Graefes Arch Clin Exp Ophthalmol DOI 10.1007/s00417-013-2390-8

MISCELLANEOUS

The effects of bupivacaine injection and oral nitric oxide on extraocular muscle in the rabbit Burak Bilgin & Huseyin Gursoy & Hikmet Basmak & Mete Ozkurt & Nese Tuncel & Funda Canaz & Serap Isiksoy & Ertugrul Colak

Received: 17 August 2012 / Revised: 20 March 2013 / Accepted: 20 May 2013 # Springer-Verlag Berlin Heidelberg 2013

Abstract Aim Nitric oxide (NO) plays a key role in muscle regeneration, which is the primary response, observed during bupivacaine-induced extraocular muscle (EOM) hypertrophy. Our aims were to investigate the effects of bupivacaine injection into the rabbit EOM and the interaction with NO. Materials and methods Superior rectus (SR) muscles of 24 New Zealand albino rabbits were studied. Single muscle twitch tension (SMTT) and tetanic muscle tensions at 50, 75, and 100 Hz were recorded using a 15 V stimuli. The rabbits were equally allocated into three groups. Measurements were performed without any drug treatments in group 1. In groups 2 and 3, bupivacaine, 0.5 ml of a 0.50 % solution, was injected into the EOM, and after 21 days, measurements were performed. Oral isosorbide dinitrate (NO donor) at 20 mg/day was given each day prior to measurements in group 3. Results SMTTs were 69.9 (66.7–77.6), 187.7 (114.9–252.1) and 204.2 (135.3–311.6) mg in groups 1, 2, and 3 respectively. SMTTs for both groups 2 and 3 were significantly higher than All the authors have full control of all primary data, and agree to allow Graefe’s Archive for Clinical and Experimental Ophthalmology to review their data upon request. The study was presented as a poster at ARVO 2012. B. Bilgin : H. Gursoy (*) : H. Basmak Department of Ophthalmology, Eskisehir Osmangazi University Medical Faculty, Eskişehir, Turkey e-mail: [email protected] M. Ozkurt : N. Tuncel Department of Physiology, Eskisehir Osmangazi University Medical Faculty, Eskişehir, Turkey F. Canaz : S. Isiksoy Department of Pathology, Eskişehir Osmangazi University Medical Faculty, Eskişehir, Turkey E. Colak Department of Biostatistics, Eskişehir Osmangazi University Medical Faculty, Eskişehir, Turkey

that for group 1 (p<0.05). Compared with group 1, group 2 exhibited a 3.8–11.7 % increase in the tetanic tensions at 50, 75, and 100 Hz, but none of these differences were statistically significant. The increase was 47.5–137.5 % in group 3 relative to group 2, and the differences were statistically significant except at 100 Hz. The enlargement of the muscle fibers after bupivacaine injection was shown histopathologically. Conclusion Bupivacaine injection increased the EOM tension in rabbits to some extent. NO augmented the effect of bupivacaine. Keywords Bupivacaine . Nitric oxide . Extraocular muscle . Rabbit

Introduction Strengthening of an extraocular muscle (EOM) without the biomechanical hazards of traditional surgery is a promising new treatment for strabismus. Injection of either insulin-like growth factor (IGF)-1 or -2 into adult rabbit superior rectus (SR) muscles resulted in significant increases in muscle mass and force generation. However, these effects are not longlasting, and increase in muscle force generation returns to control values after 14 days [1, 2]. Exogenous IGF-1 and cardiotrophin (CT)-1 resulted in an increase in EOM strength in juvenile chicks [3]. In another study in chicks, retrobulbar injection of a cocktail of growth factors resulted in an increase in EOM force [4]. Bupivacaine (BUP) injection into the EOM to improve this muscle’s function has been used clinically with some success [5–8]. The improvement in alignment is limited, at 5 to 12 prism diopters [5–8]. The effects of BUP were also studied histopathologically in monkeys and rabbits [9, 10]. Common findings in these studies were degeneration and inflammation, followed by regeneration with an increased diameter of muscle fibers [9, 10].

Graefes Arch Clin Exp Ophthalmol

Satellite cells are stem cells that are found in skeletal muscle fibers. Their activities play a key role in the promotion of muscle fibers growth, repair, and regeneration. Satellite cell activation in the EOM starts within a few hours of exposure to BUP, and these cells proliferate over the next 3 weeks to form new muscle fibers [11, 12]. Satellite cell activation is mediated by nitric oxide (NO) [13, 14], and we hypothesize that it may be possible to increase the activation of these cells by increasing the exposure of the EOM to NO. We had two aims in this experimental study. According to clinical studies, there is likely to be increased muscle tension in the EOM of the rabbit after intramuscular BUP injection, but this result has not been reported previously. Therefore, our first aim was to document the effects of BUP injection on the strength of the EOM. Our second aim was to assess the effects of combined intramuscular BUP injection and oral isosorbide dinitrate (ISD), which is an NO donor.

Materials and methods The study was approved by the Institutional Animal Care and Use Committee of Eskişehir Osmangazi University Medical Faculty (EOUMF) on the 25th of August, 2010, under the decision number 170. All of the experimental procedures were performed at the EOUMF physiology department according to the guiding principles for the care and use of animals (Anadolu University Animal Experiments Local Ethics Committee Guidelines 2011; 1.1.2). The histopathological evaluation was performed by the EOUMF pathology department. Twenty-four male adult White New Zealand rabbits were housed in single cages at the EOUMF experimental animal house, with free access to food and water. All procedures including the BUP injections and the SR muscle tension measurements were performed under general anesthesia with 10 mg/kg intramuscular ketamine hydrochloride (Ketalar, Eczacıbaşı, Turkey) and 2 mg/kg xylazine hydrochloride (Rompun, Bayer, Turkey). Proparacaine drops (Alcaine, Alcon, Turkey) were administered prior to the initiation of all procedures. The left eyes of the 24 rabbits were included in this study. Animals were divided into three groups; control (group 1; n = eight), BUP (group 2; n = eight) and NO (group 3; n = eight). Bupivacaine (Marcaine, AstraZeneca, Turkey), 0.5 ml of a 0.50 % solution, was injected into the left SR muscle of group 2 and 3 rabbits with the aid of a surgical microscope. Injections were applied at least 5 mm posterior from the insertion of the SR muscles using a 26gauge needle. The needle was left in place for an additional 30 s to reduce leakage and allow diffusion of the drug into the muscle. The SR muscle tension measurement was timeconsuming, and was performed on four rabbits per day.

Therefore, BUP was injected into four rabbits per day over four consecutive days to enable the measurement of the SR muscle tension on the 22nd day following the injection for all 16 rabbits. Rabbits were housed in single cages for 21 days following the injection. During the follow-up, group 3 rabbits were given 20 mg isosorbide dinitrate (ISD) (Isordil, Fako, Turkey) dissolved in 1 ml of distilled water orally using an insulin syringe directly into their mouth. ISD was given for 21 days, starting on the day after injection, without anesthesia. The SR muscle tension was measured in all three groups under aseptic conditions. The measurements were obtained on the 22nd day after the injection in groups 2 and 3. Two incisions were made to the upper eyelid to minimize the friction between the SR muscle and superior eye lid, which was then elevated without disinserting it totally. Also by these incisions the distal end of the SR muscle was exposed and isolated from the conjunctiva and Tenon capsule [15]. After bulbar conjunctival peritomy, the connective tissue around the SR muscle was extracted, and the SR muscle was hooked and secured with 4-0 silk suture. The SR muscle tendon was completely disinserted from the globe, and attached to isometric force transducers with 4-0 silk suture. The rabbits’ heads were fixed in a stereotactic frame to minimize any head movement. An integrated system (BIOPAC MP36, Biopac Systems, Inc., USA) composed of bipolar platinum electrodes, a voltage control panel, and software was used to measure the SR muscle tension in grams. The software can adjust the pulse frequency and duration and record the muscle tension. The positions of the force transducer and bipolar platinum contact electrodes were adjusted so that the line of force applied was perpendicular to the midline of the rabbit’s head. This position minimized the possible friction between the muscle and the surrounding tissue. Bipolar platinum contact electrodes were placed on the distal third of the SR muscle. In each SR muscle, the supra-maximum stimulation intensity was found by increasing the voltage (V) until no increase in muscle tension was recorded, using 5 ms pulses. At the supramaximum stimulation intensity, the optimum preload of each SR muscle was found by increasing the muscle’s resting length to obtain the maximum single muscle twitch force. For each SR muscle, the single muscle twitch tension and the different tetanic muscle tensions at 50, 75, and 100 Hz were recorded using 15 V stimuli with 4 min of rest between stimuli. Totally, four different measurements were taken for each muscle. All measurements were taken under similar conditions, so external factors that may affect recordings were the same for all groups. Between each impulse, one or two drops of Krebs solution were applied to the SR muscle to decrease its fatigability. For histopathological examination, at least 10 mm of the SR muscle was extracted from the insertion. The extracted

Graefes Arch Clin Exp Ophthalmol Table 1 The comparison of the mean muscle tensions at different frequencies using 15 voltage stimuli between groups 1 (control) and 2 (bupivacaine) MT in mg

Group 1

Group 2

% of increase

P value

Single-twitch tension Tetanic MT (50 Hz) Tetanic MT (75 Hz) Tetanic MT (100 Hz)

69.9 (66.7–77.6) 649.8 (386.9–797.8) 679.3 (471.6–924.9) 995.8 (515.6–1,279.7)

187.7 (114.9–252.1) 726.0 (591.0–1,476.0) 694.1 (627.2–1615.4) 1034.3 (888.1–1,646.1)

168 % 11.7 % 2.1 % 3.8 %

P<0.05* p>0.05 P>0.05 P>0.05

The 25th and 75th percentile values are given in parentheses along with the medians MT muscle tension *significant

SR muscle was fixed in 10 % neutral buffered formalin for 24 h and embedded in paraffin. Transverse 5 μm sections from each muscle specimen were stained with hematoxylin– eosin and Masson’s trichrome, and evaluated under a light microscope (Nikon Eclipse 80i, Nikon Corporation, Japan). The section which showed the most prominent changes was probably the injection site, and that was selected for histological evaluation. Histological evaluation was done with one examiner, and the examiner was blinded for the examined group. In similar studies searching histopathology effects of bupivacaine on rabbit EOM, histological evaluation of control groups showed no staining for collagen [16, 17]. Therefore, collagen staining with Masson’s trichrome between muscle fibers was used as an indication for the presence of fibrosis. The muscle fiber diameters were measured using a digital camera (Nikon Digital Sight, DS-Fi 1, Nikon Corporation, Japan) with a resolution of 2,560×1,920 pixels attached to the light microscope. Fifty fibers from the orbital layer and 50 fibers from the global layer of the muscle were randomly selected. The fibers were obtained from the part of the muscle where the histopathological changes were the most pronounced. The greatest and smallest diameters of each of 100 fibers were noted, and their mean values were calculated. After the extraction of the muscle section, the rabbits were not sacrificed. The upper eyelid and the remaining conjunctiva

were sutured in place using 6-0 Vicryl. Terramycin (Pfizer, Turkey) and Maxidex (Alcon, Turkey) ophthalmic ointments were applied. The data failed the Shapiro––Wilk test for normality; therefore, the muscle tensions of the three groups were compared using the non-parametric Kruskal–Wallis one-way analysis of variance by ranks test. The orbital and global muscle fiber diameters were compared among the three groups using ANOVA and Tukey’s HSD multiple comparisons tests. P-values<0.05 were required for statistical significance. Statistical analyses were performed using SPSS version 15.0 (SPSS Inc., Chicago, IL, USA).

Results The weights of the rabbits in grams were 2,315±485 (1,700– 2,970), 2,512±264 (2,200–2,900) and 2,540±191 (2,340– 2,900) in groups 1, 2, and 3 respectively (p>0.05). No adverse allergic, toxic, or infection-related effects were recorded in any of the rabbits in groups 2 and 3. The comparison of the muscle tensions between groups 1 and 2 is presented (Table 1). Some increase was obtained at all frequencies, but the difference was statistically significant only for the singletwitch tension.

Table 2 The comparison of the mean muscle tensions at different frequencies using 15 voltage stimuli between groups 1 (control) and 3 (nitric oxide) MT in mg

Group 1

Group 3

% of increase

P value

Single-twitch tension Tetanic MT (50 Hz) Tetanic MT (75 Hz) Tetanic MT (100 Hz)

69.9 (66.7–77.6) 649.8 (386.9–797.8) 679.3 (471.6–924.9) 995.8 (515.6–1,279.7)

204.2 (135.3–311.6) 1,265.9 (1,083.6–1,625.8) 1,614.0 (1,253.0–1,815.6) 1,420.0 (822.6–1,938.4)

192.1 % 94.8 % 137.5 % 42.5 %

P<0.05* P<0.05* P<0.05* P>0.05

The 25th and 75th percentile values are given in parentheses along with the medians MT muscle tension *significant

Graefes Arch Clin Exp Ophthalmol Table 3 The comparison of the mean muscle tensions at different frequencies using 15 voltage stimuli between groups 2 (bupivacaine) and 3 (nitric oxide) MT in mg

Group 2

Group 3

% of increase

P value

Single- twitch tension Tetanic MT (50 Hz) Tetanic MT (75 Hz) Tetanic MT (100 Hz)

187.7 (114.9–252.1) 726.0 (591.0–1,476.0) 694.1 (627.2–1,615.4) 1034.3 (888.1–1,646.1)

204.2 (135.3–311.6) 1265.9 (1,083.6–1,625.8) 1614.0 (1,253.0–1,815.6) 1,420.0 (822.6–1,938.4)

8.7 % 74.3 % 132.5 % 37.2 %

P>0.05 P>0.05 P>0.05 P>0.05

The 25th and 75th percentile values are given in parentheses along with the medians

The comparisons of the muscle tensions between groups 1 and 3 and between groups 2 and 3 are presented in Tables 2 and 3 respectively. All of the differences between groups 1 and 3 except those for 100 Hz reached statistical significance, but the considerable differences between groups 2 and 3 failed to reach statistical significance. The comparisons of the mean diameters of the orbital and global muscle fibers are presented (Table 4). Multiple comparisons between groups were performed by Tukey’s HSD test. The differences in the mean diameters of the orbital layers were significant between groups 1 and 2 (p=0.001) and between groups 1 and 3 (<0.001), whereas the differences between groups 2 and 3 were insignificant (p=0.953) (Fig. 1a and b). The differences in the mean diameters of the global layers were significant between groups 1 and 2 (p=0.001) and between groups 1 and 3 (0.002). The difference between group 2 and 3 was not significant (p=0.933). Fibrosis was not documented in any of the sections from group 1. In seven out of the eight rabbits in both group 2 and 3, fibrosis was observed (Fig. 2).

Discussion The main purposes of this study were to investigate the effects of intramuscular BUP injection and its combined effects with oral NO on the muscle tension of the rabbit SR muscle. In the present study, 2.1 to 11.7 % increases in the mean tetanic muscle tensions were obtained in response to BUP injection at all frequencies in group 2 (BUP) compared with group 1 (control), but the increases failed to reach statistical significance. However, the mean increase in single-twitch muscle tension (168 %) was statistically significant. The small increases in tetanic muscle tension were consistent with the

Table 4 The comparisons of the mean diameters of the orbital and global muscle fibers using ANOVA test *significant

reported improvements of 5 to 12 prism diopters in muscle alignment [5–8]. The oral NO given to group 3 rabbits augmented the effects of BUP on the tetanic muscle tension at all frequencies, as well as the effects on single-twitch muscle tension. The mean increases in the tetanic muscle tension in the NO group were 42.5 to 94.8 % relative to the control group. The current histopathologic findings, namely the significant increase in mean diameter of the muscle fibers and fibrosis 21 days after the injection, demonstrated the effects of BUP, but no differences were observed between the mean muscle fiber diameters of the BUP and NO groups. This last finding was not consistent with the augmentation of the effects of BUP by oral NO. NO most likely worked by activating satellite cells, which are important cells in muscle regeneration. It has been been reported that satellite cells increase the number of muscle fibers (hyperplasia) but not the crosssectional area of these fibers (hypertrophy) [14]. The numbers of muscle fibers in the different groups might also have shown the effects of NO histopathologically if the number of fibers could have been counted. In the comparison of group 2 and 3, some increase was obtained in single-twitch muscle tensions and tetanic muscle tensions at all frequencies in group 3, but the difference was not statistically significant. If we had had more rabbits in the groups, this difference might have been statistically significant. On the other hand, although the differences between group 1 and 2 in tetanic muscle tension values at frequencies of 50 Hz and 75 Hz were not statistically significant, the difference between group 1 and 3 in tetanic muscle tension values at same frequencies did reach statistical significance. We explained these results by the synergistic effect of NO and BUP. After BUP injection of muscle in laboratory animals, inflammatory cells and macrophages remove the degenerated muscle fibers over 2–10 days. At about day 2, satellite cells are activated and regeneration begins, with the muscle

Mean diameters (μ)

Group 1

Group 2

Group 3

P

Orbital fibers Global fibers

15.54±1.45 25.99±5.67

24.80±6.02 39.31±7.12

25.41±3.56 38.2±5.93

<0.001* 0.001*

Graefes Arch Clin Exp Ophthalmol

Fig. 1 The extraocular muscle (EOM) fibers from the orbital and global layers of the muscle in hematoxylin–eosin stained muscle sections under 100× magnification. a Normal EOM fibers. b EOM fibers with increased diameters in group 2

Fig. 2 The extraocular muscle fibrosis stained with Masson’s trichrome under 200× magnification in group 3. The arrows point to some of the fibrosis

reaching pre-injection size and strength around day 21. By this time, acute changes substantially recover, but nevertheless persisted [5]. Zhang et al. demonstrated significant scar formation (fibrosis) 30 days after BUP injection into rabbit EOM [16]. In a previous study on the EOM of the rabbit, muscle regeneration after intramuscular BUP injection was shown to be most prominent 1 week following the injection, while rarely detected after 2 to 4 weeks [10]. In the current study, measurements were performed 3 weeks following the injection, to show the increase in the strength of the EOM after completion of the regeneration process. Therefore, regenerating muscle fibers as an evidence of the BUP injection could not be shown. Also, in our histopathologic evaluation, we didn’t plan to show acute myotoxic changes, including inflammatory cell infiltration and myofiber destruction and regenerating muscle fibers with features of immature cells. The aim of the histopathologic evaluation of this study was to show the changes of muscle fiber diameters and presence of fibrosis. In veterinary anesthesia, ketamine is often used for its anesthetic and analgesic effects. When used as a local anesthetic, nystagmus is one of the ocular side-effects [18]. In our study, we primarily used ketamine for the induction and maintenance of general anesthesia, in combination with xylazine [10]. Under general anesthesia, there was no eye movement during the tests. The strengths of the current study are the strong hypothesis and the fact that this study was the first experimental trial of the concomitant use of an oral agent and the intramuscular injection of a drug to strengthen the EOM. However, there are some limitations, including the limitations associated with the injection technique, the methodology, effective plasma level of NO, the manner in which the muscle tension values were obtained, and the size of the study group. Most of these limitations, excluding those associated with the methodology and the number of rabbits studied, were unavoidable. The study would be statistically stronger if we had larger group size. But this was an animal study, and the SR muscles of the rabbits were excised finally, so we studied eight animals in each group with a total of 24 animals, not desiring more animals affected. Bupivacaine (1.0 to 4.5 ml of a 0.75 % solution) has been used clinically [5–8]. Zhang et al. studied the concentrationdependence of BUP in causing EOM toxicity. Comparison of 0.75 %, 0.38 % and 0.19 % concentrations of BUP have resulted in no early-stage muscle fiber injury, no inflammatory infiltrates or late-stage scar formation with doses of 0.38 % and less [16]. In our study, the amount of drug that diffused into the SR muscle could vary between the animals, and could be inadequate to obtain the expected effects. In the current study, nothing was injected into SR muscles of the control animals. However, saline injections could be performed to exclude the possible mechanical effects of

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injection when comparing the differences between the control group and the other groups. Sham injection would make the study more valuable. However, the main purpose of this study was to compare the effects of bupivacaine injection alone and bupivacaine injection with oral NO supplementation to show if there was a synergistic effect of NO and bupivacaine, so we did not add another group. In the comparison of group 2 (BUP) and group 3 (BUP + NO), trauma caused by the needle may be neglected. The control group helped us to gain experience in the measuring process of SR muscle tensions, calculating supra-maximum stimulation intensity, finding the optimum preload of each SR muscle, and recording the values obtained before testing groups 2 and 3, so the limitations associated with obtaining muscle tensions were minimized. In addition, the control group could comprise the contra-lateral eyes of the animals in the experimental groups, but one eye of each animal was spared due to animal rights considerations. A group of rabbits to which only oral ISD was given could also be included to evaluate the effects of isolated oral NO. However, the activation of muscle satellite cells, which is a fundamental step in muscle regeneration, is mediated by NO [14]. Satellite cell activation occurs as a response to myotoxic muscle injury after BUP injection [7]. NO has no effect on muscle regeneration without muscle injury [14], so we did not use NO alone in a different group. Satellite cells proliferate for 3 weeks following BUP injection [12, 13], and accordingly 20 mg/day of oral ISD for 21 days was given to the rabbits to achieve the intended effect. Isosorbide dinitrate is a lipophilic agent available in oral forms. It can be taken with or without food, because its absorption is not affected by diet [19]. Some of the NO donor agents may be given intravenously, but greater risk of systemic side-effects such as hypotension, syncope, and tachycardia exist [20]. There wasn’t any example of intraperitoneal ISD injection in rabbits in the literature. Considering the possible risk of infection when applied daily for 21 days, intraperitoneal injection was not preferred. In the previous studies, plasma concentrations of orally given nitrates reached effective doses in rabbits. Because of these factors, we preferred the oral way [21]. Intra-peritoneal ISD (30mg/kg) has been used safely in mice [14]. In humans, the preferred oral ISD dose is between 40 and 120 mg/day [22]. Our dose was higher than the dose being used clinically in patients with cardiac problems [22] but less than the dose that has been given to mice. Isosorbide dinitrate metabolites are responsible for the therapeutic effects of the drug, and have a half-life of 5 h in serum [23, 24]. The pharmacokinetics of ISD may vary in different rabbits, and therefore the formation rates of the metabolites and the half-lives may also vary accordingly. Therefore, in the present study, the fixed ISD dose given to the rabbits could produce variable tissue levels of NO.

The muscle tensions were measured primarily using isometric force transducers and bipolar platinum electrodes. All measures, such as extracting the tissues surrounding the muscle and adjusting the line of force so that it was applied perpendicularly, were taken to minimize the friction between the SR muscle and the surrounding tissues. However, it was still impossible to eliminate all of the contact between the muscle and the surrounding tissues, and this contact could have influenced our values. In conclusion, intramuscular BUP injection increased the SR muscle tension in the rabbit eye to some extent, but this increase failed to reach statistical significance. Oral NO augmented the BUP effects fairly well. The histopathological findings further demonstrated the effects of BUP but did not support the effects of NO. Further experimental studies with a greater number of animals are required to investigate the effects BUP and its combined use with oral NO.

Acknowledgments This study was supported by the Scientific and Technological Research Council of Turkey (TUBITAK 111S019). The supporting source had no involvement in (1) the study design, (2) the collection, analysis, and interpretation of the data, (3) the writing of the report, or (4) the decision to submit the report for publication Conflicts of interest The authors do not have any financial conflicts of interest related to the study.

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