Intrathecal neostigmine causes a dose-dependant analgesic effect in animal models  and humans  by inhibiting the breakdown of acetylcholine in the dorsal horn of the spinal cord. However, its clinical use is limited by side-effects (mainly nausea and vomiting) probably due to the rostral migration of the drug in the cerebrospinal fluid . Animal studies also support the existence of a peripheral antinociceptive effect of acetylcholine  and neostigmine . Controversy remains as to whether neostigmine when administered peripherally would exert an analgesic effect . A recent study , in patients undergoing arthroscopic meniscus repair, showed that intra-articular neostigmine (500 µg) produced a peripheral analgesic effect superior to that observed with intra-articular morphine (2 mg). However, Bouaziz and his colleagues were unable to support any enhancement of sensory or motor blockade with neostigmine added to mepivacaine in axillary plexus blocks . To explore the peripheral antinociceptive effects of neostigmine, we performed the present study in a carrageenan-injected rat model, a well-defined animal model of persistent inflammation .
Animal testing protocols were approved by the local animal care committee. Animals were treated according to the guidelines issued by the International Association for The Study of Pain . Rats (n = 12) (male Sprague–Dawley) were kept in individual cages on a 12-h light/dark cycle with water and food ad libitum. Hyperalgesia was determined by measuring the paw withdrawal threshold with an Analgesy-Meter (Ugo Basile, Varese, Italy). To evaluate the oedema due to carrageenan-induced paw inflammation, the paw circumference (PC) of the injected paw was measured by a thread at the metatarsal level . After assessing baseline values (T0) for the paw withdrawal threshold and paw circumference of both hind paws, the animals were briefly anaesthetized with halothane 2–3% and the right hind paw was injected with 0.2 mL of 1% carrageenan solution in 0.9% NaCl (saline) (carrageenan side). The left side was then injected with the same volume of saline (saline side). Two hours later (T1), the presence of hyperalgesia was assessed and 20 µg of neostigmine was injected in the right hind paw. This dose was chosen based on preliminary reports showing that the administration of 100 µg of the same drug via the same route was lethal for all rats tested (n = 9) and the dose of 50 µg (n = 4) was responsible for the systemic effect (sedation) with no possible interpretation of the paw withdrawal threshold.
After neostigmine injection, the paw withdrawal threshold was tested on both sides at 10 (T2), 20 (T3) and 60 min (T4). For all animals, the paw circumference was measured on both sides before (T0) and 2 h after the use of carrageenan (T1), as well as 60 min after the injection of neostigmine (T4).
Results are presented as mean ± SD. Paw withdrawal thresholds are expressed in grams (g) and paw circumference in millimetre (mm). The measured paw withdrawal threshold is presented in Figure 1 and the measured paw circumference in Table 1. Statistical significance between the groups was determined using Wilcoxon's test, whereas within each group we used an ANOVA for repeated measures followed by Bonferoni's test when appropriate.
Carrageenan-induced inflammation developed in the right hind paw with paw circumference increase reflecting oedema (Table 1) and reduction of the paw withdrawal threshold reflecting mechanical hyperalgesia (Figure 1). The intraplantar injection of neostigmine was not followed with any effect upon carrageenan-induced paw inflammation. No modification of paw circumference or paw withdrawal threshold was observed after injection of neostigmine (Tables 1 and 2 and Figure 1).
The main finding of this study was a lack of analgesic effect of peripheral neostigmine in a carrageenan-induced hyperalgesia rat model. The high incidence of lethal or systemic side-effects with greater doses (50 and 100 µg) obviously has to be considered. Neostigmine has been shown previously to produce analgesia in animals and humans by increasing the acetylcholine at central and peripheral sites of action [1–5]. Previous laboratory investigations suggest a peripheral implication of acetylcholine at the central nerve endings of small afferent fibres and have shown that acetylcholine receptors exist at the peripheral nerve level [10,11]. A recent study in rats showed a peripheral analgesic effect of neostigmine after intra-articular administration  and acetylcholine infused subcutaneously acts via the same way to produce analgesia in animal model . In vitro studies have shown that peripheral cholinergic-mediated antinociception at peripheral nerve endings is caused by a hyperpolarization of the neurones and by modulation of the nitric oxide pathways . Lack of efficiency of neostigmine added to local anaesthetic to improve the duration of the block in patients operated under plexus blockade might be due to the absence of acetylcholine release, but other studies suggested that the effect of neostigmine is more likely to occur locally in inflamed tissues [3–5]. Lower doses used in our study, in consideration of lethal or side-effects, may explain the failure of neostigmine in our model. Thus, similar systemic adverse effects, but not lethal, were reported in an intra-articular inflammatory animal model with higher doses (100 µg) . Yet the injection in a joint deletes the clearing of neostigmine and reduces the parasympathetic signs in animals and allows high intra-articular concentration of neostigmine explaining perhaps the detection of peripheral analgesic effect. The intense carrageenan-induced inflammation in our model may induce a rapid absorption of neostigmine and per se the absence of significant modification quantity of peripheral acetylcholine. In conclusion, our model does not support an analgesic effect of neostigmine upon inflammatory-induced hyperalgesia in an animal model.
The authors thank Madame R. Le Guen for technical assistance.
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