As reported in the literature, oral endotracheal intubation of rats is considered to be very difficult. Specialised equipment and complicated techniques have been ...
Oral endotracheal intubation of rats for intratracheal instillation and aerosol drug delivery Rosario Lizio1,2 , Andreas Westhof2 , Claus-Michael Lehr1 & Thomas Klenner2 1
Department of Biopharmaceutics and Pharmaceutical Technology, Saarland University, 66123 Saarbruecken, Germany and 2 ASTA Medica AG, 01277 Dresden, Germany
Summary As reported in the literat ure, oral endotracheal intubat ion of rats is considered to be very difficult. Specialised equipment and complicat ed techniques have been described to perform this procedure. In our experiment we adopted a simple method, which allowedÐwithout any complicated equipmentÐthe insertion of a relatively wide tube into the trachea of rats, allowing drug administrat ion. Keywords Endotrac heal intubat ion; rat; large tube; intrat racheal aerosol drug administrat ion
Endot racheal intubation in small laborat ory anim als is often used for studies of pulmonary absorption and the bioavailabi lity of various macromolecules (Patton e t a l. 1994, Smith e t a l. 1994, Niven e t a l. 1995, Lizio e t a l. 2000 ). Endotrac heal intubation allows the delivery of test substances both in ¯uid form (solution or suspension) as well as by aerosol (Osaier & OberdoÈrster 1997 ). Endotracheal intubation of rats is problemati c because of the narrow oral cavity and glottis, the dif®culty in visualizing the trachea, and the mobility of the larynx even under narcosis. Several techniques have been developed for endotracheal intubation in rats but most of them require special skill, e.g. blind oral tracheal intubation (Stark e t a l. 1981 ); or elaborat e and specially designed equipm ent, e.g. a miniature laryngoscope (Proctor & C o rre spond e nc e to : Dr Ro sa rio Lizio , O pera tive Rese a rc h Life -Scie nc e, Ave ntis R& T G m b H & Co KG, Industria l Pa rk Ho ec h st, 65926 Fra nk furt a m Ma in, G e rm a ny E-m a il: lizio @crt.h o e ch st.co m Accepted 27 March 2001
Fernando 1973 ), a ®bre-optic laryngoscope (Costa e t a l. 1986 ), or an otoscope (Weksler e t a l. 1994 ). Most of these methods are associated with a high failure rate and require considerable practice to ensure successful intubati on without oropharyngeal or endotracheal damage. Moreover, for endotracheal aerosol drug delivery, large tubes are needed in order to reduce aerosol condensation or deposition on the tube’s walls. T his ensures tight contact between the tube and trachea, which avoids loss of air or aerosol during application. A relatively simple intubation can be performed using a very thin cannula, as described by Yasaki and Dyck (1991) but this method is only applicable for endotracheal instillation and can produce tracheal traum a and bleeding. We have therefore developed a simple, fast and reliable method for endotracheal intubat ion in the rat that utilizes readily available normal laboratory utensils, and which allows instillation as well as delivery of aerosol for pulmonary drug studies.
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Materials and methods Ma te ria ls Male Sprague±Dawley (Mol:SD) rats weighing between 250 and 300 g were purchased from Mollegaard-Bom holtgaard (Ry, Denmark ) and used for experiments after an acclimatization period of about one week after arrival. Until the start of the experiment, the anim als were housed in Mac rolon type IV cages in groups of three, four or ®ve rats, maintained under speci®c pathogen free (SPF) conditions and given acidi®ed water (pH 3) and lab chow a d lib itu m . Standard food (Altromin pellets 1324) was purchased from Altromin GmbH (Lage, Germany). T he anim als were housed under a minimum of 15 air changes per hour at a temperature of 22 1 C with relative humidity of 55 5% in a 12 =12 light =dark cycle. T he anim al experiments were performed according to the `Principles of Laboratory Animal Care’ enforced by German Federal Regulations. T he experimental protocol has been approved by the Ethical Committ ee of the State of Hessen, Germany. Atropin used for the premedicati on was purchased from Sigma-Aldric h Chemie GmbH (Deisenhofen, Germany). Xylazi ne (Rompun 1 ) and ketam ine (Ketavet 1 ) used for anaesthesia were purchased from Bayer AG (Leverkusen, Germany) and Upjohn GmbH (Heppenheim, Germany), respectively. Oral endotracheal intubat ion was performed using a vein cat heter, 50 mm in length, 1.7 mm i.d., 2.1 mm o.d. (B. Braun Melsungen AG, Melsungen, Germany).
Meth o d T he rats were anaesthetized with a mixed solution of xylazine (8 mg=kg body weight) and ketam ine (80 mg=kg body weight) by i.m. injection. Bepanthen 1 ocular cream was applied to avoid dry corneas and subsequent eye discomfort during recovery. Ten minutes before anaesthesia the animals were medicated with at ropin by i.m. injection (50 mg=kg) under a light carbon dioxide anaesthesia, as indicat ed by Kohler e t a l. (1999 ). T his method was chosen to avoid disturbance in hormone parameters that occur when rats are injected directly, as Laboratory Animals (2001) 35
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observed by previous tests (data not shown). Atropine premedication dim inishes the sensibility and motility of the larynx, and salivary secretion, and facilitates intubation. Alternatively, local anaesthesia could be performed, as indicated by Kramer e t a l. (1998 ). On the establishment of surgical anaesthesia, as indicated by the absence of pain re¯exes, the anim als were suspended by the superior incisors on a metal frame attac hed to a board tilted at around 30 to a supine position. By using a larger inclination the abdom inal weight produces a deepening of the pre-diaphragmal region, resulting in a stretching of the respiratory tract and a light restriction of the trachea’s lumen (practical observation). A cylinder with a diameter of 1.5 cm was placed under the neck in order to position the larynx, pharynx and trachea on a straight line. T he mouth was kept open using ¯at tweezers, which contemporaneously acts to ®x the tongue. Alternati vely, as indicat ed by Yasak i and Dyck (1991 ), small forceps (iris forceps, 4-inch half-curved, Medix Inc. Madison, WI) could be used. T he trachea was visualized by percutaneous transillumination using the light of a 12 volt high-intensity lamp (Heiland GmbH, Ham burg, Germany) emitt ed from the end of a ¯exible ®ber-optic rod, positioned at the surface of the skin near the pharyngoepiglottic region of the rat’s neck. In this manner the vocal cords and the tracheal lumen were clearly visible. T he tracheal lumen appears as a luminous point where the vocal cords restrict to form a V (see Fig 1). For the intubation a modi®ed 16 gauge needle was used as a stylet for the polyethylene tube. T he needle was shortened so that it was 3 mm shorter than the catheter. T he catheter, in which the distal end was cut diagonally at a 30 angle, can be inserted using the following manoeuvre: the tip of the tube must be positioned to the right, if intubati on is to be performed from the right side of the month. T he tip of the tube can then be introduced between the vocal cords (see Fig 1). Before the complete introduction, the tube must be rotated by 90 along its axis in order to position the tip directly into the tracheal lumen, which will be found at the superior side (ventral side of the animal, see Fig 1). At this point the tube can be pushed
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experiments the anim als were autom ati cally ventilated for about 10 min with a tidal volume of 6 ml and at a frequency of 70 strokes per minute. Within this tim e an ultrasonic nebulizer connected to the system was activated for 5 min and a nebulized solution was transported by the air ¯ow to the animal’s lungs. Following completion of the procedure, the animals were kept in a box at a temperature of about 37 C until they recovered. In the subsequent period(about 72 h) the anim als did not show any sign of respiratory distress and rapidly regained their normal activity.
Discussion Fig 1 Explanation of the intubation manoeuvre. The canula is inserted between the vocal cords and then routed 90 in order to directly and exclusively reach the trachea
directly into the trachea and the needle can be withdrawn. T he orotracheal cat heter was then advanc ed into the trachea to the point at which its proximal end was level with the superior incisor. Placing a small mirror at the proximal end of the cannula, and observing the condensation of water vapour from the exhaled air enabled the correct placement of the cannula.
Results T his method of intubati on was used in more than 100 rat s for aerosol application experiments. In the majority of the cases, the intubation was performed successfully within 2± 3 min of induction of anaesthesia. Occasionally, the catheter was erroneously inserted in the oesophagus but this was attribute d to an elevated pharyngoepiglottic sensibilit y which resulted in a strong deglution re¯ex and increased mobility of this region. T his was remedied by giving more anaesthetic. In contrast to other methods, such as those previously reported by Yasaki and Dyck (1991 ), no periglottic trauma or tracheal bleeding occurred (visual observati on after extubation and lung dissection). Because of the tube’s dim ension, the catheter did not escape from the trachea during aerosol applicat ion. In our
In the last 20 years a wide variety of studies describing pulmonary absorption have been performed. A variety of methods have been described in order to deliver liquid or aerosol to the lungs of laborat ory animals, especially rats, and to determine distribution, tolerability and absorption of therapeutics (Pritchard e t a l. 1985 ). Unfortunately, in the majority of studies intubation techniques have been described inadequately, are very complicat ed or require special equipment. T his has resulted in a plethora of techniques for oral endotracheal intubation as the method of choice for pulmonary applicat ion of both liquids and aerosols. In our study we demonstrate that oral endotracheal intubation of rats is very simple to perform without any special equipment and that the adopting of a diagonally cut tube, and the use of the above described manoeuvre, allow a relatively large catheter to be easily introduced into the trachea. T he use of a large tube for intubati on allows studies of intratrac heal instillati on as described by Lizi o e t a l. (2000 ) and allows intratrach eal aerosol delivery. Ac k no w le d gm e nts T he authors would like to thank Dr D. Marx for helpful discussion on the development of this method of endotracheal intubation and Mr H. GemuÈnde for research of the materials used.
References Costa DL, Lehmann JR, Harold WN, Drew RT (1986) Transoral tracheal intubation of rodents using a ®beroptic laryngoscope. La b o ra to ry Anim a l Scie nc e 36, 256±61 Laboratory Animals (2000) 35
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Kramer K, Grimbergen JA, van Iperen DJ, van Altena K, Voss H-P, Bast A (1998) Oral endotracheal intubation of guineapigs. La b o ra to ry Anim a ls 32, 162±4 Kohler I, Meier R, Busato A, Neiger-Aeschbader G, Schatzmann U (1999) Is carbon dioxide (CO 2 ) a useful short acting anaesthetic for small laboratory animals? La b o ra to ry Anim a ls 33, 155±61 Lizio R, Klenner T, Borchard G, Romeis P, Sarlikiotis AW, Reissmann T, Lehr C-M (2000) Systemic delivery of the GnRH antagonist cetrorelix by intratracheal instillation in anesthetized rats. Euro pe a n Jo urna l o f Ph a rm a ceutica l Scie nc e 9, 253±8 Niven RW, Whitcomb KL, Shaner L, Ip AY, Kinstler OB (1995) T he pulm onary absorption of aerosolized and intratracheally instilled rhG -CSF and monoPEG ylated rhG-C SF. Ph a rm a c eutic a l Re se a rc h 12, 1343±9 Osaier M, OberdoÈrster G (1997) Intratrachel instillation vs intratracheal inhalation: differences in particle effects. Fund a m e nta l a nd Applie d To xico lo gy 40, 220±7 Patton JS, Trinchero P, Platz RM (1994) Bioavailability of pulmonary delivered peptides and proteins:
Laboratory Animals (2001) 35
Lizio et al.
a-interferon, calcitonins and parathyroid hormones. Jo urna l o f C o ntro lled Re le a se 28, 79±85 Pritc hard JN, Holmes A, Evans JC, Evans N, Evans RJ, Morgan A (1985) T he distribution of dust in the rat lung following administration by inhalation and by single intratracheal instillation. Enviro nm enta l Re se a rc h 36, 268±97 Proctor E, Fernando AR (1973) Oro-endotracheal intubation in the rat. British Jo urna l o f Ana e sth esia 45, 139±42 Smith PL, Marcello J, Chiossone DC, Orner D, Hildalgo IJ (1994) Absorption of an RGD peptide (SK & F 107760) following intratracheal administration in rats. Inte rna tio na l Jo urna l o f Ph a rm a c e utic s 106, 95±101 Stark RA, Nahwold ML, Cohen PJ (1981) Blind oral tracheal intubation of rats. Jo urna l o f Applie d Ph ysio lo gy 51, 1355±6 Weksler B, Ng B, Lenert J, Burt M (1994) A simpli®ed method for endotracheal intubation in the rat. Jo urna l o f Applie d Ph ysio lo gy 76, 1823±5 Yasaki S, Dyck PJ (1991) A simple method for rat endotracheal intubation. La b o ra to ry Anim a l Scie nc e 41, 620±2
