• 1. Alkabes SB, Adams SB, Moore GE, et al. Comparison of two tourniquets and determination of amikacin sulfate concentrations after metacarpophalangeal joint lavage performed simultaneously with intravenous regional limb perfusion in horses. Am J Vet Res 2011; 72: 613619.

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  • 2. Kelmer G, Bell G, Martin-Jimenez T, et al. Evaluation of regional limb perfusion with amikacin using the saphenous, cephalic, and palmar digital veins in standing horses. J Vet Pharmacol Ther 2013; 36: 236240.

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  • 4. Rubio-Martínez LM, Cruz AM. Antimicrobial regional limb perfusion in horses. J Am Vet Med Assoc 2006; 228: 706712.

  • 5. Rubio-Martínez LM, Elmas CR, Black B, et al. Clinical use of antimicrobial regional limb perfusion in horses: 174 cases (1999–2009). J Am Vet Med Assoc 2012; 241: 16501658.

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  • 6. Butt TD, Bailey JV, Dowling PM, et al. Comparison of 2 techniques for regional antibiotic delivery to the equine forelimb: intraosseous perfusion vs intravenous perfusion. Can Vet J 2001; 42: 617622.

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  • 9. Grice SC, Morell RC, Balestrieri FJ, et al. Intravenous regional anesthesia: evaluation and prevention of leakage under the tourniquet. Anesthesiology 1986; 65: 316320.

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  • 10. Parra-Sanchez A, Lugo J, Boothe DM, et al. Pharmacokinetics and pharmacodynamics of enrofloxacin and a low dose of amikacin administered via regional intravenous limb perfusion in standing horses. Am J Vet Res 2006; 67: 16871695.

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  • 11. Mattson S, Bouré L, Pearce S, et al. Intraosseous gentamicin perfusion of the distal metacarpus in standing horses. Vet Surg 2004; 33: 180186.

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  • 12. Rubio-Martínez LM, López-Sanromán J, Cruz AM, et al. Evaluation of safety and pharmacokinetics of vancomycin after intravenous regional limb perfusion in horses. Am J Vet Res 2005; 66: 21072113.

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  • 13. Werner LA, Hardy J, Bertone AL. Bone gentamicin concentration after intra-articular injection or regional intravenous perfusion in the horse. Vet Surg 2003; 32: 559565.

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  • 14. Baetge CL, Matthews NS, Carroll GL. Comparison of 3 total intravenous anesthetic infusion combinations in adult horses. Int J Appl Res Vet Med 2007; 5: 18.

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  • 15. Hyde RM, Lynch TM, Clark CK, et al. The influence of perfusate volume on antimicrobial concentration in synovial fluid following intravenous regional limb perfusion in the standing horse. Can Vet J 2013; 54: 363367.

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  • 16. Beccar-Varela AM, Epstein KL, White CL. Effect of experimentally induced synovitis on amikacin concentrations after intravenous regional limb perfusion. Vet Surg 2011; 40: 891897.

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  • 17. Mattson SE, Pearce SG, Bouré LP, et al. Comparison of intraosseous and intravenous infusion of technetium Tc 99m pertechnate in the distal portion of forelimbs in standing horses by use of scintigraphic imaging. Am J Vet Res 2005; 66: 12671272.

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  • 18. Mahne AT, Rioja E, Marais HJ, et al. Clinical and pharmacokinetic effects of regional or general anaesthesia on intravenous regional limb perfusion with amikacin in horses. Equine Vet J 2014; 46: 375379.

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  • 19. Errico JA, Trumble TN, Bueno AC, et al. Comparison of two indirect techniques for local delivery of a high dose of an antimicrobial in the distal portion of forelimbs of horses. Am J Vet Res 2008; 69: 334342.

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Effects of regional limb perfusion volume on concentrations of amikacin sulfate in synovial and interstitial fluid samples from anesthetized horses

Jennifer L. Godfrey DVM1, Joanne Hardy DVM, PhD2, and Noah D. Cohen DVM, MPH, PhD3
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  • 1 Department of Large Animal Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX 77845.
  • | 2 Department of Large Animal Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX 77845.
  • | 3 Department of Large Animal Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX 77845.

Abstract

OBJECTIVE To evaluate the effect of volume of IV regional limb perfusion (IVRLP) on amikacin concentrations in synovial and interstitial fluid of horses.

ANIMALS 8 healthy adult horses.

PROCEDURES Each forelimb was randomly assigned to receive IVRLP with 4 mL of amikacin sulfate solution (250 mg/mL) plus 56 mL (total volume, 60 mL) or 6 mL (total volume, 10 mL) of lactated Ringer solution. Horses were anesthetized, and baseline synovial and interstitial fluid samples were collected. A tourniquet was placed, and the assigned treatment was administered via the lateral palmar digital vein. Venous blood pressure in the distal portion of the limb was recorded. Additional synovial fluid samples were collected 30 minutes (just before tourniquet removal) and 24 hours after IVRLP began; additional interstitial fluid samples were collected 6 and 24 hours after IVRLP began.

RESULTS 30 minutes after IVRLP began, mean amikacin concentration in synovial fluid was significantly greater for the large-volume (459 μg/mL) versus small-volume (70 μg/mL) treatment. Six hours after IVRLP, mean concentration in interstitial fluid was greater for the large-volume (723 μg/mL) versus small-volume (21 μg/mL) treatment. Peak venous blood pressure after large-volume IVRLP was significantly higher than after small-volume IVRLP, with no difference between treatments in time required for pressure to return to baseline.

CONCLUSIONS AND CLINICAL RELEVANCE Study findings suggested that large-volume IVRLP would deliver more amikacin to metacarpophalangeal joints of horses than would small-volume IVRLP, without a clinically relevant effect on local venous blood pressure, potentially increasing treatment efficacy.

Abstract

OBJECTIVE To evaluate the effect of volume of IV regional limb perfusion (IVRLP) on amikacin concentrations in synovial and interstitial fluid of horses.

ANIMALS 8 healthy adult horses.

PROCEDURES Each forelimb was randomly assigned to receive IVRLP with 4 mL of amikacin sulfate solution (250 mg/mL) plus 56 mL (total volume, 60 mL) or 6 mL (total volume, 10 mL) of lactated Ringer solution. Horses were anesthetized, and baseline synovial and interstitial fluid samples were collected. A tourniquet was placed, and the assigned treatment was administered via the lateral palmar digital vein. Venous blood pressure in the distal portion of the limb was recorded. Additional synovial fluid samples were collected 30 minutes (just before tourniquet removal) and 24 hours after IVRLP began; additional interstitial fluid samples were collected 6 and 24 hours after IVRLP began.

RESULTS 30 minutes after IVRLP began, mean amikacin concentration in synovial fluid was significantly greater for the large-volume (459 μg/mL) versus small-volume (70 μg/mL) treatment. Six hours after IVRLP, mean concentration in interstitial fluid was greater for the large-volume (723 μg/mL) versus small-volume (21 μg/mL) treatment. Peak venous blood pressure after large-volume IVRLP was significantly higher than after small-volume IVRLP, with no difference between treatments in time required for pressure to return to baseline.

CONCLUSIONS AND CLINICAL RELEVANCE Study findings suggested that large-volume IVRLP would deliver more amikacin to metacarpophalangeal joints of horses than would small-volume IVRLP, without a clinically relevant effect on local venous blood pressure, potentially increasing treatment efficacy.

Intravenous regional limb perfusion is commonly used to treat horses with infections of the distal portion of the limb because the technique allows for accumulation of high antimicrobial concentrations within synovial structures, soft tissues, and bone.1–6 These concentrations reportedly exceed 50 times the MICs of antimicrobials against common equine bacterial pathogens.3,5,6 The MIC of amikacin against most equine bacterial pathogens is 16 μg/mL.3,5,6 Aminoglycosides such as amikacin are concentration-dependent antimicrobials, and attainment of the MIC is recognized as sufficient for bacterial killing. However, achievement of fluid concentrations > 10 times the MIC can increase the efficacy of treatment and discourage formation of antimicrobial resistance.7

The mechanism of movement of an antimicrobial from the intravascular space into the extravascular space after IVRLP is not completely understood. Debate exists regarding whether an increase in hydrostatic pressure (such as that achieved with a large-volume perfusate) or concentration gradient (such as that achieved with a small-volume perfusate) is the principal driving force behind antimicrobial movement. In humans, IV injection of a large-volume perfusate induces dilatation of venous capillaries with loosening of contacts between endothelial cells.8 Small gaps develop in the vessel wall that allow for enhanced filtration and diffusion of molecules.8 Presumably, antimicrobial movement during IVRLP would involve a similar mechanism. However, injection of large volumes for IVRLP can lead to complications such as tourniquet failure and perfusate (antimicrobial plus diluent) leakage into systemic circulation.1,2,4,9,10 In addition, injection of a large versus small volume for IVRLP in standing horses results in more severe signs of pain, resulting in horse movement and tourniquet displacement.4 Both pressure and concentration likely play a role in antimicrobial distribution during IVRLP.

Choice of volume for IVRLP has largely been empirically based. More information is needed to determine the optimal volume for IVRLP in horses.

Antimicrobial concentrations in synovial fluid and bone following IVRLP have been evaluated in horses.10–13 Infection of the surrounding soft tissues is expected with most joint or bone infections. Antimicrobial IVRLP is presumed to achieve high antimicrobial concentrations within those soft tissues as well. Indeed, in the only reported study10 regarding IVRLP in horses, acceptable antimicrobial concentrations were achieved within the interstitial space following IVRLP.10 Additional information is needed to determine antimicrobial concentrations achieved in the soft tissues of the distal portion of equine forelimbs via IVRLP.

The objectives of the study reported here were to determine whether injection volume would affect concentrations of antimicrobial (amikacin specifically) in synovial and interstitial fluid following IVRLP, whether venous blood pressures would differ with injection volume, and whether any relationship would exist between antimicrobial concentrations in synovial and interstitial fluid within the distal portion of the forelimb of healthy horses.

Materials and Methods

Animals

Eight healthy adult Quarter Horse mares were used in the study. All horses were deemed free of metacarpophalangeal joint disease on the basis of results of physical, lameness, and radiographic evaluations. Median body weight was 523 kg (range, 495 to 575 kg), and median age was 14 years (range, 8 to 20 years). None of the horses had received antimicrobial treatment within the past 90 days. The study protocol was reviewed and approved by the Animal Care and Use Committee of the College of Veterinary Medicine at Texas A&M University.

Experimental methods

The left forelimb of each horse was randomly allocated via coin toss to receive 1 of 2 treatments (large-volume IVRLP or small-volume IVRLP), and the right forelimb was allocated to receive the opposite treatment. The large-volume perfusate (antimicrobial plus diluent) consisted of 1 g of amikacin sulfatea (4 mL of 250 mg/mL solution) and 56 mL of lactated Ringer solution,b for a total volume of 60 mL. The small-volume perfusate consisted of 1 g of amikacin sulfate (4 mL of 250 mg/mL solution) and 6 mL of lactated Ringer solution, for a total volume of 10 mL.

Twenty-four hours prior to each IVRLP procedure, a capillary ultrafiltration probec was placed in the subcutaneous space of the treated forelimb for collection of interstitial fluid samples by use of a previously described method10 with some modifications. In preparation for probe placement, horses were restrained in stocks and sedated with detomidine hydrochloride (0.01 mg/kg, IV). Skin over the dorsolateral aspect of the midportion of the metacarpus was clipped of hair and aseptically prepared, and 3 mL of 2% lidocaine hydrochloride solution was injected under the skin. A No. 15 scalpel blade was used to create a 5-mm stab incision in the prepared region. A 70-mm-long, hollow, stainless steel cannula containing the ultrafiltration probe was introduced through the stab incision into the subcutaneous space in a proximal-to-distal direction. The hollow stainless steel cannula was then withdrawn, leaving the probe in the subcutaneous space. The probe was secured to the skin, and a blood collection tube with a 10-mL capacity was applied to the ultrafiltration collection tubing for collection of interstitial fluid samples. A sterile bandage was applied to the distal portion of the limb.

IVRLP procedure

The left forelimb procedure was performed first in all horses. Following a 2-week washout period, the right forelimb procedure was then performed. During the washout period, horses were housed on grass pasture.

In preparation for IVRLP, a temporary IV catheter was placed into a jugular vein of each horse for drug administration. A pretreatment blood sample was collected from the jugular vein into a sterile blood collection tube. Then, horses were sedated with xylazine hydrochloride (1.1 mg/kg, IV) and anesthesia was induced with ketamine hydrochloride (2.2 mg/kg, IV). Anesthesia was maintained with a standard solution containing guaifenesin, xylazine hydrochloride, and ketamine administered as a constant rate IV infusion.14 Horses were positioned on a surgery pad in lateral recumbency with the treatment limb facing upward, and the limb bandage was removed. Rolled gauze was applied in the tendon groove of the proximal aspect of the metacarpus and secured with bandage tape. A 10-cm-wide pneumatic tourniquet cuffd was placed over the bandage tape, inflated to 400 mm Hg, and left in place for 30 minutes.

A 22-gauge, 2.54-cm over-the-needle catheter was inserted into the lateral palmar digital vein in a distal-to-proximal direction at the level of the lateral proximal sesamoid bone and secured with cyanoacrylate glue. A 76.2-cm IV extension tube filled with saline (0.9% NaCl) solution was connected to the catheter and a pressure transducer for continuous blood pressure monitoring. Each limb was positioned parallel to the ground and level with the heart, and the pressure transducer was zeroed at the level of the metacarpophalangeal joint.

A winged infusion set with a 23-gauge needlee was inserted into the lateral palmar digital vein in a proximal-to-distal direction at a site just distal to the blood pressure monitoring catheter. Perfusate was then injected over a 3-minute period, and the extension tube of the winged infusion set was clamped and left in place until after tourniquet removal. Venous blood pressure was recorded every 15 seconds throughout the 30-minute procedure. Once 30 minutes had elapsed, but before the tourniquet was removed, a posttreatment blood sample was collected from the jugular vein into a blood collection tube. The tourniquet was deflated, and the blood pressure catheter and winged infusion set were removed. The bandage on the distal portion of the limb was removed, and the horse was allowed to recover without assistance. Temporary IV catheters were removed before horses were returned to their stalls.

Collection of synovial and interstitial fluid samples

Synovial fluid samples were collected from the metacarpophalangeal joint by repeated aseptic arthrocentesis with a 20-gauge, 3.8-cm needle before and 30 minutes (before tourniquet removal) and 24 hours after IVRLP began. Interstitial fluid samples were collected from the metacarpus before and 6 and 24 hours after IVRLP began by emptying the blood collection tube attached to the ultrafiltration probe. After each collection, a new blood collection tube with a 10-mL capacity was applied to the collection tubing. Following collection of the 24-hour synovial and interstitial fluid samples, the ultrafiltration probe was removed from the subcutaneous space. Horses were monitored for an additional 24 hours before they were returned to pasture.

The original study design had included collection of interstitial fluid samples 30 minutes after IVRLP began, in accordance with a reported protocol10 for consistency with timings for collection of synovial fluid samples. However, preliminary work by our research group revealed that sample collection at the 30-minute point yielded an inadequate volume of interstitial fluid for analysis. That work also revealed that the shortest period needed to collect an adequate volume for analysis (0.5 mL) was 6 hours.

Sample analysis

Blood samples were allowed to clot, then samples were centrifuged at 1,000 × g for 5 minutes and serum was harvested. Serum, interstitial fluid, and synovial fluid samples were transferred to sterile plastic microcentrifuge tubes and stored at −80°C until analyzed.

Amikacin concentrations in serum, interstitial, and synovial fluid samples were measured with a homogenous enzyme immunoassay.f This assay involves competition between the drug in the samples and the drug labeled with the enzyme glucose-6-phosphate dehydrogenase for antibody binding sites. Enzyme activity decreases with binding to the antibody, so drug concentrations in samples were measured in terms of enzyme activity. Calibration of the analyzer was performed by use of internal standards provided by the manufacturer. Serum samples were analyzed directly without modification. The limit of detection of the assay for measurement of amikacin concentration was 2.5 μg/mL.

For serum, synovial, and interstitial fluid samples, a calibration curve was made by analysis of respective blank fluid samples fortified with amikacin reference standard. The calibration curve included concentrations between 2.5 and 50 μg/mL. Synovial and interstitial fluid samples obtained during the study were analyzed in the manner described for the calibration curve. Samples containing > 50 μg of amikacin/mL were diluted with 1 to 2 parts calibration solution, and the entire assay sequence was repeated. Amikacin concentration was determined by multiplying the result by the dilution factor.g

Statistical analysis

For amikacin concentrations in synovial and interstitial fluid, data were analyzed by means of linear mixed-effects modeling, with amikacin concentration as the outcome (dependent) variable, horse as a random effect, and assessment point, treatment (large or small volume), and their interactions as categorical variables. To meet distributional assumptions of modeling, synovial and interstitial fluid concentration values were transformed by adding 1 and calculating the base-10 logarithm of that sum. The addition of a unit of 1 was necessary because amikacin concentrations for all horses before treatment were 0. Post hoc comparisons between treatments and among assessment points were performed by use of the Sidak method.

Baseline venous blood pressure data were analyzed by use of the Wilcoxon signed rank test to compare paired differences between treatments for individual horses. Time required for venous blood pressure to return to baseline was determined and compared between treatments by use of the Wilcoxon rank sum test.

For all tests, a value of P < 0.05 was used to indicate a significant difference. All analyses were performed with the aid of statistical software.h Mean values and 95% confidence intervals calculated from logarithmically transformed data were transformed back to natural values for reporting purposes.

Results

Animals

None of the 8 horses developed clinically important complications or lameness throughout the study period. The ultrafiltration probes appeared to be tolerated by all horses. Mild to moderate swelling around the region of the lateral palmar digital vein was observed in 8 forelimbs (both limbs of 1 horse and 1 limb of each of 6 horses) during IVRLP with amikacin (2 limbs during small-volume [10 mL] IVRLP and 6 during large-volume [60 mL] IVRLP). Swelling of the distal portion of the limb was treated with standard bandaging and resolved within 24 to 36 hours after IVRLP. Ultrafiltration probe malfunction resulted in no interstitial fluid collection for 12 of the 48 planned collection points.

Amikacin concentrations in synovial and interstitial fluid samples

For synovial fluid samples, significant differences in amikacin concentrations between large and small-volume treatments that were dependent on assessment point were identified by means of mixed-effects regression analysis of logarithmically transformed values (Table 1). Before IVRLP, synovial fluid samples had no detectable amikacin for either treatment. Synovial fluid amikacin concentrations 30 minutes after IVRLP began but before the tourniquet was removed were significantly greater for the large-volume versus small-volume treatment. Amikacin concentrations were > 10 times the reported MIC (ie, > 160 μg/mL) against common equine pathogens at the 30-minute assessment point for 6 of 8 limbs following large-volume treatment but for only 3 of 8 limbs following small-volume treatment. Although mean amikacin concentrations for both treatments had decreased to less than the MIC by the 24-hour assessment point, concentrations in synovial fluid samples remained significantly greater for the large-volume versus small-volume treatment.

Table 1—

Mean (95% confidence interval) amikacin concentrations in synovial and interstitial fluid samples obtained from the metacarpophalangeal joint and metacarpus, respectively, of 8 healthy horses before (0 hours) and at various points after horses received IVRLP with the same dose of amikacin sulfate (1 g) contained in a large (60 mL) or small (10 mL) volume of perfusate.

 Large volume
Assessment point (h)No. of horsesConcentration (μg/mL)No. of horsesConcentration (μg/mL)
Synovial fluid
   080a80a
   0.58459 (205–1,028)b870 (26–190)c
   24814 (7–28)d82 (0–5)a
Interstitial fluid
   060a70a
   66723 (285–1,838)b521 (5–81)c
   24612 (5–31)d62.0 (0–7)a

Values represent estimates from mixed-effects modeling of amikacin concentrations by assessment point, treatment, and assessment point-treatment interaction, which have been transformed back to natural values. Ultrafiltration probe malfunction resulted in no interstitial fluid collection for 12 of the 48 planned collection points.

Within fluid types, values with different superscript letters differ significantly (P < 0.05).

The left forelimb of each horse was randomly assigned to receive the large- or small-volume treatment first, and IVRLP was performed in that limb for all horses. Following a 2-week washout period, the right forelimb received the opposite treatment.

For the interstitial fluid samples, results obtained were similar to those for synovial fluid samples. Before IVRLP, samples had no detectable amikacin for either treatment. Interstitial fluid amikacin concentrations 6 hours after IVRLP began were significantly greater for the large-volume versus small-volume treatment (Table 1). Amikacin concentrations were > 10 times the reported MIC against common equine pathogens at the 6-hour assessment point for 6 of 8 limbs following large-volume treatment but for only 1 of 8 limbs following small-volume treatment. Mean concentrations for both treatments had decreased to below that MIC by the 24-hour assessment point, but concentrations remained significantly greater for the large-volume versus small-volume treatment.

Because of differences in collection points for synovial and interstitial fluid samples, no correlation calculations were performed. However, a general pattern was identified in comparisons between synovial and interstitial fluid amikacin concentrations. For the large-volume treatment, of the 6 limbs that had synovial fluid amikacin concentrations > 160 μg/mL at the 30-minute assessment point, 4 limbs also had interstitial fluid amikacin concentrations > 160 μg/mL at the 6-hour assessment point. For the small-volume treatment, of the 3 limbs that had synovial fluid concentrations > 160 μg/mL at the 30-minute assessment point, only 1 limb contained an interstitial fluid amikacin concentration > 160 μg/mL at the 6-hour assessment point.

Venous blood pressure measurements

No significant (P = 0.64) difference was identified between treatments in baseline venous blood pressures. Maximum venous blood pressure was identified in all horses at the end of perfusate injection (3 minutes). Maximum pressure for the large-volume treatment (median, 292.5 mm Hg; range, 242 to 320 mm Hg) was significantly (P = 0.008) greater than that for the small-volume treatment (median, 80 mm Hg; range, 40 to 139 mm Hg). For the large-volume treatment, the median time to return to baseline pressure was 18.0 minutes (range, 9.2 minutes to never returned) and for the small-volume treatment was 16.8 minutes (range, 11.0 minutes to never returned); this difference was not significant (P = 0.78).

Two horses after small-volume treatment and 3 different horses after large-volume treatment had venous blood pressures that did not return to baseline. However, the difference between final and baseline pressures was small and similar between small-volume (median, 6 mm Hg; range, 3 to 9 mm Hg) and large-volume (median, 9 mm Hg; range, 5 to 12 mm Hg) treatments.

Serum amikacin concentrations

Use of the pneumatic tourniquet at a pressure of 400 mm Hg prevented detectable movement of amikacin into systemic circulation for all horses for both the large- and small-volume treatments. All serum samples pertaining to before IVRLP and immediately prior to tourniquet removal contained no detectable concentration of amikacin.

Discussion

In the study reported here, large-volume (60 mL) IVRLP with the same amount of amikacin (1 g) as administered during small-volume (10 mL) IVRLP resulted in significantly higher amikacin concentrations in synovial and interstitial fluid samples from the forelimbs of anesthetized horses. These findings were in contrast to those in another study,15 in which no significant difference was identified in synovial fluid gentamicin concentration after IVRLP was administered to sedated horses with 10, 30, or 60 mL of gentamicin solution. In that study,15 movement was observed in all horses throughout the IVRLP procedure and several horses required additional sedation.

Horses receiving a large- versus small-volume perfusion could be anticipated to have greater increases in hydrostatic pressure in the distal portion of the limb that would result in a greater degree of nociception, thereby stimulating movement during IVRLP. Patient movement can result in leakage of perfusate under the tourniquet, leading to a decrease in the amount of antimicrobial available for diffusion into extravascular spaces.1,3,16,17 In the other study,15 serum samples were not obtained to determine whether the administered antimicrobial had entered systemic circulation. Because of the possible effects of leakage under the tourniquet, it is difficult to interpret the results of that study.15

Typically, horses are not anesthetized for performance of IVRLP unless they are already receiving anesthesia for another reason or their temperament does not allow for standing procedures to be performed. In another study18 involving IVRLP with amikacin, no significant difference was identified between standing and anesthetized horses in amikacin concentration in synovial fluid samples collected from the middle carpal joint, although mean synovial fluid amikacin concentration and regional pharmacokinetic values were higher when IVRLP was performed with horses anesthetized.18 In the present study, horses were anesthetized with an injectable agent to eliminate the variability associated with response to noxious stimulus and movement during the procedure. The lack of horse movement was likely important to maintaining tourniquet integrity.1,3,16 An anesthetized horse positioned in lateral recumbency could be reasonably expected to have a lower blood pressure throughout the distal portion of the limb than would a standing horse because its systemic blood pressure would be lower and because partial exsanguination of the limb might occur prior to tourniquet placement.9 Baseline venous blood pressures (85 to 125 mm Hg) identified in standing horses in a study19 involving various IVRLP procedures were 3 to 4 times the baseline venous blood pressures (20 to 50 mm Hg) identified in anesthetized horses in the present study. These differences suggested that tourniquet-induced leakage caused by high vascular pressure might be more likely to occur in standing versus anesthetized horses.

In the present study, large-volume IVRLP resulted in significantly greater venous blood pressure than did small-volume IVRLP; however, the higher blood pressure was not maintained throughout the procedure. There was no significant (P = 0.78) difference in the time required for pressures to return to baseline following large- or small-volume IVRLP. In humans receiving pressurized IV infusion, dilation of venous capillaries results in loosening of contacts between endothelial cells, such that small gaps develop in the vessel walls that facilitate rapid diffusion.8 In the horses of the present study, venous blood pressure following IVRLP at either volume rapidly returned to baseline. For this to have occurred, relaxation or stretching of vessel walls would have been necessary, which would presumably have resulted in gap formation between endothelial cells. In a limb injected with a larger volume, greater relaxation and stretching of the vessels are needed for pressure to return to baseline.

Ultrafiltration techniques have been used for many species, including mice, rats, dogs, cats, horses, and humans, for analysis of interstitial concentrations of drugs, electrolytes, or metabolites.10,20–22 Ultrafiltration involves a hydrophilic membrane that allows for a negative pressure gradient to be applied by a vacuum system.10,20–22 The membrane excludes proteins and other cellular matter, allowing the extracellular fluid and small molecules or ions to pass through.10,20–22 This process provides an ultrafiltrate that is well suited for analysis without the need for further extraction.10,21,22 Ultrafiltration probes have been used to measure antimicrobial concentrations in the medullary cavity and interstitial fluid following IVRLP in standing horses.10 That study revealed wide variability in antimicrobial concentrations within the medullary cavity, synovial fluid, and interstitial fluid. Proposed reasons for variability were tourniquet failure (which occurred for 11 of 12 horses) and perfusate leakage during infusion. The study reported here revealed similar variability in interstitial fluid samples; however, the large-volume treatment still resulted in a significantly greater amikacin concentration than did the small-volume treatment.

Because of mismatches between synovial and interstitial fluid in sample collection points after IVRLP in the present study, no corresponding measurements were available for correlation analysis at the second assessment points (30 minutes and 6 hours after IVRLP began, respectively), when concentrations were high. However, high concentrations of amikacin in synovial fluid samples (> 10 times the MIC) corresponded with high concentrations in interstitial fluid samples for 4 of 6 limbs following large-volume treatment, but for only 1 of 3 limbs following small-volume treatment; therefore, it is plausible that synovial fluid concentrations reflected interstitial concentrations and vice versa. Although we considered obtaining synovial fluid samples at the 6-hour assessment point to evaluate correlation with interstitial fluid samples, we elected against this because of concerns that joint fluid removal and intra-articular hemorrhage at the 6-hour point might affect the results of synovial fluid analysis at the final (24-hour) assessment. Instead, measurement of amikacin concentration 24 hours after IVRLP began was considered of prime importance because IVRLPs are typically performed every 24 hours in the clinical setting.

The Esmarch tourniquet is commonly used to perform IVRLP in clinical practice because of its ease of application and cost effectiveness. Use of this tourniquet results in less leakage of perfusate into systemic circulation during regional limb perfusion performed in standing horses.1 However, application and tourniquet pressure of the Esmarch tourniquet cannot be standardized. To reduce variability in tourniquet pressure among limbs in the present study, a pneumatic tourniquet with a 10-cm cuff set to a cuff pressure of 400 mm Hg was used. This tourniquet pressure was considerably greater than the minimum recommended tourniquet pressure of at least 100 mm Hg greater than arterial pressure.1,6,16 Serum samples in the present study were obtained prior to IVRLP and again 30 minutes after the procedure began, before the tourniquet was released. All serum samples were free of detectable amikacin concentrations, suggesting adequate tourniquet function.

Winged infusion sets with a 23-gauge needle were used to deliver the perfusate during IVRLP in the present study. Typically, we prefer to use an over-the-needle catheter to deliver the perfusate. In our experience, less damage occurs to the vein with an over-the-needle catheter than with a winged infusion set, possibly because a flexible catheter is less damaging to the vein than a needle. In preliminary evaluations, the authors (JLG and JH) found that catheterizing the medial (down-side) palmar digital vein was difficult, often resulting in hematoma formation and transient loss of access to the vein. Consequently, only the lateral palmar digital vein was used for the procedure. An over-the-needle catheter was placed in the lateral palmar digital vein for blood pressure monitoring. This catheter proved much easier and effective for use with a winged infusion set with a 23-gauge needle because of its small size and ease of placement for perfusate delivery. Following perfusate injection, the extension tube of the winged infusion set was clamped, but the needle was left in the vein to avoid inducing perfusate leakage and hematoma formation or creating a need for a pressure bandage that would alter blood pressure readings.

All horses remained free of lameness throughout the study period. Mild to moderate swelling around the sites of venipuncture was observed for 8 limbs, 6 of which received the large-volume perfusion. These swellings were likely the result of a combination of perfusate and blood leakage around the venipuncture sites. As previously mentioned, high pressures generated from large-volume perfusions can cause escape of the perfusate under the tourniquet (not observed in the present study) as well as leakage of perfusate from the venipuncture site or around the catheter used for injection.1,2,4,9,10,15 Injection of a large volume in standing, sedated horses results in pain, leading to limb movement that leads to loss of tourniquet integrity, hematoma formation, and vasculitis.1,3,4,16,17 Therefore, a greater degree of sedation than that used for small-volume perfusion is recommended for large-volume IVRLP. Repeated IVRLP becomes increasingly difficult to perform when complications such as hematoma and cellulitis develop. Therefore, vein integrity should be closely monitored and complications treated appropriately in horses receiving large-volume perfusions.

The study reported here had some additional limitations. First, only healthy horses were used. Synovial inflammation associated with infections of the distal portion of the limb results in an increase in vascularization and vessel permeability, which can affect antimicrobial movement throughout extravascular spaces.16,23 Time to maximal drug concentration is briefer and maximal drug concentration is greater after intra-articular IVRLP with amikacin in radiocarpal joints with experimentally induced synovitis versus healthy joints.16 Second, malfunctioning ultrafiltration probes resulted in a lack of some interstitial fluid samples in the present study, resulting in less statistical power. The reason for probe malfunction was not determined, but there appeared to be a relationship with the occurrence of hemorrhage during probe placement in that probe malfunction was identified more frequently in horses that had moderate hemorrhage during ultrafiltration probe placement. We hypothesized that blood clots surrounding the ultrafiltration membrane prevented collection of interstitial fluid samples.

Third, we observed marked variability in amikacin concentrations in synovial and interstitial fluid samples pertaining to both the large-volume and small-volume treatments. Variability in antimicrobial concentrations in these types of fluids is a common finding that is not specific to the present study.1,3,6,10–12,15,16,18,19 Potential causes for this variability are tourniquet failure (although not likely in the present study), blood contamination of synovial fluid from repeated arthrocentesis, variability in hematoma formation, or differences in vascular anatomy or lymphatic drainage among horses. The variability in amikacin concentration suggested that additional investigations are needed to understand the mechanisms of antimicrobial movement through tissues during and following IVRLP.

In the present study, high-volume IVRLP of the metacarpophalangeal joint resulted in significantly higher concentrations of amikacin in synovial and interstitial fluid samples obtained from the forelimbs of healthy anesthetized horses. Furthermore, venous blood pressure in the distal portion of the limb was significantly greater after injection of a large volume of perfusate versus a small volume, but pressures for both perfusate volumes returned quickly to baseline. Last, it appeared that antimicrobial concentrations in interstitial fluid could generally be expected to be similar to those in synovial fluid, but individual variability among horses must be anticipated. Study findings suggested that large-volume IVRLP would potentially increase treatment efficacy, without a clinically relevant effect on local venous blood pressure.

Acknowledgments

This manuscript represents the results of a clinical research project by Dr. Godfrey and will serve as part of the requirements to be eligible for the American College of Veterinary Surgeons certifying examination.

Supported by a grant from the Department of Large Animal Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University (RGS13–13). Dr. Cohen received support from the Link Equine Research Endowment, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University.

The authors declare that there were no conflicts of interest.

ABBREVIATIONS

IVRLP

IV regional limb perfusion

MIC

Minimal inhibitory concentration

Footnotes

a.

Amiglyde-V, Fort Dodge Animal Health, Fort Dodge, Iowa.

b.

Lactated Ringer Injection USP, Baxter Healthcare Corp, Deer-field, Ill.

c.

BASi, Purdue Research Park, West Lafayette, Ind.

d.

Scandmed, Ridgeland, Miss.

e.

Nasco, Fort Atkinson, Wis.

f.

Syva Emit amikacin assay, Siemans Healthcare Diagnostics Inc, Newark, Del.

g.

Clinical Pharmacology Laboratory, College of Veterinary Medicine, Auburn University, Auburn, Ala.

h.

S-PLUS, version 8.2, TIBCO Inc, Seattle, Wash.

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Contributor Notes

Address correspondence to Dr. Hardy (jhardy@cvm.tamu.edu).