mechanical peripheral neuropathies are the consequence of local or extrinsic compression phenomena or impingement by an anatomic neighbor causing a localized entrapment .
traumatic neuropathies are the result of either closed injuries or open injuries to peripheral nerves [ 1 , 2 ] .
both of these categories are characterized by an important inflammatory component that plays a central role in the pathogenesis of neuropathic pain .
inflammatory cells ( e.g. , macrophages ) , the production of molecules that mediate inflammation ( cytokines ) , and the production of nervous growth factors are involved [ 3 , 4 ] . in animal models
it has been demonstrated that peripheral nerve injuries induce a profound local inflammatory response that involves t cells and macrophages . in particular , in the neuropathic pain model induced by chronic constriction injury ( cci ) an important macrophage infiltrate has been described in the damaged sciatic nerve [ 57 ] and in the dorsal root ganglia [ 8 , 9 ] .
the inflammatory response paralleled with nervous tissue alterations and pain [ 7 , 10 ] .  

n - palmitoylethanolamine ( pea ) , the endogenous amide between palmitic acid and ethanolamine , belongs to the family of fatty acid ethanolamides ( faes ) , a class of lipid mediators .
pea exerts antinociceptive effects in several animal models [ 11 , 12 ] , prevents neurotoxicity and neurodegeneration [ 13 , 14 ] , and inhibits peripheral inflammation and mast cell degranulation .
anti - inflammatory effects of pea have been associated with peroxisome proliferator - activated receptor-(ppar-) activation , a nuclear receptor fundamental in the control of inflammatory responses , and expressed in various cells of the immune system [ 19 , 20 ] .
pea does not elicit anti - inflammatory effects in mutant ppar- null mice ( ppar- ) .
indeed , when assessed in either the carrageen hindpaw or phorbol ester ear pinna tests , pea reduced inflammation in wild - type , but not in ppar- , mice .
on the other hand , loverme and collegues   demonstrated the pivotal role of ppar- in the pea pharmacodynamic mechanism to relieve pain . in a mouse peripheral neuropathy model ( cci )
we evaluated the effects of repeated pea treatments on the sciatic nerve lesions responsible for neuropathic pain .
aimed to highlight the role of ppar- in pea - evoked neurorestoration during neuropathy , a morphological study has been performed in both wild - type and ppar- null mice .
all procedures met the european guidelines for the care and use of laboratory animals ( 86/609/ecc and 2010/63/ue ) , and those of the italian ministry of health ( dl 116/92 ) .
male wild - type ( wt ) and ppar- ( ko ) ( b6.129s4-svjae - pparatm1gonz ) mice , previously backcrossed to c57bl6 mice for 10 generations , were bred in our animal facility , where a colony was established and maintained by heterozygous crossing .
mice were genotyped as described on the supplier webpage ( http://jaxmice.jax.org/ ) , with minor modifications .
dna was extracted from tails using the redextract kit ( sigma - aldrich , milan , italy ) .
all animals were maintained on a 12  h light/12  h dark cycle with free access to water and standard laboratory chow .
pea was from tocris ( bristol , uk ) ; it was dissolved in peg and tween 80 2  :  1 ( sigma - aldrich ) and kept overnight under gentle agitation with a microstirring bar . before injection ,
sterile saline was added so that the final concentrations of peg and tween 80 were 20 and 10% v / v , respectively .
drug was injected subcutaneously ( s.c . ) in a dose of 30  mg  kg0.3  ml mouse  for consecutively 14 days from the day after surgery .   the sciatic nerve of 5 - 6-week - old wt and ko mice were surgically ligated as described .
in brief , the animals were anesthetized with ketamine ( 100  mg  kg i.p ) and xylazine ( 10  mg  kg i.p . ) , the left thigh was shaved and scrubbed with betadine , and a small incision ( 2  cm in length ) was made in the middle of the left thigh to expose the sciatic nerve .
the nerve was loosely ligated at two distinct sites ( spaced at a 2  mm interval ) around the entire diameter of the nerve using silk sutures ( 7 - 0 ) . behavioral tests were performed on the day 14 after surgery .
we measured mechanical hyperalgesia using a randall - selitto analgesimeter for mouse ( ugo basile , varese , italy ) .
latencies of paw withdrawal to a calibrated pressure were assessed on ipsilateral ( ligated ) paws on day 14 following ligatures .
cut - off force was set at 60  g. to assess for changes in sensation or in the development of mechanical allodynia , sensitivity to tactile stimulation was measured using the dynamic plantar aesthesiometer ( dpa , ugo basile , italy ) .
animals were placed in a chamber with a mesh metal floor covered by a plastic dome that enabled the animal to walk freely but not to jump .
the mechanical stimulus was then delivered in the mid - plantar skin of the hind paw .
the cutoff was fixed at 5  g. each paw was tested twice per session .
this test did not require any special pretraining , just an acclimation period to the environment and testing procedure .
cutoff force was set at 5  g. after the algesic test , animals were sacrificed and the ipsilateral sciatic nerves , 1 cm proximal and distal to the ligation , were explanted ; the portion containing the ligature was eliminated .
spinal cord was removed , and lumbar section was immediately frozen in liquid nitrogen .  
the tissue samples were osmicated in 1% solution of osmium tetroxide for 2  h under constant agitation . before and after osmication
, the tissue was repeatedly rinsed in 0.1  m sodium cacodylate at ph 7.4 .
after gradual dehydration in ethanol , the osmicated nerve samples were embedded in paraffin ( diapath , milan , italy ) .
transverse 5  m sections were cut on a reichert microtome ( leica , rijswijk , the netherlands ) , mounted with canada balsam , and observed under a light microscopy .
after the sacrifice , the sciatic nerve was fixed in situ using 4% formalin in phosphate buffer ( ph 7.4 ) , nerves were fixed in 4% buffered neutral formalin solution , and then the nerve was embedded in paraffin . finally 5  m sections were stained with azan - mallory for light microscopy studies and were graded for oedema and infiltrate .
the sections were semiquantified by an arbitrary scale starting from 1 , mild infiltrate and oedema , up to 10 , severe infiltrate and widespread oedema .
the procedure was carried out by an independent researcher who was masked to the experiment .
morphometry was performed on cross sections of osmium - fixed sciatic nerves 10  mm starting from the level of injury or at the corresponding level in uninjured control nerves .
the 10  mm cross section corresponded to a level distal or proximal to the injury . counts and measurements were carried out using imagej analysis software .
the first step of morphometric analysis of the sciatic nerve consisted of identifying and capturing the entire fascicle image , followed by measurement of the fascicle perimeter and area by contouring its internal epineural ( magnification : objective 20x ) .
the next step consisted of capturing sequential inner areas of the fascicle ( magnification : objective 100x ) .
the high - magnification micrographs were randomly selected in nonoverlapping areas to cover 5075% of the total cross - sectional area of the nerve .
the random selected histological images were converted into binary ( black and white ) images and cleaned of any blood vessels , degenerated nerve fibers , and artifacts , and the following parameters were then measured : total number and density of nerve fibers , axon diameter , and myelin sheath area .
the number of small fibers , defined as fibers < 4  m , and large fibers , defined as fibers  4  m , was calculated , and the myelin thickness was determined from the difference in perimeter between the fiber and the axon .
the procedure was carried out by an independent researcher who was masked to the experiment .
sections of 5  m were deparaffinized , dehydrated , and submitted to antigen retrieval .
sections were then blocked by incubation with 3% normal goat serum for 20  min .
the slides were then incubated with the primary antibodies for 18  h at 4c .
after washing in tbs , the sections were treated with the secondary antibody conjugated to alexa fluor 488 ( 1  :  1000 , invitrogen , milan , usa ) for 1  h at room temperature .
nuclei were counterstained with dapi . quantitative analysis of cd86 positive cells was performed collecting at least three independent fields through a 20  0.5  na objective , and positive cells were counted using the  cell counter 
the lumbar portion of the spinal was homogenized in lysis buffer containing 50  mm tris - hcl ph 8.0 , 150  mm nacl , 1  mm edta , 0.5% triton x-100 , complete protease inhibitor ( roche ) , and the homogenate was incubated on ice for 30 minutes .
then , the suspension was sonicated on ice using three 10-second bursts at high intensity with a 10-second cooling period between each burst .
after centrifugation ( 13000 g for 15 minutes at 4c ) aliquots containing 20  g total protein underwent to western blot analysis using a mouse anti - cox2 antiserum ( 1  :  1000 ; cell signalling , usa ) .
densitometric analysis was performed using the  imagej  analysis software , and results were normalized to -actin immunoreactivity ( 1  :  1000 rabbit antiserum , santacruz biotechnology ) as internal control .   for behavioral experiments ,
results were expressed as the mean  sem , and analyses were conducted using statistica ( statsoft , tulsa , ok , usa ) .  
histological , morphometric , and immunohistochemical analyses were performed on 5 mice per group , evaluating 6 sciatic nerve sections for each animal .
comparison , were carried out using mann - whitney nonparametric tests . in all cases ,
slides from control and experimental groups were labeled with numbers so that the person performing the image analysis was blinded as to the experimental group .
in addition , all images were captured and analyzed by an investigator other than the one who performed measures to avoid possible bias .
western blot analysis were performed on 5 mice per group ; comparisons were carried out using bonferroni posttest .
pea , ( 30  mg  kg ) s.c . daily administered starting on the day of operation , prevented pain threshold alterations elicited by cci ( figure 1 ) .
in wild - type animals 14 days after injury , pea reduced the hypersensitivity to a mechanical noxious stimulus ( randall - selitto test ; figure 1(a ) ) as well as the hypersensitivity to a nonnoxious mechanical stimulus ( figure 1(b ) ) .
pea efficacy against cci - evoked pain was lacked in ppar- knock - out ( / ) mice ( figures 1(a ) and 1(b ) ) .   on the 14th day after injury , a morphological evaluation of the sciatic nerves
5  m sections of paraffin - embedded nerve were stained by the azan - mallory procedure revealing that cci was able to induce a stronger alteration in the distal portion of the ipsilateral nerve .
the histological analysis highlighted an extensive demyelination , myelin abnormalities like characteristic aggregate , generally termed 
pathognomonic of myelin degeneration ( figure 7 , arrows ) , and an axonal damage . as shown in figure 2 the number of fibers
was significantly reduced , particularly in the distal part of the nerve of both wild - type ( + /+ ; graph ( a ) ) and ppar- null ( / ; graph ( b ) ) animals .  
morphometric evaluation of osmium - fixed tissues allowed the characterization of the alteration of the myelin sheet thickness and axonal diameter , as well as the discrimination of large against small fibers ( stratified by diameter in > 6  m for large and < 6  m for small ) .
cci was able to decrease the myelin thickness of large and small fibers in the distal portion of the ipsilateral nerve in respect to the sham both in wild - type and in knock - out mice ( figures 3(a ) and 3(b ) , large fibers ; figures 3(c ) and 3(d ) , small fibers ) .   in regard to axon diameter ,
a time - dependent decrease was revealed for all the fibers , particularly the small type , both in the distal and in the proximal portions of the ipsilateral nerve ; morphometry revealed a similar profile in ppar- ( figures 4(a ) and 4(c ) ) and ppar- ( figures 4(b ) and 4(d ) ) animals . in wild - type mice repeated pea administrations , 30  mg kg for 14 days , were able to preserve the nerve morphology .
nerve sections of pea - treated mice showed a higher number of fibers in respect to the saline - treated groups ( figure 2(a ) ) ; the myelin thickness in the distal portions of the nerve was decreased to a lesser extent ( figures 3(a ) and 3(c ) ) ; the axon diameter was protected in the pea group both in the proximal and in the distal nerve parts , even in the small fibers ( figures 4(a ) and 4(c ) ) .
pea was completely ineffective in ppar- null mice in preventing sciatic nerve alterations evaluated as number of fibers ( figure 2(b ) ) , myelin thickness ( figures 3(b ) and 4(d ) ) and axon diameters ( figures 4(b ) and 4(d ) ) .
figure 5 shows the infiltrate evaluation 14 days after ligation : inflammatory cells were present in the proximal and , at higher level , in the distal parts of both ppar- ( graph ( a ) ) and ppar- ( graph ( b ) ) mice .
30  mg  kgpea significantly prevented the cellular infiltrate number in wild - type ( figure 5(a ) ) but not in ko animals ( figure 5(b ) ) .  
moreover , both osmium fixed and azan - mallory - stained sections ( figure 7 ) allowed the observation of a massive presence of oedema among the fibers of cci animals .  
figure 6 show the quantitative oedema evaluation 14 days after operation : the alteration was more evident in the distal portion than in the proximal one without revealable differences due to knock down ppar- gene .
pea ( 30  mg  kg s.c . for 14 days ) was able to prevent the oedema induction of about 50% in cci wild - type mice ( figure 6(a ) ) .
no oedema protective effects were observable in pea - treated ppar- animals in respect to vehicle ( figure 6(b ) ) .
the immune inflammatory cells evaluated were diffusely distributed throughout the nerve tissue in all samples of the cci mice , whereas a mild cd86 positive reaction was detectable in cci mice administered with pea as well as in sham - operated animals .
ppar- mice showed a persistence of macrophage infiltrate also in the nerve of pea - treated animals ( figures 8 and 9 ) .  
cci - dependent inflammatory response in the central nervous system was evaluated measuring cox2 levels . as shown in figure 10 , in wild - type mice pea
pharmacological treatment of peripheral neuropathy is actually restricted to symptomatic drugs that are only partially able to control pain perception .
antidepressants , antincovulsants , and opioids can not intervene in nervous tissue alterations that act as a base of neuropathic pain .
the present results describe the direct protective effect of repeated pea treatment on lesioned peripheral nerves .  
cci induces morphometric alterations of the sciatic nerve that dramatically affect the portion distal from the injury and that are also able to induce severe proximal impairment . at the same dose active in pain relief
, pea prevents the reduction of myelin sheet thickness and axonal diameter and improves a characteristic degeneration of myelin as highlighted by azan - mallory staining . according to previous results [ 7 , 24 , 25 ] cci - mediated nerve architecture derangement
is accompanied by a profound local inflammatory reaction which includes oedema , infiltration of hematogenous immune cells , and induction of various soluble factors like cytokines and chemokines . in particular ,
cd86 is a phenotypic marker of the  classically activated  m1 macrophages stimulated by proinflammatory cytokines , as ifn , or by lipopolysaccharide and typically recruited after nervous system trauma .
m1 macrophages produce high levels of oxidative metabolites ( e.g. , nitric oxide and superoxide ) and proinflammatory cytokines that are essential for host defense and tumor cell killing but that also cause collateral tissue damage . the treatment with pea attenuates the degree of peripheral inflammation , reducing oedema and macrophage infiltration allowing for hypothesizing a synergism between the anti - inflammatory and the neuroprotective mechanisms of pea . on the other hand , an inflammation control mediated by pea
induces a cox-2 increase in locations of the central nervous system consistent with the neuroanatomical substrates of spinal nociception .
cox-2 produces prostaglandins that contribute to the development and maintenance of spinal hyperexcitability after peripheral nerve injury [ 28 , 29 ] . reducing cox-2 levels
, pea seems able to intervene also in these central mechanisms of pain chronicization . to note that a direct inhibition of pro - inflammatory cytokines , using a tnf- antibody , for instance , attenuates pain - related behavior but has no effect on nerve regeneration .
pea is a naturally occurring amide between palmitic acid and ethanolamine it is a lipid messenger known to mimic several endocannabinoid - driven actions even though pea does not bind cb1 , cb2 , and abn - cbd receptors .
so far , numerous actions of pea on immune cells such as inhibition of mast cell degranulation , attenuation of leukocyte extravasation , and modulation of cytokine release from macrophages have been described [ 16 , 32 ] .
anti - inflammatory effects of pea have been associated with peroxisome proliferator - activated receptor ( ppar)-alpha activation .
ppar- , well known for its role in lipid metabolism , controls transcriptional programs involved in the development of inflammation through mechanisms that include direct interactions with the proinflammatory transcription factors , nf-b and ap1 , and modulation of ikb function .
pharmacological studies have demonstrated that ppar- agonists are therapeutically effective in rodent models of inflammatory and autoimmune diseases .
our results show that in a neuropathic pain model the ppar- genetic ablation determines a loss of pea effectiveness in reducing oedema prevention and cd-86 positive infiltrating cells .
on the other hand , the recruitment of reactive inflammatory cells and subsequent proinflammatory cascades offers a prime target for neuroprotective agents .
agonists of ppar- such as fenofibrate and wy-14643 protect against cerebral injury by antioxidant and anti - inflammatory mechanisms and reduce the incidence of stroke in mice [ 35 , 36 ] . using a spinal cord injury model , genovese et al .   demonstrated that dexamethasone utilizes ppar- to reduce inflammation and tissue injury in a rat model of spinal cord trauma . on the contrary ,
the ppar- agonist gemfibrozil does not promote tissue preservation and behavioral recovery after spinal contusion injury in mice .
our study shows the obligatory role of ppar- for the neuroprotective effect of pea in peripheral neuropathy . in the sciatic nerve of cci mice
pea exerts a widespread protective effect on both myelin , and axons throughout a ppar--mediated mechanism , since pea treatment fails to rescue nerve tissue in ppar- knock - out animals .
the neuroprotective pea properties were suggested by skaper et al .   since dose dependently pea protected cerebellar granule cells from glutamate toxicity in neuronal single cell cultures and prevented histamine - induced cell death in hippocampal cultures . these effects were elicited without involvement of cb receptors .
more recently koch et al .   described the protective effect of pea on dentate gyrus granule cells in excitotoxically lesioned organotypic hippocampal slice cultures ; the specific ppar- antagonist gw6471 blocked these effects .
pea exerts neuroprotective activities in neurodegenerative diseases . in a mouse model of alzheimer 's and parkinson 's diseases ,
pea reduced oxidative and apoptotic damages and improve behavioral dysfunctions by a ppar--mediated mechanism [ 14 , 41 ] . in a cellular model ,
pea was able to blunt -amyloid - induced astrocyte activation and , subsequently , to improve neuronal survival through selective ppar- activation .  
in neuropathic conditions ppar- seems to join the antihyperalgesic , anti - inflammatory , and neuroprotective effects of pea . on the other hand
the inflammatory response to a damage is crucial for both pain sensation and tissue alterations ; the importance of inflammatory mediators is well demonstrated in the pathogenesis of neuropathic pain , where infiltrating macrophages and schwann cells may be involved in the modulation of these mediators in response to nerve injury .
starting from the relevance of the ppar- in pea antineuropathic properties , the misunderstood role of peroxisome is intriguing .
peroxisomes fulfill multiple tasks in metabolism and adapt contents and functions according to cell type , age , and organism . among the metabolic reactions that take place in peroxisomes , oxygen metabolism , -oxidation of a number of carboxylates that can not be handled by mitochondria , -oxidation of 3-methyl - branched chain and 2-hydroxy fatty acids , ether lipid synthesis , and detoxification of glyoxylate
patients with peroxisomal dysfunction present with severe and diverse neurological anomalies , including neuronal migration defects , dysmyelination and inflammatory demyelination , macrophage infiltration , and axon damage , proving that these organelles are indispensible for the normal development and maintenance of the nervous system [ 4547 ] .
thereafter , the peroxisome stimulation could be a broad spectrum approach to prevent nervous tissue damage , and a pea , ppar--mediated , increase in peroxisome number and/or functionality could be also hypothesized .
a preclinical study   showed that pea - mediated reduction of spinal cord damage was paralleled by an induction of ppar- expression and an up - regulation of potential ppar- target genes , but a clear relationship between ppar- activation and peroxisome boosting is still lacking .
the present results demonstrate the neuroprotective properties of pea in a preclinical model of neuropathic pain .
antihyperalgesic and neuroprotective properties are related to the anti - inflammatory effect of pea and its ability to prevent macrophage infiltration in the nerve .
 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  kg1 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- 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--mediated mechanism , can directly intervene in the nervous tissue alterations responsible for pain , starting to prevent macrophage infiltration . 
1. Introduction
2. Materials and Methods
3. Results 
4. Discussion 
5. Conclusions 