The Effects of Bupivacaine and Neostigmine on Articular Cartilage and Synovium in the Rabbit Knee Joint

Transcription

1 The Journal of International Medical Research 2004; 32: The Effects of Bupivacaine and Neostigmine on Articular Cartilage and Synovium in the Rabbit Knee Joint N DOGAN 1, AF ERDEM 1, Z ERMAN 2 AND M KIZILKAYA 1 1 Department of Anaesthesia and 2 Department of Pathology, Medical School, Atatürk University, Erzurum, Turkey We investigated the effects of intraarticular injections of bupivacaine and neostigmine on articular cartilage and the synovial membrane of rabbit knee joints. Saline, bupivacaine or neostigmine were each administered intra-articularly into 15 knee joints. Five joints per drug treatment were prepared for histopathological examination 24 h, 48 h and 10 days after injection. A pathologist examined the histological samples for inflammation of the articular cartilage, inflammatory cell infiltration, hypertrophy and hyperplasia of the synovial membrane, in a blinded manner. There were no histopathological changes in the saline-treated control joints. Joints treated with bupivacaine and neostigmine showed significantly more histopathological changes than control joints. Joints treated with neostigmine showed significantly more histopathological changes than those treated with bupivacaine, except for articular cartilage inflammation on day 10. We conclude that intra-articular bupivacaine and neostigmine cause histopathological changes in rabbit knee joints, with neostigmine having a greater effect than bupivacaine. KEY WORDS: BUPIVACAINE; NEOSTIGMINE; ANAESTHETICS; INTRA-ARTICULAR ANALGESIA; ARTICULAR CARTILAGE; SYNOVIAL MEMBRANE; KNEE JOINT; HISTOPATHOLOGICAL CHANGES Introduction Patients undergoing arthroscopy are usually not hospitalized during the pre-operative and post-operative periods, as out-patient care with local anaesthesia is preferred. Intra-articular drug administration is generally given for postoperative analgesia, and ketorolac, morphine, bupivacaine, sufentanil and neostigmine are the most frequently used drugs. 1 3 The histopathological effects of any drug administered locally are very important. Intra-articularly administered drugs have different histopathological effects on articular cartilage and synovium. 4 Intraarticular ketorolac and morphine may cause inflammatory cell infiltration, hyperplasia and hypertrophy of the cells of the synovial membrane, or inflammation of the articular cartilage of the knee joint. 4 We evaluated and compared the shortand long-term histopathological effects of bupivacaine and neostigmine in a rabbit knee joint model. 513

2 Materials and methods ANIMALS This study was carried out in the Experimental Research Laboratory at Atatürk University (Erzurum, Turkey) with prior approval of the Animal Research Committee (Atatürk University). The experiments were conducted in accordance with institutional guidelines. Thirty New Zealand male rabbits with a mean weight of 3.1 ± 0.2 kg were used. The rabbits were provided by the Veterinary Faculty of Ataturk University (Erzurum, Turkey) and were kept in the Animal Research Laboratory of Ataturk University. JOINT INJECTIONS After a 6-h period of fasting, animals were anaesthetized with intra-muscular ketamine hydrochloride (10 mg/kg Ketalar ; Eczacibasi Warner Lambert, Istanbul, Turkey). The right and left hind knee joints were used. Aseptic conditions were observed and the drug administered via a 38 gauge insulin injector (Hayat G mm, Hayat Tibbi Aletler, Istanbul, Turkey). Fifteen animals were randomly chosen to form a control group. They received intraarticular saline (0.9% NaCl) into the right hind knee joint and were placed into the first cage. Bupivacaine (0.5%, 0.25 ml Marcaine, AstraZeneca, Istanbul, Turkey) was injected into the right hind knee joint of a further 15 animals, and neostigmine (0.05 mg, 0.25 ml Neostigmin, Adeka, Samsun, Turkey) injected into the left hind knee joints of the same animals. These animals were kept in a second cage. JOINT PREPARATION AND EVALUATION Five rabbits were randomly selected from each cage 24 h, 48 h and 10 days after intra-articular administration and killed with an intraperitoneal thiopental injection (150 mg/kg; Pental Sodyum Enjectabl Flakon 1 g, I.E. ULAGAY, Istanbul, Turkey). Under aseptic conditions the skin of the injected knee joint was peeled back to expose the joint, without damaging the joint capsule. The bones forming the joint were excised by osteotomy about 1 cm from the joint. The five joints that received the same treatment were collected in the same pot of 10% formalin at each time-point. These were labelled according to the treatment received and time after injection when the rabbit was killed: C, control (saline); B, bupivacaine; N, neostigmine; group 1, 24 h after injection; group 2, 48 h after injection; and group 3, 10 days after injection. The nine pots were therefore labelled C 1, B 1, N 1 C 2, B 2, N 2, C 3, B 3 or N 3 and the groups referred to by this notation. The joint specimens were fixed with 10% formalin, and then decalcified in ethylenediaminetetraacetate (14%). The samples were embedded in paraffin and 5 µm sections cut and stained with haematoxylin and eosin. A pathologist, who was blinded to the treatment groups, examined all of the sections on the same day for inflammation of the articular cartilage, inflammatory cell infiltration in the synovial membrane, synovial membrane cell hypertrophy and cell hyperplasia. Inflammatory changes in the articular cartilage were graded on a scale of 1 5: 1, no inflammation; 2, minimal inflammation (mild congestion and oedema); 3, mild inflammation (congestion and oedema, few neutrophils); 4, moderate inflammation (neutrophils and macrophages, synoviocyte hyperplasia); and 5, severe inflammation (neutrophils and macrophages, fibrin exudation, synoviocyte hyperplasia). 4 Synovial membrane cell hyperplasia, synovial membrane cell hypertrophy and 514

3 synovial membrane inflammatory cell infiltration were each graded from 0 to 3 using a system modified from Mankin et al. 5 : 0, normal; 1, minimal or mild; 2, moderate; and 3, severe. STATISTICAL ANALYSIS Statistical analysis used the Mann-Whitney U-test to compare different treatments at each time-point and to compare the same treatment at different time-points. A P-value < 0.05 was considered significant. Results A total of 45 rabbit knee joints were examined during the study and Table 1 shows the results of the histopathological evaluation of each knee joint. There were no significant differences between the extent of articular cartilage inflammation, synovial membrane inflammatory cell infiltration, synovial membrane cell hyperplasia and hypertrophy when comparing the results for each drug treatment at the three time-points. This indicated that the histopathological changes did not get worse with time for each drug treatment. The joints treated with saline showed no significant histopathological changes. Overall, the joints that received bupivacaine and neostigmine demonstrated significantly more inflammation of the articular cartilage than the joints treated with saline (P < 0.005). More inflammation of the articular cartilage was observed in the joints of animals in group N 1 compared with B 1, although after 10 days more inflammation was observed in joints in group B 3 compared with N 3 (P < 0.005). The joints of animals in group N 2 showed more inflammation of the articular cartilage than those in group B 2 (P < 0.005). Minimal inflammatory cell infiltration of the synovial membrane was observed in only one joint in the control groups. Less inflammatory cell infiltration was observed in the joints of animals in the control groups than in those animals in the drug-treated groups (P < 0.005). There was less inflammatory cell infiltration in the joints of animals in groups B 1 and B 2 than in those animals in groups N 1 and N 2 (P < 0.005). All joints treated with bupivacaine showed minimal or mild changes in synovial membrane cell hyperplasia. There was less cell hyperplasia in the joints treated with bupivacaine than in joints treated with neostigmine (P < 0.005). The joints that received either bupivacaine or neostigmine showed significantly more cell hyperplasia than saline-treated joints (P < 0.005). All of the joints treated with neostigmine showed moderate synovial membrane cell hypertrophy. All joints that received neostigmine or bupivacaine showed significantly more cell hypertrophy than salinetreated joints (P < 0.005). There was more cell hypertrophy observed in the joints of animals in groups N 1 and N 2 than in those in groups B 1 and B 2 (P < 0.005). Figure 1 shows representative light microscopic images of the histopathological changes observed 10 days after injection in knee joints treated with saline (Fig. 1A), neostigmine (Fig. 1B) and bupivacaine (Fig. 1C). Discussion A number of drugs are used to control pain, especially following arthroscopic knee surgery. Bupivacaine is an amide group local anaesthetic; it is the most frequently used local anaesthetic for intra-articular postoperative analgesia. Intra-articular bupivacaine, a combination of intra-articular bupivacaine and ketorolac, and intravenous ketorolac were compared as treatments for post-operative analgesia. A combination of intra-articular bupivacaine and ketorolac 515

4 TABLE 1: Incidence of histopathological changes in rabbit knee joints following intra-articular treatment with saline (C), bupivacaine (B) and neostigmine (N) measured by routine histology at three time-points after treatment (n = 5 per group) Synovial membrane changes Articular cartilage Inflammatory cell Cell Cell Treatment inflammation infiltration hyperplasia hypertrophy group Group 1 C (24 h) B N Group 2 C (48 h) B N Group 3 C (10 days) B N Articular cartilage inflammation was scored by a pathologist blinded to the treatment groups using the following scale: 1, no inflammation; 2, minimal inflammation (mild congestion and oedema); 3, mild inflammation (congestion and oedema, small number of neutrophils); 4, moderate inflammation (neutrophils and macrophages, synoviocyte hyperplasia); and 5, severe inflammation (neutrophils and macrophages, fibrin exudation, synoviocyte hyperplasia). Inflammatory cell infiltration, cell hyperplasia and cell hypertrophy in the synovial membrane were scored using the following scale modified from Mankin et al. 5 : 0, normal; 1, minimal or mild; 2, moderate; 3, severe. 516

5 A B C FIGURE 1: Haematoxylin and eosin stained 5 µm sections of rabbit knee joints treated with an intra-articular injection of (A) saline, (B) neostigmine, and (C) bupivacaine. Samples were obtained 10 days post-injection. A, magnification 200; B and C, magnification 100 was shown to be more effective than intravenous ketorolac. 1 6 Intra-articular bupivacaine was also an effective analgesic when used in combination with tenoxicam 7 and morphine. 8,9 Many studies have shown that bupivacaine has a good analgesic effect when used intra-articularly. There is the potential for bupivacaine to cause inflammatory responses in articular cartilage and synovial membrane cells, however. When compared with saline solution, bupivacaine has been shown to cause structural changes in chondrocytes, 10 but we failed to find a published study that investigated the effects of bupivacaine on the joint surface. In our study, intra-articular bupivacaine treatment caused more inflammation than saline treatment, but less inflammation in articular cartilage, and less inflammatory cell infiltration, hyperplasia and hypertrophy in the synovial membrane compared with intra-articular neostigmine treatment. The histopathological changes were minimal at 24 h after intra-articular injection, but had increased slightly by 48 h and 10 days after treatment. Our results show that bupivacaine is an agent that can be used safely as an intraarticular injection. There have been a few case reports, however, indicating that bupivacaine can lead to a delayed hypersensitivity reaction 11 and toxicity after intraarticular use. 12 In this case, the histopathological changes on the joint brought about by bupivacaine might be important. A combination of bupivacaine, morphine and methylprednisolone has proved to be an effective intra-articular analgesic. 13 In Rasmussen and Kehlet s study, 13 bupivacaine 517

6 had been used in combination with methylprednisolone, but histopathological evaluation was not performed. In the present study, we evaluated inflammation by histopathological examination. Neostigmine is a synthetic reversible inhibitor of cholinesterase, which directly influences nicotinic receptors and blocks acetylcholinesterase activity. The cholinergic system has been shown to play a role in the perception and transmission of pain. 14 The analgesic effect of neostigmine is possibly associated with cerebral cholinergic and noradrenergic pathways, 15,16 and is more associated with muscarinic receptors than with nicotinic receptors. 17 In view of these features, the analgesic efficacy of intraarticular neostigmine has been confirmed. 2 Neostigmine, when combined with clonidine, failed to produce a greater analgesic effect than when either of the drugs were used alone. 18 It has also been shown that the peripheral use of neostigmine was less effective than epidural use. 19 There have been no published studies on the histopathological effects of intraarticular neostigmine in knee joints. In our study, intra-articular neostigmine treatment caused more histopathological changes in the synovial membrane and articular cartilage of the rabbit knee joint compared with intra-articular saline or bupivacaine treatment. This effect was seen at all three time-points after intra-articular injection. The intra-articular doses of bupivacaine used in humans are 0.25% in 40 ml and 0.5% in 30 ml. 6,7 The intra-articular doses of neostigmine used in humans for postoperative analgesia after knee surgery are 125 µg, 250 µg or 500 µg. 2 There have been no previous studies on animals that indicate what doses of bupivacaine and neostigmine should be used in rabbits for joint analgesia. As the drugs are administered intraarticularly in about ml in humans, we decided to use around 1/100 to 1/120 of the volume for the knee joints of rabbits. In this study, we only investigated the histopathological effects of one dose of each drug. Our histopathological findings in rabbit knee joints may not be directly comparable with human joints, but as it is difficult to investigate human joints, rabbits are used as a convenient animal model for toxicology investigations. We can only assume that the histopathological changes we have demonstrated might be observed in other animals. Our results would suggest that physicians should be cautious when administering intra-articular bupivacaine and neostigmine. We conclude that intra-articular bupivacaine and neostigmine caused histopathological changes in the articular cartilage and synovial membrane of the rabbit knee joint, with neostigmine having a greater effect than bupivacaine. Received for publication 29 March 2004 Accepted subject to revision 6 April 2004 Revised accepted 14 June 2004 Copyright 2004 Cambridge Medical Publications References 1 Reuben SS, Connelly NR: Postoperative analgesia for outpatient arthroscopic knee surgery with intraarticular bupivacaine and ketorolac. Anesth Analg 1995; 80: Yang LC, Chen LM, Wang CJ, Buerkle H: Postoperative analgesia by intra-articular neostigmine in patients undergoing knee arthroscopy. Anesthesiology 1998; 88: Richardson MD, Bjorksten AR, Hart JA, McCullough K: The efficacy of intra-articular morphine for postoperative knee arthroscopy analgesia. Arthroscopy 1997; 13: Irwin MG, Cheung KM, Nicholls JM, 518

7 Thompson N: Intra-articular injection of ketorolac in the rat knee joint: effect on articular cartilage and synovium. Br J Anaesth 1998; 80: Mankin HJ, Dorfman H, Lippiello L, Zarins A: Biochemical and metabolic abnormalities in articular cartilage from osteo-arthritic human hips. II. Correlation of morphology with biochemical and metabolism data. J Bone Joint Surg Am 1971; 53: Smith I, Shively RA, White PF: Effects of ketorolac and bupivacaine on recovery after outpatient arthroscopy. Anesth Analg 1992; 75: Cook TM, Tuckey JP, Nolan JP: Analgesia after day-case knee arthroscopy: double-blind study of intra-articular tenoxicam, intra-articular bupivacaine and placebo. Br J Anaesth 1997; 78: Khoury GF, Chen AC, Garland DE, Stein C: Intraarticular morphine, bupivacaine, and morphine/ bupivacaine for pain control after knee videoarthroscopy. Anesthesiology 1992; 77: Ruwe PA, Klein I, Shields CL: The effect of intraarticular injection of morphine and bupivacaine on postarthroscopic pain control. Am J Sports Med 1995; 23: Nole R, Munson NM, Fulkerson JP: Bupivacaine and saline effects on articular cartilage. Arthroscopy 1985; 1: Craft DV, Good RP: Delayed hypersensitivity reaction of the knee after injection of arthroscopy portals with bupivacaine (Marcaine). Arthroscopy 1994; 10: Liguori GA, Chimento GF, Borow L, Figgie M: Possible bupivacaine toxicity after intraarticular injection for postarthroscopic analgesia of the knee: implications of the surgical procedure. Anesth Analg 2002; 94: Rasmussen S, Kehlet H: Intraarticular glucocorticoid, morphine and bupivacaine reduce pain and convalescence after arthroscopic ankle surgery: a randomised study of 36 patients. Acta Orthop Scand 2000; 71: Buerkle H, Boschin M, Marcus MA, Brodner G, Wusten R, Van Aken H: Central and peripheral analgesia mediated by the acetylcholinesteraseinhibitor neostigmine in the rat inflamed knee joint model. Anesth Analg 1998; 86: Naguib M, Yaksh TL: Antinociceptive effects of spinal cholinesterase inhibition and isobolographic analysis of the interaction with mu and alpha 2 receptor systems. Anesthesiology 1994; 80: Hood DD, Eisenach JC, Tuttle R: Phase I safety assessment of intrathecal neostigmine methylsulfate in humans. Anesthesiology 1995; 82: Yaksh TL, Grafe MR, Malkmus S, Rathbun ML, Eisenach JC: Studies on the safety of chronically administered intrathecal neostigmine methylsulfate in rats and dogs. Anesthesiology 1995; 82: Gentili M, Enel D, Szymskiewicz O, Mansour F, Bonnet F: Postoperative analgesia by intraarticular clonidine and neostigmine in patients undergoing knee arthroscopy. Reg Anesth Pain Med 2001; 26: Lauretti GR, de Oliveira R, Perez MV, Paccola CA: Postoperative analgesia by intraarticular and epidural neostigmine following knee surgery. J Clin Anesth 2000; 12: Address for correspondence Dr N Dogan Atatürk University, Lojmanlary 31.Blok, Daire:1, Erzurum, Turkey. nazdogan@hotmail.com 519