Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

The ₂-adrenoceptor agonists (₂-agonists) are becoming increasingly popular in veterinary and human medicine for use as anxiolytics, analgesics, and preanesthetic sedatives (Maze and Tranquilli, 1991). In veterinary practice, the relatively new ₂-agonist, (4-[1-(2,3-dimethylphenyl)ethyl]-1H-imadazole) hydrochloride (medetomidine), is widely used in Europe and Australia for sedation and to reduce the general anesthetic requirement (Cullen, 1996). This drug is 7 times more selective for ₂-adrenergic receptors than the prototype ₂-agonist, clonidine (Virtanen, 1989). The dextroisomer of medetomidine, dexmedetomidine, is being developed for use in human anesthetic practice. Dexmedetomidine has been used in the perioperative period to provide sedation and anxiolysis (Aantaa et al., 1990a), to reduce opioid, thiopental, and inhalation anesthetic requirements (Aantaa et al., 1990b), and to reduce hemodynamic instability in humans (Aantaa et al., 1990b).

It has been known for some time that the ₂-agonist xylazine decreases partial pressure of oxygen in arterial blood (PaO₂) in cattle (DeMoor and Desmet, 1971), goats (Kumar and Thurmon, 1979), and sheep (Doherty et al., 1986; Nolan et al., 1986). The degree of hypoxemia in sheep after sedation with clonidine (Eisenach, 1988) or xylazine (Nolan et al., 1986; Doherty et al., 1986) is quite severe. We recently reported that three newer ₂-agonists (detomidine, medetomidine, and romifidine) also produce severe hypoxemia when administered i.v. at equipotent sedative doses in conscious sheep (Celly et al., 1997a,b). The level of hypoxemia was similar with the five ₂-agonists studied, irrespective of their differences in selectivity for ₂- versus ₁-adrenoceptors (Celly et al., 1997a). A selective ₂-agonist that does not cross the blood brain barrier, 2-(2,6-diethylphenylamino)-2-imidazol (ST-91), also produced a comparable level of hypoxemia, suggesting involvement of a peripheral component in the development of hypoxemia (Eisenach, 1988; Celly et al., 1997b). The hypoxemia was not caused by hypoventilation and was not due to postural changes after drug administration (Celly et al., 1997a,b). It was accompanied by significant changes in respiratory frequency and in the maximum change in transpulmonary pressure (Ppl) required for tidal volume (V_T) breathing; heart rate (HR) and mean arterial pressure (MAP) were less affected. Others have reported that the development of hypoxemia occurs even when the animal is ventilated artificially (Nolan et al., 1986; Eisenach, 1988). Although these studies suggest a drug-induced increase in the proportion of blood flow going through the lungs without becoming oxygenated, i.e., intrapulmonary shunt fraction (Q_s/Q_t), as one possible mechanism underlying ₂-agonist-induced hypoxemia, the origin of this increase in shunt fraction remains unclear.

From the studies reported to date, it is not possible to separate the relative contribution of pulmonary and cardiovascular alterations with respect to the development of hypoxemia in sheep. The doses used were clinically useful sedative doses, and these doses produce significant circulatory alterations (Campbell et al., 1979; Cullen, 1996). It is quite possible that a fall in cardiac output (t) and mixed venous oxygen saturation magnified the apparent degree of venous admixture associated with any ventilation/perfusion mismatch (McDonell, 1996).

The present study was conducted to investigate the relative contribution of the cardiovascular and respiratory systems to ₂-agonist-induced hypoxemia in ruminants. To test the hypothesis that the pulmonary alterations are paramount and that these effects can occur without central ₂- receptor involvement, the effect of incremental doses of the central and peripheral acting ₂-agonist, medetomidine, the peripherally acting ₂-agonist, ST-91, and a saline placebo were studied in halothane anesthetized, ventilated sheep.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

Experimental Animals. Five adult female Arcot sheep weighing 80 to 90 kg [mean body weight 84 ± 1.7 S.E.] were used in the study. Each animal was used on three different occasions to study the cardiopulmonary effects of placebo (physiological saline), medetomidine, and ST-91. A minimum of 7 days separated each experiment and the order of treatment was randomized. The study was approved by the institutional Animal Care Committee and followed the guidelines of the Canadian Council on Animal Care.

Instrumentation. The sheep were positioned in a custom designed restraint device that served to minimize the positional effects of anesthesia on pulmonary function. The base of the wooden stock had four holes cut to permit the animal's legs to protrude such that the sheep rested comfortably on its sternum and abdomen on a pad of foam 15 cm thick. Sternal recumbency is associated with less interference of gas exchange in anesthetized large mammals than lateral or dorsal recumbency (McDonell, 1996). Anesthesia was induced with pentobarbital sodium (20 mg/kg i.v.), a cuffed endotracheal tube (diameter 9.5-10.5 mm) was inserted, and halothane/O₂ was used for maintenance of anesthesia with intermittent positive pressure ventilation (IPPV) to maintain eucapnia. Muscle paralysis was induced with 0.2 mg/kg atracurium (Tracrium, Burroughs Wellcome, Research Triangle Park, NC) administered i.v., and the level of muscle relaxation was monitored with a peripheral nerve stimulator (Innervator, Fisher & Paykel Electronics Ltd., Auckland, NZ) using electrodes applied to the ulnar nerve. Total absence of muscle response to a train of four 20-mA stimuli was maintained throughout the study by the supplemental administration of atracurium (usually 0.1 mg/kg i.v.) as needed. Airway gas concentrations (inspired O₂ and end-tidal halothane and CO₂) were monitored on a breath-to-breath basis using a rapid response monitor (Criticare 1100 Patient Monitor, Criticare System Inc., Waukesha, WI). The gas analyzer was calibrated at the beginning and end of each experiment using known concentrations of halothane, O₂, and CO₂ (Anesthesia calibration gas, Criticare System Inc., Waukesha, WI). A constant volume, electronically cycled, sinusoidal airflow ventilator (Harvard pump, Harvard Apparatus Co., Inc., Dover, MA) was inserted into the inspiratory side of the anesthetic rebreathing circuit to produce constant volume IPPV. The pump was set to deliver a constant V_T (700 ml) with respiratory frequency at approximately 8/min. Breathing frequency was adjusted as needed to maintain eucapnia [partial pressure of carbon dioxide in arterial blood (PaCO₂) ranging between 40-45 mm Hg], whereas V_T was kept constant.

Drugs. The drugs tested were the relatively selective ₂-adrenoceptor agonists medetomidine (molecular weight = 236.7) and ST-91 (molecular weight = 253.8). Medetomidine was available as a 1 mg/ml solution (Domitor, Orion Corporation, Farmos, Turku, Finland), whereas ST-91 (Boehringer Ingelheim, Ridgefield, CT) was dissolved in 0.9% saline to make a final concentration of 1 mg/ml. All doses have been expressed as salts.

Experimental Protocol. After the onset of anesthesia and instrumentation, more than 1 h was provided as a stabilization period. Any gas accumulation in the rumen was expressed and then the lungs were "sighed" as follows to ensure a previous volume history for pulmonary mechanics measurements (Wheeler et al., 1990). The animal was disconnected from the pneumotachograph and anesthetic machine/ventilator at the level of the endotracheal tube and any secretions in the upper airways were suctioned out. The endotracheal tube was then attached to a second anesthetic circuit containing 1% halothane in oxygen and a 3-l rebreathing bag. Using this circuit, the sheep's lungs were expanded to an inflation pressure of 30 cm of H₂O over 3 to 5 s, after which passive exhalation to functional residual capacity (FRC) was permitted. This maneuver was repeated three times before the animal was reconnected to the pneumotachograph and primary anesthetic circuit. A 2-min period of constant volume IPPV was permitted to provide for stabilization of cardiovascular, respiratory, and end-tidal gas measurement before baseline (pretreatment) measurements of respiratory data, vascular pressures, and t were obtained. At this time, arterial and mixed venous blood samples were collected simultaneously for subsequent blood gas analysis and TXB₂ determination. After baseline sampling, the first dose of the test drug (0.5 µg/kg for medetomidine, 1.5 µg/kg for ST-91, or 2.0 ml saline) was then given i.v. diluted to a 2.0 ml volume. Thereafter, cardiovascular measurements were made at 3, 10, and 20 min, and respiratory measurements were made at 2, 5, 10, and 20 min.

Statistical Analysis. A general survey of the data showed that, after administration of each dose, the peak drug effect for the variables in individual animals was either 3 or 5 min postdrug administration, irrespective of the variable. The effects tended to decrease in intensity by 20 min. To represent the overall response of a variable after each dose administration, a weighted mean was calculated from the values at 3, 10, and 20 min postdrug administration for cardiovascular variables and from values calculated at 2, 5, 10, and 20 min postdrug administration for respiratory variables. This resulted in five sampling interval values; i.e., pretreatment baseline values and weighted mean values after dose I, II, III, and IV. The data were then subjected to two-way ANOVA for repeated measures to test for significance (p  .05) of treatment over time, as well as for differences between treatments and the placebo (Dawson-Saunders and Trapp, 1990). When a significant effect of treatment was observed, comparisons were performed between treatments using one-way ANOVA and a post hoc least significant difference (LSD) test. To account for repeated measures in the experimental design, the LSD was calculated using  values corrected by Bonferroni's method to control the overall level of significance (p  .05; Dawson-Saunders and Trapp, 1990). The results have been presented as the average of weighted mean ± S.E.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

Placebo-Treated Group. No significant changes were observed in any of the variables throughout the length of the experiment in the placebo group; however, C_dyn tended to decrease, whereas R_L tended to increase over the period of the experiment (Fig. 1).

View larger version (21K): [in this window] [in a new window]   Fig. 1.   The effect of four doses of saline (), medetomidine (), and ST-91 () on Cdyn (A), RL (B), and Ppl (C). Baseline measurements were obtained in the halothane-anesthetized ventilated sheep (n = 5) before administration of dose I. Doses I to IV for medetomidine were 0.5, 1.0, 2.0, and 4.0 µg/kg; for ST-91 were 1.5, 3.0, 6.0, and 12 µg/kg; and for the placebo treatment were 2.0 ml saline. Significant differences (*) between values obtained with placebo treatment and either medetomidine or ST-91 treatment at each dose level are shown (p  .05; two-way repeated measures ANOVA followed by LSD test with Bonferroni's correction).

Medetomidine-Treated Group. A significant decrease in C_dyn was seen within 2 min of dose I; this response was repeated after doses II, III, and IV (Fig. 1). An increase in Ppl and R_L occurred with dose I and subsequent doses (Fig. 1). A significant decrease in PaO₂ was observed with all doses (Fig. 2), with the maximum decrease after the first dose (to 214 mm Hg after 0.5 µg/kg medetomidine i.v. versus 509 mm Hg in placebo-treated animals). The decrease in PaO₂ was accompanied by a significant increase in PaCO₂ after doses I, II, and III, with a maximum increase to 50.3 mm Hg after dose I (Fig. 2). A significant increase in P(A-a)O₂ was present after all doses (Fig. 2). No significant changes were seen in PVR, Ppa, and Ppaw after any dose of medetomidine (Fig. 3). There was a significant decrease in MAP after doses I and II, but not after doses III and IV (Fig. 4), whereas HR did not change. Cardiac index and SV did not change, whereas SVR decreased after doses I and II, but was unchanged after doses III and IV (Fig. 4). Shunt fraction was increased after each dose of medetomidine; however, the greatest increase (27%) occurred after dose I (Fig. 4). No changes were seen in arterial pH, packed cell volume, TPP, and base excess. Similarly PO₂ did not change after medetomidine, and there were no significant changes in arterial and mixed venous blood concentrations of TXB₂ when compared with the placebo-treated animals.

View larger version (22K): [in this window] [in a new window]   Fig. 2.   The effect of four doses of saline (), medetomidine (), and ST-91 () on PaO2 (A), PaO2 (B), and P(A-a)O2 (C). Baseline measurements were obtained in the halothane-anesthetized ventilated sheep (n = 5) before administration of dose I. Doses I to IV for medetomidine were 0.5, 1.0, 2.0, and 4.0 µg/kg; for ST-91 were 1.5, 3.0, 6.0, and 12 µg/kg; and for the placebo treatment were 2.0 ml saline. Significant differences (*) between values obtained with placebo treatment and either medetomidine or ST-91 treatment at each dose level are shown (p  .05; two-way repeated measures ANOVA followed by LSD test with Bonferroni's correction).

View larger version (20K): [in this window] [in a new window]   Fig. 3.   The effect of four doses of saline (), medetomidine (), and ST-91 () on mean Ppa (A), PVR (B), and Ppaw (C). Baseline measurements were obtained in the halothane-anesthetized ventilated sheep (n = 5) before administration of dose I. Doses I to IV for medetomidine were 0.5, 1.0, 2.0, and 4.0 µg/kg; for ST-91 were 1.5, 3.0, 6.0, and 12 µg/kg; and for the placebo treatment were 2.0 ml saline. Significant differences (*) between values obtained with placebo treatment and either medetomidine or ST-91 treatment at each dose level are shown (p  .05; two-way repeated measures ANOVA followed by LSD test with Bonferroni's correction).

View larger version (20K): [in this window] [in a new window]   Fig. 4.   The effect of four doses of saline (), medetomidine (), and ST-91 () on MAP (A), SVR (B), and Qs/Qt (C). Baseline measurements were obtained in the halothane-anesthetized ventilated sheep (n = 5) before administration of dose I. Doses I to IV for medetomidine were 0.5, 1.0, 2.0, and 4.0 µg/kg; for ST-91 were 1.5, 3.0, 6.0, and 12 µg/kg; and for the placebo treatment were 2.0 ml saline. Significant differences (*) between values obtained with placebo treatment and either medetomidine or ST-91 treatment at each dose level are shown (p  .05; two-way repeated measures ANOVA followed by LSD test with Bonferroni's correction).

ST-91-Treated Group. Treatment with ST-91 produced similar but smaller alterations of pulmonary mechanic variables than observed after medetomidine. A significant decrease in C_dyn occurred after doses III and IV, whereas R_L and Ppl values were not significantly different from the placebo treatment at any dose (Fig. 1). A trend toward lower PaO₂ levels and elevated P(A-a)O₂ gradients was noticed after all doses; however, this decrease was only significant after dose IV (Fig. 2). The lowest level of PaO₂ after the fourth dose of ST-91 was 271 mm Hg, whereas it was 487 mm Hg in placebo-treated animals. The drop in PaO₂ was not accompanied by any change in PaCO₂ (Fig. 2) or PO₂.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The most important finding of the study was the fall in PaO₂ and increase in P(A-a)O₂ after i.v. medetomidine at a dose (0.5 µg/kg) that is 1/20 the sedative dose. This decrease in PaO₂ was associated with rapid onset of changes in R_L, C_dyn, and Ppl, without changes in CI, MAP, HR, PVR, Ppa, or Ppaw. On the other hand, ST-91 only produced significant changes in PaO₂ at the highest dose. After ST-91, the decrease in PaO₂ was accompanied by a decrease in CI and an increase in MAP, Ppa, and Ppaw, without significant alterations of R_L and Ppl. These findings suggest that the mechanism(s) by which medetomidine and ST-91 produced lower PaO₂ levels were different.

Baseline cardiovascular and blood gas values were similar to normal sheep anesthetized with halothane (Fujimoto and Leuchan, 1985). Pretreatment PaO₂ levels were above 450 mm Hg, and mean Q_s/Q_t values were between 6 and 11%. Breath-by-breath repeatability of pressure-volume loops, C_dyn, R_L, and Ppl was excellent and the values were similar to measurements previously reported for conscious, standing sheep with a nasal endotracheal tube (Wanner and Reinhart, 1978; Wheeler et al., 1990). No significant changes were observed with the placebo treatment, although C_dyn tended to decrease and R_L and CI to increase. The temporal increase in CI during constant depth halothane anesthesia was expected, probably due to a gradual increase in blood volume over time (Dunlop et al., 1987).

The complexity of the animal model and ethical consideration of animal numbers prevented doing a classic dose-response study. The elimination half-life of medetomidine is 37.9 ± 2.8 min in sheep (Muge et al., 1996) and there undoubtedly was only partial metabolism of the first dose by the time the fourth dose was administered. In our previous studies with medetomidine in conscious sheep (Celly et al., 1997a), an i.v. dose of 10 µg/kg produced sedation for 30 to 45 min and bradycardia for 30 min with no significant change in blood pressure. The effects of prior metabolism might be different for ST-91 because less is known about its elimination kinetics. However, the fractionated doses we used produce variations in threshold for respiratory and circulatory changes that we hoped to achieve.

Halothane was selected as the anesthetic for this study because halothane has been used by others to facilitate ₂-agonist evaluations in sheep (Nolan et al., 1986) and it can be precisely monitored. It is also reported that halothane minimizes the effect of minor changes in lung volume on R_L (Joyner et al., 1992). One can only speculate on the influence of halothane in the present study. If there was any effect, it would probably be to reduce the change in pulmonary mechanics and gas exchange associated with a given dose of ₂-agonist.

Medetomidine Group. Medetomidine produced a significant decrease in PaO₂, a marked increase in R_L and Ppl, and a 3-fold reduction in C_dyn at the lowest dose. Although the results are presented as a weighted mean of the 20-min interval, actual onset of changes in pulmonary mechanics were evident at 2 min and reached a peak by 5 min in all cases. The magnitude of the changes in Ppl produced by 0.5 µg/kg medetomidine was similar to those observed in conscious sheep using a sedative dose, i.e., 10 µg/kg (Celly et al., 1997a). A subsedative dose of xylazine (20 µg/kg) produced a 3-fold increase in airway pressure in halothane anesthetized, ventilated sheep (Nolan et al., 1986), whereas Gustin et al. (1989) observed a dose-related increase in respiratory resistance and Ppl after nonsedative doses of xylazine in conscious calves.

ST-91-Treated Group. Respiratory responses to ST-91 tended to be biphasic with increasing doses, whereas changes in vascular pressures, PVR, and SVR were dose-related. With the initial low dose, C_dyn decreased by 50% whereas R_L, Ppl, and P(A-a)O₂ doubled and Q_s/Q_t tripled. These changes were not significant due at least in part to interanimal variability. The decrease in PaO₂ was less pronounced (509 mm Hg with placebo; 349 mm Hg with ST-91). Cardiac index remained stable until dose IV, whereas PO₂ was not altered at any dose. The pulmonary changes were sustained, but not increased, by increasing ST-91 doses, and only the fall in C_dyn was statistically significant. The fall in PaO₂ and increase in P(A-a)O₂ and Q_s/Q_t became significant with the fourth dose; at this dose, Ppa, PVR, Ppaw, MAP, and SVR were markedly elevated.