Effects of a single intravenous bolus injection of alfaxalone on canine splenic volume as determined by computed tomography

Abstract

The purpose of this study was to evaluate the effects of a single intravenous dose of alfaxalone on canine splenic volume. In 6 adult beagle dogs the splenic volume [mean ± standard error (SE)] was determined by computed tomography to be 0.17 ± 0.02 L before alfaxalone administration and 0.24 ± 0.02 L (P = 0.0091) and 0.23 ± 0.02 L (P = 0.0268) 15 and 30 min, respectively, after alfaxalone administration. Hematocrits (mean ± SE) obtained at the same times were, respectively, 46.3% ± 1.3%, 40.6% ± 1.3% (P = 0.0015), and 41.7% ± 1.3% (P = 0.0057). In conclusion, alfaxalone caused relaxation of the canine splenic capsule and an increase in the splenic volume, along with a decrease in the hematocrit in these dogs.

Introduction

Causes of naturally occurring splenomegaly in dogs include splenic hypertrophy, portal hypertension, infection, primary or infiltrative neoplasia, congestion or enlargement of the spleen due to metabolic disorders, and immune-mediated disease (1–5). Anesthetic drugs can also have significant effects on various vital parameters such as the hematocrit and on organ systems when administered to dogs (3). Some anesthetics and sedatives can cause splenomegaly after their administration (1–3,6). Since enlargement of the spleen is often the primary indicator of splenic disease, drug-induced splenomegaly could result in intraoperative misdiagnosis (1,3). Drug-induced splenomegaly could also complicate surgical approaches to the abdomen, limit exposure of abdominal organs, and increase the risk of accidental splenic laceration, resulting in additional hemorrhage and surgical complications (1,6).

The exact mechanism of changes in splenic size secondary to anesthetic drug administration in dogs is not known but is suggested to be secondary to changes in smooth muscle tone and changes in systemic blood pressure and cardiac output, which alter blood flow (1–3,7). Smooth muscle relaxation, as seen with various anesthetics and sedatives, is suspected to result in relaxation of the muscle fibers in the splenic capsule (1,3). This could permit secondary engorgement of the spleen with blood and subsequent splenomegaly and erythrocyte sequestration (1,3).

Various drugs have been reported to influence canine splenic size (1–3,6,8–10). Barbiturates, such as thiopental, are known to cause splenomegaly (1,2,8,10). Acepromazine administration was reported to result in increased splenic volume, whereas hydromorphone caused no change in splenic volume (1). Reduction of splenic volume was reported with xylazine (9), but no change was observed with dexmedetomidine (1). Propofol (2,6-diisopropylphenol), an anesthetic induction agent, interacts with γ-aminobutyric acid (GABA) receptors and inhibits N-methyl-D-aspartate receptors (11). Use of propofol was reported to result in splenomegaly, evident on computed tomography (CT), after a single bolus injection in healthy dogs (1). Owing to these changes, the authors of that report discouraged the use of propofol for procedures in which splenomegaly should be avoided (1). Although the mechanism by which thiopental and propofol cause splenic capsule relaxation is unknown, it is assumed to be related to these drugs’ systemic sympatholytic effects.

Techniques employed to measure splenic size include radiography, nuclear scintigraphy, ultrasonography, CT, and tracing of the spleen onto translucent paper during laparotomy (1,5,6). Two-dimensional radiographic and ultrasonographic techniques often lack objective criteria for determining if splenomegaly is present owing to patient positioning and the location selected for measurement of splenic size (2). However, CT rapidly provides radiographic images and allows for 3-dimensional reconstruction, which is considered the most accurate way to measure organ volume in vivo (1,5,12–14). Also, this technique allows for baseline measurement in the awake animal before any drug administration. The area of an organ can be determined on each CT slice and then multiplied by the slice thickness to determine the volume. The sum of the volumes of all the slices reflects organ volume (1,13).

Alfaxalone (3α-hydroxy-5α-pregnane-11,20-dione) is a synthetic neuroactive steroid used for the induction of anesthesia by intravenous (IV) administration (15–22). Its use and properties are similar to those of propofol (18,23). Because its cardiovascular effects are similar to those of propofol and it also causes skeletal muscle relaxation via GABA receptors in the central nervous system (15–22), we hypothesized that alfaxalone may have similar effects on the canine splenic capsule, resulting in splenomegaly. To our knowledge, the effect of alfaxalone on canine splenic size was unknown. The purpose of this study was to use CT to measure changes in splenic volume caused by alfaxalone when administered as a single IV bolus to healthy dogs.

Materials and methods

Six adult purpose-bred beagles, 1 female and 5 male, aged 7.33 ± 4.13 y and weighing 11.4 ± 1.65 kg (means ± standard deviations), were included in this prospective study. They were assessed as healthy from the results of physical examination, a complete blood count, and serum biochemical analyses. They had been acclimatized to handling for several months before the study. The dogs were housed in groups in a climate-controlled room with a light cycle of 12 h of light and 12 h of darkness and acclimatized to a plastic dog crate during the week before treatment and data collection. Food was withheld for 12 h before anesthesia and data collection. Ad libitum access to water was permitted. The study was approved Animal Use Form (AUF): 201609448 by the Institutional Animal Care and Use Committee, University of Florida, Gainesville, Florida, USA.

On the day of the study a 22-gauge IV catheter was placed in either cephalic vein without prior sedation. The catheter was connected to a 53-cm-long extension line primed with 3 mL of 0.9% saline (Baxter International, Deerfield, Illinois, USA). The dogs were placed in a narrow crate on the CT table with the extension line extending outside of the crate to allow for drug administration without interaction with the dog. Saline solution (3 mL) was administered and a baseline CT image obtained. Then 4 mg/kg body weight (BW) of alfaxalone (Alfaxan; Jurox Pty, Rutherford, New South Wales, Australia) was administered through the IV catheter and the extension line flushed.

A multislice CT scanner (Aquilion Prime; Toshiba American Medical Systems, Tustin, California, USA) was used to obtain images at baseline and then 10, 15, and 30 min after alfaxalone injection. Images were acquired by a body standard axial mild filter with the following parameters: slice thickness 3 mm, collimator pitch PF 0.813/HP 65 (Pitch Factor 0.813/Helical Pitch 65 rows) to improve image quality, 120 kV, modulated mA (50 mA on average), and field large enough to obtain images of the entire abdomen. The images were imported into commercially available software (Mimics ×64, version 14; Materialise, Plymouth, Michigan, USA). The splenic area was manually delineated on each slice by a board-certified veterinary radiologist (C.R.B.). Splenic volume was calculated by the software program.

Blood samples for measurement of the hematocrit were collected at the time of catheter insertion and by direct venous puncture of the contralateral cephalic vein 15 and 30 min after alfaxalone administration. The samples were placed in heparinized capillary tubes and spun in a microcentrifuge at 3325 × g for 12 min. The hematocrit was measured immediately after centrifugation. The values obtained before and at 15 and 30 min after drug administration were compared.

A multiple-comparisons analysis of variance (ANOVA) with post-hoc Student’s t-test for all pairwise comparisons was used to evaluate the drug’s effects on splenic volume and hematocrit. Results were considered significant if the P-value was 0.05 or less. All data were analyzed with commercial statistical software (JMP PRO 13; SAS Institute, Cary, North Carolina, USA). Normality was assessed with use of the Kolmogorov–Smirnov and Shapiro–Wilk tests.

Results

The data were normally distributed. The mean splenic volume [± standard error (SE)] significantly increased after alfaxalone administration (Figure 1), from 0.17 ± 0.02 L at baseline to 0.24 ± 0.02 L after 15 min (P = 0.0091) and 0.23 ± 0.02 L after 30 min (P = 0.0268). There was no significant difference in splenic volume between 15 min and 30 min after alfaxalone administration (P = 0.5420).

Figure 1.

Mean ± standard error in hematocrit measurements and splenic volume as determined by computed tomography in beagles before, 15 min after, and 30 min after intravenous administration of a single bolus of alfaxalone (4 mg/kg BW).

* Value differs significantly (P ≤ 0.05) from the baseline value.

Alfaxalone also induced a significant decrease in the mean hematocrit (± SE) (Figure 1), from 46.3% ± 1.3% at baseline to 40.6% ± 1.3% after 15 min (P = 0.0015) and 41.7% ± 1.3% after 30 min (P = 0.0057). There was no significant difference in hematocrit between 15 min and 30 min after alfaxalone administration (P = 0.4353).

Discussion

In this study, administration of a single IV bolus of alfaxalone resulted in a significant increase in canine splenic volume as measured by CT. To our knowledge, this is the first report of splenomegaly due to alfaxalone administration. Previous studies have observed increases in splenic volume secondary to administration of barbiturates, acepromazine, and propofol (1,6,8). Although there have been previous conflicting reports of the effects of propofol on splenic volume, a recent study also reported a significant increase in splenic volume after administration of a single bolus of propofol (1). Although the mechanism for the changes in splenic volume induced by propofol remains unknown, those investigators suggested that it may be secondary to blood redistribution after systemic hypotension paired with the direct effects of the drug on smooth muscle, potentially resulting in relaxation of the splenic capsule and subsequent splenomegaly (1). Alfaxalone administration results in a dose-dependent decrease in arterial blood pressure, which could also lead to blood redistribution, as observed with propofol (19). Barbiturates, such as thiopental, are known to result in splenomegaly, which may be secondary to the same depressive effect on systemic arterial blood pressure due to vasodilation and smooth muscle relaxation (11). Additionally, propofol has an antagonistic effect on α-adrenergic receptors (24), and perhaps this is the mechanism of splenomegaly seen after administration of propofol, thiopental, alfaxalone, and acepromazine. Acepromazine is a known α₁-adrenergic antagonist that causes effects similar to those of alfaxalone observed in our study. Acepromazine causes a decrease in vascular tone and hematocrit, as well as splenic enlargement (25). Although it is not known if alfaxalone can act directly on the splenic capsule, we hypothesize that its administration caused an increase in splenic volume and a decrease in hematocrit by depressive mechanisms acting on vascular and nonvascular smooth muscle similar to those reported for propofol, thiopental, and acepromazine.

Administration of a single IV bolus of alfaxalone also resulted in a significant decrease in the hematocrit, the first such report, to our knowledge. Previous publications reported a reduction in hematocrit after propofol administration (1,26), and others have attributed the reduction in hematocrit after thiopental administration to be secondary to splenic relaxation and erythrocyte sequestration in the spleen (8,27). Baldo et al (1) observed a significant decrease from baseline in the hematocrit after propofol administration as well as after the administration of acepromazine and thiopental. Although the observed changes were not clinically significant in healthy patients, they could further compromise anemic patients.

Our study was intended to look at the effect of a single IV dose of alfaxalone on the size of the spleen, but other factors can affect this organ. Transport and exposure to the CT room may have resulted in excitement and stress before drug testing. Stress increases the release of adrenaline and noradrenaline, which can result in splenic contraction (28). To reduce this possibility, we ensured that the dogs used in this study had been acclimatized to handling for several months and were acclimatized to the dog crate over the course of a week before the study. Increases in stress hormone levels have been associated with procedures such as venipuncture and catheter placement (28). Release of these hormones can occur rapidly and could have reduced splenic volume before testing (28). However, sedation before catheterization would have added an unwanted variable. The dogs were permitted to recover after injection of the single bolus of alfaxalone. Excitement during emergence from anesthesia, identified as paddling and dysphoric vocalization, was observed around 20 min after alfaxalone administration in all the dogs. This excitement could have resulted in increased release of adrenaline and noradrenaline, which may have limited the full effect of alfaxalone on splenic size and caused the decrease in size at 30 min. Therefore, it is not certain if alfaxalone reached its maximum effect on splenic size and whether the results would have been different if anesthesia had been maintained with a continuous infusion of the drug until the CT scan at 30 min. Restraint would have introduced another variable and might have exacerbated the stress during recovery. No injuries occurred during recovery and no treatment was needed.

The dogs tolerated being confined to the crate during CT image acquisition; however, the crate did allow the dogs mobility within the crate. If considerable motion was observed on review of the CT images immediately after acquisition, CT was done again until images acceptable for accurate determination of splenic volume were obtained. All studies were completed within 4 min of the designated time. The longer time required to acquire the images and the motion may have limited the full extent of splenomegaly because of drug metabolism and increased catecholamine levels during the recovery phase.

Use of CT for determination of organ volume has been found to be reliable and accurate (5,13). When ultrasonography is used, patient position and operator can affect the measurement of visceral volume. A study comparing ultrasonography with CT found the latter to be superior in measuring organ volume. However, even using CT, hand-outlining of organs requires dexterity and expertise to accurately delineate the organ when there are edges of low contrast (5). For this reason, outlines of the spleens in our study were done by a single Board-certified radiologist. A previous study, however, found low interobserver and intraobserver variability in the evaluation of organ volume from CT images (13).

In this study, administration of a single IV bolus of alfaxalone to healthy dogs resulted in significant splenic enlargement and reduction in hematocrit. These changes may not be clinically significant in healthy dogs but may compromise animals with such conditions as anemia and diaphragmatic herniation. Also, the increase in splenic size can make it easier to puncture the spleen with a tro-car during laparoscopic procedures. It is unknown whether these effects would be maintained while the patient is under general anesthesia and whether these changes would persist until the time of surgical exploration. It is also unknown whether these effects would be observed with different alfaxalone dosages or in drug combinations. Furthermore, the impact on and clinical implications for subsequent anesthesia maintenance with inhalant anesthetics after induction with alfaxalone are unknown. In addition, conditions that may affect splenic size, including neoplasia, rickettsial diseases, and high sympathetic output, may alter the effects of alfaxalone.

In conclusion, a single IV bolus of alfaxalone produced splenic enlargement and decreased hematocrit in our canine population. Therefore, alfaxalone should not be administered if splenomegaly is to be avoided.