A new method for induction of the cough reflex.

A new method for artificially inducing the cough reflex in the unrestrained and unanesthetized dog has been devised. The present method can be utilized for testing the relative effectiveness of antitussive drugs. A monopolar electrode is in serted into the dog tracheal mucosa in such a way as to minimize surgical stress. The most successful electrical stimulation has been obtained from square-wave pulses which were produced by a 1-3 volt, 20 Hz. The duration of the square-wave pulse was 1.0 msec and the stimulation was applied for 5 sec. This stimulation usually produced 3-5 cough reflex. It has been found that electrical stimulation of the tracheal mucosa induces the cough reflex and this phenomenon can be successfully reproduced at inter vals of at least 5 minutes duration. Our method allows for a lower threshold voltage for cough induction than those previously published, a greater utilization of each dog and a reduction of time required to standardize the cough reflex and the drug dosage for each animal. As the dogs are caged, there is more freedom of movement, thus the drug effect on posture and general behaviour can be better observed. The method described may be applicable to induction of the cough reflex in a variety of experimental animals. Following Craigie's first experiments (I) on the artificially induced cough reflex in the dog, Larsell & Burget (2) and Ernst (3) further investigated mechanical and chemical sti mulation of the tracheal mucosa to induce coughing in experimental animals. The known methods for cough induction were developed only after extensive research. These can be classified as due to electrically (4-14), mechanically (2. 3, 9, 15-21), and chemically (2, I6-18, 20, 22-25) induced stimulation. The receptors for cough induction can be divided into: a) the external mechanical stimulation receptor, which is situated in the upper tra cheal mucosa; b) the chemical stimulation receptor, located in the trachea and throughout the bronchial trees (26). Since coughing is also induced by inhalation of hot or cold air, the cough reflex cannot be attributed solely to mechanical or chemical stimulation of the receptors. It is thus feasible to consider coughing as originating in the trachea regardless of the stimulation. It is necessary that any artificially induced coughing closely resemble the mechanism of that which occurs naturally. Previously reported experimental methods have not proven satisfactory and to induce a reproducible cough and minimize injury to the trachea, various methods of cough induction have been investigated in our laboratory (4, 5). In the present study, the cough reflex was induced electrically, in the unrestrained and unanesthitized dog, by mean of a flexible electrode. This method induces a cough which can be easily reproduced and facilitates study of the effectiveness of antitussive drugs. 1. Experimental animals The animals used in these experiments were healthy mongrel dogs weighing 7-10 kg and guinea-pigs weighing 500-800 g. Both males and females were used. Food and water were provided ad libiturn and the animals were maintained in a constant environment. The temperature and humidity were kept at 21'C and 60%, respectively. 2. Stimulating electrode The electrodes were inserted in the dog trachea before each experiment. They were made of stainless steel wire (0.1 mm diameter) and coated with a thin polyvinyl film (Fig. 1). This coated electrode was sheathed by a stainless steel needle (size No. 1,12, Natsume). An adhesive Alon-alpha (Toagosei Chemical) was used for fixation of the polyvinyl coated electrode and the stainless steel needle sheath and also as insulation. Before use, the resis tance of each electrode was tested. Each electrode was allowed to stand in water for a few minutes, after which it was wiped dry with gauze and the resistance between the needle sheath and the central electrode was measured by a galvanometer. Elec trodes with a resistance greater than 25 MSJ between the central core and stainless steel sheath were used. Electrodes used for guinea-pigs were unsheathed since the guinea-pig trachea is too narrow to accommodate the same type of electrode used for dogs. A stainless steel needle (size No. 113, Natsume) placed arbitrarily into the muscle behind the ear was used as an indiffereni electrode against the electrode inserted intc the trachea of animals. FIG. 1. Electrode for stimulation of the tra cheal mucosa in the dog. Fic. 2. Electrode introduction into the dog trachea. 3. Induction of cough reflex Dogs: The sheathed electrode was further threaded through a guiding cannula (needle of size No. 1 / 1, Natsume) until the tip touched the trachea (Fig. 2). The elec trode was introduced through a shaved portion of the neck 4 cm below the bottom of the thyroid cartilage. The tip of the electrode was bent at an angle of 30 -60 before insertion into the guiding cannula (Fig. 1). The central core of the electrode was made of stainless steel wire with moderate flexibility to enable the tip of the electrode to pierce the tracheal mucosa without injuring the trachea. After insertion and placement of the electrode were completed, at least 5 minutes were allowed to pass as a short period of time is required to allow for the induction of coughing due to insertion. The guiding cannula was pulled out as soon as the electrode had been inserted into the trachea. Guinea-pigs: Since the guinea-pig trachea is narrower than that of a dog, the electrode used was unsheathed and was covered only with a guiding cannula (needle size No. 1/2, Natsume). The non-anesthetized animals were fixed in a supine position and the electrode was introduced into the trachea. General operation to induce cough reflex to the tracheal mucosa: The parameters of electrical stimulation necessary to induce 3-5 coughs were a square-wave pulse with a 20 Hz frequency, the duration of pulse 1.0 msec and the duration of application 5 seconds. The specific voltage necessary for cough stimulation and the site of electrode placement were carefully determined for each individual animal. When stimulation caused excessive cough ing, the site of placement of the electrode was shifted upward (toward the throat). This prevented a too rapid exhaustion of the animal and prolonged the time during which coughs could be reproduced. When the site of optimal stimulation was obtained, the electrode was fixed to the neck with vinyltape. Macroscopical examination of the stimulation site of the tracheal mucosa of the dogs and guinea-pigs was performed on the same day as stimulation, and three, four and seven days after stimulation. 4. Recording of f the cough reflex A respiratory pick up (Nihon Kohden, MCR-2TA) was applied to the thorax of the dogs or guinea-pigs, and coughing was induced utilizing electric current. The respiratory pick up was connected to a pen recorder (Nihon Kohden, RM-150) through an amplifier (Nihon Kohden, RB-2). The amplitude of the pen movement was found to be in parallel with the severity of the cough. Studies were conducted in a shielded cage (Fig. 3). 5. Standard criteria for evaluation of the autitussive effect of the drugs Fic. 3. Unrestrained and non-anesthetized dog in a shielded cage. The amplitude and frequency of coughs were recorded continuously both before and after drug administration, on a pen recorder (Nihon Kohden, RM-150). Prior to drug administration, the amplitude of each cough was measured with a cm scale, and this average was multiplied by the number of coughs. At intervals of 5, 10, 15, 30, 45, 60, 90 and 120 min after drug administration, determination of the ampli tude was made and the number of coughs counted. The efficacy of the drug was defined as the percent decrease of amplitude multiplied by the number of coughs as a function of time after drug administration when a drug was found to be effective. The degree of antitussive activity of each drug was assessed by plotting the percentage of response inhibition as a function of time for the first 30 min after drug administration and determining the area under the curve. Maximal antitussive drug activity occurred during the 30 min period after drug administration and in order to determine the effectiveness of each drug, it was compared to the cough severity before the drug had been given. ED50 was calculated by the method of Litchfield and Wilcoxon (27). 6. Drugs used In the present experiment, the following antitussive drugs were investigated, morphine hydrochloride (Takeda), codeine phosphate (Sankyo), N-2-Pyridylmethyl-N-phenyl-N-2 piperidinoethyl amine hydrochloride (picoperidamine hydrochloride, Takeda), 1-(2-hydroxy ethoxyethyl)-4-p-chlorobenzylpiperidine hydrochloride (piclobetol, Sankyo) and 14-hy droxydihydro-6fl-thebainol-4-methyl ether (oxymethebanol, Sankyo).