Palmitoylethanolamide is a disease-modifying agent in peripheral neuropathy: pain relief and neuroprotection share a PPAR-alpha-mediated mechanism

Abstract

Neuropathic syndromes which are evoked by lesions to the peripheral or central nervous system are extremely difficult to treat, and available drugs rarely joint an antihyperalgesic with a neurorestorative effect. N-Palmitoylethanolamine (PEA) exerts antinociceptive effects in several animal models and inhibits peripheral inflammation in rodents. Aimed to evaluate the antineuropathic properties of PEA, a damage of the sciatic nerve was induced in mice by chronic constriction injury (CCI) and a subcutaneous daily treatment with 30 mg kg(-1) PEA was performed. On the day 14, PEA prevented pain threshold alterations. Histological studies highlighted that CCI induced oedema and an important infiltrate of CD86 positive cells in the sciatic nerve. Moreover, osmicated preparations revealed a decrease in axon diameter and myelin thickness. Repeated treatments with PEA reduced the presence of oedema and macrophage infiltrate, and a significant higher myelin sheath, axonal diameter, and a number of fibers were observable. In PPAR- α null mice PEA treatment failed to induce pain relief as well as to rescue the peripheral nerve from inflammation and structural derangement. These results strongly suggest that PEA, via a PPAR- α -mediated mechanism, can directly intervene in the nervous tissue alterations responsible for pain, starting to prevent macrophage infiltration.

Figure 1

Chronic treatment effects on pain behaviour. Comparison of PEA-effects in PPAR-α ^+/+ and  ^−/− mice. (a) Response to a mechanical noxious stimulus on the ipsilateral paw 14 days after CCI evaluated by Randall-Selitto test. (b) Response to a mechanical nonnoxious stimulus evaluated by Von Frey test. PEA (30 mg kg⁻¹) was daily s.c. administered for 14 days starting from the day of operation. Each value represents the mean of 2 experiments with 12 mice per group. **P < 0.01 versus vehicle-treated mice.

Figure 2

Morphometry: number of fibers. 5 μm nerve sections of osmium-fixed tissues were analyzed. The distal and the proximal tract of the ipsilateral ligated nerve (CCI) was compared with the contralateral and with the sciatic nerve of sham-operated animals (sham). Effect of repeated PEA administrations (30 mg kg⁻¹ s.c. for 14 days) on the number of nervous fibers in respect to vehicle-treated CCI animals and vehicle-treated sham animals. (a) PPAR-α ^+/+ mice versus (b) knock-out animals. Quantitative analysis was performed evaluating 5 animals for each group. *P < 0.05 was considered as significantly different from sham, vehicle-treated mice. °P < 0.05 was considered as significantly different from CCI, vehicle-treated mice.

Figure 3

Morphometry: myelin thickness. Nerve sections (5 μm) of osmium-fixed tissues were analyzed. The distal and the proximal tract of the ipsilateral ligated nerve (CCI) was compared with the contralateral and with the sciatic nerve of sham-operated animals (sham). Myelin thickness of large and small fibers of PEA-treated (30 mg kg⁻¹ s.c. for 14 days) PPAR-α ^+/+ ((a) and (c)) and PPAR-α ^−/− ((b) and (d)) mice in respect to saline-treated CCI and saline-treated sham animals. Quantitative analysis was performed evaluating 5 animals for each group. *P < 0.05 was considered as significantly different from sham, vehicle-treated mice. °P < 0.05 was considered as significantly different from CCI, vehicle-treated mice.

Figure 4

Morphometry: axon diameters. Nerve sections (5 μm) of osmium-fixed tissues were analyzed. The distal and the proximal tract of the ipsilateral ligated nerve (CCI) was compared with the contralateral and with the sciatic nerve of sham-operated animals (sham). Axon diameters of large and small fibers of PEA-treated (30 mg kg⁻¹ s.c. for 14 days) wild-type ((a) and (c)) and PPAR-α null ((b) and (d)) mice in respect to saline-treated CCI and saline-treated sham animals. Quantitative analysis was performed evaluating 5 animals for each group. *P < 0.05 was considered as significantly different from sham, vehicle-treated mice. °P < 0.05 was considered as significantly different from CCI, vehicle-treated mice.

Figure 5

Infiltrate evaluation. On the 14th day after CCI, 5 μm nerve sections of formalin-fixed tissues were analyzed by Azan-Mallory stain, and the presence of inflammatory infiltrate was evaluated and quantified by an arbitrary scale starting from 1, mild infiltrate, up to 10, severe infiltrate. Effect of PEA repeated treatments (30 mg kg⁻¹ s.c. daily) was observed in (a) PPAR-α ^+/+ and in (b)  ^−/− mice where the distal and the proximal tract of the ipsilateral ligated nerve of CCI was compared with the contralateral and with the sciatic nerve of sham-operated animals. Quantitative analysis was performed evaluating 5 animals for each group. *P < 0.05 was considered as significantly different from sham, vehicle-treated mice. °P < 0.05 was considered as significantly different from CCI, vehicle-treated mice.

Figure 6

Oedema evaluation. On the 14th day after CCI, 5 μm nerve sections of formalin-fixed tissues were analyzed by Azan-Mallory stain and the presence of oedema infiltrate was evaluated and quantified by an arbitrary scale starting from 1, mild oedema, up to 10, widespread oedema. Effect of PEA repeated treatments (30 mg kg⁻¹ s.c. daily) was observed in (a) PPAR-α ^+/+ and in (b)  ^−/− mice where the distal and proximal tract of the ipsilateral ligated nerve of CCI was compared with the contralateral and with the sciatic nerve of sham-operated animals. Quantitative analysis was performed evaluating 5 animals for each group. *P < 0.05 was considered as significantly different from sham, vehicle-treated mice. °P < 0.05 was considered as significantly different from CCI, vehicle-treated mice.

Figure 7

Light micrographs from 5 μm transverse sections of mouse sciatic nerve stained by Azan-Mallory, comparison between PPAR-α ^+/+ and PPAR-α ^−/− animals; 14th day. Sham: section of sciatic nerve from sham animals; CCI + vehicle: distal part of the sciatic nerve of injured vehicle-treated animals; CCI + PEA distal part of the sciatic nerve of CCI mice s.c. treated with 30 mg kg⁻¹ PEA daily for 14 days starting from the surgery. Ovoids are evidenced by arrows. Original magnification 20x.

Figure 8

CD86 positive cells evaluation in sciatic nerve. 14 days after CCI, 5 μm sections of the formalin-fixed distal part of the sciatic nerve underwent immunohistochemical staining for CD86. Effect of PEA repeated treatments (30 mg kg⁻¹ s.c. daily) was evaluated in (a) PPAR-α ^+/+ and in (b) PPAR-α ^−/− mice, and quantitative analysis was performed evaluating 5 animals for each group. *P < 0.05 was considered as significantly different from sham, vehicle-treated mice. °P < 0.05 was considered as significantly different from CCI, vehicle-treated mice.

Figure 9

CD86 positive cells evaluation in sciatic nerve. 14 days after CCI, 5 μm sections of the formalin-fixed distal part of the sciatic nerve underwent immunohistochemical staining for CD86. Effect of PEA repeated treatments (30 mg kg⁻¹ s.c. daily) was evaluated in PPAR-α ^+/+ and in  ^−/− mice, and representative images are showed, and a detailed image is shown in the insert. Original magnification 20x.

Figure 10

COX-2 expression levels in spinal cord. On the 14th day the spinal cord of PPAR-α ^+/+ and PPAR-α ^−/− mice was analyzed by western blot. Upper panel shows the densitometric quantification of COX-2 levels in CCI vehicle-treated animal in comparison with CCI mice repetitively treated with PEA (30 mg kg⁻¹ s.c. daily). Values were normalized to β-actin immunoreactivity. Data are expressed as the mean ± SEM of triplicate determinations performed on 5 animals for each group. **P < 0.01 was considered as significantly different from vehicle (+/+).