Efecto de la pregabalina sobre la expresión de Fos en el tronco del encéfalo y la médula espinal en dos modelos de dolor neuropático

In recent decades, there have been significant advances in the search and discovery of new therapeutic strategies against pain. This has been possible thanks to, in large part, a greater knowledge of the underlying mechanisms of pain. Particularly significant have been the data from the anatomical substrate of pain transmission and its endogenous modulation. Based on these anatomical data, pain has been classified in different types: neuropathic pain, when the source of pain is located in the nervous system itself, and non-neuropathic pain, when the origin of pain is located in non-neural anatomical structures, as the skin (superficial nociceptive pain), musculoskeletal system structures (deep nociceptive pain), or internal organs (visceral pain). However, the above mentioned advances are not equally distributed among the types of pain that we have mentioned. Neuropathic pain response to analgesic treatment, or in many other cases even with opioids, is still far from being satisfactory The lack of response of neuropathic pain to analgesic treatment, or in many cases even to opioids, has led therapists to look for other pharmacological solutions. Of all the proposals, the one that is currently be claimed with more strength, is the use of antiepileptic drugs, actually proving to be a valid alternative to treat neuropathic pain (McQuay et al., 1995). The mechanism of action of these anticonvulsant drugs is different from traditional analgesics; while the latter reduce the afferent signals from both healthy and injured tissue, antiepileptic drugs could be called antinociceptive rather than antihyperalgesic, as long as they do not have a per se effect but to reduce the excitability of dorsal horn neurons induced by the damaged of the tissue. One of the antiepileptic drugs first tested in the treatment of neuropathic pain was gabapentin (Dirks et al., 2002, Turan et al., 2004). However, at present, this drug is being replaced by new by-products to increase its efficiency and reduce its adverse effects, among which pregabalin highlights. Pregabalin is the pharmacologically active S-enantiomer of 3-aminomethyl-5-methyl-hexanoic, which is similar to its predecessor gabapentin. Its mechanism of action is not known yet exactly. There are several cellular mechanisms that have been proposed to explain its effect in the treatment of neuropathic pain: acting on NMDA receptors, calcium channels, sodium channels, monoaminergic pathways, opioid and nonopioid systems, … Despite its name and the one of its predecessor molecule, gabapentin has no direct gabaergic action, neither blocks GABA uptake nor its metabolism. Pregabalin, like gabapentin, binds to the alpha2-delta protein subunit of channel voltage-dependent calcium , which might explain its antihyperalgesic effect (Schwarz et al., 2005). Pregabalin has demonstrated antiallodynic and antihyperalgesic activity in several models of neuropathic pain in rodents (Lauria-Horner and Pohl, 2003, Field et al., 1999, Chen and Pan, 2001), these effects being observed at doses of 2 to 4 times lower to those of gabapentin. It also presents a higher tolerability than with other anticonvulsants, including gabapentin, which makes it a valid therapeutic option for the treatment of these patients. The most frequently shown adverse events are: dizziness, drowsiness, peripheral edema, headache, blurred vision and constipation, being all of them usually mild to moderate. Although pregabalin has been used in different types of neuropathic pain, such as post-herpetic neuralgia (Dworkin et al., 2003; Sabatowski et al., 2004), at present its interest is focussed on its effectiveness in post-surgical pain (Dahl et al., 2004). In fact, post-surgical pain can be considered as a type of neuropathic pain that could be considered temporary or reversible. Preclinical studies have shown that surgical incision in the paw of the rat is followed by a prolonged thermal and mechanical hyperalgesia, as a clinical result of central sensitization (Whiteside et al., 2004). These data show that sensitization of dorsal horn of the spinal cord, though reversible in most surgical patients, may be an important algesic mechanism in the first post-interventional days or weeks. Antihyperalgesic drugs, such as pregabalin, thus become post-surgical analgesic, attractive because they offer the possibility to block pathological pain, at the same time that they would allow physiological pain, which would be useful for its protective and alertness function. In the same way, the action of pregabalin would be useful in post-surgical pain if we consider that the mechanism of action of these anticonvulsant drugs differs entirely from that of opiates, thus preventing the decrease in intestinal motility, one of the major adverse effects of opioids in post-surgical patients (Field et al., 1997). These experimental data have been corroborated in the only randomized, double-blind, placebo-controlled trial in postoperative dental pain, and where the results showed statistically significant differences between the group treated with 300 mg of pregabalin and placebo-treated group (Hill et al., 2001). Even though all the experimental studies in animals with pregabalin (Field et al., 1999; Eutamene et al., 2000, Hurley et al., 2002) and human clinical trials that support the clinical use of buprenorphine in neuropathic pain, we were not able to find any work that deepened in the anatomical substrate in which the analgesic effect of this antiepileptic substance underlies at a central nervous system level. The only work that we found studying the effect that the administration of these anticonvlulsant substances, particularly gabapentin, has on the expression of protooncogene c-fos at the level of central Nervis system, is focused on the spinal cord, forgetting the supraspinal level study (Kaneko et al., 2000). Our literature review about the topic has not even showed a single work that pretend to identify the supraspinal neural structures that are activated or at least are altered after the administration of pregabalin in healthy animals, or with different types of neuropathic pain. However, it seems that the identification of brainstem centers altering its neuronal activity by the administration of these substances, would be useful for a better understanding of their mechanisms of action, which would improve their effectiveness both individual use and in combination with other analgesics. OBJECTIVE Identify the brainstem centers involved in the pain modulation circuit, in which the administration of pregabalin provokes significant changes in neuronal activity. The interest of achieving the proposed objectives resides in the idea that clinical studies show that pregabalin has an analgesic effect in those cases in which it neuropathic pain is shown. However, we do not know the nerve centers through which such antihyperalgesic action is shown. Traditional analgesics show their effects modulating the neuronal activity of the different brainstem centers, integrated in the endogenous pain control circuit. To use in a more safe and effective way these new drugs, it is important to identify the neural centers whose activity, measured as changes in the expression of c-fos proto-oncogene in the nucleus of its cells (Harris, 1998), changes after administration of these substances. The study about different algesic stimulation conditions, would also allow us to know how much the substance of our trial, pregabalin, may block at the posterior horn of the spinal Fos expression, due to the arrival at that level of the primary painful afferents. MATERIAL AND METHODS The methodology we will use to develop and achieve the objectives of this project is based on the detection with immunocytochemical techniques of Fos expressing neurons in the brainstem centers involved in pain control circuit, in different experimental situations, as well as the posterior horn of the lumbar segment of the spinal cord. In a more detailed way, the experimental procedure would be the following: A) EXPERIMENTAL ANIMAL This work will be performed in 50 albino rats of Sprague-Dawley, male, weighing approximately 250 g. The animals will be kept under standard laboratory conditions for at least a week before the start of the test: cycles of 12 hours light – 12 hours darkness (the light switches on at 8:00 am and switches off at 8:00 pm ), temperature (15 º C) and constant humidity, standard diet and free access to food and water. B) EXPERIMENTAL GROUPS Animals will be distributed in the following experimental groups: B.1) Physiological control group (CFS) (n = 10): Animals in this group will be used to the handling maneuvers that might be necessary later on during the experiment. These manipulations would be the following: - Take the animal out of the cage and weight it. - Inmobilizate the animal, for 5 seconds, holding it with one hand under its front legs, while they are crossed in saltire in front of its face, and holding its tail with the other hand. This inmobilization maneuver will be performed twice to each animal: the first, once it has been weighed, and the second, half hour later. During the first inmobilization, 1 ml of saline will be injected intraperitoneally (ip). Between one inmobilization and the other, animals will remain in their cages. The animals will be sacrificed 2 hours after the saline ip injection, ie within 2 hours of the second inmobilization. The purpose of this group is to reject that neither the stress of detention, nor the possible pain from ip injection, or even the abdominal distension caused by the introduction of a 1 ml ip volume, shows a pattern of Fos expression that could mask the Fos expression induced by the applied stimulus, analgesic or painful. B.2) Control group Simulated Neuropathically simulated control group(CSN) (n = 10): Similar to the CSF group, except in that a week before, all surgical maneuvers necessary to get the exposure of the sciatic nerve will be made to the animals of this group, so that it may be comparable to the group on which the test of sustained constriction of the sciatic nerve will be applied (Taber et al., 1969), but always avoiding injuries. The purpose of this group is to dismiss that neither the surgical maneuvers necessary to apply the model of neuropathic pain proposed by Taber et al. (1969) will mask the Fos expression induced by the constriction of the nerve. B.3) Pregabalin control group (CPG) (n=10): Rats in this group are injected, on the day of the experiment, 1 ml ip, equal to a dose of 30 mg / kg morphine (Eutamene et al., 2000) during the first immobilization, 2 hours and a half before the slaughter. B.4) Neuropathic Pain Control Group (CRC) (n = 10): Similar to the CSN group, but with the difference that the animals in this group received a painful neuropathic stimulus by the constriction of the sciatic nerve. As mentioned before, the neuropathic pain model chosen in this project is the one proposed by Taber et al. (1969), consisting on the provocation of a sustained constriction of the sciatic nerve. It is performed by 4 ligatures spaced 1 mm on the sciatic nerve trunk at the height of the biceps femoris, with a 4-0 silk. The animals are slaughtered a week after surgery. B.5) Pregabalin + Neuropathic Pain Group (PGDN) (n = 10): The animals in these groups receive a neuropathic painful stimulus, and the day after are injected 1 ml ip analgesic, daily for one week, and after then they are slaughtered. The handling of the animals will be done following the recommendations given by the Ethics Committee of the University of Medicine, in Valencia, as well as those proposed by Zimmermann (1983) for researchs on experimental pain in conscious animals. C) ANESTHESIA AND PERFUSION The animals were sacrificed with an pentobarbital ip overdose (120 mg / kg), starting with the maneuvers of the process of perfusion, once it was checked that the animal was deeply anesthetized (absence of corneal reflex), and the heart was still beating. The whole process of anesthesia and perfusion in any case exceed 15 minutes, to avoid iatrogenic c-fos induction as a result of the handling or the anesthesia itself (Herdegen et al., 1991, Takayama et al., 1994). The infusion started in all cases with a wasing of the vascular tree with 100-200 ml of isotonic heparinized saline (15,000 IU / liter) for then, injecting the fixing solution consisting of phosphate buffer and 4% paraformaldehyde. Once the perfusion process finished, the animals were decapitated, and we started to descaloting and removing the brain and the lumbar spinal cord. D) OBTAINING OF THE SECTIONS Pieces were stored at 4 ° C in the same fixative solution overnight. After this time, they were transferred to a cryoprotective solution with 30% sucrose, in which they remained 24 hours. Once cryoprotected, the pieces were frozen and sectioned with a cryostat in coronal sections of 40 microns thick. E) IMMUNOCYTOCHEMISTRY REVELATION OF THE C-FOS PROTO-ONCOGENE After three washes with PBS to remove sucrose, we proceed to detect the immunocytochemical of the protein encoded by the proto-oncogene object of our study, c-fos, by the ABC method (Hsu et al., 1981). The sections go through successive solutions (Valverde-Navarro et al., 1996). Mounting of the sections: The sections are mounted on gelatinized slides with a solution of 0.5% gelatin and chrome alum 0.05%. Then they are let to dry overnight at room temperature, and the next day, they are dehydrated in increasing ethanol dilutions (70 º, 96 º and 100 º, Panreac), cleared with xylene and covered with Merckoglas. F) SELECTION OF THE LEVELS OF STUDY The levels corresponding to both the spinal lumbar and the brain stem are selected using the Paxinos and Watson atlas (2005). At least 2 sections are selected by each of the chosen levels (Murphy et al., 1995). G) OBTAINING OF DISTRIBUTION PATTERNS OF FOS-POSITIVE NEURONS The distribution pattern of inmurreactivas cells against Fos is obtained from drawings in which the Fos-positive neurons are plucked manually, using a Zeiss camera attached to a Axioskopp Zeiss microscope with a 20x objective. These drawings are made by an experimenter unaware of the experimental group where the preparations belong when they are pointed. H) OBTAINING OF DIGITAL IMAGES OF THE PREPARATIONS Images for the picture study are captured by a NIKON video camera (DXM 1200) attached to a Nikon Eclipse E600 microscope with direct light. I) STATISTICAL STUDY Statistical analysis of data will be carried out using SPSS program, version 15. The analysis will begin with a description including the media, the number of cases and the standard deviation for the original variables. The descriptive analysis will be attached to a graph with the estimated means for each original variable within each of the experimental groups. Then, there will be an analysis of variance (ANOVA) of a factor for the variable, the factor being the different treatment received by each of the experimental groups. In case that the analysis would detect differences between the groups, multiple comparisons would be useful to determine homogeneous sets of experimental groups that would share the same mean. As previously known, no single technique for performing these comparisons exists, depending on the outcome that we have used. In our case, if necessary, we would use the Tukey and Scheffe methods that produce extreme situations, particularly the last one, the most conservative, and that always leads to a smaller number of homogeneous groups (Sokal and Rholf, 1995). RESULTS At the level of the spinal dorsal horn, the pregabalin produces a marking reduction of fos-expression induced by both painful stimuli. Pregabalin induces expression of c -fos at both rostrocaudal levels of the nucleus of the solitary tract (NTS) , regardless of the application or not of a painful stimulus . As in the NTS, pregabalin also induces expression of c-fos in both levels of the lateral portion of the parabrachial nucleus (LPB) , regardless of the application or not of a painful stimulus . In the locus coeruleus (LC) pregabalin, nociceptive pain stimuli induce the expression of c -fos , but not neuropathic pain stimuli. By contrast, pregabalin did not triggered intense Fos immunoreaction in the dorsal raphe nucleus ( DR ), whereas reduced the expression of c-fos induced by the nociceptive pain stimulus. CONCLUSIONS 1. – At the level of the dorsal horn of the spinal cord , pregabalin blocks the expression of c-fos induced by both somatic and neuropathic painful stimuli. 2. - Administration of pregabalin causes significant activation of neurons in the nucleus of the solitary tract, especially in the medial (m), center (c) and ventro-lateral (vl) subnuclei. By contrast, these neurons are activated only after a painful stimulus, regardless of whether it is somatic nociceptive or neuropathic . 3 . - Administration of pregabalin causes significant activation of neurons in the lateral parabrachial nucleus , especially in the external (elPB) and center (clPB) subnuclei. Also, the application of a painful stimulus (both somatic nociceptive and neuropathic) also induce an activation of neurons in the LPB , mainly in the dorsal subnucleus (dlPB) and in the area that remains outside the central subnucleus (clPB), although in a lesser extent than pregabalin. 4 - The administration of pregabalin causes a significant activation of neurons especially in the caudal portion of the locus coeruleus (LC) . The effect of an algesic stimuli on LC varies depending on the nature of the stimulus: somatic nociceptive one significantly actives LC neurons , whereas neuropathic one not. 5 . - Administration of pregabalin causes significant activation of neurons in the dorsal raphe nucleus, such as a somatic nociceptive pain stimulus. By contrast, the application of a neuropathic pain stimulus fails to activate neurons in the LC.