Traumatic wounds in the limbs of horses are common and frequently involve synovial structures that can affect an animal's athletic career and even be life-threatening.1–3 Regional limb perfusion with antimicrobials is a simple procedure that allows effective concentrations of antimicrobials to be achieved locally with minimal systemic effects.4,5
Vascular isolation of the area to be perfused is achieved by use of a tourniquet to compress blood vessels proximal to that area. Effectiveness of the tourniquet is measured by its ability to prevent leakage of the perfusate into the systemic circulation. Numerous studies have been conducted to examine tourniquet characteristics, including tourniquet width,6 site of injection,7 volume of perfusate,8 and prior exsanguination,9 to determine the most efficacious manner for performing IVRLP. When performing regional limb perfusion, tourniquet application of up to 25 or 30 minutes has been used,6,10–12 and such application has been practical and effective for achieving appropriate synovial concentrations of antimicrobials. In 2 recent studies, duration of tourniquet application was evaluated, and there was no significant difference in mean synovial concentrations between groups when the tourniquet was maintained for 20 minutes versus 30 minutes13 or 10 minutes versus 30 minutes.14 However, the authors are not aware of any study that has been conducted to determine the actual Cmax and Tmax for synovial fluid following instillation of the perfusate in healthy joints to identify the ideal amount of time for maintaining a tourniquet to corroborate findings for those studies.13,14
Aminoglycosides are concentration-dependent drugs; therefore, a higher Cmax-to-MIC ratio is associated with a greater bactericidal effect.12 Studies15,16 conducted to evaluate systemic administration of aminoglycosides for the treatment of gram-negative sepsis in humans have revealed that the peak systemic aminoglycoside Cmax-to-MIC ratio should be at least between 8 to 1 and 10 to 1 to maximize the effect of these drugs. High concentrations are essential when treating infections that may be caused by less susceptible pathogens17; therefore, the goal of treatment should be optimization of Cmax by use of the highest possible nontoxic dose.16 A shorter duration of tourniquet application could greatly reduce discomfort of treated horses and the degree of sedation required to perform procedures in standing animals.
The purpose of the study reported here was to determine the Cmax in the DIP joint and the Tmax after IVRLP of 2 g of amikacin sulfate by use of a cephalic vein. We hypothesized that Tmax would be less than the typical duration of tourniquet application of 30 minutes.
Materials and Methods
Animals
Nine horses (3 Thoroughbreds, 2 warmbloods, 2 Quarter Horses, 1 Arabian, and 1 American Paint Horse) were selected from the research herd at the Center for Equine Health of the University of California-Davis. Horses were 7 to 19 years old (mean, 10 years old), and body weight ranged from 490 to 579 kg (mean, 523 kg). Horses had no signs of lameness while walking and had no evidence of vascular abnormalities, effusion of the DIP joint, or obvious pathological changes to the forelimbs, as determined on the basis of results of physical and lameness examinations. Horses were housed in stalls and monitored twice daily (particularly with regard to swelling of the DIP joint, localized swelling at the site of perfusion, or lameness while walking in a straight line and in circles to the left and right) during the sample collection period and for 2 days after collection of the last sample. After the end of this initial period, horses were monitored closely by caretakers for another 5 days before being placed in a pasture. The protocol was approved by the University of California-Davis Institutional Animal Care and Use Committee.
IVRLP
Amikacin sulfatea was administered to a forelimb of each horse by use of IVRLP. The choice of forelimb for each horse was determined with a random number generator.b Horses were sedated by IV administration of detomidine hydrochloridec (0.01 mg/kg) and butorphanol tartrated (0.01 mg/kg). Additional amounts of the sedatives were administered during the procedure if a horse had signs of discomfort (shifting weight, lifting the limb, pawing, or walking).
Median, ulnar, and medial cutaneous antebrachial nerve blocks of the selected forelimb were performed with mepivicainee (total, 20 mL) to desensitize the medial aspect of the limb to allow catheter insertion into the cephalic vein and reduce discomfort from the tourniquet. Additionally, an abaxial sesamoid nerve block was also performed (6 mL of mepivacaine) to ensure there was no skin sensation when samples were collected from the DIP joint and to reduce discomfort associated with the procedures.
The area over the cephalic vein of the selected forelimb was clipped and aseptically prepared. A pneumatic tourniquetf (11.6-cm cuff) was applied 10 cm proximal to the accessory carpal bone. The same clinician (JEN) placed the tourniquet for each horse to ensure standardized tourniquet placement. A gauze roll was placed beneath the tourniquet at a point overlying the cephalic vein, and the tourniquet was insufflated to 450 mm Hg. A 22-gauge, 2.5-cm catheterg was placed in the cephalic vein distal to the tourniquet at the level of the chestnut (torus carpeus). Amikacin sulfate (2 g diluted to a volume of 60 mL with saline [0.9% NaCl] solution) was infused through the cephalic catheter over a 3-minute period via extension tubing by use of a roller pump.h After the infusion was completed (end of infusion = time 0), the catheter was removed, and a pressure bandage was placed over the injection site. The tourniquet was removed at 20 minutes.
Sample collection
Venous blood samples (5 mL) were collected in lithium heparin tubes by jugular venipuncture at 5, 10, 15, 19 (1 minute before tourniquet release), 21 (1 minute after tourniquet release), 25, and 30 minutes after completion of the infusion. Samples of synovial fluid (0.5 mL) were aseptically collected in lithium heparin tubes via arthrocentesis of the DIP joint by use of the dorsal approach at 5, 10, 15, 20, 25, and 30 minutes after completion of the infusion. A light bandage consisting of soft, elastic roll gauze secured with an elastic bandage was applied around the DIP joint after collection of the final sample of synovial fluid; the bandage was maintained for 24 hours. Horses received 2 g of phenylbutazonei IV on the day of perfusion. Phenylbutazone was administered after the final sample of synovial fluid was collected.
Analysis of serum and synovial fluid
Samples were centrifuged (1,700 × g for 5 minutes) immediately after collection. Serum and synovial fluid supernatant were stored in plastic tubes and frozen at −80°C until analysis. Amikacin concentrations were determined by use of a fluorescence polarization immunoassayj (kinetic interaction of microparticles in solution as measured by changes in light transmission) with an analyzer,k as reported elsewhere.9,18
The limit of detection for the assay was < 0.6 μg/mL. Testing was performed at the Biochemical Laboratory of the William R. Pritchard Veterinary Medical Teaching Hospital.
Data analysis
Data were evaluated for normality with the Shapiro-Wilk test by use of commercial statistical software.l Data were reported as median, range, mean, and 95% confidence intervals. The Cmax and Tmax for each horse were determined by visual inspection of data for the amikacin concentration in serum and synovial fluid. To detect differences in amikacin concentrations in synovial fluid over time, the nonparametric Friedman test was used. Significance was set at values of P < 0.05.
Results
Two horses (10- and 6-year-old warmblood mares) were excluded from the study because we were unable to retrieve an adequate amount of synovial fluid (at least 0.3 mL) from the DIP joint at multiple time points. There were no complications associated with application of the tourniquet for 20 minutes. No complications associated with the IVRLP or arthrocentesis were observed.
Amikacin concentrations in synovial fluid obtained at 5, 10, and 30 minutes were not normally distributed. Median amikacin concentrations in synovial fluid collected from the DIP joint at the various times were summarized (Table 1).
Concentration of amikacin (μg/mL) in synovial fluid of the DIP joint of 7 horses after IVRLP by use of the cephalic vein.
| Time (min) | Median | Range | Mean | 95% CI |
|---|---|---|---|---|
| 5 | 20 | 3–1,785 | 335 | 0–943 |
| 10 | 410* | 17–2,420 | 628 | 0–1,410 |
| 15 | 550* | 37–2,167 | 812 | 35–1,588 |
| 20 | 490 | 31–2,134 | 677 | 21–1,333 |
| 25 | 360† | 34–1,408 | 473 | 42–904 |
| 30 | 171†‡ | 27–1,067 | 262 | 0–599 |
A tourniquet was placed over the cephalic vein, and amikacin sulfate (2 g diluted to a volume of 60 mL with saline [0.9% NaCl] solution) was infused through a cephalic vein catheter over a 3-minute period (end of infusion = time 0).
Value differs significantly (P < 0.001) from the value at 5 minutes.
Value differs significantly (P < 0.05) from the value at 20 minutes.
Value differs significantly (P < 0.05) from the values at 15 and 25 minutes.
CI = Confidence interval.
Median Cmax for synovial fluid collected from the DIP joint was 600 μg/mL (range, 37 to 2,420 μg/mL). Median Tmax was 15 minutes (range, 10 to 20 minutes). The Cmax and Tmax, respectively, for each of the 7 horses were 2,420 μg/mL and 10 minutes, 2,167 μg/mL and 15 minutes, 790 μg/mL and 20 minutes, 600 μg/mL and 15 minutes, 550 μg/mL and 15 minutes, 180 μg/mL and 15 minutes, and 37 μg/mL and 15 minutes. There was a significant (P = 0.001) difference in amikacin concentration in synovial fluid over time.
Serum amikacin concentrations were determined (Table 2). Concentrations were below the limit of detection before removal of the tourniquet in only 2 of 7 horses. Those 2 horses had the highest peak amikacin concentrations in synovial fluid (2,420 and 2,167 μg/mL), and the Tmax for those 2 horses was 10 and 15 minutes.
Concentration of amikacin (μg/mL) in serum obtained from 7 horses after IVRLP by use of the cephalic vein.
| Time (min) | Median | Range | Mean | 95% CI |
|---|---|---|---|---|
| 5 | 14 | 0–18 | 10 | 3–18 |
| 10 | 12 | 0–16 | 9 | 3–15 |
| 15 | 10 | 0–15 | 7 | 2–13 |
| 19* | 9 | 0–13 | 6 | 2–12 |
| 21* | 12 | 0–30 | 14 | 4–25 |
| 25 | 19 | 8–22 | 17 | 12–22 |
| 30 | 13 | 7–19 | 14 | 10–18 |
A tourniquet placed over the cephalic vein was removed at 20 minutes (end of infusion = time 0); thus, blood samples were collected 1 minute before and 1 minute after tourniquet removal.
Discussion
Results of the present study corroborated our hypothesis that the Tmax for the DIP joint was less than the routinely used duration for tourniquet application in horses undergoing IVRLP of the distal aspect of a limb.6,8,10–12 Despite the fact the tourniquet remained in place, the median Cmax did not increase at > 15 minutes, which indicated that application of the tourniquet for 15 minutes should have been sufficient to achieve peak concentrations of antimicrobials in the DIP joint when performing IVRLP.
Most of the studies1,10–12 conducted to evaluate IVRLP in horses have involved a tourniquet application time of 30 minutes. However, a recent study14 revealed that concentrations of amikacin in standing sedated horses were similar after a tourniquet application time of 10 or 30 minutes. In an earlier study,12 investigators found that the mean peak amikacin concentration in synovial fluid was at 15 minutes after the beginning of perfusion, which is similar to results for the study reported here. However, in that previous study,12 samples were not obtained until 15 minutes after the beginning of perfusion, and it is possible that peak concentrations were achieved earlier during perfusion. Interestingly, the mean peak amikacin concentration in synovial fluid did not increase from 15 to 30 minutes despite maintaining application of the tourniquet in that study12; instead, the amikacin concentration actually decreased from 15 to 30 minutes. There were similar findings in the present study, which reiterated that prolonged tourniquet application is not necessary for clinical situations. One possible limitation of the present study, as determined by the fact that the Tmax in 1 horse was 20 minutes, is that the tourniquet could have been left in place for 30 minutes.
The Cmax achieved in synovial structures of the distal portion of limbs by use of IVRLP is extremely variable. Studies10,12,14,19,20 of regional perfusion have found high variability in mean antimicrobial concentrations in synovial fluid. The variation in Cmax in the study reported here may have been attributable to loss of perfusate, variations in position and pressure application of the tourniquet, and variation in the antimicrobial dose-to-body weight ratio. Even though the tourniquet was applied to all horses by the same investigator (JEN), unavoidable variations in placement could have altered the volume of tissue perfused. Only 2 of 7 horses had undetectable concentrations of amikacin in the serum prior to tourniquet release, which indicated a partial failure of the tourniquet for the other 5 horses. Interestingly, these 2 horses had the highest Cmax. All horses, except for 1, had synovial fluid amikacin concentrations ≥ 160 μg/mL (ie, 10 times the MIC of 16 μg/mL). That horse had the highest serum amikacin concentration, which indicated that tourniquet failure played a major role when attempting to achieve adequate amikacin concentrations in the synovial fluid.
Previous studies13,14,20 performed by our laboratory group yielded lower mean synovial fluid amikacin concentrations than has been reported in other studies.6,8,10 To potentially improve the peak concentrations in the synovial structures of the distal portion of a limb, we instilled the perfusate with a peristaltic pump over a 3-minute period in an attempt to minimize hydrostatic pressure within the vessel adjacent to the tourniquet, which could result in leakage into the systemic circulation. A total perfusate volume of 60 mL was used because that is the volume routinely used in our veterinary hospital. In 6 of 7 horses, we achieved a target synovial fluid concentration of 160 μg/mL (ie, 10 times the MIC), compared with achieving that target concentration in only 3 of 7 horses undergoing IVRLP with the same 60-mL volume in another study.8 One potential reason for this may have been the longer time of 3 minutes for instillation of the perfusate volume in the present study, which we hypothesize may have helped reduce the hydrostatic pressure within the vessel and thereby reduced leakage of the perfusate at the level of the tourniquet. It should be mentioned that investigators of that previous study8 measured concentrations in the metacarpophalangeal joint, compared with the DIP joint in the present study, which does not allow a direct comparison to be made. Investigators of another study6 reported that a pneumatic tourniquet placed proximal to the carpus was significantly more efficient for regional limb perfusion than was a wide rubber tourniquet. Although wide rubber tourniquets may be used more frequently in clinical practice, the pneumatic tourniquet was chosen for the present study to allow standardization of the pressure applied.
An advantage of administering an antimicrobial drug via IVRLP rather than systemically is the avoidance of adverse systemic effects because concentrations in the systemic circulation will be lower.4,7 It is important to mention that the median Cmax in serum was 19 μg/mL, which was close to the MIC of 16 μg/mL, at 25 minutes after the completion of the infusion, which indicated that IVRLP may offer some systemic antimicrobial effects in addition to local effects. This might not necessarily be beneficial in animals with limb trauma in which the blood supply may be compromised.
Some limitations of the present study included the variability in the amikacin concentrations, both in serum and synovial fluid samples, among horses, which is similar to results of previous studies6,8–10,12 of IVRLP. In addition, the study was conducted on healthy horses without evidence of joint disease; therefore, findings may not have reflected results for bacterial sepsis conditions. Pharmacokinetics and pharmacodynamics of amikacin may change in in-flamed joints.11 In that study,11 inflammation within the synovial structures actually resulted in a higher Cmax and shorter Tmax.
Another limitation was the use of the DIP joint as our source of synovial fluid samples. Two horses had to be excluded from the study because we were unable to collect an adequate volume of synovial fluid at various time points. The DIP joint was chosen to allow sampling of a synovial structure of the distal portion of a limb without having to manipulate the limb and to avoid movements resulting from those manipulations that could have affected efficacy of the tourniquet; however, difficulty retrieving synovial fluid from this joint was a limitation of the study.
Another potential limitation was that measuring amikacin concentrations in synovial fluid of the DIP joint may not have been representative of concentrations in other joints of the distal portion of the limb, as has been reported elsewhere.19 In addition, a larger number of horses may have offset some of the variability in the results. Also, it should be mentioned that the use of the pneumatic tourniquet and peristaltic pump may not have been clinically relevant because neither currently is commonly used in practice; however, they were used in the present study to reduce some of the human error and variability encountered with application of a wide rubber tourniquet and manual infusion of the perfusate.
Application of a tourniquet for 15 minutes while performing IVRLP was sufficient to achieve peak amikacin concentrations in synovial fluid of the DIP joint in the present study. Use of a shorter duration for tourniquet application (ie, 15 minutes vs 30 minutes) may allow practitioners to perform IVRLPs in a more efficient manner and likely reduce the discomfort of horses and the degree of sedation required to perform these procedures in standing horses.
Acknowledgments
Supported by The Center for Equine Health at the University of California-Davis with funds provided by contributions from private donors.
ABBREVIATIONS
| Cmax | Maximum concentration |
| DIP | Distal interphalangeal joint |
| IVRLP | IV regional limb perfusion |
| MIC | Minimum inhibitory concentration |
| Tmax | Time to maximum concentration |
Footnotes
TEVA Parenteral Medications Inc, Irvine, Calif.
True random number generator, Randomness and Integrity Services Ltd, Dublin, Ireland. Available at: www.random.org. Accessed Jul 24, 2016.
Dormosedan, Zoetis, Florham Park, NJ.
Torbugesic, Fort Dodge Animal Health, Fort Dodge, Iowa.
Carbocaine-V, Zoetis, Florham Park, NJ.
Delfi PTS portable tourniquet system, Delfi Medical Innovations Inc, Vancouver, BC, Canada.
BD Insyte, Becton Dickinson Infusion Therapy Systems Inc, Sandy, Utah.
Minipuls 3, Gilson Inc, Middleton, Wis.
VetOne, Boise, Idaho.
Roche kinetic interaction of microparticles in solution screening assay, Roche Diagnostics GmBH, Mannheim, Germany.
Cobas c311 analyzer, Roche Diagnostics GmBH, Mannheim, Germany.
SPSS Statistics for Windows, version 19.0, IBM Corp, Armonk, N Y.
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