Materials and Methods

Horses—To be included in this study, stallions had to be at least 2 years old, weigh > 300 kg, and have testicles with grossly normal anatomic features. Prior to admission to the study, a physical examination was performed to ensure that the horse was healthy. Twenty-eight stallions were available for participation in the study; prior to inclusion of each horse, informed consent was obtained from the owner or trainer. Among the horses, there were 17 Standardbreds, 6 Norwegian Coldblooded Trotters, 2 Thoroughbreds, 1 Quarter Horse, 1 Icelandic horse, and 1 mixed-breed horse. The mean ± SD age of the horses was 4.2 ± 2.8 years (age range, 2 to 14 years), and mean weight was 466 ± 62 kg (weight range, 322 to 620 kg).

Anesthesia and instrumentation—Food was withheld from each horse for at least 12 hours prior to anesthesia, although free access to water was permitted. A catheter was placed in a jugular vein. Acepromazine (20 µg/kg, IM) was administered while each horse was in the stable; prior to the horse being walked to the induction area, sodium penicillin (10 × 10⁶ units, IV) dissolved in sterile water and flunixin (1.1 mg/kg, IV) was administered. Romifidine (50 µg/kg, IV) and butorphanol (20 µg/kg, IV) were administered to each horse in the induction area. Anesthesia was induced by use of ketamine (2.5 mg/kg, IV) and midazolam (50 µg/kg, IV). After orotracheal intubation, each horse was placed in dorsal recumbency and the endotracheal tube was connected to a circle anesthesia system. Isoflurane vaporized in oxygen and air was administered; the gas mixture was continuously drawn from the rostral end of the endotracheal tube into an anesthetic monitor^a that analyzed inspiratory and expiratory isoflurane, O₂, and CO₂ concentrations. Systolic, diastolic, and mean arterial blood pressures and pulse rate were measured directly through a catheter that was placed in a facial artery and connected to a pressure transducer^b (zeroed at the level of the thoracic inlet). Intermittent positive pressure ventilation was started and adjusted to keep end-tidal CO₂ concentration between 5% and 6%. Dobutamine^c was infused with an infusion pump^d at a rate required to maintain MABP > 60 mm Hg. A polyionic balanced electrolyte solution^e was administered IV. End-tidal isoflurane was stabilized at 1.7% for the duration of the experimental period. The point at which isoflurane concentration became stable was designated T0 minutes. After a minimum of 10 minutes after the end-tidal isoflurane concentration was stabilized (ie, T10 minutes), the dobutamine infusion rate, fluid infusion rate, and ventilation were held constant (to avoid bias in blood pressure and pulse rate measurements) for the duration of the experimental period. Also at T10 minutes, a 0.8 × 40-mm needle was inserted in the middle of each testicle; via these needles, either no injection was made (control group) or 15 mL of 2% lidocaine^f (lidocaine group) was injected into the testicles. Both testicles in each horse received the same treatment. In 2 horses, a solution of lidocaine labeled with radioactive carbon (C¹⁴) was used. All needle placements and injections were performed by the same anesthetist (HAH).

Surgery—All surgeries were performed by 1 of 2 designated surgeons (SL, TR). The skin over each testicle was incised, and the left incision advanced down to the common vaginal tunic. The subcutaneous tissue was stripped proximally with a gauze swab. The spermatic cord was clamped with crushing forceps before placement of a transfixing ligature proximal to the forceps. An Ochsner forceps was placed further distally on the spermatic cord to avoid leakage of blood from the testicle when it was removed; the spermatic cord was then transected with a scalpel immediately distal to the crushing forceps. The crushing forceps was kept closed for at least 5 minutes after transection of the spermatic cord. The procedure was repeated on the right testicle, after which the subcutaneous tissue and skin at each incision site were sutured with absorbable suture.

Recording of data—In each horse, the left testicle was always removed first; the data collected regarding blood pressures, pulse rate, and cremaster muscle tension were associated with removal of this testicle. The exact times of needle insertion, skin incision, and clamping and transection of the left spermatic cord were recorded. Values of SABP, DABP, MABP, and pulse rate were obtained from the anesthetic monitor; values were recorded 15 minutes after the end-tidal isoflurane concentration was stabilized (ie, T15 minutes) and at 30-second intervals thereafter. Baseline values were calculated as mean values of measurements made between T15 and the start of surgery. Surgery was commenced 20 minutes after the end-tidal isoflurane concentration was stabilized (ie, T20 minutes), and data were recorded until 5 minutes after the left crushing forceps was applied; the shortest data collection period extended from T20 minutes to T26.5 minutes, and the longest data collection period extended from T20 minutes to T29 minutes. Immediately after completion of the surgery, the surgeon scored the degree of cremaster muscle tension encountered during removal of the left testicle by use of a 10-cm VAS; 0 cm represented no cremaster muscle tension, and 10 cm represented extreme cremaster muscle tension that prevented completion of surgery. For each horse, rectal temperature and general well-being were assessed and surgical wounds were examined on the day after surgery and on subsequent days if the horse was not returned home. Three weeks after surgery, each client was contacted and interviewed by use of standardized questions to obtain information regarding possible postoperative complications.

Autoradiography—Radiolabeled [C¹⁴]lidocaine^g (0.24 mg in 0.5 mL of ethanol; specific activity, 56 mCi/mmol) was evaporated to dryness by use of a gentle stream of N₂ gas. Two milliliters of lidocaine^f (20 mg/mL) was then added and vortexed, and the resultant solution was transferred back to the 20-mL vial of lidocaine. This resulted in a [C¹⁴]lidocaine solution with a radioactivity level of 1.25 µCi/mL. In each of 2 horses, radiolabeled lidocaine was injected in the left testicle and the spermatic cord was clamped at 12.3 or 12.7 minutes after injection. The injected testicle and spermatic cord were removed from each horse and immediately frozen in liquid nitrogen. The Oschner forceps was kept closed to avoid loss of blood before the specimen was frozen. The testicle and spermatic cord were embedded in a 1% (wt/vol) gel of carboxymethylcellulose and frozen at −75°C in a bath of hexane and dry ice. Sagittal sections (30 µm thick) from different levels of the samples were prepared by use of a cryomicrotome^h and collected at −20°C on adhesive tapeⁱ according to the method of Ullberg.⁷ After freeze-drying at −20°C for 24 hours, the sections were apposed to radiographic film.^j Following exposure at −20°C for 80 days, the radiographic films were developed.

Randomization and statistical analysis—The study was conducted as a prospective, randomized, partially blinded clinical study. On the basis of previous data,^4,8 a power analysis was performed. The analysis was based on a 2-sided test and a significance level of 5%, with the intention of detecting a difference of 12 mm Hg in MABP response between groups. Inclusion of at least 28 stallions was estimated to give a power > 90%. Randomization of horses to either the control or lidocaine group was done in blocks of 4 prior to the study. To have balanced group sizes, 2 horses were allocated to each treatment in each block. Just prior to the needle insertion, a numbered, sealed envelope was opened to reveal which treatment the horse was to receive; after the envelope was opened, no adjustments of infusion rates or ventilator settings were permitted. The surgeons were unaware of the treatment administered. For data analyses, a significance level of 5% and 2-sided tests were used throughout the study. Individual mean pulse rates and SABP, DABP, and MABP values for the baseline period (T15 to T20 minutes) and the intraoperative mean values during the 5-minute period after the left spermatic cord was clamped were calculated. Differences between these individual means were calculated. Prior to further analysis, all data were tested for normality by use of a Shapiro-Wilk test. The baseline means for pulse rate, SABP, DABP, and MABP were tested between groups by use of Student t tests. The individual differences in pulse rate, SABP, DABP, and MABP were analyzed within and between groups by use of Student t tests. The influence of surgeon on the cremaster muscle tension data was evaluated by visual inspection of the data and use of a Mann-Whitney test. To avoid surgeon-associated bias affecting the cremaster muscle tension data, the results were transformed to a common percentage scale; the results from each surgeon were corrected directly as a percentage by setting the largest observation of that surgeon to 100%. A comparison between groups was then performed by use of a Mann-Whitney test. Commercially available software^k,l was used for data handling and statistical analyses. Results are given as mean ± SD unless otherwise stated.

Results

Of the 28 horses in the study, 14 were allocated to the control group and 14 were allocated to the lidocaine group. In the control group, there were 9 Standardbreds, 3 Norwegian Coldblooded Trotters, 1 Thoroughbred, and 1 mixed-breed horse; the mean age of these horses was 3.5 ± 1.7 years, and the mean weight was 460 ± 47.6 kg. Horses in the control group received dobutamine at an infusion rate of 1.1 ± 0.47 µg/kg/min. Of the 2 surgeons involved in the study, 1 surgeon castrated 9 control horses and the other castrated 5 control horses. In the lidocaine group, there were 8 Standardbreds, 3 Norwegian Coldblooded Trotters, 1 Thoroughbred, 1 Quarter Horse, and 1 Icelandic horse; the mean age of these horses was 4.9 ± 3.5 years, and the mean weight was 471 ± 75 kg. Horses in the lidocaine group received dobutamine at an infusion rate of 1.1 ± 0.53 µg/kg/min. Of the 2 surgeons involved in the study, each castrated 7 lidocaine-treated horses.

In the lidocaine group, the dose of lidocaine administered was 1.3 ± 0.13 mg/kg. Induction of anesthesia, surgery, maintenance of anesthesia, and recovery from anesthesia of all horses proceeded without complications. No horse in either group moved during surgery, and no additional bolus of anesthetics was given to any horse. In the control group, 1 horse had mild colic postoperatively and in 1 horse, dehiscence of the right suture line developed a few hours after surgery. In the lidocaine group, 3 horses had high rectal temperatures the first morning after surgery (range, 38.6° to 39.3°C) and in 1 horse, the right surgical incision was opened 6 days after surgery because of swelling of the right part of the scrotum. The horse with colic was treated with metamizole (dipyrone; 32 mg/kg, IV, once), 20 L of polyionic balanced electrolyte solution IV, and 8 L of water through a nasogastric tube. The horse with dehiscence of the right suture line was treated for 3 days with procaine penicillin (20,000 U/kg, IM, q 12 h). The horse with a rectal temperature of 39.3°C postoperatively was treated for 3 days with flunixin (1.1 mg/kg, IV, q 24 h) and procaine penicillin (20,000 U/kg, IM, q 12 h), and the other 2 horses with high rectal temperature were not given any specific treatment. The horse in which the right suture line was opened was treated with trimethoprim (5 mg/kg, PO, q 12 h) and sulfadiazine (25 mg/kg, PO, q 12 h) for 5 days. The rest of the horses were in good general condition postoperatively. On the basis of information received from owners, all complications in horses of both groups resolved within 3 weeks after surgery. No significant differences in blood pressure and pulse rate baseline values between the 2 groups were detected.

Cremaster muscle tension was not normally distributed, and a significant (P = 0.03) difference in uncorrected VAS scores between surgeons was identified. Median (25% to 75% quantiles) uncorrected VAS scores for the control and lidocaine groups were 4.3 cm (range, 2.8 to 5.7 cm) and 1.0 cm (range, 0.2 to 2.1 cm), respectively. Median (25% to 75% quantiles) corrected VAS scores for the control and lidocaine groups were 69% (range, 52% to 94%) and 14% (range, 5% to 31%), respectively. There was a significant difference (P = 0.009) in corrected VAS scores between groups.

Visual evaluation of the autoradiograms of testicles that were injected with [C¹⁴] lidocaine prior to removal from 2 of the horses revealed poor distribution of radiolabeled lidocaine within the testicular parenchyma and cremaster muscle but diffuse distribution of lidocaine to and within the spermatic cord (Figure 1).

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Photograph (A) and corresponding autoradiogram (B) of a sagittal section from a testicle that was removed from a stallion after the testicle had been injected with 15 mL of 2% lidocaine labeled with radioactive carbon (performed 12.7 minutes prior to clamping of the testicular cord). After removal from the horse, the testicle was immediately frozen and tissue sections prepared. To obtain autoradiograms, sections were apposed to radiographic film for 80 days; films were subsequently developed. In panel A, notice the testicular parenchyma (T), epididymidis (E), spermatic cord (S), and cremaster muscle (C). In panel B, blackened areas are indicative of the presence of radiolabeled lidocaine in those testicular structures. Bar = 5 cm.

Citation: American Journal of Veterinary Research 67, 3; 10.2460/ajvr.67.3.403

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Discussion

In the study of this report, castration induced significant increases in blood pressure measurements in stallions anesthetized with isoflurane (1.7% end-tidal isoflurane concentration), compared with baseline values, but there were no significant changes in pulse rate. Compared with findings in the control group, intratesticular injections of lidocaine prior to castration reduced the magnitude of these surgery-induced increases in blood pressure measurements, illustrating the analgesic effect of the treatment. Cremaster muscle tension may affect the ease with which castration can be performed; after an intratesticular lidocaine injection, the surgeons involved in the present study detected significantly less cremaster muscle tension, compared with subjective findings in untreated control horses. This suggests that intratesticular administration of lidocaine may facilitate the castration procedure.

Isoflurane has little analgesic effect,⁹ as demonstrated by the increase in blood pressure measurements during castration in the horses of the present study, and analgesic supplementation is warranted. Intratesticular administration of lidocaine improved anesthetic quality by increasing muscle relaxation, providing additional analgesia, and attenuating autonomic reflexes, which together with loss of consciousness are considered to be the properties of balanced anesthesia.¹⁰ Volatile agents induce dose-dependent cardiovascular and respiratory depression in horses⁵; thus, decreases in the amounts of those volatile agents required by patients undergoing various procedures are probably beneficial. Administration of other agents that provide analgesia may decrease the requirement for volatile anesthetic agents. In the study of this report, intratesticular administration of lidocaine appeared to provide a noticeable analgesic effect in stallions undergoing castration. The injection is easily performed, and because the injection site is removed during the surgery, there is little associated risk of interference with surgery or wound healing. The present study did not reveal any increased risk of complications after intratesticular injection of lidocaine in horses.

In several studies^11–13 of halothane-anesthetized horses, it has been found that different surgical stimuli induce an increase in MABP (compared with preoperative values) that is mediated by an increase in systemic vascular resistance; the increased resistance to blood flow results in a decreased cardiac output. This finding has also been determined in ponies when castration was applied as a surgical stimulus during anesthesia with halothane.² In the present study, the intratesticular lidocaine injection decreased the castration-induced increase in MABP detected in control horses; the increase in MABP may be associated with a similar reduction in cardiac output during isoflurane anesthesia.

The testis, epididymidis, and content of the spermatic cord are innervated by visceral fibers that extend distally within the spermatic cord, whereas the scrotal skin, spermatic fascia, and cremaster muscle are innervated by somatic fibers that originate from outside the spermatic cord.¹⁴ During castration of unmedicated young pigs, cutting of the spermatic cord was the part of the procedure associated with most signs of pain¹⁵; analgesia of the spermatic cord is thus warranted. Via an intratesticular injection, the local anesthetic is administered distal to the spermatic cord, which may seem to be an illogical treatment. In the present study, the autoradiograms of testicles treated with an intratesticular injection of radiolabeled lidocaine indicated that the drug was readily transported proximally in the spermatic cord and subsequently distributed diffusely within the spermatic cord. Distribution within the testicular parenchyma was sparse. Lidocaine did not cross the parietal layer of the tunica vaginalis into the cremaster muscle to any noticeable extent. In the horses of the lidocaine group, there was a significant blood pressure response to castration, which indicates that the local anesthetic block was not complete. Autoradiography revealed that the observed analgesic effects were most likely caused by inhibition of visceral afferent nerve fibers in the spermatic cord, whereas the somatic nociception from the skin incision and clamping of the cremaster muscle was not affected. The autoradiograms indicated that neither the cremaster muscle nor its innervation contained an appreciable amount of radiolabeled lidocaine; the reduction in cremaster muscle tension detected by the surgeons in association with intratesticular administration of lidocaine was probably caused by a decrease in visceral nociception and not because of direct paralysis of the cremaster muscle.

Extracellular fluid in the testicular parenchyma is absorbed into lymphatic capillaries, which drains into larger lymphatic vessels in the spermatic cord.¹⁶ In rams, the rate of lymph flow from each testis is high, compared with flow per unit weight from other parts of the body.¹⁶ Increased hydrostatic pressure that results from lidocaine injection into the testicular parenchyma probably increases lymph flow further, facilitating distribution of lidocaine into the spermatic cord. A rapid lymphatic transport of methylene blue after intratesticular injection has been identified in horses; 1.5 minutes after injection, methylene blue was detected 20 cm proximal to the testis.¹⁷ An autoradiographic study¹⁸ in young pigs in which 1% lidocaine with 5 µg of adrenalin/mL was injected into testicles revealed that there was a higher concentration of lidocaine in the spermatic cord 3 minutes after intratesticular injection, compared with the amount present in the spermatic cord 10 minutes after injection. This finding illustrates the speed of distribution of lidocaine into the spermatic cord after intratesticular administration. These results indicate that the maximum analgesic effect of lidocaine may have been attained sooner than 10 minutes after intratesticular injection in horses.

Systemically administered lidocaine may be used for analgesia during anesthesia of equids.¹⁹ In the present study, there was a rapid distribution of lidocaine into the spermatic cord of stallions, and a previous study¹⁷ revealed rapid systemic absorption of various drugs after intratesticular administration in different mammals. Systemic effects may have caused some of the analgesia detected in horses in our study. When lidocaine is administered IV for analgesia in ponies, a starting bolus dose of 2.5 to 5 mg/kg administered IV has been suggested.¹⁹ The mean ± SD dose of lidocaine given to the horses in our study was 1.3 ± 0.13 mg/kg. On evaluation of the autoradiograms of tissue sections from testicles removed during castration, it was evident that there was still an appreciable amount of lidocaine in the testicles at that time. We believe that the systemic analgesic effect of lidocaine in the horses of the present study was of minor importance and that the effects on blood pressure measurements were mainly a result of local analgesia.

In our investigation, broad inclusion criteria were used to achieve a representative sample of the hospital equid population; therefore, breed, age, and weight varied among the horses included in the study. This variation among horses could potentially have biased the data collected in our study; however, because the breeds, age, and weights of horses and the dobutamine infusion rates did not differ much between the lidocaine and control groups, a possible bias was considered unlikely. Ideally, 1 surgeon should have performed all surgeries, but this was not possible in our situation. The shortcoming of having 2 surgeons perform the castrations was reduced because both surgeons contributed almost equally in both groups. Cremaster muscle tension was scored subjectively; as a consequence, the influence of the surgeon on the results was investigated further and a difference between surgeons was identified. To overcome this, each surgeon’s VAS scores were corrected directly as a percentage of the greatest observation of that surgeon, thereby reducing variability between surgeons.

A needle insertion without subsequent injection was chosen as the control treatment because pilot investigations revealed that bleeding after the needle insertion revealed whether an injection had been made or not. Injection of saline solution was not used in our study because we intended to compare the use of lidocaine injection with a clinical alternative, namely the injection of no fluid. Masking of treatments to the anesthetist who administered them was not considered necessary because when treatment allocation for each horse was revealed, no adjustments that could influence cardiovascular variables were permitted. A crossover study design in which each horse would receive an injection of lidocaine in one testicle and not the other was considered for our study but was not implemented because of concerns regarding carryover effects between removals of the 2 testicles.

In the present study, cardiovascular responses were considered to be indicators of nociception; such responses have traditionally been used as indicators of nociception during anesthesia. In a previous study⁴ in isoflurane-anesthetized horses, cardiovascular responses were compared with electroencephalographic responses, and cardiovascular responses were found to be more sensitive indicators of nociception. Experimental work performed in rodents revealed that blood pressure measurements and pulse rate may increase or decrease in response to nociception.²⁰ During ocular or genital surgery, a vasovagal response may also decrease blood pressure and pulse rate in other species.^21,22 In the present study, castration induced an increase in blood pressure measurements in all horses in both groups. A possible reason is that all horses were anesthetized at the same end-tidal isoflurane concentration and the intention was to attain similar physiologic conditions in all horses prior to castration. No significant difference in pulse rate response to castration was detected within or between groups. However, results of a previous study⁴ indicated that pulse rate decreased from baseline values during castration of isoflurane-anesthetized horses. A possible explanation for the difference between these 2 studies may be that in that previous study, an end-tidal isoflurane of 1.4% was used; therefore, the CNS of those horses was less depressed and thus may have been more responsive to nociception that could facilitate a vasovagal response. Another difference was that glycopyrrolate was administered to the horses in the previous study,⁴ which may have confounded the results. In the present study, we believe it is fair to assume that the smaller increase in blood pressure measurements in stallions during castration indicated comparatively less intense nociception.

Our data suggest that following intratesticular administration, lidocaine distributes into the spermatic cord of horses in concentrations that are sufficient to decrease nociception through visceral nerves, whereas nociception from the cremaster muscle, scrotal skin, and spermatic fascia that is conducted through somatic afferent nerve fibers is probably unaffected. An intratesticular injection of lidocaine in isoflurane-anesthetized horses prior to castration appears to decrease cardiovascular responses to the surgical stimulus and cause less cremaster muscle tension during surgery.