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The anti-nociceptive effects of Porphyromonas gingivalis lipopolysaccharide
Archives of Oral Biology 102 (2019) 193–198
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Archives of Oral Biology journal homepage: www.elsevier.com/locate/archoralbio
The anti-nociceptive effects of Porphyromonas gingivalis lipopolysaccharide a,⁎
b
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d
e,f
Junad Khan , Bollama Puchimada , Daniel Kadouri , Tali Zusman , Fawad Javed , Eli Eliav
T
g
a
Orofacial Pain and Temporomandibular Joint Disorders, Eastman Institute for Oral Health, University of Rochester, 625 Elmwood Avenue, Rochester, NY, 14620, USA Department of Diagnostic Sciences, Rutgers School of Dental Medicine, NJ, USA Oral Biology, Rutgers School of Dental Medicine, NJ, USA d Department of Diagnostic Sciences, Rutgers School of Dental Medicine, NJ, USA e Department of Periodontology, Stony Brook University, Stony Brook, NY, USA f Laboratory for Periodontal-, Implant-, Phototherapy (LA-PIP), School of Dental Medicine, Stony Brook University, Stony Brook, NY, USA g Eastman Institute for Oral Health, University of Rochester, NY, USA b c
A R T I C LE I N FO
A B S T R A C T
Keywords: Pain Cytokine Inflammation Virulence
Objective: The objective of this study was to assess the effect of Porphyromonas gingivalis lipopolysaccharide (PG LPS) on acute pain-related behaviour induced in rats and to measure its impact on the levels of pro-inflammatory cytokines (IL-1β, IL-6) and anti-inflammatory (IL-10) cytokines. Design: The Brennan model was used to induce acute pain like signs in rats' hind paw. Twenty-four hours following the surgery the rats were divided into 5 groups and the affected paws were injected with 0.2 m l of one of three commercialized forms PG LPS doses (high - 1 mg/ml, medium - 0.6 mg/m l and low - 0.2 mg/m l), diclofenac sodium (1 mg/kg) or saline. Tactile allodynia, mechanical hyperalgesia, body temperature and paw swelling were assessed at baseline, 24 h postoperatively and 2 h after the paw injection. The affected and contralateral paw tissue was assessed for the mentioned above cytokines levels employing enzyme-linked immunosorbent assay. Results: This study may suggest that PG LPS can reduce pain like behaviour via increased levels of anti-inflammatory cytokine IL-10 (5900 ± 748, p < 0.05). The high PG LPS dose and diclofenac reduced the tactile allodynia and mechanical hyperalgesia significantly (42.2 ± 4 and1.6 ± 0.3, p < 0.05). PG LPS high dose increase IL-10 levels while diclofenac reduces IL-1β levels significantly (5900 ± 748 and 1760 ± 271.2). The LPS administration had no effect on paw swelling and did not increase rat’s body temperature. Conclusion: The results demonstrated that PG LPS local application could possess anti- nociceptive properties, which at least in part is mediated by an increase in IL-10 levels.
1. Introduction Inflammation plays an important role in various pain conditions and is known to be associated with increased pain and hypersensitivity (Khan, Hassun, Zusman, Korczeniewska, & Eliav, 2017; Matsuda, Huh, & Ji, 2019; Scheff et al., 2017). It is widely accepted that pain induced by inflammation is an adaptive response intended to protect the organism from further injury. However, not all inflammatory conditions are associated with increased sensitivity or pain. One such example is periodontitis, a complex disease characterized by inflammation and infection of the tissues that support the teeth. Periodontal disease is a multifactorial condition elicited by bacteria that interact with host tissues and cells leading to the destruction of the periodontal structures, including the tooth-supporting tissues, alveolar bone and periodontal ligament. Although chronic periodontitis is an inflammatory condition,
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pain rarely manifests as part of the disease phenotype. Periodontal disease usually results from a host-mediated inflammatory response to pathogenic microflora present in periodontal pocket. Porphyromonas gingivalis (PG) is a Gram-negative rod-shaped anaerobe highly prevalent in those affected with periodontitis compared to healthy individuals. PG is considered a major etiological agent of periodontitis (Darveau, 2009; Kulkarni, Bhat, Thomas, Bhat, & Kulkarni, 2018; Leira et al., 2019; Socransky, Haffajee, Cugini, Smith, & Kent, 1998) for acute and chronic forms of the disease (Van Winkelhoff, Loos, Van Der Reijden, & Van Der Velden, 2002). Like other pathogenic bacteria, PG produces a substantial array of putative virulence factors, such as adherence factors, proteases, toxic metabolites and cellular constituents that are able to induce an immune response (de Diego et al., 2013; Murakami et al., 2004; Tebloeva, Revazova, Fabrikant, Dmitrieva, & Gurevich, 2014). Furthermore, like other gram-negative bacteria, PG contains
Corresponding author. E-mail address: [email protected] (J. Khan).
https://doi.org/10.1016/j.archoralbio.2019.04.012 Received 18 January 2019; Received in revised form 15 April 2019; Accepted 19 April 2019 0003-9969/ © 2019 Elsevier Ltd. All rights reserved.
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Institutional Animal Care and Use Committee at Rutgers University, Newark, NJ (Protocol number 10077E113). Experiments were in accordance with federal law, the regulations of the National Institute of Health and the guidelines of the International Association for the Study of Pain (Zimmermann, 1983).
lipopolysaccharide (LPS) that is found in the microbe’s outer membrane (Coughlin, Haug, & McGroarty, 1983; Raetz & Whitfield, 2002). It has been shown that compared to other LPS, LPS of PG exhibits reduced systemic but similar local induction of inflammatory markers (Liu, Desta, Raptis, Darveau, & Graves, 2008). Furthermore, PG LPS has been shown to play a role in bone resorption and soft tissue destruction by stimulating macrophages, monocytes, and fibroblasts to release a variety of compounds such as prostaglandins, cytokines and metalloproteases (Bramanti, Wong, Weintraub, & Holt, 1989; Holt & Bramanti, 1991; Lindemann, Economou, & Rothermel, 1988; Roberts, Richardson, & Michalek, 1997; Sismey-Durrant & Hopps, 1991; Yamaji et al., 1995). Although the PG LPS can stimulate host immune response, it is thought to be a weaker innate immune stimulator than other Gram-negative bacteria. (Jain & Darveau, 2010; Pulendran et al., 2001). As periodontal disease symptoms usually involve inflammation of the periodontium but are rarely associated with elevated pain symptoms, it is possible to hypothesize that PG, a major etiological agent of periodontal disease, may possess certain anti-nociceptive effects. Rodent models of orofacial pain conditions can be divided into three major groups: Inflammatory (acute or chronic), nerve injury and dental injury. To induce inflammatory pain, compounds such as Complete Freund’s Adjuvant (CFA), carrageenan, formalin, mustard oil or capsaicin are injected to the TMJ area, the masseter muscle or perioral regions (Borsani, Bernardi, Albertini, Rezzani, & Rodella, 2009; Neubert et al., 2005; Rohrs, Neubert, Caudle, & Allen, 2018; Shinoda et al., 2011; Tambeli, Seo, Sessle, & Hu, 2001). Neuropathic pain can be induced by infra orbital or mental nerves manipulations, such as nerve ligation, transection or exposure to inflammatory adjuvant (Nomura et al., 2002, Benoliel, Wilensky, Tal, & Eliav, 2002; Vos, Strassman, & Maciewicz, 1994). The dental pain models usually involve mechanical pulp exposure and often inflammatory adjuvants administration to the exposed pulp (Khan et al., 2008; Raoof et al., 2018; Zhang et al., 2006). The pain intensity is assessed mainly by the rodent behaviour, response to mechanical and thermal stimuli and loss of weight over time (Krzyzanowska & Avendano, 2012). In dental injury model changes in bite force intensity and pattern can be assessed as well (Khan et al., 2008). None of the rodent pain models exactly mirror the human condition. However the relatively severe pain induced can support trigeminal system pain pathways research. The existing orofacial pain models are not ideal for assessing PG LPS potential anti nociceptive effect. The chronic inflammatory process characterizing gingivitis or periodontitis is not comparable to the robust inflammation and severe acute pain induced inflammatory adjuvants administration. The nerve injury orofacial pain models are also not relevant, gingivitis or periodontitis will hardly affect major nerves. The intraoral dental injury pain models are useful for dental pain but not for pain related to soft tissue. Therefore, to explore the potential analgesic effect of PG LPS on acute pain, we used the Brennan model (Brennan, Vandermeulen, & Gebhart, 1996), a validated post-surgical pain model. Although the injury is not in the oral cavity, the pain in this model is mild to moderate and has a substantial inflammatory component. Moreover, pain assessment is simpler and has been validated in numerous studies (Krzyzanowska & Avendano, 2012). In addition to PG LPS effect on pain we evaluated its effects on the levels of interleukin-1 beta (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor alpha (TNF-α), major pro-inflammatory cytokines known to induce pain, as well as interleukin-10 (IL-10), an anti-inflammatory cytokine involved in pain reduction (de Oliveira, Sakata, Issy, Gerola, & Salomao, 2011; Dinarello, 2000; Jiang, Peng, Liu, & Kuo, 2013; Khan et al., 2015; Noma et al., 2011; Ramesh, MacLean, & Philipp, 2013; Shubayev, Kato, & Myers, 2010; Wieseler-Frank, Maier, & Watkins, 2005).
2.1. Experimental design A total of 50 Sprague Dawley rats were used in this study. The rats were habituated to the laboratory and testing apparatus for 4 days followed by baseline recordings of tactile-allodynia and mechanicalhyperalgesia, body temperature and paw swelling. Twenty four hours following left paw skin and fascia incision (Brennan incision model) (Brennan et al., 1996), the rats were evaluated again for the same parameters. A total of 50 rats underwent the surgical procedure and developed signs of pain like behaviour, 24 h post-surgery. These rats were then randomly divided into 5 groups, 10 rats per group. The control groups were locally injected with 0.2 ml of either diclofenac sodium (1 mg/kg) or saline. The experimental groups were locally injected with 0.2 m l of commercialized forms of PG LPS (Ultrapure LPS from P. gingivalis ATCC 33277, Catalog# tlrl-pglps; InvivoGen, San Diego, CA) suspension in saline in one of three different doses; High 1 mg/m l, medium - 0.6 mg/m l and low - 0.2 mg/m l. The effect of PG LPS on pain has not been studied before. Therefore we used three doses that were within the range of doses that had been used in previous rodent studies (Chiang, Kyritsis, Graves, & Amar, 1999). Tactile-allodynia and mechanical-hyperalgesia, body temperature and paw swelling were recorded 2 h post injection. The rats were then euthanized and paw tissues were collected bilaterally to perform Enzyme-Linked Immunosorbent Assay (ELISA) for pro-inflammatory (IL-1β and IL-6) and anti-inflammatory cytokine (IL-10) levels.
2.2. Animals Adult male Sprague Dawley rats weighing approximately 250–300 g at the time of surgery were used. The rats were ordered from a single source (Taconic Farms) and kept in the animal facility under direct veterinary observation. During the entire period, the rats were maintained on standard rodent chow, reverse osmotically-treated water and maintained on a twelve-hour day and night cycle.
2.3. Brennan model of incisional (acute) pain A cocktail of ketamine (50 mg/kg) and xylazine (7.5 mg/kg) solution were injected intraperitoneally to anaesthetize the rats. Following verification of the anaesthesia, a 1 cm longitudinal incision was made in the plantar surface of the hind paw beginning 0.5 cm from the end of the heel. The skin, planar fascia, and underlying muscle were incised. The skin was closed with 5.0 nylon sutures. A single calibrated investigator (JK) performed all the surgeries. During the surgical procedure, the eyes were lubricated and the rats were kept on a warming pad to maintain constant body temperature. Behavioural testing was done 24 h following the incision, using the method described below.
2.4. Tactile allodynia behavioural testing Tactile allodynia was tested with von Frey hairs/Semmes-Weinstein monofilaments (Stoelting, Wood Dale, IL) (Tal & Bennett, 1994). The rats were placed on a perforated metal floor and a monofilament applying 26 g of force was applied in the mid-plantar region of the hind paw 5 times with a 1 s interval. A percentage response was calculated, a higher percentage of withdrawals indicated increased tactile allodynia (Flatters & Bennett, 2004).
2. Materials & methods All procedures and experimental protocols were approved by the 194
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2.5. Mechanical hyperalgesia behavioural testing Mechanical hyperalgesia was evaluated by a blunted acupuncture needle (pinprick test) (Tal & Bennett, 1994). The rat was placed on a perforated floor and a blunted acupuncture needle was applied in the area of the mind-plantar hind paw. The duration of paw nursing was timed for up to 15 s. Normal responses of brief duration were assigned a duration of 0.5 s. An increase in the withdrawal duration was determined as increased nociceptive behaviour.
Fig. 1. Percentage response to 26 g stimuli applied to the surgery and contralateral paws were used to assess tactile-allodynia. The tests were performed at baseline, 24 h post-surgery and 2 h following the injection of one of the following: diclofenac, low dose PG LPS, medium dose PG LPS, high dose PG LPS or saline to the surgery area. In the surgery side, significant increase in the % response compared to baseline was demonstrated 24 h following the surgery. Two hours post injection, significant reductions in the % response were observed in the diclofenac and the high dose PG LPS groups compared to the saline group (p < 0.05) and the % response prior injection (p < 0.05). In the contralateral side, no significant changes were observed in all groups at all time points. Data are presented as median and the error bars represent interquartile range.
2.6. Body temperature Temperature was recorded by placing a digital thermometer in the right ear lobe of the rat. (Bioseb Infrared thermometer: BIO = IRB153) 2.7. Swelling Paw swelling was recorded using a calibrated gauge with a digital display (Mitutoyo 293–349, Coolant Proof LCD Micrometer). The rat paw was placed in between the immovable and the movable part of the gauge, one end proximal to the metacarpal pad and the other on the dorsal part of the paw. The knob was moved until the paw could not be easily pulled away and then the screen displayed the number. The paw was marked with a non-removable marker to ensure consistent points of measurement.
saline. In the contralateral side, no significant changes were observed at baseline, post-surgery, or post-injection (Fig. 1).
3.1.2. Mechanical hyperalgesia At baseline, no significant difference was found between the groups in either the ipsilateral or contralateral paws. All groups demonstrated a significant increase in paw nursing time (sec) 24 h post-surgery when compared to baseline (p < 0.05). Two hours post-injection, a significant reduction in paw nursing time was observed in the groups receiving either diclofenac or high dose PG compared to saline. In the contralateral side, no significant changes were observed at baseline, post-surgery, or post-injection (Fig. 2).
2.8. Enzyme-linked immunosorbent assay (ELISA) Two hours following injection of the LPS, a 2 cm piece of the ipsilateral and contralateral paw tissue were collected while rats were under deep pentobarbital anaesthesia. The tissue samples were weighed and placed in a medium containing 300 μ l of Cell Lytic-MT mammalian tissue lysis/extraction reagent (Sigma Chemical Co.), and 0.25 μ l of protease inhibitor cocktail (Sigma Chemical Co.). After centrifuging at 13,000×g for 10 min, the medium was collected and the TNF α, IL-6 and IL-1β and IL-10 levels were assayed with a site enzyme-linked immunoassay (ELISA, R&D Systems Inc., MN, USA). Using a curve plotted from standard solutions, cytokine levels were calculated as pg. (cytokine)/mg (tissue)/m l (medium).
3.2. Paw swelling At baseline, no significant changes were observed in the groups. 24 h post surgery, all groups had developed significant swelling compared to baseline with no significant differences between the groups. The same trend was observed post-injection, in which case there were no significant changes observed amongst the groups. The contralateral side showed no difference at baseline, and no change post injury or post-injection (Fig. 3).
2.9. Data analysis Statistical analyses were performed using JMP Pro 13.0 statistical software (SAS Institute Inc., Cary NC, USA). The sample size analysis was based on previous studies performed on similar pain models (Farrar, Young, LaMoreaux, Werth, & Poole, 2001; Khan et al., 2017). (Alpha for significance is 0.05 and required power is 80%). The outcome distribution was tested for normality using the Shapiro-Wilk test, showing mechanical hyperalgesia, tactile allodynia and cytokines level outcomes to be significantly skewed. Therefore, nonparametric approaches, such as Wilcoxon rank sum test was used to compare the medians of the groups. To assess body temperature and swelling, repeated measures ANOVA was used. P-values less than 0.05 were considered statistically significant.
Fig. 2. Withdrawal duration following pin prick to the surgery and contralateral paws was used to assess mechano-hyperalgesia. The tests were performed at baseline, 24 h post-surgery and 2 h following the injection of one of the following: diclofenac, low dose PG LPS, medium dose PG LPS, high dose PG LPS or saline to the surgery area. In the surgery side, significant increase in the withdrawal duration compared to baseline was demonstrated 24 h following the surgery (p < 0.05). Two hours post injection, significant reduction in the withdrawal duration was observed in the diclofenac and the high dose PG LPS groups compared to the saline group (p < 0.05) and the withdrawal duration prior injection (p < 0.05). In the contralateral side, no significant changes were observed for all groups at all time points. Data are presented as median and the error bars represent interquartile range.
3. Results 3.1. Behavior 3.1.1. Tactile allodynia At baseline, no significant difference was found between the groups in either the ipsilateral or contralateral paws. All groups demonstrated a significant elevation in percentage response when compared to baseline 24 h following the surgery (p < 0.05). Two hours post-injection, a significant reduction in the percent of responses was observed in the groups receiving either diclofenac or high dose of PG, compared to 195
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Fig. 3. Paw swelling was recorded from the surgery and contralateral paws, at baseline, 24 h post-surgery and 2 h following the injection of one of the following: diclofenac, low dose PG LPS, medium dose PG LPS, high dose PG LPS or saline to the surgery area. No significant changes were observed between the groups at baseline, post-surgery and post injection. Data are presented as mean ± SEM.
reduction in pain-related behaviour and increase in IL-10 levels induced by local administrated of PG LPS to the rats’ paw. The LPS administration had no effect on paw swelling and did not increase the rats’ body temperature. PG plays an important role in acute and chronic forms of periodontitis (Van Winkelhoff et al., 2002), it comprises many virulence factors. However its membrane Lipopolysaccharide (LPS) is probably the most significant component. LPS is a virulence factor found in gramnegative bacteria outer cell membrane, that is recognized by the immune system as an indication for the detection of bacterial invasion (Coughlin et al., 1983; Raetz & Whitfield, 2002). Gram-negative bacteria LPS is known to elicit its virulent effects and activate the innate defence system through Toll-Like Receptors (TLR), especially TLR4 which are pattern recognition cells and detect specific bacteria. These in turn can activate inflammatory pathways and induce inflammation via pro-inflammatory cytokines, which can explain the presence of extensive inflammation and pro-inflammatory cytokines such as IL-6, IL-1β and IL-8 in patients suffering from periodontitis gingival crevicular fluids and epithelium (Eskan, Hajishengallis, & Kinane, 2007; Takeuchi & Akira, 2010). PG LPS has distinctive characteristics, it induces relatively weaker inflammatory response compare to other bacteria such Escherichia coli (Kanaya, Nemoto, Ogawa, & Shimauchi, 2004; Liu et al., 2008) and it may activate TLR2 receptors in addition to TRL4 receptors (Darveau et al., 2004; Diya, Lili, Shenglai, Zhiyuan, & Jie, 2008). The anti-nociceptive effect of the PG LPS in the present study, was comparable to that induced by diclofenac sodium administration. However the two treatment effects on the measured cytokines were different. Diclofenac is a nonsteroidal anti-inflammatory drug that is also known to have an effect on N-methyl-D-aspartate (NMDA) receptors (Gan, 2010). Two hours following administration diclofenac reduced significantly the paw IL-1β level, but had no significant effect on the levels of the other two pro-inflammatory cytokines studied (IL-6 and TNFα). This selective effect may be related to the short time after administration the tissue was harvested, it is possible that with time an effect on other cytokines could be observed. Pro-inflammatory cytokines are known to have a significant role in inducing and maintaining pain (Eliav, Benoliel, Herzberg, Kalladka, & Tal, 2009), and the lower IL-1β level may have a role in the anti-nociceptive effect. PG LPS had no effect on the pro-inflammatory cytokines, however it induced a significant increase in the paw IL-10 levels. IL-10 is a potent anti-inflammatory cytokine with an apparent anti-nociceptive effect (Khan et al., 2015). IL-10 also have interesting interactions with PG LPS. It has been demonstrated that human fibroblasts IL-6 production induced by PG LPS is inhibited by IL-10 secretion (Wang et al., 1999). IL-10 knockout mice are highly susceptible to PG-induced alveolar bone loss (Sasaki et al., 2004), and it was suggested that IL-10 have a protective role on bone loss induced by PG (Sasaki et al., 2004). This may at least in part, explain the absence of pain in the majority of patients suffering from periodontal diseases. We tested the PG LPS palliative effect on the rat paw and not intraorally or in the orofacial region. As elaborated in the introduction, the available orofacial pain models induce robust inflammation, severe pain or acute dental pain that are significantly different from the PGinduced periodontal disease. Instead, we used incision-induced pain
Fig. 4. Body temperature was recorded by placing a digital thermometer in the right ear lobe of the rat, at baseline, 24 h post-surgery and 2 h following the injection of one of the following: diclofenac, low dose PG LPS, medium dose PG LPS, high dose PG LPS or saline. No significant changes were observed in body temperature in all the groups at all time points. Data are presented as mean ± SEM.
3.3. Body temperature There were no significant changes in body temperature seen at baseline, post-surgery or post-injury in any of the groups (Fig. 4). The LPS, saline, and diclofenac did not show any effect on body temperatures. 3.4. Cytokine levels 3.4.1. TNF-α Two hours post-injection, no significant differences were found in TNF-α levels between the groups in the ipsi and contralateral nerves (Fig. 5A). 3.4.2. IL-6 Two hours post-injection, no significant differences were found in Il6 levels between the groups in the ipsi and contralateral nerves (Fig. 5B). 3.4.3. IL-1β Two hours post-injection, no significant differences were found in IL-1β levels between the groups in the ipsi and contralateral nerves. However, a reduction was observed in the ipsilateral nerve of the group treated with diclofenac (Fig. 5C). 3.4.4. IL-10 Two hours post-injection, IL-10 levels were significantly higher in the ipsilateral nerves of the group treated with high dose of PG LPS compared to all the other groups. The contralateral IL-10 levels demonstrated a trend of elevation compared to the other groups but did not approach significance (Fig. 5D). 4. Discussion The most noteworthy findings of the study were the significant 196
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Fig. 5. Surgery and contralateral paws tissue samples were harvested 2 h following administration of diclofenac, low dose PG LPS, medium dose PG LPS, high dose PG LPS or saline to the surgery area. The tissue’s TNF α, IL-6 and IL-1β and IL-10 levels were assayed with an enzyme-linked immunoassay. No significant differences in TNF-α (A) or IL-6 (B) were found between the groups. Treatment with diclofenac (C) reduced the surgery side IL-1β compared to the other groups (p < 0.05). The high dose PG LPS (D) increases the IL-10 levels in the surgery side significantly compared to the other groups (p < 0.05). Data are presented as median and the error bars represent interquartile range.
model that is commonly used to assess acute postoperative pain like behaviour. The pain in this model is mild to moderate and strongly associated with local inflammatory process. Diclofenac had an inhibitory effect on pro inflammatory cytokine while PG LPS may have subdued the inflammatory response by enhancing anti-inflammatory cytokine level. LPS being an external pyrogen that can increase body temperature and induce fever in rats (Raetz & Whitfield, 2002). However the doses employed in this study did not have effect on body temperature or swelling. The PG LPS was administrated locally, and the temperature was measured only 2 h following administration, it is possible that following systemic administration and extended duration the outcome would have been different. The PG LPS effect was found to be dose dependent, both the anti-nociceptive effect and the increase in IL-10 levels occurred only following the higher dose administration. PG LPS dose association, other LPS and virulence factors should be further studied to learn more about the association between the anti-nociceptive effect, cytokines levels and undesirable side effects following PG LPS administration.
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5. Conclusion The results of this study demonstrate that PG LPS local application could possess anti nociceptive properties, which at least in part is mediated by an increase in IL-10 levels. This was tested on incisional acute pain model and further studies should evaluate the effect on additional models, in the orofacial region as well as chronic pain models. Conflict of interest and financial disclosure The authors acknowledge and have no personal or financial conflict of interest. No funding was received for this project. References Benoliel, R., Wilensky, A., Tal, M., & Eliav, E. (2002). Application of a pro-inflammatory agent to the orbital portion of the rat infraorbital nerve induces changes indicative of ongoing trigeminal pain. Pain, 99(3), 567–578. Borsani, E., Bernardi, S., Albertini, R., Rezzani, R., & Rodella, L. F. (2009). Alterations of
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Archives of Oral Biology journal homepage: www.elsevier.com/locate/archoralbio
The anti-nociceptive effects of Porphyromonas gingivalis lipopolysaccharide a,⁎
b
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d
e,f
Junad Khan , Bollama Puchimada , Daniel Kadouri , Tali Zusman , Fawad Javed , Eli Eliav
T
g
a
Orofacial Pain and Temporomandibular Joint Disorders, Eastman Institute for Oral Health, University of Rochester, 625 Elmwood Avenue, Rochester, NY, 14620, USA Department of Diagnostic Sciences, Rutgers School of Dental Medicine, NJ, USA Oral Biology, Rutgers School of Dental Medicine, NJ, USA d Department of Diagnostic Sciences, Rutgers School of Dental Medicine, NJ, USA e Department of Periodontology, Stony Brook University, Stony Brook, NY, USA f Laboratory for Periodontal-, Implant-, Phototherapy (LA-PIP), School of Dental Medicine, Stony Brook University, Stony Brook, NY, USA g Eastman Institute for Oral Health, University of Rochester, NY, USA b c
A R T I C LE I N FO
A B S T R A C T
Keywords: Pain Cytokine Inflammation Virulence
Objective: The objective of this study was to assess the effect of Porphyromonas gingivalis lipopolysaccharide (PG LPS) on acute pain-related behaviour induced in rats and to measure its impact on the levels of pro-inflammatory cytokines (IL-1β, IL-6) and anti-inflammatory (IL-10) cytokines. Design: The Brennan model was used to induce acute pain like signs in rats' hind paw. Twenty-four hours following the surgery the rats were divided into 5 groups and the affected paws were injected with 0.2 m l of one of three commercialized forms PG LPS doses (high - 1 mg/ml, medium - 0.6 mg/m l and low - 0.2 mg/m l), diclofenac sodium (1 mg/kg) or saline. Tactile allodynia, mechanical hyperalgesia, body temperature and paw swelling were assessed at baseline, 24 h postoperatively and 2 h after the paw injection. The affected and contralateral paw tissue was assessed for the mentioned above cytokines levels employing enzyme-linked immunosorbent assay. Results: This study may suggest that PG LPS can reduce pain like behaviour via increased levels of anti-inflammatory cytokine IL-10 (5900 ± 748, p < 0.05). The high PG LPS dose and diclofenac reduced the tactile allodynia and mechanical hyperalgesia significantly (42.2 ± 4 and1.6 ± 0.3, p < 0.05). PG LPS high dose increase IL-10 levels while diclofenac reduces IL-1β levels significantly (5900 ± 748 and 1760 ± 271.2). The LPS administration had no effect on paw swelling and did not increase rat’s body temperature. Conclusion: The results demonstrated that PG LPS local application could possess anti- nociceptive properties, which at least in part is mediated by an increase in IL-10 levels.
1. Introduction Inflammation plays an important role in various pain conditions and is known to be associated with increased pain and hypersensitivity (Khan, Hassun, Zusman, Korczeniewska, & Eliav, 2017; Matsuda, Huh, & Ji, 2019; Scheff et al., 2017). It is widely accepted that pain induced by inflammation is an adaptive response intended to protect the organism from further injury. However, not all inflammatory conditions are associated with increased sensitivity or pain. One such example is periodontitis, a complex disease characterized by inflammation and infection of the tissues that support the teeth. Periodontal disease is a multifactorial condition elicited by bacteria that interact with host tissues and cells leading to the destruction of the periodontal structures, including the tooth-supporting tissues, alveolar bone and periodontal ligament. Although chronic periodontitis is an inflammatory condition,
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pain rarely manifests as part of the disease phenotype. Periodontal disease usually results from a host-mediated inflammatory response to pathogenic microflora present in periodontal pocket. Porphyromonas gingivalis (PG) is a Gram-negative rod-shaped anaerobe highly prevalent in those affected with periodontitis compared to healthy individuals. PG is considered a major etiological agent of periodontitis (Darveau, 2009; Kulkarni, Bhat, Thomas, Bhat, & Kulkarni, 2018; Leira et al., 2019; Socransky, Haffajee, Cugini, Smith, & Kent, 1998) for acute and chronic forms of the disease (Van Winkelhoff, Loos, Van Der Reijden, & Van Der Velden, 2002). Like other pathogenic bacteria, PG produces a substantial array of putative virulence factors, such as adherence factors, proteases, toxic metabolites and cellular constituents that are able to induce an immune response (de Diego et al., 2013; Murakami et al., 2004; Tebloeva, Revazova, Fabrikant, Dmitrieva, & Gurevich, 2014). Furthermore, like other gram-negative bacteria, PG contains
Corresponding author. E-mail address: [email protected] (J. Khan).
https://doi.org/10.1016/j.archoralbio.2019.04.012 Received 18 January 2019; Received in revised form 15 April 2019; Accepted 19 April 2019 0003-9969/ © 2019 Elsevier Ltd. All rights reserved.
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Institutional Animal Care and Use Committee at Rutgers University, Newark, NJ (Protocol number 10077E113). Experiments were in accordance with federal law, the regulations of the National Institute of Health and the guidelines of the International Association for the Study of Pain (Zimmermann, 1983).
lipopolysaccharide (LPS) that is found in the microbe’s outer membrane (Coughlin, Haug, & McGroarty, 1983; Raetz & Whitfield, 2002). It has been shown that compared to other LPS, LPS of PG exhibits reduced systemic but similar local induction of inflammatory markers (Liu, Desta, Raptis, Darveau, & Graves, 2008). Furthermore, PG LPS has been shown to play a role in bone resorption and soft tissue destruction by stimulating macrophages, monocytes, and fibroblasts to release a variety of compounds such as prostaglandins, cytokines and metalloproteases (Bramanti, Wong, Weintraub, & Holt, 1989; Holt & Bramanti, 1991; Lindemann, Economou, & Rothermel, 1988; Roberts, Richardson, & Michalek, 1997; Sismey-Durrant & Hopps, 1991; Yamaji et al., 1995). Although the PG LPS can stimulate host immune response, it is thought to be a weaker innate immune stimulator than other Gram-negative bacteria. (Jain & Darveau, 2010; Pulendran et al., 2001). As periodontal disease symptoms usually involve inflammation of the periodontium but are rarely associated with elevated pain symptoms, it is possible to hypothesize that PG, a major etiological agent of periodontal disease, may possess certain anti-nociceptive effects. Rodent models of orofacial pain conditions can be divided into three major groups: Inflammatory (acute or chronic), nerve injury and dental injury. To induce inflammatory pain, compounds such as Complete Freund’s Adjuvant (CFA), carrageenan, formalin, mustard oil or capsaicin are injected to the TMJ area, the masseter muscle or perioral regions (Borsani, Bernardi, Albertini, Rezzani, & Rodella, 2009; Neubert et al., 2005; Rohrs, Neubert, Caudle, & Allen, 2018; Shinoda et al., 2011; Tambeli, Seo, Sessle, & Hu, 2001). Neuropathic pain can be induced by infra orbital or mental nerves manipulations, such as nerve ligation, transection or exposure to inflammatory adjuvant (Nomura et al., 2002, Benoliel, Wilensky, Tal, & Eliav, 2002; Vos, Strassman, & Maciewicz, 1994). The dental pain models usually involve mechanical pulp exposure and often inflammatory adjuvants administration to the exposed pulp (Khan et al., 2008; Raoof et al., 2018; Zhang et al., 2006). The pain intensity is assessed mainly by the rodent behaviour, response to mechanical and thermal stimuli and loss of weight over time (Krzyzanowska & Avendano, 2012). In dental injury model changes in bite force intensity and pattern can be assessed as well (Khan et al., 2008). None of the rodent pain models exactly mirror the human condition. However the relatively severe pain induced can support trigeminal system pain pathways research. The existing orofacial pain models are not ideal for assessing PG LPS potential anti nociceptive effect. The chronic inflammatory process characterizing gingivitis or periodontitis is not comparable to the robust inflammation and severe acute pain induced inflammatory adjuvants administration. The nerve injury orofacial pain models are also not relevant, gingivitis or periodontitis will hardly affect major nerves. The intraoral dental injury pain models are useful for dental pain but not for pain related to soft tissue. Therefore, to explore the potential analgesic effect of PG LPS on acute pain, we used the Brennan model (Brennan, Vandermeulen, & Gebhart, 1996), a validated post-surgical pain model. Although the injury is not in the oral cavity, the pain in this model is mild to moderate and has a substantial inflammatory component. Moreover, pain assessment is simpler and has been validated in numerous studies (Krzyzanowska & Avendano, 2012). In addition to PG LPS effect on pain we evaluated its effects on the levels of interleukin-1 beta (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor alpha (TNF-α), major pro-inflammatory cytokines known to induce pain, as well as interleukin-10 (IL-10), an anti-inflammatory cytokine involved in pain reduction (de Oliveira, Sakata, Issy, Gerola, & Salomao, 2011; Dinarello, 2000; Jiang, Peng, Liu, & Kuo, 2013; Khan et al., 2015; Noma et al., 2011; Ramesh, MacLean, & Philipp, 2013; Shubayev, Kato, & Myers, 2010; Wieseler-Frank, Maier, & Watkins, 2005).
2.1. Experimental design A total of 50 Sprague Dawley rats were used in this study. The rats were habituated to the laboratory and testing apparatus for 4 days followed by baseline recordings of tactile-allodynia and mechanicalhyperalgesia, body temperature and paw swelling. Twenty four hours following left paw skin and fascia incision (Brennan incision model) (Brennan et al., 1996), the rats were evaluated again for the same parameters. A total of 50 rats underwent the surgical procedure and developed signs of pain like behaviour, 24 h post-surgery. These rats were then randomly divided into 5 groups, 10 rats per group. The control groups were locally injected with 0.2 ml of either diclofenac sodium (1 mg/kg) or saline. The experimental groups were locally injected with 0.2 m l of commercialized forms of PG LPS (Ultrapure LPS from P. gingivalis ATCC 33277, Catalog# tlrl-pglps; InvivoGen, San Diego, CA) suspension in saline in one of three different doses; High 1 mg/m l, medium - 0.6 mg/m l and low - 0.2 mg/m l. The effect of PG LPS on pain has not been studied before. Therefore we used three doses that were within the range of doses that had been used in previous rodent studies (Chiang, Kyritsis, Graves, & Amar, 1999). Tactile-allodynia and mechanical-hyperalgesia, body temperature and paw swelling were recorded 2 h post injection. The rats were then euthanized and paw tissues were collected bilaterally to perform Enzyme-Linked Immunosorbent Assay (ELISA) for pro-inflammatory (IL-1β and IL-6) and anti-inflammatory cytokine (IL-10) levels.
2.2. Animals Adult male Sprague Dawley rats weighing approximately 250–300 g at the time of surgery were used. The rats were ordered from a single source (Taconic Farms) and kept in the animal facility under direct veterinary observation. During the entire period, the rats were maintained on standard rodent chow, reverse osmotically-treated water and maintained on a twelve-hour day and night cycle.
2.3. Brennan model of incisional (acute) pain A cocktail of ketamine (50 mg/kg) and xylazine (7.5 mg/kg) solution were injected intraperitoneally to anaesthetize the rats. Following verification of the anaesthesia, a 1 cm longitudinal incision was made in the plantar surface of the hind paw beginning 0.5 cm from the end of the heel. The skin, planar fascia, and underlying muscle were incised. The skin was closed with 5.0 nylon sutures. A single calibrated investigator (JK) performed all the surgeries. During the surgical procedure, the eyes were lubricated and the rats were kept on a warming pad to maintain constant body temperature. Behavioural testing was done 24 h following the incision, using the method described below.
2.4. Tactile allodynia behavioural testing Tactile allodynia was tested with von Frey hairs/Semmes-Weinstein monofilaments (Stoelting, Wood Dale, IL) (Tal & Bennett, 1994). The rats were placed on a perforated metal floor and a monofilament applying 26 g of force was applied in the mid-plantar region of the hind paw 5 times with a 1 s interval. A percentage response was calculated, a higher percentage of withdrawals indicated increased tactile allodynia (Flatters & Bennett, 2004).
2. Materials & methods All procedures and experimental protocols were approved by the 194
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2.5. Mechanical hyperalgesia behavioural testing Mechanical hyperalgesia was evaluated by a blunted acupuncture needle (pinprick test) (Tal & Bennett, 1994). The rat was placed on a perforated floor and a blunted acupuncture needle was applied in the area of the mind-plantar hind paw. The duration of paw nursing was timed for up to 15 s. Normal responses of brief duration were assigned a duration of 0.5 s. An increase in the withdrawal duration was determined as increased nociceptive behaviour.
Fig. 1. Percentage response to 26 g stimuli applied to the surgery and contralateral paws were used to assess tactile-allodynia. The tests were performed at baseline, 24 h post-surgery and 2 h following the injection of one of the following: diclofenac, low dose PG LPS, medium dose PG LPS, high dose PG LPS or saline to the surgery area. In the surgery side, significant increase in the % response compared to baseline was demonstrated 24 h following the surgery. Two hours post injection, significant reductions in the % response were observed in the diclofenac and the high dose PG LPS groups compared to the saline group (p < 0.05) and the % response prior injection (p < 0.05). In the contralateral side, no significant changes were observed in all groups at all time points. Data are presented as median and the error bars represent interquartile range.
2.6. Body temperature Temperature was recorded by placing a digital thermometer in the right ear lobe of the rat. (Bioseb Infrared thermometer: BIO = IRB153) 2.7. Swelling Paw swelling was recorded using a calibrated gauge with a digital display (Mitutoyo 293–349, Coolant Proof LCD Micrometer). The rat paw was placed in between the immovable and the movable part of the gauge, one end proximal to the metacarpal pad and the other on the dorsal part of the paw. The knob was moved until the paw could not be easily pulled away and then the screen displayed the number. The paw was marked with a non-removable marker to ensure consistent points of measurement.
saline. In the contralateral side, no significant changes were observed at baseline, post-surgery, or post-injection (Fig. 1).
3.1.2. Mechanical hyperalgesia At baseline, no significant difference was found between the groups in either the ipsilateral or contralateral paws. All groups demonstrated a significant increase in paw nursing time (sec) 24 h post-surgery when compared to baseline (p < 0.05). Two hours post-injection, a significant reduction in paw nursing time was observed in the groups receiving either diclofenac or high dose PG compared to saline. In the contralateral side, no significant changes were observed at baseline, post-surgery, or post-injection (Fig. 2).
2.8. Enzyme-linked immunosorbent assay (ELISA) Two hours following injection of the LPS, a 2 cm piece of the ipsilateral and contralateral paw tissue were collected while rats were under deep pentobarbital anaesthesia. The tissue samples were weighed and placed in a medium containing 300 μ l of Cell Lytic-MT mammalian tissue lysis/extraction reagent (Sigma Chemical Co.), and 0.25 μ l of protease inhibitor cocktail (Sigma Chemical Co.). After centrifuging at 13,000×g for 10 min, the medium was collected and the TNF α, IL-6 and IL-1β and IL-10 levels were assayed with a site enzyme-linked immunoassay (ELISA, R&D Systems Inc., MN, USA). Using a curve plotted from standard solutions, cytokine levels were calculated as pg. (cytokine)/mg (tissue)/m l (medium).
3.2. Paw swelling At baseline, no significant changes were observed in the groups. 24 h post surgery, all groups had developed significant swelling compared to baseline with no significant differences between the groups. The same trend was observed post-injection, in which case there were no significant changes observed amongst the groups. The contralateral side showed no difference at baseline, and no change post injury or post-injection (Fig. 3).
2.9. Data analysis Statistical analyses were performed using JMP Pro 13.0 statistical software (SAS Institute Inc., Cary NC, USA). The sample size analysis was based on previous studies performed on similar pain models (Farrar, Young, LaMoreaux, Werth, & Poole, 2001; Khan et al., 2017). (Alpha for significance is 0.05 and required power is 80%). The outcome distribution was tested for normality using the Shapiro-Wilk test, showing mechanical hyperalgesia, tactile allodynia and cytokines level outcomes to be significantly skewed. Therefore, nonparametric approaches, such as Wilcoxon rank sum test was used to compare the medians of the groups. To assess body temperature and swelling, repeated measures ANOVA was used. P-values less than 0.05 were considered statistically significant.
Fig. 2. Withdrawal duration following pin prick to the surgery and contralateral paws was used to assess mechano-hyperalgesia. The tests were performed at baseline, 24 h post-surgery and 2 h following the injection of one of the following: diclofenac, low dose PG LPS, medium dose PG LPS, high dose PG LPS or saline to the surgery area. In the surgery side, significant increase in the withdrawal duration compared to baseline was demonstrated 24 h following the surgery (p < 0.05). Two hours post injection, significant reduction in the withdrawal duration was observed in the diclofenac and the high dose PG LPS groups compared to the saline group (p < 0.05) and the withdrawal duration prior injection (p < 0.05). In the contralateral side, no significant changes were observed for all groups at all time points. Data are presented as median and the error bars represent interquartile range.
3. Results 3.1. Behavior 3.1.1. Tactile allodynia At baseline, no significant difference was found between the groups in either the ipsilateral or contralateral paws. All groups demonstrated a significant elevation in percentage response when compared to baseline 24 h following the surgery (p < 0.05). Two hours post-injection, a significant reduction in the percent of responses was observed in the groups receiving either diclofenac or high dose of PG, compared to 195
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Fig. 3. Paw swelling was recorded from the surgery and contralateral paws, at baseline, 24 h post-surgery and 2 h following the injection of one of the following: diclofenac, low dose PG LPS, medium dose PG LPS, high dose PG LPS or saline to the surgery area. No significant changes were observed between the groups at baseline, post-surgery and post injection. Data are presented as mean ± SEM.
reduction in pain-related behaviour and increase in IL-10 levels induced by local administrated of PG LPS to the rats’ paw. The LPS administration had no effect on paw swelling and did not increase the rats’ body temperature. PG plays an important role in acute and chronic forms of periodontitis (Van Winkelhoff et al., 2002), it comprises many virulence factors. However its membrane Lipopolysaccharide (LPS) is probably the most significant component. LPS is a virulence factor found in gramnegative bacteria outer cell membrane, that is recognized by the immune system as an indication for the detection of bacterial invasion (Coughlin et al., 1983; Raetz & Whitfield, 2002). Gram-negative bacteria LPS is known to elicit its virulent effects and activate the innate defence system through Toll-Like Receptors (TLR), especially TLR4 which are pattern recognition cells and detect specific bacteria. These in turn can activate inflammatory pathways and induce inflammation via pro-inflammatory cytokines, which can explain the presence of extensive inflammation and pro-inflammatory cytokines such as IL-6, IL-1β and IL-8 in patients suffering from periodontitis gingival crevicular fluids and epithelium (Eskan, Hajishengallis, & Kinane, 2007; Takeuchi & Akira, 2010). PG LPS has distinctive characteristics, it induces relatively weaker inflammatory response compare to other bacteria such Escherichia coli (Kanaya, Nemoto, Ogawa, & Shimauchi, 2004; Liu et al., 2008) and it may activate TLR2 receptors in addition to TRL4 receptors (Darveau et al., 2004; Diya, Lili, Shenglai, Zhiyuan, & Jie, 2008). The anti-nociceptive effect of the PG LPS in the present study, was comparable to that induced by diclofenac sodium administration. However the two treatment effects on the measured cytokines were different. Diclofenac is a nonsteroidal anti-inflammatory drug that is also known to have an effect on N-methyl-D-aspartate (NMDA) receptors (Gan, 2010). Two hours following administration diclofenac reduced significantly the paw IL-1β level, but had no significant effect on the levels of the other two pro-inflammatory cytokines studied (IL-6 and TNFα). This selective effect may be related to the short time after administration the tissue was harvested, it is possible that with time an effect on other cytokines could be observed. Pro-inflammatory cytokines are known to have a significant role in inducing and maintaining pain (Eliav, Benoliel, Herzberg, Kalladka, & Tal, 2009), and the lower IL-1β level may have a role in the anti-nociceptive effect. PG LPS had no effect on the pro-inflammatory cytokines, however it induced a significant increase in the paw IL-10 levels. IL-10 is a potent anti-inflammatory cytokine with an apparent anti-nociceptive effect (Khan et al., 2015). IL-10 also have interesting interactions with PG LPS. It has been demonstrated that human fibroblasts IL-6 production induced by PG LPS is inhibited by IL-10 secretion (Wang et al., 1999). IL-10 knockout mice are highly susceptible to PG-induced alveolar bone loss (Sasaki et al., 2004), and it was suggested that IL-10 have a protective role on bone loss induced by PG (Sasaki et al., 2004). This may at least in part, explain the absence of pain in the majority of patients suffering from periodontal diseases. We tested the PG LPS palliative effect on the rat paw and not intraorally or in the orofacial region. As elaborated in the introduction, the available orofacial pain models induce robust inflammation, severe pain or acute dental pain that are significantly different from the PGinduced periodontal disease. Instead, we used incision-induced pain
Fig. 4. Body temperature was recorded by placing a digital thermometer in the right ear lobe of the rat, at baseline, 24 h post-surgery and 2 h following the injection of one of the following: diclofenac, low dose PG LPS, medium dose PG LPS, high dose PG LPS or saline. No significant changes were observed in body temperature in all the groups at all time points. Data are presented as mean ± SEM.
3.3. Body temperature There were no significant changes in body temperature seen at baseline, post-surgery or post-injury in any of the groups (Fig. 4). The LPS, saline, and diclofenac did not show any effect on body temperatures. 3.4. Cytokine levels 3.4.1. TNF-α Two hours post-injection, no significant differences were found in TNF-α levels between the groups in the ipsi and contralateral nerves (Fig. 5A). 3.4.2. IL-6 Two hours post-injection, no significant differences were found in Il6 levels between the groups in the ipsi and contralateral nerves (Fig. 5B). 3.4.3. IL-1β Two hours post-injection, no significant differences were found in IL-1β levels between the groups in the ipsi and contralateral nerves. However, a reduction was observed in the ipsilateral nerve of the group treated with diclofenac (Fig. 5C). 3.4.4. IL-10 Two hours post-injection, IL-10 levels were significantly higher in the ipsilateral nerves of the group treated with high dose of PG LPS compared to all the other groups. The contralateral IL-10 levels demonstrated a trend of elevation compared to the other groups but did not approach significance (Fig. 5D). 4. Discussion The most noteworthy findings of the study were the significant 196
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Fig. 5. Surgery and contralateral paws tissue samples were harvested 2 h following administration of diclofenac, low dose PG LPS, medium dose PG LPS, high dose PG LPS or saline to the surgery area. The tissue’s TNF α, IL-6 and IL-1β and IL-10 levels were assayed with an enzyme-linked immunoassay. No significant differences in TNF-α (A) or IL-6 (B) were found between the groups. Treatment with diclofenac (C) reduced the surgery side IL-1β compared to the other groups (p < 0.05). The high dose PG LPS (D) increases the IL-10 levels in the surgery side significantly compared to the other groups (p < 0.05). Data are presented as median and the error bars represent interquartile range.
model that is commonly used to assess acute postoperative pain like behaviour. The pain in this model is mild to moderate and strongly associated with local inflammatory process. Diclofenac had an inhibitory effect on pro inflammatory cytokine while PG LPS may have subdued the inflammatory response by enhancing anti-inflammatory cytokine level. LPS being an external pyrogen that can increase body temperature and induce fever in rats (Raetz & Whitfield, 2002). However the doses employed in this study did not have effect on body temperature or swelling. The PG LPS was administrated locally, and the temperature was measured only 2 h following administration, it is possible that following systemic administration and extended duration the outcome would have been different. The PG LPS effect was found to be dose dependent, both the anti-nociceptive effect and the increase in IL-10 levels occurred only following the higher dose administration. PG LPS dose association, other LPS and virulence factors should be further studied to learn more about the association between the anti-nociceptive effect, cytokines levels and undesirable side effects following PG LPS administration.
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