Damage to peripheral nerves often produces a condition of neuropathic pain, characterized by an increase in painful sensitivity, such as hyperalgesia and allodynia. Furthermore, damage to the nerves produces immunological and neuronal changes and also in the expression of genes and proteins even at the level of the spinal cord [
26]. Moreover, the compression of the peripheral nerves is often also associated with the loss of motor function, mainly due to an insufficient regeneration of the nerve. The therapeutic approach to this type of pathology and in particular to neuropathic pain, is a clinical problem [
27]. In this study, we used a sciatic nerve crush model, one of the most used models for studying cellular and molecular mechanisms in the peripheral nerve [
28], to study the effects of PEA-OXA on pain inhibition and pathological processes after crush to the sciatic nerve. PEA-OXA is the oxazoline (2-pentadecyl-2-oxazoline) of palmitoylethanolamide (PEA), a fatty acid amide belonging to the family of
N-acylethanolamines (NAEs). Numerous studies show that PEA is an important endogenous mediator in controlling the inflammatory and analgesic phenomena [
8,
29] with neuroprotective effect [
6]. In particular, a recent clinical study has highlighted the role of PEA as a valid therapeutic strategy for the treatment of nerve compression pain: such as the treatment of sciatic nerve pain and carpal tunnel syndrome; in addition, clinical trials have shown that no adverse effects are reported for PEA, making PEA a valid and safe alternative to opioids and co-analgesics in the treatment of neuropathic pain [
9]. Recently, it has been seen that the pharmacological inhibition of NAAA produces a marked analgesic and anti-inflammatory effect [
30]; therefore, treatment with PEA-OXA combines the known effects of PEA with those of NAAA inhibition, which has among other effects, the increase of endogenous endocannabinoid levels [
25]. Therefore, this particular mechanism of action enhances the already known therapeutic properties of PEA, and in fact, a recent study has shown that compared to PEA the same dose of PEA-OXA produces a significant improvement in analgesic and anti-inflammatory action [
25]. So, in this study, the histological analysis of the sciatic nerve showed that 14 days after the sciatic nerve crush, there is a significant presence of edema, infiltration, and degradation of the myelin layer. Instead, compared with vehicle, the PEA-OXA treatment at dose of 10 mg\kg daily for 14 days following the sciatic nerve crush has had a beneficial effect. In fact, the histological analysis shows a significant reduction of edema and infiltration and therefore a beneficial effect in axonal regeneration. As an indicator of an efficient reparative process, we evaluated the expression of β-III-tubulin both at the spinal level and in the sciatic nerve [
31,
32], our results showed a significant reduction in the expression of β-III-tubulin both at the spinal level and in the sciatic nerve, following the sciatic nerve crush. When compared to the vehicle group, daily treatment with PEA-OXA at a dose of 10 mg\kg has significantly increased the expression of β-III-tubulin both at the spinal level and the damaged sciatic nerve. Subsequently, we assessed the presence of mast cells following the damage and if the treatment with PEA-OXA was able to have a protective effect. In fact, numerous evidence suggest that mast cells play a key role in the development of the inflammatory process and in the generation of neuropathic pain following the peripheral nerve injury [
33]. It has been seen that mast cells accumulate near the area of the injured nerve [
34], where they are responsible for the release of various proinflammatory mediators such as TNF-α and IL-1β [
35], and these factors are also responsible for the recruitment of leukocytes. In addition, the activated mast cells are responsible for the release of substances that sensitize the nociceptors and contribute to hyperalgesia among these in particular histamine [
33]. In fact, blockers of histamine receptors alleviate or they inhibit neuropathic pain [
36]. Our results show an increase in mast cells 14 days after sciatic nerve crush, whereas daily treatment with PEA-OXA at a dose of 10 mg\kg significantly reduced the number of mast cells in the injured nerve. In fact, we have found that the sciatic nerve crush produces a significant diminution of the nociceptive threshold to mechanical and thermal stimuli. Daily treatment for 14 days with PEA-OXA at a dose of 10 mg\kg was able to significantly increase the pain threshold to mechanical and thermal stimuli. Another way to monitor the activation of the nociceptive pathway is by measuring the levels of c-fos [
37], and in fact, c-fos is recognized as a marker of activity of neurons excited by algesic stimuli and has been seen to increase chased to damage to the peripheral nerves together with the mechanic allodynia and thermal hyperalgesia [
38]. In fact, at the level of the lumbar portion of the spinal cord, we found a significant increase in the expression of c-fos, 14 days after the sciatic nerve crush, while treatment with PEA-OXA significantly reduced the expression of c-fos, consistently with the decrease of the mechanical allodynia and thermal hyperalgesia that we observed. In addition, the mechanism of the analgesic effect of PEA-OXA involves the combination of the already known analgesic effects of PEA with those of the inhibition of the main enzyme responsible for its degeneration NAAA [
25], it has also been seen that the inhibition of NAAA produces an analgesic effect [
39]. In fact, we found at the spinal level a significant increase in the expression of NAAA following sciatic nerve crush, whereas the daily treatment for 14 days with PEA-OXA at a dose of 10 mg\kg significantly reduced the expression of NAAA compared with the vehicle group. We subsequently assessed whether sciatic nerve damage could produce changes in protein and gene expression even at the spinal level. In particular, by western blot analysis of the lumbar portion of the spinal cord, we first evaluated the involvement of the inflammatory process, nerve damage has been shown to produce an important inflammatory response, and inhibition of this process is a strategy for the treatment of neuropathic pain [
40]. Our results showed that 14 days after sciatic nerve crush, at the spinal cord level of the mice from the vehicle group, there was a significant increase in Ikb-α degradation and therefore a consequent increase in Nf-κB translocation, and the activation of the Nf-κb pathway is a pivotal event in a whole series of changes, as in gene expression, and in the increase of the expression of different proteins (such as cytokines and chemokines) neurotransmitters and therefore in painful hypersensitivity [
41]. Ours results showed a significant increase in Ikb-α degradation and consequent Nf-kB translocation into the nucleus, 14 days after sciatic nerve crush, whereas PEA-OXA treatment exerts a significantly protective effect; furthermore, at the lumbar spinal level, from the vehicle group, we found a significant increase in the expression of the proinflammatory cytokines TNF-α and IL-β while PEA-OXA treatment daily for 14 days following the sciatic nerve crush significantly reduces the expression of proinflammatory cytokines TNF-α and IL-1β. A typical sign of damage to the central nervous system is the increase in reactive astrocytes and of microglia as indicated by an increase in GFAP and Iba-1 expression [
42,
43]. In addition, several evidence show how microglia and astrocyte are activated, at the spinal level, in the neuropathic pain state [
44,
45]. Our results show that both at the damaged nerve level and at the spinal cord level, there is a significant increase in the expression of GFAP and Iba-1, in the vehicle group compared to sham mice. The treatment with PEA-OXA has significantly reduced, compared to the vehicle group, the expression of GFAP and Iba-1 both at the level of the sciatic nerve and at the spinal level. After the damage to the peripheral nerves following different events such as the inflammatory response that we have observed, the Wallerian degeneration and neuronal apoptosis, in particular the neuronal apoptosis, plays a key role as an excessive activation of this process can compromise the efficiency of the reparative processes [
46]. Therefore, we evaluated the expression of caspase-3 as a marker of the process of programmed cell death (apoptosis) [
47]. At the spinal level, we found that 14 days after sciatic nerve crush there was a significant increase in caspase-3 expression, whereas daily PEA-OXA treatment significantly reduced caspase-3 expression. Furthermore, to indicate an increase in the apoptotic process we found in the vehicle group an increase in the ratio between Bax and Bcl-2, two factors involved in the regulation of this process [
48]; also in this case, the PEA-OXA treatment had a protective effect. Moreover, through TUNEL staining, we evaluated the apoptotic process in the sciatic nerve tissue. Fourteen days after sciatic nerve crush, the vehicle group showed a significant increase in the intensity of TUNEL staining. Instead, when compared to the vehicle, treatment with PEA\OXA 10 mg\kg significantly reduced the number of apoptotic cells. In opposition to the apoptotic process, there are the reparative processes of neuronal growth, and in these type of processes, a key role is played by the growth factors like the nerve growth factor (NGF) [
49]. Therefore, consistent with what was observed for the apoptotic process, at the spinal level, we found a significant reduction in NGF expression 14 days after the sciatic nerve crush, while daily treatment with PEA-OXA significantly increased the expression of NGF when compared to the vehicle group. This is a key step in neuroprotective action of the PEA-OXA. Indeed, NGF plays a fundamental role in the function and survival of neurons in the peripheral nervous system [
50]. Moreover, it has been seen that a deficit in NGF is often associated with various neurodegenerative diseases [
51], and in particular, neurodegenerative processes together with neuroinflammation play a key role in the pathogenesis and presence of neuropathic pain [
52]. Finally, we evaluated the events described so far from a macroscopic point of view, and in fact, we evaluated the recovery of motor functions, by evaluating the ability of mice to cross a wooden beam (beam walking test) and the sciatic functional index (SFI). The calculation of SFI is often used to evaluate Functional recovery following sciatic nerve injury [
53]. The SFI is scaled such that − 100 represents a complete nerve injury and 0 represents normal function. Our results show how when compared to the vehicle, treatment with PEA-OXA significantly improves functional recovery following sciatic nerve injury from both 7 and 14 days. We observed both 7 and 14 days after the sciatic nerve crush that the mice of the vehicle group took a significantly longer time to cross the beam and also with greater difficulty as indicated by a greater number of foot fall from the beam. Instead, treatment with daily PEA-OXA at a dose of 10 mg\kg produced a significant improvement in locomotor ability.