Tracheal intubation of anaesthetised rats to maintain the airway for adequate gas exchange is an essential step in many in-vivo experiments. However, this procedure is a considerable challenge to researchers due to the difficult anatomical characteristics of these animals which include a large tongue, long molars, small larynx, narrow trachea, rapidly mobile vocal cords and protruding soft palate.1 Accordingly, a number of methods and instruments have been devised to accomplish this procedure,2–6 yet most involve the application of complex techniques or the use of expensive devices with failure rates of up to 20% and high risk of periprocedural animal deaths.6 We wished to develop an easy and reliable method of tracheal intubation as a prerequisite for conducting subsequent in-vivo research. The aim of this study was to investigate the feasibility and safety of a novel method for orotracheal intubation in anaesthetised rats using currently available equipment and devices. We aimed to devise a technique, which produced wide opening of the oral cavity for clear visualisation of the vocal cords allowing intubation using a guidewire, and to compare this with a widely used technique7 from which our new method was modified.
All animal procedures and experimental protocols were approved by the Institutional Animal Care and Use Committee (IACUC) of the Taichung Veterans General Hospital (protocol number: La-97571; IACUC Chairman: Jaw-Ji Tsai; date of approval: 08 January 2009) and were carried out in accordance with the guiding principles of the American Physiological Society for the care and use of animals in research and teaching.
Sprague-Dawley rats weighing 250 to 350 g which were to undergo open-chest surgery in another research protocol (left coronary artery ligation to induce myocardial ischaemia) were obtained from the National Animal Center, Taipei, Taiwan, and housed under controlled conditioning (25°C constant temperature, 55% relative humidity, 12-h light cycle) and received standard pelleted diet and free water during the study period. All rats were anaesthetised with intraperitoneal pentobarbital (60 mg kg−1) and then placed supine on a specially designed platform for orotracheal intubation.
A specially designed oropharyngeal intubation wedge (roof 1 cm, bottom 2.5 cm, wedge angle 25°; Fig. 1a) was made from a 3-ml syringe (Perfect Medical Industry, CHANG HWA, Taiwan) to expand and maintain the oropharyngeal space, as previously described.7 A 16-gauge intravenous catheter (length 45 mm, internal diameter 1.7 mm, hub 20 mm; BD Angiocath Plus, Becton Dickinson Korea Ltd, Korea; Fig. 1B) was used as the tracheal tube for intubation. A 70-mm-long needle stylet with a 30-mm hub with the distal 5 mm (the sharp tip) removed (Fig. 1c), or a 0.025-inch (0.64-mm diameter) guidewire (part of the Radifocus Introducer II Standard Kit, Terumo Europe N.V., Leuven, Belgium) cut to 20 cm from the default 45 cm length (Fig. 1d) was used to guide the tracheal tube. A torque device (Radifocus Torque device, Terumo Europe N.V.; Fig. 1e) was used to manipulate the guidewire across the vocal cords. A 1-W light-emitting diode flashlight was used as a light source to visualise the oropharynx. A specially designed plastic platform (30 × 20 cm) was as attached to the head mount of a tripod head and fixed on a stand (Fig. 2). The platform angle could be freely adjusted or fixed to suit the user's needs.
Orotracheal intubation was initially performed using the Jou method.7 Briefly, rats (group Jou) were anaesthetised and placed flat in the supine position. The oral cavity was opened using the oropharyngeal wedge (Fig. 1a), and the vocal cord was illuminated using the torch aimed at the ventral area of the animal's neck. The trachea was intubated using the 16-gauge intravenous catheter (Fig. 1b) over a 70-mm stiff, malleable needle (Fig. 1c). For the new technique, anaesthetised rats (group New) were placed in dorsal recumbent position on the platform. The upper incisors were hooked and fixed by an elastic band onto the proximal end of the platform which was then elevated to an angle of 60 to 70° (Fig. 2) allowing the user to clearly visualise the oropharyngeal cavity and to help straighten the curve between the trachea and larynx. The rat mouth was opened with a vascular clamp and the oropharyngeal intubation wedge was inserted. The rim of the outer part of the intubation wedge was placed firmly against the inner side of upper incisors. The tongue was pulled laterally using atraumatic forceps and the torch was shone so that it illuminated the orifice of the trachea. The oropharyngeal intubation wedge was then adjusted anterosuperiorly to displace the soft palate in order to better facilitate intubation. When the vocal cords were clearly visualised, the 0.025-inch guidewire (Fig. 1d), controlled by a torque device (Fig. 1e), was advanced through the intubation wedge into the trachea (Fig. 3). The torque device was removed and the 16-gauge catheter was advanced over the wire into the trachea. The feel of the tracheal cartilage rings encountered while advancing the catheter helped to confirm the passage of the catheter in the trachea rather than the oesophagus. The wire and the oropharyngeal intubation wedge were then withdrawn.
After intubation, the catheter hub was connected to a rodent ventilator (Model 141, NEMI Scientific Inc., Framingham, Massachusetts, USA) delivering 55 to 60 breaths min−1 at a volume of 1.5 ml per 100 g. Correct tracheal positioning of the catheter was confirmed by the rise and fall of the bilateral chest wall.
Assessment of feasibility and safety
Feasibility of tracheal intubation was evaluated using the time for completion of the procedure, the number of attempts needed, the difficulties encountered during the procedure (vigorous gag reflex interrupting intubation, excess salivary secretion hindering clear vision of the larynx and unusual resistance restricting smooth advancement of the tube into trachea) and the overall success rate. Safety was assessed by recording oesophageal malpositioning, macroscopic laryngeal injury, massive oral bleeding or procedure-related mortality. Finally, after completion of the open-chest surgical procedure and removal of the heart, the chest cavity was opened and the trachea exposed. The position of the tip of the tracheal tube was determined using an inverted microscope at ×5 magnification. The integrity of the trachea between the vocal cords and the catheter tip position was examined histologically by a researcher blinded to group allocation using hematoxylin–eosin staining.
Continuous variables were expressed as mean (±SD) or median (interquartile range). Normally distributed continuous data were compared between groups using unpaired Student's t-test, whereas nonparametric continuous data were compared using the Mann–Whitney U-test. Statistical significance was defined as a P value less than 0.05. All analyses were performed using SPSS software version 10.1 (SPSS Inc., Chicago, Illinois, USA).
Each technique for intubation was practiced on two rats as a learning exercise. The techniques were then used in 35 consecutive rats in group Jou and then 50 consecutive rats in group New. The characteristics of animals and the consequences of orotracheal intubation are summarised in Table 1. With both techniques the vocal cords of all animals were clearly visible. When the new, modified method was used, it was completed more smoothly, rapidly and successfully in all 50 animals at the first attempt without intra-oesophageal malpositioning, oral cavity bleeding or other major complications compared with Jou's method (Table 1). All animals in group New completed the open-chest surgery without limb cyanosis or air leakage from the lungs. There were no complications with the new, modified intubation technique.
At autopsy, the tip position of the tracheal tube was located at 12 to 16 mm from the tracheal carina in all animals in both groups without any identifiable gross tracheal mucosa injury. Microscopic examination of the tracheal segment near the tip of the tracheal tube indicated that the epithelial lining was focally interrupted and cilia damaged in 17 of 33 rats in group Jou (Fig. 4a), but was intact in all animals in group New (P < 0.001, Fig. 4b).
Blind tracheal intubation was the prevailing practice for laboratory rats several decades ago,8,9 but has largely been abandoned in recent years due to the relatively low rate of success and the high incidence of complications. Currently, most orotracheal intubation techniques involve vocal cord visualisation before intubation of the trachea which appears to be the key to technical success, yet these techniques usually require a longer learning curve and the use of complex, bulky or expensive devices. The instruments used to facilitate tracheal orifice identification include those used for opening the oral cavity space, such as paediatric laryngoscope blades,10 specially designed laryngoscopes with a spatula,11 otoscope cones12 and modified nasal speculum,13 as well as instruments used for directly revealing the location of tracheal entrance, such as videoendoscopes.14,15 However, the main disadvantage of almost all devices to open the oral cavity is that they obscure direct visualisation of the vocal cords and trigger pharyngeal–laryngeal reflexes, resulting in an overall success rate (80 to 100%) no better than that which can be obtained with other conventional and simpler methods. Videoendoscopic equipment, on the contrary, is costlier and takes longer to master compared with simpler tools. In addition, the operator must overcome the geometric mismatch between the real oral cavity and the video images, something which requires experience.
The use of a hollow oropharyngeal wedge made of a 3-ml syringe to keep the oropharyngeal cavity open was introduced by Jou et al. Using this device, the vocal cords can be directly visualised through the inner lumen of the syringe with the assistance of a light pointed onto the rat's neck.7 In their report, this method allowed completion of tracheal intubation within 30 s in all rats. However, in our hands (experienced animal researchers using tracheal intubation routinely for in-vivo experiments), only 86% of rats could be intubated after an average of three attempts using their method. The relatively low success rate and the high incidence of periprocedural complications were mostly attributable to the stiff guiding stylet which stimulated brisk gag and salivary reflexes, but provided poor guidance for the tracheal tube to cross the vocal cords. In contrast, these shortcomings were overcome with our new method. The thin (0.025 inch) guidewire offers advantages over a 0.035-inch guidewire6 or other 20-cm long, 1-mm thick guidewire12 used elsewhere, due to its soft tip and thinner shaft which can easily and less traumatically cross the rat's rapidly vibrating vocal cords (50 to 70 Hz) and the narrow opening (1.5 to 2-mm diameter)9 without triggering vigorous gag and salivary reflexes. In addition, the torque device (usually used for delicate human percutaneous coronary intervention) enhances the manoeuvrability of the guidewire as it enters the trachea. Furthermore, the oropharyngeal cavity of the rats can be visualised by directly illuminating it with a light source which helps to prevent inadvertent injury and malpositioning of the guidewire in the oesophagus. With the rat placed in the dorsal recumbent position at 60 to 70° tilt, the operator was able to easily visualise the larynx and vocal cords from above without needing to adopt an uncomfortable posture. With the upper incisors fixed by an elastic band, the weight of the rat's lower body stretches the torso downwards, helping to open the oropharyngeal cavity and further straightening the angle between the trachea and larynx and facilitate the introduction of the tracheal tube.
In summary, tracheal intubation in rats was accomplished more safely and reliably with a slanted platform, an oropharyngeal intubation wedge, a guidewire and torque device and a modified tracheal tube. All of these components can be easily assembled in a typical animal laboratory which conducts cardiovascular experiments, or can be acquired at low cost. The entire intubation procedure can be completed quickly by a single person without the aid of assistants.
Tracheal intubation in anaesthetised undergoing experimental surgical procedures was easily and rapidly performed using a new, modified technique and conventional instruments with a 100% success rate and no complications. This technique may be considered the method of choice to quickly establish a secure airway in rats undergoing experimental procedures.
Assistance with the study: none declared.
Financial support and sponsorship: this work was supported in part by the grants from Taichung Veterans General Hospital (TCVGH-996301C and TCVGH-993106C) and National Science Council of the Republic of China Research Grant (NSC 99–2314B-075A-007-MY3).
Conflicts of interest: none declared.
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