table>

anesthesia and intubation of preadolescent mouse pups for cardiothoracic surgery

Murine surgical models play an important role in preclinical research. Mechanistic insights into myocardial regeneration after cardiac injury may be gained from cardiothoracic surgery models in 0-14-day-old mice, the cardiomyocytes of which, unlike those of adults, retain proliferative capacity. Mouse pups up to 7 days old are effectively immobilized by hypothermia and do not require intubation for cardiothoracic surgery. Preadolescent (8-14-day-old) mouse pups, however, do require intubation, but this is challenging and there is little information regarding anesthesia to facilitate intubation.&#160;Here, we present dosage regimens of ketamine/xylazine/atropine in 10-day-old C57BL/6J mouse pups that allow endotracheal intubation, while minimizing animal mortality. Empirical titration of ketamine/xylazine/atropine dosage regimens to body weight indicated that the response to anesthesia of mouse pups of different weights was non-linear, whereby doses of 20/4/0.12 mg/kg, 30/4/0.12 mg/kg, and 50/6/0.18 mg/kg facilitated intubation of pups weighing between 3.15-4.49 g (n = 22), 4.50-5.49 g (n = 20), and 5.50-8.10 g (n = 20), respectively. Lower-body-weight pups required more intubation attempts than heavier pups (p &#60; 0.001). Survival post-intubation correlated with body weight (59%, 70%, and 80% for low-, mid-, and high-weight groups, respectively, R2 = 0.995). For myocardial infarction surgery after intubation, a surgical plane of anesthesia was induced with 4.5% isoflurane in 100% oxygen and maintained with 2% isoflurane in 100% oxygen. Survival post-surgery was similar for the three weight groups at 92%, 86%, and 88% (p = 0.91). Together with refinements in animal handling practices for intubation and surgery, and minimizing cannibalization by the dam post-surgery, overall survival for the entire procedure (intubation plus surgery) correlated with body weight (55%, 60%, and 70% for low-, mid-, and high-weight groups, respectively, R2 = 0.978). Given the difficulty encountered with intubation of 10-day old pups and the associated high mortality, we recommend cardiothoracic surgery in 10-day-old pups be restricted to pups weighing at least 5.5 g.

Anesthesia of 10-day-old mice. The10-day-old pups can be anesthetized with 4.5% isoflurane in 4-5 min; however, they recover from anesthesia in the process of preparation for intubation. Due to their small size, intubation under isoflurane anesthesia delivered by a standard nose cone is not feasible. We have previously used a ketamine/xylazine/atropine anesthetic regimen of 100/13/0.5 mg/kg, respectively, for cardiothoracic surgery in 15- and 21-day-old pups and adults4,</sup...

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Anesthesia and Intubation of Preadolescent Mouse Pups for Cardiothoracic Surgery

Cardiothoracic surgical models in mice &gt;7 days old require intubation, but this is challenging for preadolescent (8-14-day-old) mouse pups and there is little information on anesthetic regimens for intubation. Here, we present dosage regimens of ketamine/xylazine/atropine in 10-day-old C57BL/6J mouse pups that allow endotracheal intubation, while minimizing animal mortality.

Murine surgical models play an important role in preclinical research. Mechanistic insights into myocardial regeneration after cardiac injury may be ...

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2.2K Views.  Victor Chang Cardiac Research Institute. Cardiothoracic surgical models in mice >7 days old require intubation, but this is challenging for preadolescent (8-14-day-old) mouse pups and there is little information on anesthetic regimens for intubation. Here, we present dosage regimens of ketamine/xylazine/atropine in 10-day-old C57BL/6J mouse pups that allow endotracheal intubation, while minimizing animal mortality.

Video: Anesthesia and Intubation of Preadolescent Mouse Pups for Cardiothoracic Surgery

Survivable Stereotaxic Surgery in Rodents 

The ability to measure extracellular basal levels of neurotransmitters in the brain of awake animals allows for the determination of effects of different systemic challenges (pharmacological or physiological) to the CNS. For example, one can directly measure how the animal's midbrain dopamine projections respond to dopamine-releasing drugs like d-amphetamine or natural stimuli like food. In this video, we show you how to implant guide cannulas targeting specific sites in the rat brain, how to insert and implant a microdialysis probe and how to use high performance liquid chromatography coupled with electrochemical detection (HPLC-EC) to measure extracellular levels of oxidizable neurotransmitters and metabolites. Local precise introduction of drugs through the microdialysis probe allows for refined work on site specificity in a compound s mechanism of action. This technique has excellent anatomical and chemical resolution but only modest time resolution as microdialysis samples are usually processed every 20-30 minutes to ensure detectable neurotransmitter levels. Complementary ex vivo tools (i.e., slice and cell culture electrophysiology) can assist with monitoring real-time neurotransmission.

Survivable Stereotaxic Surgery in Rodents

The monitoring of extracellular neurotransmitter levels in distinct brain regions of freely moving animals offers insights on the link between neurotransmitter release and behavior. In vivo microdialysis coupled with electrochemical detection provides excellent anatomical and chemical resolution; and information on how basal neurotransmission is altered by pharmacological or physiological manipulations.

The ability to measure extracellular basal levels of neurotransmitters in the brain of awake animals allows for the determination of effects of different ...

Human In-Vivo Bioassay for the Tissue-Specific Measurement of Nociceptive and Inflammatory Mediators

This in-vivo human bioassay can be used to study human volunteers and patients. Samples are collected from pertinent tissue sites such as the skin via aseptically inserted microdialysis catheters (Dermal Dialysis, Erlangen, Germany). Illustrated in this example is the collection of interstitial fluid from experimentally inflamed skin in human volunteers. Sample collection can be combined with other experimental tests. For example, the simultaneous assessment of locally released biochemicals and subjective sensitivity to painful stimuli in experimentally inflamed skin provides the critical biochemical-behavioral link to identify biomarkers of pain and inflammation. Presented assay in the living human organism allows for mechanistic insight into tissue-specific processes underlying pain and/or inflammation. The method is also well suited to examine the effectiveness of existing or novel interventions - such as new drug candidates - targeting the treatment of painful and/or inflammatory conditions.
This article will provide a detailed description on the use of microdialysis techniques for collecting interstitial fluid from experimentally inflamed skin lesion of human study subjects. Interstitial fluid samples are typically processed with aid of multiplex bead array immunoassays allowing assaying up to 100 analytes in samples as small in volume as 50 microliters.


A technique is presented for the in-vivo collection of interstitial fluid samples from pertinent tissue sites (here, experimentally inflamed skin) for the measurement of biochemicals mediating pain and inflammation.

This in-vivo human bioassay can be used to study human volunteers and patients. Samples are collected from pertinent tissue sites such as the skin via ...

Determining heat and mechanical pain threshold in inflamed skin of human subjects

In a previous article in the Journal of Visualized Experiments we have demonstrated skin microdialysis techniques for the collection of tissue-specific nociceptive and inflammatory biochemicals in humans. In this article we will show pain-testing paradigms that are often used in tandem with microdialysis procedures. Combining pain tests with microdialysis provides the critical link between behavioral and biochemical data that allows identifying key biochemicals responsible for generating and propagating pain.
Two models of evoking pain in inflamed skin of human study participants are shown. The first model evokes pain with aid of heat stimuli. Heat evoked pain as described here is predominantly mediated by small, non-myelinated peripheral nociceptive nerve fibers (C-fibers). The second model evokes pain via punctuated pressure stimuli. Punctuated pressure evoked pain is predominantly mediated by small, myelinated peripheral nociceptive nerve fibers (A-delta fibers). The two models are mechanistically distinct and independently examine nociceptive processing by the two major peripheral nerve fiber populations involved in pain signaling.
Heat pain is evoked with aid of the TSA II, a commercially available thermo-sensory analyzer (Medoc Advanced Medical Systems, Durham, NC). Stimulus configuration and delivery is handled with aid of specific software. Thermodes vary in size and shape but in principle consist of a metal plate that can be heated or cooled at various rates and for different periods of time. Algorithms assessing heat-evoked pain are manifold. In the experiments shown here, study participants are asked to indicate at what point they start experiencing pain while the thermode in contact with skin is heated at a predetermined rate starting at a temperature that does not evoke pain. The thermode temperature at which a subject starts experiencing pain constitutes the heat pain threshold.
Mechanical pain is evoked with punctuated probes. Such probes are commercially available from several manufacturers (von Frey hairs). However, the accuracy of von Frey hairs has been criticized and many investigators use custom made punctuated pressure probes. In the experiments shown here eight custom-made punctuated probes of different weights are applied in consecutive order, a procedure called up-down algorithm, to identify perceptional deflection points, i.e., a change from feeling no pain to feeling pain or vice versa. The average weight causing a perceptional deflection constitutes the mechanical pain threshold.

Algorithms assessing heat and mechanical pain thresholds in experimentally inflamed skin of human study subjects are shown. The two pain testing paradigms independently examine nociceptive processing by the two major peripheral nerve fiber populations transmitting pain, i.e., non-myelinated C fibers and small myelinated A-delta fibers.

In a previous article in the Journal of Visualized Experiments we have demonstrated skin microdialysis techniques for the collection of tissue-specific ...

Imaging In-Stent Restenosis: An Inexpensive, Reliable, and Rapid Preclinical Model

Preclinical models of restenosis are essential to unravel the pathophysiological processes that lead to in-stent restenosis and to optimize existing and future drug-eluting stents.
A variety of antibodies and transgenic and knockout strains are available in rats. Consequently, a model for in-stent restenosis in the rat would be convenient for pathobiological and pathophysiological studies. 
In this video, we present the full procedure and pit-falls of a rat stent model suitable for high throughput stent research. We will show the surgical procedure of stent deployment, and the assessment of in-stent restenosis using the most elegant technique of OCT (Optical Coherence Tomography). This technique provides high accuracy in assessing plaque CSAs (cross section areas) and correlates well with histological sections, which require special and time consuming embedding and sectioning techniques. OCT imaging further allows longitudinal monitoring of the development of in-stent restenosis within the same animal compared to one-time snapshots using histology.

This video demonstrates how to use a preclinical inexpensive and reliable model to study pathobiological and pathophysiological processes of in-stent restenosis development. Longitudinal in vivo monitoring using OCT (Optical Coherence Tomography) and analysis of OCT images are also demonstrated.

Preclinical models of restenosis are essential to unravel the pathophysiological processes that lead to in-stent restenosis and to optimize existing and ...

Tracking Dynamics of Muscle Engraftment in Small Animals by In Vivo Fluorescent Imaging

Muscular dystrophies are a group of degenerative muscle diseases characterized by progressive loss of contractile muscle cells. Currently, there is no curative treatment available. Recent advances in stem cell biology have generated new hopes for the development of effective cell based therapies to treat these diseases. Transplantation of various types of stem cells labeled with fluorescent proteins into muscles of dystrophic animal models has been used broadly in the field. A non-invasive technique with the capability to track the transplanted cell fate longitudinally can further our ability to evaluate muscle engraftment by transplanted cells more accurately and efficiently.
In the present study, we demonstrate that in vivo fluorescence imaging is a sensitive and reliable method for tracking transplanted GFP (Green Fluorescent Protein)-labeled cells in mouse skeletal muscles. Despite the concern about background due to the use of an external light necessary for excitation of fluorescent protein, we found that by using either nude mouse or eliminating hair with hair removal reagents much of this problem is eliminated. Using a CCD camera, the fluorescent signal can be detected in the tibialis anterior (TA) muscle after injection of 5 x 105 cells from either GFP transgenic mice or eGFP transduced myoblast culture. For more superficial muscles such as the extensor digitorum longus (EDL), injection of fewer cells produces a detectable signal. Signal intensity can be measured and quantified as the number of emitted photons per second in a region of interest (ROI). Since the acquired images show clear boundaries demarcating the engrafted area, the size of the ROI can be measured as well. If the legs are positioned consistently every time, the changes in total number of photons per second per muscle and the size of the ROI reflect the changes in the number of engrafted cells and the size of the engrafted area. Therefore the changes in the same muscle over time are quantifiable. In vivo fluorescent imaging technique has been used primarily to track the growth of tumorogenic cells, our study shows that it is a powerful tool that enables us to track the fate of transplanted stem cells.

Tracking Dynamics of Muscle Engraftment in Small Animals by In Vivo Fluorescent Imaging

We describe an in vivo fluorescence imaging protocol to monitor muscle regeneration by GFP-labeled myoblasts after transplantation into skeletal muscles of both healthy and dystrophic mice. This protocol can be adapted to study muscle regeneration by transplantation of other types of cells and in other muscular conditions as well.

Muscular dystrophies are a group of degenerative muscle diseases characterized by progressive loss of contractile muscle cells. Currently, there is no ...

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Human cancer and response to therapy is better represented in orthotopic animal models. This paper describes the development of an orthotopic mouse model of ovarian cancer, treatment of cancer via oral delivery of drugs, and monitoring of tumor cell behavior in response to drug treatment in real time using in vivo imaging system. In this orthotopic model, ovarian tumor cells expressing luciferase are applied topically by injecting them directly into the mouse bursa where each ovary is enclosed. Upon injection of D-luciferin, a substrate of firefly luciferase, luciferase-expressing cells generate bioluminescence signals. This signal is detected by the in vivo imaging system and allows for a non-invasive means of monitoring tumor growth, distribution, and regression in individual animals. Drug administration via oral gavage allows for a maximum dosing volume of 10 mL/kg body weight to be delivered directly to the stomach and closely resembles delivery of drugs in clinical treatments. Therefore, techniques described here, development of an orthotopic mouse model of ovarian cancer, oral delivery of drugs, and in vivo imaging, are useful for better understanding of human ovarian cancer and treatment and will improve targeting this disease.

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Orthotopic animal models of ovarian cancer replicate better human disease and therefore enhance our understanding of cancer progression and tumor response to therapy. A mouse model receives an intrabursal injection of luciferase-expressing ovarian tumor cells. Treatment is administered via oral gavage. Tumor growth is monitored by in vivo imaging system.

Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...

Principles of Rodent Surgery for the New Surgeon

For both scientific and animal welfare reasons, training in basic surgical concepts and techniques should be undertaken before ever seeking to perform surgery on a rodent. Students, post-doctoral scholars, and others interested in performing surgery on rodents as part of a research protocol may not have had formal surgical training as part of their required coursework. Surgery itself is a technical skill, and one that will improve with practice. The principles of aseptic technique, however, often remain unexplained or untaught. For most new surgeons, this vital information is presented in piecemeal fashion or learned on the job, neither of which is ideal. It may also make learning how to perform a particular surgery difficult, as the new surgeon is learning both a surgical technique and the principles of asepsis at the same time. This article summarizes and makes recommendations for basic surgical skills and techniques necessary for successful rodent surgery. This article is designed to supplement hands-on training by the user's institution.

Before attempting surgery, a new surgeon should have training in basic surgical techniques and concepts. This article will present basic surgical considerations with an emphasis on rodents.

For both scientific and animal welfare reasons, training in basic surgical concepts and techniques should be undertaken before ever seeking to perform ...

Assessing Murine Resistance Artery Function Using Pressure Myography

Pressure myograph systems are exquisitely useful in the functional assessment of small arteries, pressurized to a suitable transmural pressure. The near physiological condition achieved in pressure myography permits in-depth characterization of intrinsic responses to pharmacological and physiological stimuli, which can be extrapolated to the in vivo behavior of the vascular bed. Pressure myograph has several advantages over conventional wire myographs. For example, smaller resistance vessels can be studied at tightly controlled and physiologically relevant intraluminal pressures. Here, we study the ability of 3rd order mesenteric arteries (3-4 mm long), preconstricted with phenylephrine, to vaso-relax in response to acetylcholine. Mesenteric arteries are mounted on two cannulas connected to a pressurized and sealed system that is maintained at constant pressure of 60 mmHg. The lumen and outer diameter of the vessel are continuously recorded using a video camera, allowing real time quantification of the vasoconstriction and vasorelaxation in response to phenylephrine and acetylcholine, respectively.
	To demonstrate the applicability of pressure myography to study the etiology of cardiovascular disease, we assessed endothelium-dependent vascular function in a murine model of systemic hypertension. Mice deficient in the &alpha;1 subunit of soluble guanylate cyclase (sGC&alpha;1-/-) are hypertensive when on a 129S6 (S6) background (sGC&alpha;1-/-S6) but not when on a C57BL/6 (B6) background (sGC&alpha;1-/-B6). Using pressure myography, we demonstrate that sGC&alpha;1-deficiency results in impaired endothelium-dependent vasorelaxation. The vascular dysfunction is more pronounced in sGC&alpha;1-/-S6 than in sGC&alpha;1-/-B6 mice, likely contributing to the higher blood pressure in sGC&alpha;1-/-S6 than in sGC&alpha;1-/-B6 mice. 
	Pressure myography is a relatively simple, but sensitive and mechanistically useful technique that can be used to assess the effect of various stimuli on vascular contraction and relaxation, thereby augmenting our insight into the mechanisms underlying cardiovascular disease.

In pressure myography, an intact small segment of a vessel is mounted onto two small cannulas and pressurized to a suitable luminal pressure. Here, we describe the method to measure vasorelaxation response of the mouse 3rd order mesenteric arteries in c57 and sGC&alpha;1-/- mice using pressure myography.

Pressure myograph systems are  exquisitely useful in the functional assessment of small arteries, pressurized  to a suitable transmural pressure. The  ...

Intravital Microscopy of the Microcirculation in the Mouse Cremaster Muscle for the Analysis of Peripheral Stem Cell Migration

In the era of intravascular cell application protocols in the context of regenerative cell therapy, the underlying mechanisms of stem cell migration to nonmarrow tissue have not been completely clarified. We describe here the technique of intravital microscopy applied to the mouse cremaster microcirculation for analysis of peripheral bone marrow stem cell migration in vivo. Intravital microscopy of the M. cremaster has been previously introduced in the field of inflammatory research for direct observation of leucocyte interaction with the vascular endothelium. Since sufficient peripheral stem and progenitor cell migration includes similar initial steps of rolling along and firm adhesion at the endothelial lining it is conceivable to apply the M. cremaster model for the observation and quantification of the interaction of intravasculary administered stem cells with the endothelium. As various chemical components can be selectively applied to the target tissue by simple superfusion techniques, it is possible to establish essential microenvironmental preconditions, for initial stem cell recruitment to take place in a living organism outside the bone marrow.

Intravital microscopy of the mouse M. cremaster microcirculation offers a unique and well-standardized in vivo model for the analysis of peripheral bone marrow stem cell migration.

In the era of intravascular cell application protocols in the context of regenerative cell therapy, the underlying mechanisms of stem cell migration to ...

Rapid Isolation And Purification Of Mitochondria For Transplantation By Tissue Dissociation And Differential Filtration

Previously described mitochondrial isolation methods using differential centrifugation and/or Ficoll gradient centrifugation require 60 to 100 min to complete. We describe a method for the rapid isolation of mitochondria from mammalian biopsies using a commercial tissue dissociator and differential filtration. In this protocol, manual homogenization is replaced with the tissue dissociator&#8217;s standardized homogenization cycle. This allows for uniform and consistent homogenization of tissue that is not easily achieved with manual homogenization. Following tissue dissociation, the homogenate is filtered through nylon mesh filters, which eliminate repetitive centrifugation steps. As a result, mitochondrial isolation can be performed in less than 30 min. This isolation protocol yields approximately 2 x 1010 viable and respiration competent mitochondria from 0.18 &#177; 0.04 g (wet weight) tissue sample.

A method for rapid isolation of mitochondria from mammalian tissue biopsies is described. Rat liver or skeletal muscle preparations were homogenized with a commercial tissue dissociator and mitochondria were isolated by differential filtration through nylon mesh filters. Mitochondrial isolation time is &#60;30 min compared to 60 - 100 min using alternative methods.

Previously described mitochondrial isolation methods using differential centrifugation and/or Ficoll gradient centrifugation require 60 to 100 min to ...

Anesthesia and Intubation of Preadolescent Mouse Pups for Cardiothoracic Surgery

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Chapters in this video

Survivable Stereotaxic Surgery in Rodents

Human In-Vivo Bioassay for the Tissue-Specific Measurement of Nociceptive and Inflammatory Mediators

Determining heat and mechanical pain threshold in inflamed skin of human subjects

Imaging In-Stent Restenosis: An Inexpensive, Reliable, and Rapid Preclinical Model

Tracking Dynamics of Muscle Engraftment in Small Animals by In Vivo Fluorescent Imaging

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Principles of Rodent Surgery for the New Surgeon

Assessing Murine Resistance Artery Function Using Pressure Myography

Intravital Microscopy of the Microcirculation in the Mouse Cremaster Muscle for the Analysis of Peripheral Stem Cell Migration

Rapid Isolation And Purification Of Mitochondria For Transplantation By Tissue Dissociation And Differential Filtration