Materials and Methods

Animals—Six neonatal foals (4 males and 2 females) that were 1 to 3 days old and that weighed 52.9 to 66.5 kg were used in the study. All foals were university owned and had been born without complications; results of physical examination, CBC determination, and serum biochemical analysis were used to confirm their health status prior to inclusion in the study. Adequate transfer of passive immunity was confirmed prior to initiation of the study by measurement of plasma IgG concentration via a commercial immunoassay.^a The Institutional Animal Care and Use Committee of the University of Florida approved the study.

Anesthesia and instrumentation—The day before or the day of the experiment, a 7-F, 30-cm doublelumen catheter^b was placed in a jugular vein of each foal and its tip directed into the right atrium for measurement of central venous pressure and collection of central venous blood. Radiography was used to verify correct placement of the catheter. Anesthesia was induced via administration of diazepam^c (0.05 mg/kg, IV) followed by ketamine^d (2.0 mg/kg, IV), and the foal was positioned in left lateral recumbency. A nasotracheal tube (internal diameter, 10 mm) was placed for administration of isoflurane^e (2% to 3% inspired concentration) vaporized in oxygen via a circle system with an oxygen flow of 2 to 3 L/min; ETI and ETCO₂ concentrations were monitored by use of an infrared gas analyzer^f that was calibrated before each experiment with the recommended standardized calibration gas mixture.^g Foals were allowed to breathe spontaneously throughout the study. Measurements of f_R, V_T, V_E, and I:E ratio were obtained via spirometry.^h Values of SAP, DAP, and MAP were obtained from a 20-gauge, 1.88-inch catheter that was placed in the right metatarsal artery and attached to an electronic pressure transducer positioned and zeroed at the level of the sternal manubrium. The pressure transducer was calibrated against a mercury manometer. Arterial blood pressures, ECG trace, and heart rate were monitored^f continuously and displayed throughout the experiment. A polyionic crystalloid fluid with 5% dextrose was administered IV at a rate of 5 mL/kg/h during anesthesia to prevent hypoglycemia and maintain normovolemia.ⁱ Esophageal temperature was monitored electronically and maintained at 37.5° to 39°C by use of a circulating water blanket and cotton blankets.

Measurements of cardiac output were determined by use of LiDCO. A LiDCO computer^j was used as previously described.¹⁴ The lithium chloride sensor^j was attached to the side port of a 3-way stopcock that was connected to the arterial catheter. Extension tubing was used to attach the sensor to a blood collection bag, and blood passed through a peristaltic pump to ensure a blood flow of 4 mL/min across the sensor.^k The hemoglobin and serum sodium concentrations required by the LiDCO computer for calculation of cardiac output were determined immediately prior to obtaining cardiac output measurements by use of a blood gas analyzer.^l The dose of lithium chloride^m (0.16 to 0.28 mmol) was held in an extension set attached to the jugular vein catheter, and 8 seconds after starting the injection phase on the LiDCO computer, the extension set was flushed with 12 mL of saline (0.9% NaCl) solution containing heparin. Measurements of cardiac output were obtained in duplicate to verify < 20% variation between the 2 measurements, and the mean value was used for data analysis.

Experimental design—Each foal received 1 of 3 treatments (saline solution, doxapram, and caffeine) on 3 consecutive days; thus, each foal received all 3 treatments. Each drug was administered at 2 doses (termed low and high doses). The doses were selected to represent upper and lower limits of those cited in the veterinary medical literature.^11,13 At least 24 hours elapsed between administrations of each drug. Three foals received doxapram on the first day of the study (day 1), and 3 foals received saline solution on day 1 on a rotational basis. Because of its long elimination half-life (10 to 21 hours), caffeine was always administered on day 3 for any foal. Following an initial 0.5 mg/kg loading dose, doxapramⁿ was administered at a constant rate infusion of 0.03 mg/kg/min for 20 minutes (low dose) and 0.08 mg/kg/min for 20 minutes (high dose). Caffeine citrate° was administered IV as a bolus at a dose of 10 mg/kg (5 mg of caffeine base/kg; low dose) followed by a second bolus (10 mg/kg [5 mg of caffeine base/kg; high dose]). Administration of the second bolus after only 20 minutes constituted the high dose (ie, 20 mg of caffeine citrate/kg or 10 mg of caffeine base/kg) because of drug accumulation as a result of the long elimination half-life. Saline solution was used as a placebo; this control treatment was included in the study design to account for the possible effects of prolonged anesthesia on measured and calculated variables. All solutions were prepared by dilution in saline solution to a concentration that resulted in the same total volume of each infusion and were administered via a syringe infusion pump.^p

On each of the 3 days, each foal was anesthetized and instrumented; inspired isoflurane concentration was titrated to achieve a deep level of anesthesia with an ETCO₂ concentration of 70 to 80 mm Hg (duration of titration, approx 30 minutes). The ETI concentration was recorded and kept constant for the remainder of the experiment. The foal was maintained at this steady state of anesthesia for an additional period of 20 minutes prior to baseline data acquisition. Measurements of f_R, V_T, V_E, I:E ratio, cardiac output, SAP, DAP, MAP, heart rate, central venous pressure, ETI concentration, PaCO₂, PaO₂, central venous partial pressure of oxygen, arterial HCO₃⁻ and lactate concentrations, and cerebral blood flow velocities were recorded. From these variables, V_T index, V_D/V_T, cardiac index, stroke volume index, SVR, V˙O₂, DO₂, and ERO₂ were calculated (Appendix).

Immediately after baseline measurements were obtained, a low dose of saline solution, doxapram, or caffeine was administered, and all variables were recorded a second time after a period of 20 minutes. The high-dose treatment was then administered, and all variables were recorded a third time after a period of 20 minutes. All instrumentation was removed once the experiment was concluded each day, with the exception of jugular catheters that were left in place with a protective bandage applied to the neck between treatment days to avoid repetitive placement. Foals were assisted throughout the recovery from anesthesia, returned to their stall once fully awake, and observed for normal suckling and behavior.

Measurement of cerebral flow velocity—In a previous study¹⁵ in ponies, it was determined that changes in internal carotid artery blood flow are representative of changes in total cerebral blood flow. The internal carotid artery was assessed ultrasonographically in a longitudinal view at its course caudal to the ramus of the mandible; blood flow velocities of the internal carotid artery were measured via transcutaneous 2-dimentional Doppler ultrasonography with a 10-MHz transducer.^q Only homogeneous flow waves were analyzed. The system software was used to calculate Vps, Ved, TAM, and VTI. From these values, resistive and pulsative indices were calculated (Appendix).

Caffeine and doxapram concentrations—For the experiments involving caffeine or doxapram, serum was obtained from blood samples for measurement of caffeine and theophylline or doxapram at each data collection time point (baseline, low-dose, and high-dose assessments). By use of a modification of a previously published method,¹⁶ concentrations of caffeine and its major metabolite, theophylline, were measured via HPLC with detection by UV absorption. Ten milliliters of 0.2M phosphate buffer (pH, 6.5) was added to each thawed 2-mL serum sample. The samples were then applied to a preconditioned ion-exchange–reverse-phase, solid-phase extraction cartridge.^r After the sample had passed, the cartridge was washed with water (7 mL), dried under vacuum (1 minute), and eluted with methanol (7 mL). The extract was evaporated under nitrogen (45°C) and redissolved in 100 μL of mobile phase. Analysis was performed via HPLC with a variable wavelength UV detector.^s The analytes were introduced in a 50-μL injection and separated across a column^t that was maintained at 28°C. Separation was isocratic in a 94:4:2 (vol:vol:vol) phosphate buffer (10mM [pH, 4.8]):acetonitrile:tetrahydrofuran mixture with a flow rate of 1 mL/min. Ultraviolet detection at 275 nm was used. Peak areas for both compounds had a linear relationship with drug concentration over the ranges of 25 to 3,333 ng/mL (caffeine) and 2.5 to 10,000 ng/mL (theophylline), with a correlation coefficient value ≥ 0.999. The limit of quantification of the assay was 1.875 ng/mL for caffeine and 0.188 ng/mL for theophylline. Each sample was run in duplicate, and drug concentrations were estimated by comparison of peak areas against linear standard curves for each analyte. For duplicate quality-control samples (10 and 250 ng/mL) prepared at the time of study sample preparation, recoveries ranged from 85% to 101%.

Concentrations of doxapram were measured via HPLC with detection by UV absorption on the basis of a previously published method.¹⁷ To each thawed serum sample (0.5 mL), 2.5 mL of 10mM ammonium dihydrogenphosphate buffer (pH, 4.7) and 15 mL of 9:1 (vol:vol) chloroform:2-propanol were added. After centrifugation at 3,000 × g for 5 minutes, the upper aqueous layer was aspirated. The organic layer was evaporated under nitrogen (40°C) and redissolved in buffer (100 μL). Analysis and separation were as described for caffeine and theophylline assessments. Ultraviolet detection at 214 nm was used. Peak areas had a linear relationship with drug concentration over the ranges of 10 to 25,000 ng/mL, with a correlation coefficient value ≥ 0.999. The limit of quantification of the assay was 4.0 ng/mL. Each sample was run in duplicate, and drug concentrations were estimated by comparison of peak areas against linear standard curves. For duplicate quality-control samples (400 and 2,000 ng/mL) prepared at the time of study sample preparation, recoveries ranged from 96% to 99%.

Data analysis—Normality and equality of variance of the data were assessed by use of the Kolmogorov-Smirnov and Levene tests, respectively. Because an orthogonal design could not be used, the effect of the order of drug administration (days 1, 2, and 3) on each measured or calculated variable at baseline was assessed in preliminary analyses by use of a 1-way ANOVA for repeated measures. Data for variables that were not influenced by the day of the study are expressed as mean values ± SD. In rare instances when there was a significant effect of the study day on baseline data, the data are presented as the difference from baseline (value of a given variable minus value of the same variable at baseline) ± SD. A 2-way ANOVA for repeated measures was used to determine the effects of treatment (saline solution, caffeine, and doxapram), dose (baseline, low dose, and high dose), and the interaction between treatment and dose on each measured and calculated variable. Variables that did not meet the assumptions of the ANOVA were rank transformed prior to analysis. When appropriate, multiple pairwise comparisons were performed by use of the Holm-Sidak test. The Spearman coefficient of rank correlation was used to determine the strength of the relationship between PaCO₂, PaO₂, arterial HCO₃⁻ concentration, f_R, V_T,V_E, I:E ratio, and serum caffeine and doxapram concentrations. For all analyses, a value of P < 0.05 was considered significant.

Results

All 6 foals completed the study without adverse effects and had apparently normal behavior throughout the experimental period. Mean ± SD serum concentrations of doxapram were 1,127 ± 522 ng/mL (range, 688 to 2,143 ng/mL) during the low-dose data collection period and 3,246 ± 2,142 ng/mL (range, 1,185 to 6,206 ng/mL) during the high-dose data collection period. Mean serum concentrations of caffeine were 4,860 ± 386 ng/mL (range, 4,189 to 5,250 ng/mL) during the low-dose data collection period and 10,379 ± 647 ng/mL (range, 9,224 to 10,964 ng/mL) during the high-dose data collection period. Theophylline was not detected in any serum sample. There was a significant decrease in baseline arterial lactate concentration and a significant increase in baseline heart rate across study days (data not shown); therefore, these data were expressed as a change from baseline values. Among highdose treatments, the difference in arterial lactate concentration from baseline value was significantly greater during administration of doxapram than that during administration of caffeine or saline solution (Table 1). The difference in arterial lactate concentration from baseline value was also significantly greater during administration of the high dose of doxapram, compared with that associated with the low dose. There was no significant effect of the order of drug administration or day of the study on any other measured or calculated baseline variable.

Table 1—

Mean ± SD heart rate and arterial lactate concentration in 6 anesthetized neonatal foals with isoflurane-induced respiratory acidosis before and after administration of saline (0.9% NaCl) solution, doxapram, or caffeine (1 treatment on each of 3 consecutive days). Each treatment included administration of a low and high dose in succession: doxapram (initial 0.5 mg/kg loading dose, followed by a constant IV infusion at a rate of 0.03 mg/kg/min for 20 minutes [low dose] and 0.08 mg/kg/min for 20 minutes [high dose]), caffeine citrate (10 mg/kg IV [low dose] followed by a second 10 mg/kg bolus [high dose] after 20 minutes), and equivalent volumes of saline solution administered IV.

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At baseline, no significant differences were detected in any of the measured or calculated variables among treatment groups. At both infusion rates, administration of doxapram resulted in significant increases in f_R, V_E, I:E ratio, arterial blood pH, and PaO₂, compared with baseline values, whereas administration of saline solution or caffeine had no effects on these variables (Table 2). For both the low- and high-dose treatments, these variables were also significantly higher during doxapram administration than they were during administration of caffeine and saline solution. There was a significant dose-dependent decrease in arterial HCO₃⁻ concentration and PaCO₂ during doxapram treatment. In contrast, PaCO₂ increased from baseline values after administration of saline solution or caffeine. The PaCO₂ was significantly lower during doxapram treatment than it was during treatment with caffeine or saline solution. Compared with findings during treatment with saline solution, respiratory, blood gas, and co-oximetry variables during caffeine treatment did not differ significantly. Serum doxapram concentrations were significantly correlated with PaCO₂, PaO₂, f_R, V_T index, V_E, and I:E ratio (Table 3). In contrast, serum caffeine concentrations were not significantly correlated with those variables.

Table 2—

Mean ± SD values of respiratory, blood gas, and co-oximetry variables in 6 anesthetized neonatal foals with isoflurane-induced respiratory acidosis before and after administration of low and high doses of saline solution, doxapram, or caffeine.

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Table 3—

Correlation between PaCO₂, PaO₂, HCO₃⁻, f, V, V, I:E ratio, and serum caffeine and doxapram concentrations in 6 anesthetized neonatal foals with isoflurane-induced respiratory acidosis.

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Administration of doxapram resulted in a significant increase in DAP, SAP, and MAP (Table 4). Both SAP and MAP were significantly greater in foals receiving doxapram than in foals receiving saline solution or caffeine. There were no effects of treatment or significant differences among treatment groups for other measured or calculated cardiovascular variables. Administration of doxapram to the foals resulted in a significant decrease in internal carotid artery Vps, compared with baseline values (Table 5). However, Vps during administration of doxapram was not significantly different from that determined during administration of saline solution. The Vps associated with caffeine administration was not significantly different from that determined at baseline, but it was significantly higher than the value determined during administration of saline solution or doxapram. Compared with the baseline value, internal carotid artery TAM significantly increased following caffeine treatment; however, the TAM determined in caffeine-treated foals was not significantly different from that determined in foals receiving doxapram or saline solution.

Table 4—

Mean ± SD cardiovascular variables in 6 anesthetized neonatal foals with isoflurane-induced respiratory acidosis before and after administration of low and high doses of saline solution, doxapram, or caffeine.

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Table 5—

Mean ± SD internal carotid blood flow velocity variables in 6 anesthetized neonatal foals with isoflurane-induced respiratory acidosis before and after administration of low and high doses of saline solution, doxapram, or caffeine.

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Discussion

To the authors' knowledge, there is no proven technique for experimental induction of hypoxic-ischemic encephalopathy in foals. The extent of the monitoring required (as illustrated by the present study) in combination with the variability of the severity of the disease in naturally affected foals makes acquisition of meaningful data from a clinical study extremely difficult. In many species, including horses, the use of inhalant agents for anesthesia results in progressive CO₂ retention without a concomitant increase in V_E, suggesting a progressive loss of the central response to CO₂.^18,19 As a result, inhalation anesthesia represents a convenient and noninvasive means with which to assess the effects of pharmacologic respiratory support in neonatal foals with decreased responsiveness to CO₂. However, the effects of inhalation anesthesia on respiratory control may not be identical to the effects of respiratory depression caused by CNS damage because inhalation anesthesia likely suppresses the muscles of respiration in addition to the respiratory center. In addition, administration of 100% oxygen during the experiments of the present study may have affected the foals' response to caffeine and doxapram because the possible hypoxic drive in some foals with hypoxic-ischemic encephalopathy could result in less effective changes in V_E in response to pharmacologic intervention.

Caffeine has become the treatment of choice for apnea associated with prematurity in human infants because of its wide therapeutic index and ease of oncedaily administration.^4,7 Intravenous administration of caffeine to infants with apnea, newborn lambs, cats, and healthy adult humans results in significant increases in V_E and V_T, with no significant effects on f_R.^20-23 Administration of caffeine to humans also results in a significant decrease in ETCO₂ concentration in response to inhaled CO₂.²⁴ These effects on respiratory function can be detected within 10 minutes after IV administration of a bolus of caffeine.^22,24

Targeted therapeutic concentrations in infants treated with caffeine range from 5,000 to 20,000 ng/mL.^5,25 However, in a study²² in infants with neonatal apnea, peak increases in V_E were detected at plasma caffeine concentrations as low as 2,900 ng/mL, whereas restoration of the respiratory pattern is optimal only at concentrations of approximately 10,000 ng/mL. Despite the fact that serum caffeine concentrations were well within the therapeutic range for humans²² and lambs,²¹ no effects on respiratory and arterial blood gas variables were detected in the equine neonates of the present study. It is possible that higher serum concentrations of caffeine are required in foals, compared with the requirement of other species. Alternatively, caffeine may not increase V_E and facilitate CO₂ elimination in foals.

Oral administration of caffeine has been widely recommended to increase CO₂ responsiveness (and thus decrease PaCO₂) in foals with respiratory acidosis resulting from central respiratory depression.^2,12,26 By linear extrapolation from clinical experience in human infants, a loading dose of 10 mg/kg administered orally followed by a maintenance dose of 2.5 to 3.0 mg/kg, PO, every 24 hours has been recommended for foals.¹³ In adult horses, caffeine has an oral bioavailability of only 39% and peak serum concentrations are achieved 2 hours following administration.²⁷ To our knowledge, the pharmacokinetics of caffeine have not been studied in neonatal foals. In human neonates, caffeine is almost completely absorbed after oral administration.²⁸ Even if orally administered caffeine is completely absorbed by equine neonates, plasma concentrations after the recommended oral loading dose would not be higher than those achieved via IV administration of the total dose used in the present study. Therefore, it is unlikely that a single loading dose of 10 mg of caffeine/kg administered orally would promote ventilation and lower PaCO₂ in foals.

In adult horses, the half-life of caffeine ranges from 10 to 21 hours and caffeine is detectable in both plasma and urine for as long as 9 days after dosing.^27,29,30 The elimination half-life of caffeine decreases and clearance increases exponentially with postnatal age in children.³¹ Therefore, caffeine may accumulate with repeated administration in equine neonates. However, in a clinical setting, hypercapnia must be corrected rapidly to prevent cerebral acidosis and cerebral vasodilation.³ A delay of several days would not be clinically acceptable. Further studies will be required to investigate the pharmacokinetics of caffeine and determine whether plasma caffeine concentrations that are higher than those achieved in the foals of the present study have effects on respiratory function in equine neonates.

The long elimination half-life of caffeine was the factor that precluded the use of an orthogonal design in the present study and the reason that caffeine was always administered on the third day of the study. The use of an orthogonal experimental design would have required a washout period of at least 10 days between treatments. Because the respiratory and hemodynamic variables change rapidly within the first weeks after birth, such a prolonged washout period would have resulted in a profound age effect and would have prevented a rational comparison of each treatment.^32,33 In the present study, with the exception of heart rate and arterial lactate concentration, there was no measurable effect of the order of drug administration on baseline values of the variables evaluated. During the first few days after a foal's birth, arterial lactate concentration decreases progressively, whereas heart rate increases progressively.^33,34 As a result, the small but significant differences in baseline arterial lactate concentration and heart rate detected in the foals of the present study were more likely an effect of increasing age than a cumulative effect of repeated episodes of anesthesia.

In contrast to caffeine, administration of doxapram to foals in the present study resulted in a significant increase in f_R, V_E, I:E ratio, arterial blood pH, and PaO₂ as well as significant dose-dependent decreases in arterial HCO₃⁻ concentration and PaCO₂. Administration of doxapram also resulted in a significant increase in SAP and MAP. These effects in the study foals are similar to those detected in adult horses.^35-37 Following IV administration of the drug to adult horses, plasma doxapram concentrations decrease rapidly with an elimination half-life of approximately 2 hours.³⁸ In adult horses, the duration of the effects of a bolus of doxapram is only 5 minutes; thus, a continuous IV infusion of the drug is required.³⁷

The therapeutic range of plasma doxapram concentrations in infants is not firmly established, but concentrations ranging between 1,500 and 4,000 ng/mL have been used successfully.^39,40 These concentrations are similar to those achieved in the foals of the present study, in which mean serum doxapram concentrations following low- and high-dose treatments were 1,127 and 3,246 ng/mL, respectively. Administration of the high dose of doxapram did not result in a significant increase in f_R, V_E, I:E ratio, arterial blood pH, or PaO₂, compared with findings associated with administration of the low dose; in contrast, arterial HCO₃⁻ concentration and PaCO₂ were significantly lower during administration of the high dose. These differences may be attributable to a dose-dependent effect or simply to a time-dependent effect on CO₂ elimination as a result of a longer period with a high V_E. Acute changes in PaCO₂ result in acute changes in arterial HCO₃⁻ concentration because of electrochemical neutrality and the law of mass action.⁴¹ In the present study, doxapram concentrations varied substantially among foals. This finding was not unexpected because the pharmacokinetics of doxapram vary widely among human neonates.⁴² In the foals of the present study, administration of doxapram at the high dose resulted in a small (0.17 ± 0.12 mmol/L) but significant increase in arterial lactate concentration. This finding is unlikely to be of clinical relevance because the increase in arterial lactate concentration was not associated with a decrease in DO₂ or changes in V˙O₂ or ERO₂.

In 2 studies,^6,43 administration of caffeine impaired cerebral and intestinal blood flow velocities in preterm infants, and similar findings following administration of doxapram have been reported.^44,45 Results of a study¹⁵ in ponies indicated that 95% of the blood flowing through the internal carotid artery supplies the brain and that changes in internal carotid artery blood flow are representative of changes in total cerebral blood flow. The high dose of doxapram administered to the foals in the present study resulted in a significant decrease in Vps, compared with the baseline value. A similar decrease in Vps following administration of doxapram in preterm infants has also been reported.⁴⁵ However, it is unknown whether the decrease in Vps is the result of the hemodynamic effects of doxapram or simply the result of the marked decrease in PaCO₂ achieved during administration of doxapram.⁴⁵ The lack of change in the foals' VTI after treatment with caffeine or doxapram has suggested that overall internal carotid artery blood flow (hence, cerebral perfusion) was not altered substantially in the present study. Application of more sensitive techniques for assessment of cerebral blood flow and metabolism, such as positron emission tomography, single photon emission computed tomography, and magnetic resonance imaging, may provide more insights on the effects of these drugs on brain perfusion in both equine and human neonates.⁴⁶

Results of the present study indicated that doxapram restored ventilation in a dose-dependent manner in neonatal foals with isoflurane-induced hypercapnia. In contrast, the effects of caffeine on respiratory function were indistinguishable from those of a placebo. Caffeine is unlikely to improve ventilation and decrease hypercapnea in equine neonates. Additional studies will be required to determine the minimum effective dose of doxapram and assess the long-term effects of this drug in equine neonates with hypoxic-ischemic encephalopathy in a clinical setting.