Role of cyclooxygenase-2 in lipopolysaccharide-induced hyperalgesia in formalin test

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1 Indian Journal of Experimental Biology Vol. 43, January 25, pp. 53- Role of ylooxygenase-2 in lipopolysaharide-indued hyperalgesia in formalin test Satyanarayana S V Padi & Shrinivas K Kulkarni * Pharmaology Division, University Institute of Pharmaeutial Sienes, Panjab University, Chandigarh 114, India Reeived 24 Otober 23; revised 2 September 24 Lipopolysaharide (LPS)-indued hyperalgesia and the role of ylooxygenase (COX) isoforms in aute and hroni noiepti ve assays have been well established. However, the role of COX isoforms in LPS-indued hyperalgesia in the formalin test is not lear. Thus, the present study was undertaken to haraterize the time ourse of form alin-i ndued noieptive response in LPS-pretreated mie and to investigate possi ble effets of COX inhibitors to address the potential role of COX isoforms in LPS-indued hyperalgesia in the formalin test. All the animals showed typial biphasi response to formalin hallenge. At hr (immediately) and 4 hr after LPS pretreatment, animals did not show any alteration in formalinindued toni pain. However, 12 and 16 hr after LPS pretreatment, there was a signifiant inrease in the late phase of formalin-indued noi fen sive response as ompared to ontrol mie. Treatment with intravenously admi ni stered ketorola (a nonseletive COX inhibitor) signi fiantly and dose-dependently inhibited the late phase of formalin-indued noieptive behaviour in saline and LPS-pretreated mie. In ontrast, pareoxib (prodrug of valdeoxib, a seletive COX-2 inhibitor) or dexamethasone (COX-2 transription inhibitor), when administered intravenously or intraperitoneally, respetively, did not show antinoiepti ve effet in the formalin test in saline-pretreated mie. However, both the agents signifiantly and dosedependently dereased the late phase noieptive behaviour of the formalin test in LPS-pretreated mie to the level of the animals that reeived saline pretreatment. These resul ts suggest that indution of COX-2 by proinfl ammatory mediators and subsequent release of prostaglandins ould be responsible fo r LPS enhanement of formalin-indued noifensive behaviour and supports an important role of COX-2 in LPS-indued hyperalgesia in the formalin test. Keywords: Cylooxygenase isoforms, Formalin test, Hyperalgesia, Lipopolysaharide. Peripheral ti ssue injury or inflammation auses exaggerated pain behaviour that inludes hyperalgesia, an inreased responsiveness to noxious stimuli. Prostaglandins (PGs) speifially in the spinal ord, have long been thought to play a key role in inflammatory proess, sensitisation of noieptors, generation of pain and noieptive proessing 1 2 Nonsteroidal anti-inflammatory drugs (NSAIDs) at by inhibiting ylooxygenase (COX), whih is the ratelimiting enzyme, that atalyses onversion of arahidoni aid to generate PGs. There are two isoforms of COX, namely COX-1 and COX-2. COX I is onstitutively expressed in most ells for housekeeping funtions, while COX-2 is present in low levels in physiologial onditions, but is rapidly indued by ytokines, growth fators and inflammatory stimuli 2 3 Reently, COX-3 has been identified but its physiologial and pathologial roles are yet to be haraterized 4. *Correspondent author Telefax : Lipopolysaharide (LPS), also known as endotoxin, a major onstituent of the outer membrane of the ell wall of gram-negative bateria, is reported to produe number of pathophysiologial hanges in the organism 5 ' 6. It is also known to release proinflammatory ytokines suh as interleukin-1 (IL-l), IL-6, and tumour nerosis fator-alpha (TNF-a) by ativated monoytes and marophages 5-7 In addition to proinflammatory ytokines,. the subsequent release of nitri oxide (NO) and prostaglandins (PGs) has been found to indue long-lasting hyperalgesia after entral, systemi and loal administration of LPS 8 11 Reently, many studies have been performed using various lasses of pharmaologial agents inluding COX inhibitors to haraterize and delineate possible underlying mehanisms involved in LPS-indued hyperalgesia in various noieptive assays It is generally agreed that the formalin test reprodues various aspets of aute inflammatory pain analogous to human postoperative pain. Further, the pain intensity in this noieptive assay is a reproduible and quantifiable behavioural response

2 54 INDIAN 1 EXP BIOL, JANUARY 25 and has been used for the evaluation of the analgesi ativity of various pharmaologial agents The diluted formalin when injeted into hind paw of mie and rats shows harateristi biphasi liking and biting behaviours to ontinuous (toni) noxious stimuli and this omplex noieptive patterns last for approximately min. The first or aute phase lasts for about 5 min whih is followed by a longer-lasting, more persistent phase (about 4 min) that is haraterized by shaking or liking and biting behaviours of the paw 17. It is generally agreed that the first phase results at least in part from diret ativation of primary afferent fibres, both low-threshold mehanoreeptive and noieptive types, whereas the seond phase reflets a failitated state of entral sensitisation driven by the persistent primary afferent inputs and this ongoing ativity releases exitatory amino aids and neuropeptides that are neessary for the development of the seond phase 1 18 The role of COX isoforms and the effets both peripherally and entrally administered COX inhibitors in formalin-indued noifensive behaviour have been well establishedl.l 9 2 Despite extensive studies reported on LPS-indued hyperalgesia, the role of COX isoforms in LPS-indued hyperalgesia in the formalin test is not known. Thus, the present study has been arried out to examine time ourse and harateristis of LPS-indued hyperalgesia in the formalin test and to investigate the effets of COX inhibitors to address the role of COX isoforms on LPS enhaned formalin-indued noifensive behaviour in animals. Materials and Methods Materials-Pareoxib sodium (Panaea Biote Ltd., India) and ketorola tromethamine (Ketanov 15 mg/ml) intravenous injetion (Ranbaxy Ltd., India), dexamethasone (Unihem Labs, India), lipopolysaharide from Salmonella typhimurium (Sigma, USA), and formalin (37% formaldehyde) (SD Fine Chemials, India) were used in this study. Pareoxib sodium, 2% formalin and lipopolysaharide were freshly prepared by dissolving in normal saline to suitable onentration. All the drugs were administered in a dose volume of 1 mill g body weight of mie. Experimental animals-albino Swiss mie (2-25 g) of either sex (bred in Central Animal House of Panaea Biote Ltd., Punjab) were housed under standard onditions of light and dark yle with food and water ad libitum. The protool was approved by the Institutional Animal Ethis Committee of Panjab University, Chandigarh and arried out in aordane with the guidelines of the Indian National Siene Aademy. Newly aquired mie were alimatised to laboratory onditions 2 hr before being used in the experiment. Eah animal was used for a single treatment and sarified by euthanasia after experimentation. Eah group onsisted of six animals. Formalin-indued toni pain-the mouse paw formalin test was arried out aording to a previous repore 1 Briefly, mie were injeted with of 2% formalin solution in normal saline subutaneously into the plantar surfae of the left paw with a 26- guage needle fitted to a mirosyringe. Pain response was quantified by ounting the time spent in liking and biting of the injeted paw for 5 min periods from -45 min. Two phases of spontaneous liking was observed after formalin injetion. The interval from - 1 min was defined as early phase and the interval 1-45 min as late phase, respetively. Experimental design-lps dissolved in normal saline and injeted 5 -Lg/.1 ml/mouse was administered intraperitoneally at different time intervals. Control animals reeived normal saline.1 ml/mouse. The LPS pretreated and ontrol animals were subjeted to formalin-indued toni pain at hr (immediately), 4, 12, or 16 hr after LPS administration. Ketorola (nonseletive COX inhibitor) or pareoxib (seletive COX-2 inhibitor) (I, 5 or 1 mg/kg) was administered intravenously 3 min before assessing formalin-indued toni pain in saline or LPS-pretreated mie. A single injetion of dexamethasone (.5 mg/kg) was administered intraperitoneally 2 hr before saline or LPS pretreatment to mie and another InJetion of dexamethasone (.5 mg/kg, ip) was administered 2 hr before formalin hallenge. Saline pretreated and LPS pretreated ontrol animals also reeived equivalent volume of normal saline intravenously 3 min before formalin hallenge. In all these animals, formalinindued noieptive responses were observed 12 hr after saline or LPS pretreatment. Statistial analysis-all the values were expressed as mean ±SE. The mean sum of liking and biting behaviour between two groups was analysed by unpaired Student's!-test. The mean sum of liking and biting behaviour in both early and late phases of the formalin test was analysed by one-way analysis of variane with Dunnett's!-test for multiple

3 PADI & KULKARNI: ROLE OF CYCLOOXYGENASE-2 IN LIPOPOLYSACCHARIDE 55 omparisons between different groups. A value of P <.5 was onsidered as statistially signifiant. Results Effet of lipopolysaharide pre-treatment on the formalin-indued noieption in mie-all the mie subutaneously hallenged with formalin into hind paw showed harateristi biphasi response with an early and a late phase (Fig. 1). A preliminary study was performed to assess any gross differene in formalin-indued noieptive behaviours of the LPSinjeted mie ompared to saline injeted mie immediately after the treatment ( hr). No signifiant differene between in time ourse of formalinindued noieptive response was observed in various ontrol groups of animals tested at various time intervals after saline injetion i.e., at, 4, 12, and 16 hr, respetively indiating onsisteny and reproduibility of the measurements among groups (Fig. 1). LPS pretreatment at hr (immediately) and for 4 hr did not show any signifiant differene in the sum of liking and biting behaviour responses in both early (Fig. 2A) and late phases (Fig. 2B) of the 12 -<>-Saline ( hr) -{}--- Saline (4 hr) loll! 4 " g 2 --i!r- LPS ( hr) -<>-- LPS (4 hr) " o (1 -<>-Saline (12 hr) -{}--- Saline ( 16 hr) " " OD = :g ::i i!r- LI'S (1 2 hr) -<>-- LPS ( 16 hr) Minutes after injetion offommlin Fig.!-Time ourse of formalin response in mie pretreated with lipopolysaharide (LPS) or saline (A) and 4 hr and (B) 12 and 16 hr before th,. noieptive test. Points represent the mean ±S.E. of formalin-indued liking and biting responses in seonds during 5 min intervals observed for 45 min. formalin hallenge as ompared to ontrol mie that reeived saline pretreatment. In ontrast, LPS pretreatment for 12 and 16 hr markedly inreased the mean liking and biting responses during the time ourse of the formalin test resulting in a state of toni hyperalgesia (Fig. lb). A signifiant enhanement of formalin-indued noieptive response as ompared to saline pretreatment group was observed during the late phase (1-45 min) but not in the early phase (-1 min) in mie with LPS pretreatment for 12 hr or more (Fig. 2). Therefore, in the subsequent experiments, formalin-indued noifensive behaviour in saline or drug treated animals was observed 12 hr after LPS pretreatment. Effet of ylooxygenase inhibitors in lipopolysaharide enhaned formalin-indued noieption in mie--intravenous administration of ketorola (1, 5, or 1 mg/kg) signifiantly and dose u "' 4 2 "... oo E : " 35 " OD 3 :;<... E ::l VJ 25 2 ISO 5 '---'-- Late Phase Eariy Phase 4,.I. Saline *!lllps 12 Time after LPS adminislrntion (hr) 16 '--- * Fig. 2-Duration of noieptive behaviour during (A) early phase ( - 1 min) and (B) late phase (1-45 min) of the formalin test in mie pretreated with lipopolysaharide (LPS) or saline, 4, 12, and 16 hr before formalin hallenge. The data represent the mean ± S.E. of sum of formalin-indued liking and biting responses in seonds during the early and late phase. * P <.5 as ompared to orresponding saline pretreated group (t-test).

4 56 INDIAN J EXP BIOL, JANUARY 25 dependently dereased formalin-indued noieptive behaviour as ompared to saline treatment in ontrol or LPS-pretreated mie (Fig. 3B). However, it did not alter noieptive response in early phase of the formalin test in saline or LPS-pretreated mie (Fig. 3A). Both, pareoxib (1, 5 or 1 mg/kg, iv) and dexamethasone (.5 mg/kg, ip) showed noieptive responses similar to saline-treated mie following formalin injetion (Figs 4 and 5). On the ontrary, pareoxib signifiantly and dose-dependently redued the enhaned number of liking and biting behaviour in late phase of the formalin test in LPS-pretreated mie to the level of the animals that reeived only saline, however it had no effet on early phase of the formalin test (Fig. 4). Pretreatment with dexamethasone before LPS pretreatment signifiantly inhibited the enhanement of late phase, but not early phase formalin-indued noifensive behaviour (Fig. 5). Disussion In the present study, systemi administration of 14 Early phase 14 Early phase u ". "',_ on g 4 "' o : = Vl 25 2 * Late phase Saline 1.PS u.; 4 2 o : -'--'-----"" ' : o 4 : E Vl * Late phase 5 Control Ket I Ket 5 Treatment (mgll<g) Ket 1 Control Pare.I Pare 5 Pare 1 Treatment (mg/lq,:) Fig. 3--Effet of ketorola (Ket) on noieptive behaviour during (A) early phase (-1 min) and (B) late phase (1-45 min) of the formalin test in mie pretreated with saline or lipopolysaharide (LPS) 12 hr before formalin hallenge. Ketorola was intravenously administered 3 min before noieptive assay. The data represent the mean ± S.E. of sum of formalin-indued liking and biting responses in seonds during the early and late phase. * P <.5 as ompared to orresponding saline pretreated group (ttest). " P < as ompared to LPS-pretreated ontrol animals (one way ANOVA followed by Dunnett's test). Fig. 4--Effet of pareoxib (Pare) on noieptive behaviour during (A) early phase (-1 min) and (B) late phase (1-45 min) of the formalin test in mie pretreated with saline or lipopolysaharide (LPS) 12 hr before formalin hallenge. Pareoxib was intravenously administered 3 min before noieptive assay. The data represent the mean ±S.E. of sum of formalin-indued liking and biting responses in seonds during the early and late phase. * P <.5 as ompared to orresponding saline pretreated group (t-test). " P < as ompared to LPSpretreated ontrol animals (one way ANOVA followed by Dunnett's test).

5 PAD! & KULKARNI: ROLE OF CYCLOOXYGENASE-2 IN LIPOPOLYSACCHARIDE 57 '-'.; = "... OD =. " "' ';ll ::;:... E " "' ISO 5 * Control Early phase Late phase Saline LPS DEX.5 rngikg Fig. 5--Effet of dexamethasone (Dex) on noieptive behaviour during (A) early rhase (-1 min) and (B) the late phase (1-45 min) of the formalin test in mie pretreated with saline or lipopolysaharide (LPS) 12 hr before formalin hallenge. Dexamethasone was intraperitoneally administered 2 hr before LPS pretreatment. The data represent the mean ± S.E. of sum of formalin-indued liking and biting responses in seonds during the early and late phase. * P <.5 as ompared to orresponding saline pretreated group (t-test). P < as ompared to LPSpretreated ontrol animals (one way ANOVA followed by Dunnett' s test). LPS enhaned formalin-indued liking and biting behaviours in the late phase, whih is indiative of hyperalgesi response. The important aspet in this study is the time ourse of hyperalgesia that was most evident and onsistent in all the LPS-pretreated animals. Although there was no signifiant differene in the toni pain behaviour at hr and after 4 hr, but there was a marked and signifiant potentiation of noieptive response at 12 and 16 hr after LPS pretreatment in mie hallenged with formalin resulting in a state of hyperalgesia. Although dosedependent LPS potentiation of formalin-indued toni pain was not studied, the same onentration of LPS was employed whih was previously reported to ause hyperalgesia in mie 13 ' 22. Indeed, the late phase represents failitated state of entral sensitisation, however, marked enhanement of noieptive response was observed in late phase following LPS pretreatment suggesting that inreased release of noieptive mediators and/or release of ertain mediators other than those normally involved in formalin noieptive responses are speifially indued by LPS pretreatment. Systemi administration of ketorola, a nonseletive COX inhibitor, but not pareoxib (a prodrug of valdeoxib, a seletive COX-2 inhibitor) or dexamethasone (COX-2 transription inhibitor) showed antinoieptive effet in the formalin test in saline-pretreated mie. Seletive COX-1 and nonseletive COX inhibitors, but not seletive COX-2 inhibitors redued formalin-indued noieptive responses : 2. Consistent with previous reports, the results of the present study well support the role of COX-I derived PGs for failitation of noieptive proessing that ours in inflammatory late phase of the formalin test. Importantly, ketorola markedly dereased the late phase noieptive behaviour of the formalin test in LPS-pretreated mie. Further, both pareoxib and dexamethasone also redued LPSindued hyperalgesia. However, both these agents redued only the enhanement of noieptive behaviour by LPS to the level of the animals that reeive only saline treatment. These results impliate that PGs derived from COX-1 but not by COX-2 play a role in this aute noieptive test in normal animals whereas PGs derived speifially from COX-2 may be involved in LPS potentiation of formalin-indued noieptive responses. In the reent past, a number of studies attempted to unravel the mehanisms underlying in LPS-indued hyperalgesia LPS, when administered intraperitoneally does not ross blood-brain barrier, however, it stimulates the expression and release of various immunologial fators, ytokines suh as ILl, IL-6, and TNF-a by ativated monoytes and marophages and proinflarnrnatory mediators in the periphery and in the entral nervous system. In addition, these ytokines inrease the expression of inos, and COX-2, and also indue the expression and release of various proinflammatory mediators inluding PGs and neuropeptides 1 ' It is well known that LPS regulates COX isozymes expression differentially with downregulation of COX-1 and

6 58 INDIAN J EXP BIOL, JANUARY 25 upregulation of COX-2 (refs. 25, 26). Previous studies have shown that the dramati inrease in levels of COX-2 isoform in response to proinflammatory ytokines in synovial JOints, marophages, monoytes, and spinal ord 14 " 27 " 28. Further, various irulating fators inrease the expression of COX-2 in the neuronal and nonneuronal elements besides marophages and fibroblasts within 2 hr and subsides by 4-24 hr following LPS administration, but similar hanges were also observed after peripheral tissue injury and inflammation 14 " In the spinal ord, LPS ativates the signalling pathways suh as nulear fator kappab (NF-KB), AP-1, CREB, mitogen ativating protein kinase (MAPK) asade led to transriptional ativation of COX-2 expression in astroytes, miroglia, endothelial ells, and leptomeningeal ells 14 " 32. Inreased expression of COX-2 that atalyses arahidoni aid into PGH 2 and PGE synthase that produes PGE 2 from PGH 2 in skin, dorsal root ganglia, white and gray matter of spinal ord or synovioytes was observed 4-24 hr after IL-l or systemi LPS pretreatment, respetively The results of the present study are oherent to time ourse of inreased expression of COX-2 where timedependent hyperalgesia was observed hr after LPS pretreatment. Importantly, the inreased expression of COX-2 inreases basal and evoked PGs release. These PGs sensitise peripheral nerve endings and failitate entral noieptive proessing in spinal ord resulting in exaggerated pain behaviour (hyperalgesia). These data indiate that the elevated COX-2 ould be a major ontributor to hypersensitivity after LPS administration. In the present study, the effet of seletive inhibition of COX-2 prior to LPS pre-treatment was not evaluated beause marked hyperalgesia was observed 12 hr after LPS administration. Moreover, formalin-indued biting and liking for min would not be suffiient for ativation of COX-2 mrna and generation of COX-2. In addition, COX-1 is present onstitutively and readily releases PGs in response to tissue injury and noxious stimuli whereas COX-2 is induible and not present onstitutively in the periphery and in spinal ord where sensitisation of noieptors and noieptive proessing ours, respetively 2 3. Indeed, seletive COX-2 inhibitors have no role in the ativation of COX-2 mrna and generation of new enzyme. It is likely that COX-2 may not be assoiated with spinal prostanoid synthesis autely or with failitated noieption, whih ours within limited time frame of aute analgesi tests. Although, COX-2 expression was not measured, the administration of dexamethasone, an inhibitor of COX-2 expression prior to LPS injetion signifiantly prevented hyperalgesia in the present study. Further, lear inhibition of COX-2 expression by dexamethasone has been demonstrated in both in vitro and in vivo In various studies, enhanement of the late phase of formalin noieptive responses have also been reported in rats that were already in a state of neuropathy-indued hyperalgesia due to spinal nerve ligation and diabetes It has also been reported that hyperalgesia in these neuropathy models is dependent on COX-2 and enhaned release of PGs in spinal ord Further, seletive inhibition of COX-2 produed signifiant antihyperalgesi effet against hroni pain in experimental animals in whih there is a marked inrease in COX-2 mrna and PGs in spinal ord Similarly, seletive inhibition of COX-2 reversed ytokine and LPS-indued hyperalgesia in various peripheral and entral noieptive assays 8 ' 1 ' Taken together, the results provide support to the onept that the PGs released by COX-2 involve in entral noieptive proessing that results in hyperalgesi behaviour in LPSpretreated animals. In onlusion, the above results indiate that LPS potentiated formalin-indued noieptive response with marked hyperalgesia produed in the late phase. Further, LPS-mediated indution of COX-2 ontributes to the development of inflammatory pain hypersensitivity in the formalin test. Aknowledgement Grant support to SSVP by M/s Panaea Biote Ltd., Lalru, Punjab, India is appreiated. Referenes Malmberg A B & Yaksh T L, Antinoieptive ations of spinal non-steroidal anti-inflammatory agents on the formalin test in the rat, J Pharmaal Exp Ther, 263 ( 1992) Willingale H L, Gardiner N J, MLymont N, Giblet S & Grubb B D, Prostanoids synthesized by ylooxygenase isoforms in the rat spinal ord and their ontribution to the development of neuronal hyperexitability. Br J Phannaal, 122 (1997) Kulkarni S K, Jain N K & Singh A, Cylooxygenase isoenzymes and newer therapeuti potential for seletive COX-2 inhibition, Methods Find Exp Clin Pharmaal, 22 (2) Chandrasekharan N V, Dai H, Tarepu Roos K L. Evanson N

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