This study focused on the combination of neostigmine with the non-selective Cox inhibitor, diclofenac, or the selective Cox-2 inhibitor, celecoxib. This rationale was based on the facts that the anti-nociceptive effect of NSAIDs is Cox mediated as stated by Miranda et al. (4) On the other hand, neostigmine, being an acetylcholinesterase inhibitor, should possess anti-inflammatory properties as seen in several studies examining the cholinergic anti-inflammatory pathway (9, 11).
Comparing both combinations in the tail clip test of this study, Neo + Cele reveals a boosting onset of analgesic action within 15 min, but short duration, fading after less than 30 min. In contrast, Neo + Diclo reveals rapid onset with lesser magnitude and decrease in analgesia right after 30 min. Measuring β-endorphin serum level after 60 min has proven the loss of analgesic activity of all treated groups, while writhing tests has proven the effective analgesia of both combinations within 15 min period. This outcome proves previous observation by Miranda et al. (4) that selective Cox-inhibition is not yet enough to identify the strong analgesic property of NSAIDs. Many others studies have shown that celecoxib has less analgesic effect than other NSAIDs and that its effect depends on the duration of administration, where it is preferred for short-term treatment periods due to reversibility of its effect (25–27).
The fact that Neo + Diclo in this study didn’t show any additive or synergistic analgesic effect compared to Neo alone, came in contradiction to the study by Miranda et al.(28) who proved a synergistic analgesic effect of combining neostigmine with diclofenac, attributing this effect to the increased acetylcholine concentration in the synaptic cleft, which in turn gives a signal of supraspinal antinociception. Yet, adding celecoxib to neostigmine caused a clear, instant synergism in the tail clip, a result that needs more investigation and could be of important clinical implications for rapid manipulation of acute pain states.
Formalin injection is a well-known model of persistent inflammatory pain in experimental animals, which is characterized by edema and inflammation. The release of histamine, bradykinin, serotonin, substance P and prostaglandins induce the inflammatory reaction. These chemical substances cause an increase in vascular permeability, which promote the accumulation of fluid in interstitial tissue (29). Injection of the rat paw with formalin causes tissue damage and evokes a biphasic spinal release of prostaglandin E2 that increases in concentration of 110% in the 0–10 min period and then 83% in the 20–30 min period after formalin injection, with no further increase after 30–60 min of formalin injection (30). It is well known, that NSAIDs reduce the nociceptive behavior during the second phase (20–30 min), while the first phase is not affected (29).
Accordingly, in this study, the anti-inflammatory properties of neostigmine have shown a clear synergistic effect within 24 h when added to celecoxib (Neo + Cele), which then declined after 5 days of the study, an effect that is attributed to Neo rather than Cele. This outcome comes in accordance with the study of Nishyama (31), who has shown the same effect upon studying celecoxib in acute formalin test. Surprisingly, the Neo + Diclo combination had an opposite outcome, as it didn’t show a significant effect after 24 h, but caused clear potentiation after 5 days. However, analyzing serum samples after 5 days, both combinations Neo + Cele and Neo + Diclo have shown an increase in the anti-inflammatory properties of neostigmine as seen in the results of both TNF-α, NF-кB and HS-CRP.
Histopathological examination of the inflamed hind paws after 5 days came in accordance with the biochemical data, where all drugs have shown decreased inflammation. Diclofenac and celecoxib monotherapy have shown much better effect than neostigmine. However, combination therapy has shown delayed histopathological improvement compared to neostigmine monotherapy. This discrepancy from the biochemical data is attributed to the short treatment protocol of this study (5 days), where histopathological improvements require chronic treatment (32). Moreover, a study by Álvarez-Soria et al. (33) matched the outcome of our combination therapy, where treatment with celecoxib and classic NSAID improved joint pain and function, decreased Cox-2 expression, but synovial macrophage infiltration were less affected. As specified by Tsuboi et al. (34) inflammatory cell infiltration and vascularization continues even with improved clinical outcome of the joint in RA patients, indicating a subclinical inflammation.
The positive feedback loop of the Cox-2-PGE2-EP2-NF-κB signaling pathway is well known to initiate and preserve inflammation (35). Activated Cox-2 increase the production of PGE2, that binds to the EP2 receptor and consequently activates NF-κB; the important inducer of pro-inflammatory cytokine (35, 36). More than twenty-five cytokines are controlled directly by NF-κB. On the other hand, high levels of TNF-α promptly activates Cox-2 and NF-κB in a positive feedback loop (37). In a study of Walker et al. (38) celecoxib has shown a similar Cox-2 selectivity ratio to diclofenac. In contrast, comparing in vivo findings of both drugs, same therapeutic concentrations have shown different effect on Cox-1. Accordingly, in our study, diclofenac (monotherapy and in combination with Neo) has shown more decrease in Cox-1 activity than celecoxib. Both drugs, however, had same effect on Cox-2 activity.
Expanding to the previous findings, a mouse model of Saccular Intracranial Aneurysms has shown that endothelia cells-activated NF-κB activity was suppressed with the administration of the selective Cox-2 inhibitor celecoxib (35). Moreover, the results of the Cox isoforms activity study in the present work come in accordance with a previous work from Nishiyama et al. (39), where intrathecal administration of celecoxib resulted in a dose dependent inhibition of the flinch response of the formalin test, but had no significant effect on the tail flick latency. With doses up to 200 mg, no hemodynamic changes or behavioral side-effects were observed (39).
Clinically, neostigmine is being used for its analgesic effect in both the perioperative and postoperative phases with different patients proving high efficacy (40, 41). It is well documented, that any surgical procedure causes perioperative immunosuppression and neuroendocrine stress, exerted by activation of the autonomic nervous system and the hypothalamic-pituitary-adrenal axis (42). Moreover, intraoperative blood pressure management, blood transfusion, hyperglycemia, hypothermia and the anesthetic agent itself cause also perioperative immunosuppression, which persist for at least few days post operation (43).
Numerous studies demonstrated that commonly used anesthetic agents in surgery and intensive care units possess anti-inflammatory properties and could impair the inflammatory response process either directly or indirectly by modulating the stress response (44, 45). However, anti-inflammatory properties of anesthetic agents is therapeutically beneficial in only very distinct situations, where it’s immunosuppressive properties affect the long term outcomes of most patients after surgery (46, 47).
According to the current study, adding a combination of Neo + Diclo or Neo + Cele to the anesthetic agent in the perioperative phase might promote the anti-inflammatory characteristics of neostigmine adding to the anti-inflammatory effect of the anaesthetic agent. The clinical implication of such combinations in anesthesia requires more in vivo studies.
The nephrotoxicity of the conventional NSAIDs as well as selective Cox-2 inhibitors is well known, where both Cox isoforms play a crucial role in maintaining renal function homeostasis (48). Accordingly, all treated groups of this study seemed to disturb some kidney functions as seen in the results of serum urea and creatinine, however with normal histopathological appearance of the kidney, which may relate to the low dose of diclofenac and celecoxib used in this study. This comes in accordance with Izhar et al. who claims that the side effects seen by diclofenac and celecoxib are related to their dosing frequency rather than their sensitivity (49).
The effect of Diclo on the liver in the current study has shown, surprisingly, complete safety as reflected on the serum AST, serum ALT and histopathological examination results. Known that diclofenac is usually categorized of being high toxic (50), the normal appearance in this study might be related to the low dose used.
Neostigmine as well as celecoxib appears to disturb liver functions moderately as seen in the serum AST/ALT results and the histopathological pictures. However, combining both drugs has shown to be safer than each drug alone. The hepatotoxicity of celecoxib within therapeutic doses has been intensively studied earlier and it was recommended to be used with caution in patients with liver insufficiency (51).