The aim of our study was to evaluate the effects of NM in behavioral improvement in neuropathic rats, and to evaluate NGF and GFAP levels in the DRG and spinal cord after NM.
Behavioral data confirmed the onset of neuropathic pain syndrome 3 days after CCI, which stabilized by the 14th day after injury, as shown by reduced mechanical, thermal and allodynic thresholds in the ipsilateral side of injury. We demonstrated those ten NM sessions each other day restored mechanical, thermal and allodynic sensitivity, as well as all behavior parameters that were associated with the neuropathic condition.
There is a growing body of evidence that a unilateral nerve injury induces contralateral changes. The effect of the unilateral peripheral nerve lesion on contralateral non-lesioned structures was reviewed by many authors. In general, the responses to contralateral injuries are usually in smaller magnitude and with a briefer time course compared to ipsilateral changes [21, 22]. However, our results demonstrated an effect at the unilateral nerve injury which corroborates with others studies showing the same response [23–27]. Methodological variables could account for this discrepancy, such as different strains of rats, different methodologies or different suture material used for different authors .
The impact of halothane anesthesia does not interfere with behavior responses, since the nociceptive experiments were realized on subsequent day after NM sessions. In addition, studies demonstrated that from inhalation anesthetics, halothane was able to decrease nociceptive reactions (returned to basal levels) faster than other inhalatory anesthesics (enflurane, isoflurane, and desflurane) . Furthermore, studies have shown that the use of inhalational anesthetic during nine sessions of physical therapy in rats did not change expression of glial cells markers . Suggesting that this anesthetic does not influence in chronic models of sensitization [30, 31].
Our behavioral data are in agreement with studies showing that NM is efficient as a therapeutic approach in clinical applications [1, 3, 11]. Studies realized recently, demonstrated the reversion of hyperalgesia, as well as nerve regeneration and decreased of glial cells expression such as microglia and astrocytes in the spinal cord, after joint mobilization in a model of sciatic nerve crush injury . In addition, Malisza, K.L. et al. (2003) examine neuronal activation in the spinal cord due the secondary hyperalgesia resulting from intrajoint capsaicin injection .
On the other hand, studies realized by Vicenzino et al. (1998) and Sterling et al. (2001) using spinal manual therapy, showing a hypoalgesia effect but no influence on thermal hyperalgesia, suggesting an effect in inhibitory pathway from the dorsal periaquedutal gray area [33, 34].
In this regard, we also evaluated the levels of NGF and GFAP in the DRG and spinal cord after NM treatment in CCI-injured animals. We demonstrated an increase of NGF expression in CCI-injured rats, and a decrease to normal levels after NM treatment in DRG tissues. On the other hand, no difference was observed in lumbar spinal cord tissue analyzed. This indicates that the increase of NGF occurs at time points that are coincident with its role in pain-related behaviors, suggesting that NGF is involved in the nociceptive effects induced by neuropathic pain. Our data are in agreement with several studies showing that NGF injections by systemic and peripheral routes induce nociception [15–17, 19]. Indeed, NGF seems to be a critical mediator of all types of pain, including short-term pain , surgical pain , inflammatory pain  visceral pain  and neuropathic pain [19, 38].
The pain induced by peripheral inflammation or neuropathic pain is currently being linked to the involvement of glial cells or satellite cells located in the spinal cord and DRG, respectively [39, 40]. Our studies demonstrate through Western Blot technique an increase in optical density of the bands marked with astrocytes and satellite cells (GFAP) in spinal cord and DRG, respectively, in animals with chronic constriction sciatic nerve (CCI). Animals with CCI and treated with NM, we observed a decrease in GFAP-IR in both tissue analyzed and a reversal of hyperalgesia and allodynia. Our findings corroborate Garrison et al. 1991 showed an increased density of these cells, specifically astrocytes, in the spinal cord after induction of ligatures in the sciatic nerve . Nerve injury activates glial cells, which produce and release inflammatory mediators that act on glial cells and neurons, sensitizing the dorsal horn and facilitating pain transmission [39, 42]. Here we demonstrated an increase of NGR-IR and GFAP-IR in DRG neurons after CCI-injury, which was decreased after NM treatment. This suggests an involvement of NGF and glial cells in our experimental model, which remains to be fully understood. Our observations were generally consistent with the literature regarding the time course and localization of astrocytes and satellite cells activation . Nerve injury and inflammation have been shown to lead to proliferation and hypertrophy of satellite cells, and to upregulate other molecules, including neurotrophins, tumor necrosis factor α, and functional gap junctions [17, 43].
Glial cells appear to be a pivotal component of the neuroinflammatory process, and it is known that this type of cell, in the spinal cord, reacts to peripheral nerve injury and inflammation by activation, proliferation, and by releasing messengers that contribute to pathological pain. Glial cells participate in synaptogenesis and maintenance of synaptic activity and expression of receptors, transporters, ionic channels, at least through the release of neurotrophins such as NGF [44–46].
In summary, our data reveal a reversal of the NGF and GFAP increase in DRG by NM treatment in CCI animals. On the other hand, we observed the involvement of GFAP only in spinal cord. We also demonstrated that NM sessions are able to improve behavioral responses, providing evidence of the importance of NM in restoring the changes caused by CCI injury, which contribute to neuropathic pain behavior. These findings suggest that NM reverses the nociceptive behavior of neuropathic animals, and suggest the involvement of glial cells and NGF in such an effect.