Name: anesthesia and intubation of preadolescent mouse pups for cardiothoracic surgery
Uploaded: 2022-06-02T14:30:00.000+00:00
Duration: 9 min 47 s
Description: Watch this Scientific Journal Video about Anesthesia and Intubation of Preadolescent Mouse Pups for Cardiothoracic Surgery at JoVE.com

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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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><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&amp;nbsp;</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&amp;nbsp;</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&amp;nbsp;</td><td>BD Insyte&amp;nbsp;</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 &amp;mu;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&amp;nbsp;</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

Use of a Hanging Weight System for Coronary Artery Occlusion in Mice

Murine studies of acute injury are an area of intense investigation, as knockout mice for different genes are becoming increasingly available 1-38. Cardioprotection by ischemic preconditioning (IP) remains an area of intense investigation. To further elucidate its molecular basis, the use of knockout mouse studies is particularly important 7, 14, 30, 39. Despite the fact that previous studies have already successfully performed cardiac ischemia and reperfusion in mice, this model is technically very challenging. Particularly, visual identification of the coronary artery, placement of the suture around the vessel and coronary occlusion by tying off the vessel with a supported knot is technically difficult. In addition, re-opening the knot for intermittent reperfusion of the coronary artery during IP without causing surgical trauma adds additional challenge. Moreover, if the knot is not tied down strong enough, inadvertent reperfusion due to imperfect occlusion of the coronary may affect the results. In fact, this can easily occur due to the movement of the beating heart.
Based on potential problems associated with using a knotted coronary occlusion system, we adopted a previously published model of chronic cardiomyopathy based on a hanging weight system for intermittent coronary artery occlusion during IP 39. In fact, coronary artery occlusion can thus be achieved without having to occlude the coronary by a knot. Moreover, reperfusion of the vessel can be easily achieved by supporting the hanging weights which are in a remote localization from cardiac tissues.
We tested this system systematically, including variation of ischemia and reperfusion times, preconditioning regiments, body temperature and genetic backgrounds39. In addition to infarct staining, we tested cardiac troponin I (cTnI)
as a marker of myocardial infarction in this model. In fact, plasma levels of cTnI correlated with infarct sizes (R2=0.8). Finally, we could show in several studies that this technique yields highly reproducible infarct sizes during murine IP and myocardial infarction6, 8, 30, 40, 41. Therefore, this technique may be helpful for researchers who pursue molecular mechanisms involved in cardioprotection by IP using a genetic approach in mice with targeted gene deletion. Further studies on cardiac IP using transgenic mice may consider this technique.

A murine model for myocardial ischemia and ischemic preconditioning is an important tool study cardioprotective mechanisms in vivo. Here, we report an easy applicable in situ model for cardiac IP using a hanging-weight system for coronary artery occlusion.

Murine studies of acute injury are an area of intense investigation, as knockout mice for different genes are becoming increasingly available 1-38. ...

Medicine

Endotracheal Intubation in Mice via Direct Laryngoscopy Using an Otoscope

Mice, both wildtype and transgenic, are the principal mammalian model in biomedical research currently. Intubation and mechanical ventilation are necessary for whole animal experiments that require surgery under deep anesthesia or measurements of lung function. Tracheostomy has been the standard for intubating the airway in these mice to allow mechanical ventilation. Orotracheal intubation has been reported but has not been successfully used in many studies because of the substantial technical difficulty or a requirement for highly specialized and expensive equipment. Here we report a technique of direct laryngoscopy using an otoscope fitted with a 2.0 mm speculum and using a 20 G&#160;intravenous catheter as an endotracheal tube. We have used this technique extensively and reliably to intubate and conduct accurate assessments of lung function in mice. This technique has proven safe, with essentially no animal loss in experienced hands. Moreover, this technique can be used for repeated studies of mice in chronic models.

Endotracheal Intubation in Mice via Direct Laryngoscopy Using an Otoscope

We have developed a simple, reliable, and relatively inexpensive method for endotracheal intubation in mice via direct laryngoscopy using an otoscope with a 2.0 mm speculum. This technique is atraumatic and can be used for repeated measurements in chronic experiments. We find it superior to tracheostomy or previously reported nonsurgical techniques.

Mice, both wildtype and transgenic, are the principal mammalian model in biomedical research currently. Intubation and mechanical ventilation are ...

In vivo Measurement of the Mouse Pulmonary Endothelial Surface Layer

The endothelial glycocalyx is a layer of proteoglycans and associated glycosaminoglycans lining the vascular lumen. In vivo, the glycocalyx is highly hydrated, forming a substantial endothelial surface layer (ESL) that contributes to the maintenance of endothelial function. As the endothelial glycocalyx is often aberrant in vitro and is lost during standard tissue fixation techniques, study of the ESL requires use of intravital microscopy. To best approximate the complex physiology of the alveolar microvasculature, pulmonary intravital imaging is ideally performed on a freely-moving lung. These preparations, however, typically suffer from extensive motion artifact. We demonstrate how closed-chest intravital microscopy of a freely-moving mouse lung can be used to measure glycocalyx integrity via ESL exclusion of fluorescently-labeled high molecular weight dextrans from the endothelial surface. This non-recovery surgical technique, which requires simultaneous brightfield and fluorescent imaging of the mouse lung, allows for longitudinal observation of the subpleural microvasculature without evidence of inducing confounding lung injury.

In vivo Measurement of the Mouse Pulmonary Endothelial Surface Layer

The endothelial glycocalyx/endothelial surface layer is ideally studied using intravital microscopy. Intravital microscopy is technically challenging in a moving organ such as the lung. We demonstrate how simultaneous brightfield and fluorescent microscopy may be used to estimate endothelial surface layer thickness in a freely-moving in vivo mouse lung.

The endothelial glycocalyx is a layer of proteoglycans and associated glycosaminoglycans lining the vascular lumen. In vivo, the glycocalyx is highly ...

Mouse Pneumonectomy Model of Compensatory Lung Growth

In humans, disrupted repair and remodeling of injured lung contributes to a host of acute and chronic lung disorders which may ultimately lead to disability or death. Injury-based animal models of lung repair and regeneration are limited by injury-specific responses making it difficult to differentiate changes related to the injury response and injury resolution from changes related to lung repair and lung regeneration. However, use of animal models to identify these repair and regeneration signaling pathways is critical to the development of new therapies aimed at improving pulmonary function following lung injury. The mouse pneumonectomy model utilizes compensatory lung growth to isolate those repair and regeneration signals in order to more clearly define mechanisms of alveolar re-septation. Here, we describe our technique for performing mouse pneumonectomy and sham pneumonectomy. This technique may be utilized in conjunction with lineage tracing or other transgenic mouse models to define molecular and cellular mechanism of lung repair and regeneration.

Mouse pneumonectomy is a commonly employed model of compensatory lung growth. This procedure can be used in conjunction with lineage tracing or transgenic mouse models to elucidate underlying mechanisms.

In humans, disrupted repair and remodeling of injured lung contributes to a host of acute and chronic lung disorders which may ultimately lead to ...

A Closed-chest Model to Induce Transverse Aortic Constriction in Mice

Research on cardiac hypertrophy and heart failure is frequently based on pressure overload mouse models induced by TAC. The standard procedure is to perform a partial thoracotomy to visualize the transverse aortic arch. However, the surgical trauma caused by the thoracotomy in open-chest models changes the respiratory physiology as the ribs are dissected and left unattached after chest closure. To prevent this, we established a minimally invasive, closed chest approach via lateral thoracotomy. Herein we approach the aortic arch via the 2nd intercostal space without entering the chest cavities, leaving the mouse with a less traumatic injury to recover from. We perform this operation using standard laboratory settings for open chest TAC procedures with equal survival rates. Apart from maintaining physiological breathing patterns due to the closed chest approach, the mice seem to benefit by showing rapid recovery, as the less invasive technique appears to facilitate a fast healing process and to reduce immune response after trauma.

Here, we present a protocol of Transverse Aortic constriction (TAC) via a lateral thoracotomy. This technique is a minimally invasive, closed chest surgical procedure aiming to simulate pressure overload and heart failure in mice utilizing standard TAC laboratory settings.

Research on cardiac hypertrophy and heart failure is frequently based on pressure overload mouse models induced by TAC. The standard procedure is to ...

A Mouse Model of Orotracheal Intubation and Ventilated Lung Ischemia Reperfusion Surgery

Ischemia reperfusion (IR) injury frequently results from processes that involve a transient period of interrupted blood flow. In the lung, isolated IR permits the experimental study of this specific process with continued alveolar ventilation, thereby avoiding the compounding injurious processes of hypoxia and atelectasis. In the clinical context, lung ischemia reperfusion injury (also known as lung IRI or LIRI) is caused by numerous processes, including but not limited to pulmonary embolism, resuscitated hemorrhagic trauma, and lung transplantation. There are currently limited effective treatment options for LIRI. Here, we present a reversible surgical model of lung IR involving first orotracheal intubation followed by unilateral left lung ischemia and reperfusion with preserved alveolar ventilation or gas exchange. Mice undergo a left thoracotomy, through which the left pulmonary artery is exposed, visualized, isolated, and compressed using a reversible slipknot. The surgical incision is then closed during the ischemic period, and the animal is awakened and extubated. With the mouse spontaneously breathing, reperfusion is established by releasing the slipknot around the pulmonary artery. This clinically relevant survival model permits the evaluation of lung IR injury, the resolution phase, downstream effects on lung function, as well as two-hit models involving experimental pneumonia. While technically challenging, this model can be mastered over the course of a few weeks to months with an eventual survival or success rate of 80%-90%.

A mouse surgical model to create left lung ischemia reperfusion (IR) injury while maintaining ventilation and avoiding hypoxia.

Ischemia reperfusion (IR) injury frequently results from processes that involve a transient period of interrupted blood flow. In the lung, isolated IR ...

Immunology and Infection

In vivo Calcium Imaging in Mouse Inferior Olive

Inferior olive (IO), a nucleus in the ventral medulla, is the only source of climbing fibers that form one of the two input pathways entering the cerebellum. IO has long been proposed to be crucial for motor control and its activity is currently considered to be at the center of many hypotheses of both motor and cognitive functions of the cerebellum. While its physiology and function have been relatively well studied on single-cell level in vitro, presently there are no reports on the organization of the IO network activity in living animals. This is largely due to the extremely challenging anatomical location of the IO, making it difficult to subject to conventional fluorescent imaging methods, where an optic path must be created through the entire brain located dorsally to the region of interest.
Here we describe an alternative method for obtaining state-of-the-art -level calcium imaging data from the IO network. The method takes advantage of the extreme ventral location of the IO and involves a surgical procedure for inserting a gradient-refractive index (GRIN) lens through the neck viscera to come into contact with the ventral surface of the calcium sensor GCaMP6s-expressing IO in anesthetized mice. A representative calcium imaging recording is shown to demonstrate the feasibility to record IO neuron activity after the surgery. While this is a non-survival surgery and the recordings must be conducted under anesthesia, it avoids damage to life-critical brainstem nuclei and allows conducting large variety of experiments investigating spatiotemporal activity patterns and input integration in the IO. This procedure with modifications could be used for recordings in other, adjacent regions of the ventral brainstem.

In vivo Calcium Imaging in Mouse Inferior Olive

We present a protocol to expose the brainstem of adult mouse from the ventral side. By using a gradient-refractive index lens with a miniature microscope, calcium imaging can be used to examine the activity of inferior olive neural somata in vivo.

Inferior olive (IO), a nucleus in the ventral medulla, is the only source of climbing fibers that form one of the two input pathways entering the ...

Neuroscience

Quantification of Biventricular Function and Morphology by Cardiac Magnetic Resonance Imaging in Mice with Pulmonary Artery Banding

Right ventricular (RV) function and failure are major determinants of outcome in acquired and congenital heart diseases, including pulmonary hypertension. Assessment of RV function and morphology is complex, partly due to the complex shape of the RV. Currently, cardiac magnetic resonance (CMR) imaging is the golden standard for noninvasive assessment of RV function and morphology. The current protocol describes CMR imaging in a mouse model of RV pressure load induced by pulmonary artery banding (PAB). PAB is performed by placing a 6-0 suture around the pulmonary artery over a 23 G needle. The PAB gradient is determined using echocardiography at 2 and 6 weeks. At 6 weeks, the right and left ventricular morphology and function is assessed by measuring both end-systolic and end-diastolic volumes and mass by ten to eleven cine slices 1 mm thick using a 9.4 T magnetic resonance imaging scanner equipped with a 1,500 mT/m gradient. Representative results show that PAB induces a significant increase in RV pressure load, with significant effects on biventricular morphology and RV function. It is also shown that at 6 weeks of RV pressure load, cardiac output is maintained. Presented here is a reproducible protocol for the quantification of biventricular morphology and function in a mouse model of RV pressure load and may serve as a method for experiments exploring determinants of RV remodeling and dysfunction.

To understand the pathophysiology of right ventricular (RV) adaptation to abnormal loading, experimental models are crucial. However, assessment of RV dimensions and function is complex and challenging. This protocol provides a method to perform cardiac magnetic resonance imaging (CMR) as a noninvasive benchmark procedure in mice subjected to RV pressure load.

Right ventricular (RV) function and failure are major determinants of outcome in acquired and congenital heart diseases, including pulmonary hypertension. ...

Cardiac Response to &#946;-Adrenergic Stimulation Determined by Pressure-Volume Loop Analysis

Determination of the cardiac function is a robust endpoint analysis in animal models of cardiovascular diseases in order to characterize effects of specific treatments on the heart. Due to the feasibility of genetic manipulations the mouse has become the most common mammalian animal model to study cardiac function and to search for new potential therapeutic targets. Here we describe a protocol to determine cardiac function&#160;in vivo using pressure-volume loop measurements and analysis during basal conditions and under &#946;-adrenergic stimulation by intravenous infusion of increasing concentrations of isoproterenol. We provide a refined protocol including ventilation support taking into account the positive end-expiratory pressure to ameliorate negative effects during open-chest measurements, and potent analgesia (Buprenorphine) to avoid uncontrollable myocardial stress evoked by pain during the procedure. All together the detailed description of the procedure and discussion about possible pitfalls enables highly standardized and reproducible pressure-volume loop analysis, reducing the exclusion of animals from the experimental cohort by preventing possible methodological bias.

Here we describe a cardiac pressure-volume loop analysis under increasing doses of intravenously infused isoproterenol to determine the intrinsic cardiac function and the &#946;-adrenergic reserve in mice. We use a modified open-chest approach for the pressure-volume loop measurements, in which we include ventilation with positive end-expiratory pressure.

Determination of the cardiac function is a robust endpoint analysis in animal models of cardiovascular diseases in order to characterize effects of ...

Tracheotomy-Based Endotracheal Intubation Protocol for Non-Survival Rat Models

Endotracheal Intubation via Tracheotomy and Subsequent Thoracotomy in Rats for Non-Survival Applications

Endotracheal intubation and subsequent ventilation are often basic requirements for translational research in rat models for various interventions that require controlled or high ventilation pressures or access to the thoracic cavity and organs. Conventional endoorotracheal intubation using the anatomically existing route through the mouth is well suited for survival experiments. However, this procedure poses some challenges, including generally higher levels of the required experience and technical skill, more advanced equipment, and greater time effort with relevant intubation failure rates and complications such as tracheal perforation, temporary systemic hypooxygenation, and relevant aerial leakage.
This manuscript, therefore, presents a detailed step-by-step protocol for endotracheal intubation through tracheotomy in non-survival rat models when guaranteed intubation success, constant oxygenation levels, high ventilation pressures, or open thoracotomy are required.
The protocol emphasizes the importance of meticulous surgical technique to ensure consistent and reliable outcomes, especially for researchers who are inexperienced or lack routine in the technique of endoorotracheal intubation via direct laryngoscopy. This procedure is, therefore, expected to minimize animal suffering and unnecessary animal losses.

Here we introduce a standardized procedure for endotracheal intubation via tracheotomy followed by thoracotomy in rats, aimed at enhancing the precision and reproducibility of non-survival applications requiring invasive ventilation and exposure of thoracic organs in in vivo rat models.

Endotracheal intubation and subsequent ventilation are often basic requirements for translational research in rat models for various interventions that ...

Anesthesia and Intubation of Preadolescent Mouse Pups for Cardiothoracic Surgery

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Chapters in this video

Use of a Hanging Weight System for Coronary Artery Occlusion in Mice

Endotracheal Intubation in Mice via Direct Laryngoscopy Using an Otoscope

In vivo Measurement of the Mouse Pulmonary Endothelial Surface Layer

Mouse Pneumonectomy Model of Compensatory Lung Growth

A Closed-chest Model to Induce Transverse Aortic Constriction in Mice

A Mouse Model of Orotracheal Intubation and Ventilated Lung Ischemia Reperfusion Surgery

In vivo Calcium Imaging in Mouse Inferior Olive

Quantification of Biventricular Function and Morphology by Cardiac Magnetic Resonance Imaging in Mice with Pulmonary Artery Banding

Cardiac Response to β-Adrenergic Stimulation Determined by Pressure-Volume Loop Analysis

Endotracheal Intubation via Tracheotomy and Subsequent Thoracotomy in Rats for Non-Survival Applications