endotoxin - induced uveitis ( eiu ) is an animal model of acute ocular inflammation .
usually , this experimental pathology lasts up to 72 hours and has similarities with the human pathology .
cytokines , chemokines , nitric oxide ( no ) , and impairment of stress - sensitive enzymes such as heme oxygenase-1 increase the inflammatory response to eiu .
augmentation of local recruitment of pmn promotes the inflammation and cell injury of the ciliary bodies in this pathology .
thus , prospectively pharmacological agents that promote endogenous defensive responses , reduce the burden of inflammatory mediators released within the eye structures or reinforce the resolution of the inflammation that may exert cytoprotection and good outcome for uveitis .  
a growing body of evidence indicates that a good resolution of the inflammation could be achieved by means of resolvins .
these are a family of potent lipid mediators derived that promote the resolution of the inflammatory response back to a noninflamed state .
resolvin d1 ( rvd1 ) is the major actor of the resolvins family ; it is produced physiologically from the sequential oxygenation of dha by 15- and 5-lipoxygenase   and it has effects on important components of the inflammation .
resolvin d1 reduces human polymorphonuclear leukocyte ( pmn ) transendothelial migration , the earliest event in acute inflammation , and exhibits a dose - dependent reduction in leukocyte infiltration in a murine model of peritonitis with a maximal inhibition of ~35% at a 10100  ng dose .
rvd1 also acts as a scavenger of cytokines and chemokines from the inflamed site and inhibits the production of pmn - derived free radicals [ 58 ] .
other biological actions have been reported , with a therapeutical potential such as a reduction in inflammatory pain .   on this base , we have investigated whether the stimulation of the resolutive phase of the inflammation through the use of the resolvin d1 ameliorates the immunoinflammatory profile of the rat eye following experimental uveitis .
male sprague - dawley rats ( 180220  g ) were injected in one footpad with 200  g of lipopolysaccharide ( lps , salmonella minnesota , sigma , st louis , mo , usa ) in 0.1  ml of sterile pyrogen - free saline   for the induction of eiu .
the rats were treated with vehicle , lps or lps + resolvin d1 ( rvd1 ) ( n = 6 for each ) and killed 24  h after treatment .
the doses of rvd1 ( 101001000  ng / kg ) were chosen in the range of those used in murine models of inflammation ; they were injected by intravenous bolus ( in 200  l of sterile saline ) via the tail vein 1  h following lps injection .
clinical manifestations of eiu were graded from 0 to 4 in a blinded fashion according to the previously reported scoring system [ 10 , 11 ] : 0 = no inflammatory reaction ; 1 = discrete dilation of iris and conjunctival vessels ; 2 = moderate dilation of iris and conjunctival vessels with moderate flare in the anterior chamber ; 3 = intense iridal hyperemia with intense flare in the anterior chamber ; and 4 = same clinical signs as 3 with presence of fibrinoid exudation in the pupillary area and miosis .
no signs of uveitis were observed in the animals at the beginning of each experiment .
eiu clinical data shown were representative of 3 sets of experiments and presented as mean  sem of 6 observations . immediately after the biomicroscope examination , the animals were killed with an overdose of anesthesia .
aqueous humor ( aqh ) was collected immediately from both eyes by an anterior chamber puncture ( 3040  l / rat ) , using a 30-gauge needle under a surgical microscope and stored in ice water until testing .
one unit of mpo activity has been reported to be equivalent to approximately 2  10 pmn [ 12 , 13 ] .
after 24  h of eiu , the eyes were harvested and cut in two halves .
one half of each eye was immediately fixed by immersion in 10% buffered formalin and paraffin - embedded for immunohistochemistry .
sections were serially cut at 5  m , placed on lysine - coated slides , and stained with hematoxylin and eosin and with the trichrome method .
the other half of each eye was immediately frozen in liquid nitrogen and stored at 80c for the later biochemical assays described below .
subsequently the frozen tissues were homogenized in a solution containing 0.5% hexa - decyl - trimethyl - ammonium bromide dissolved in 10  mm potassium phosphate buffer ( ph 7 ) and centrifuged for 30  min at 4,000  g at 4c .
paraffin - embebbed eye samples were treated with an xylene substitute ( hemo - de ; fisher scientific ) in order to remove the paraffin , and tissue sections were rehydrated with ethanol gradient washes .
tissue sections were quenched sequentially in 3% hydrogen peroxide aqueous solution and blocked with pbs 6% nonfat dry milk ( biorad , milan , italy ) for 1  h at room temperature .
sections were then incubated with specific antibodies anti - cd11b , anti - cd4 , and anti - cd8 ( santa cruz biotec , usa ) .
specific labelling was detected with a biotin - conjugated goat anti - rabbit igg and avidin - biotin peroxidase complex ( dba , milan , italy ) .
the specimens were analyzed by an expert pathologist ( intraobserver variability 6% ) blinded to the experimental protocol .
six distinct preparations for each group of animals were done and 20 microscopic fields were analyzed in each preparation at 400x magnification .
tnf - alpha levels in tissue homogenates ( 50  l ) were determined using a commercially available elisa specific for the rats cytokine , purchased from r&d systems ( abingdon , uk ) .
briefly , tissue supernatant aliquots ( 50  l ) were assayed for tnf - alpha and compared to a standard curve constructed with 0 - 1  ng / ml of the standard cytokine .
the elisa showed negligible ( < 1% ) cross - reactivity with several murine cytokines and chemokines ( data as furnished by manufacturer ) .
a similar procedure was followed for determination of the chemokines cxcl8 and rantes by elisa ( r&d systems , uk ) and used according to the manufacturer instructions .  
all values are expressed as mean  sem of number ( n ) of rats for the in vivo experiments .
statistical analysis was assessed either by student 's t - test ( when only two groups were compared ) or one - way anova followed by dunnett 's test ( more than two experimental groups ) .
a probability p value less than 0.05 was considered significant to reject the null hypothesis .
24  h after the administration of 200  g lps into the footpad of sprague - dawley rats , severe changes of the structure of the eye occurred with a clinical score of 3.90  0.3 attributed ( figure 1 ) .
rvd1 ( 101001000  ng / kg ) dose - dependently attenuated the development of the ocular inflammation caused by lps and improved the clinical score attributed in a dose - dependent manner ( figure 1 ) .
particularly , the insurgence of eiu was effectively reduced ( 22.6  1.4% less ) by the intermediate dose of rvd1 , while the highest dose protected the eye until the 74  3% ( figure 1 ) .
the uveitis clinical scores for these two doses were significantly reduced to 1.02  0.2 ( p < 0.05 ) and 2.98  0.3 ( p < 0.01 ) compared with vehicle - treated group .
concomitantly , a severe inflammatory and immune response rose within the eye of the rats .
the immunohistochemistry performed on the eye revealed tissues were largely oedematous and telangiectatic with an oblong profile of the blood vessels ( figure 2 ) , caused by the development of the oedema that tissues were largely oedematous and telangiectatic with an oblong profile of the blood vessels ( figure 2 ) .
particularly , both the external fibrous ( sclera ) and vascular median tunics ( choroid ) were markedly infiltrated of flogistic elements ( figure 2 ) .
these were composed predominantly of pmn leukocytes cd11b and t - lymphocytes cd4 and cd8 .
in fact , figure 3 showed that rvd1 reduced the number of infiltrated cd4 and cd8 particles within the perivascular tissue . at 24  h , the number of cd4 particles for lps + rvd1 treated animals were significantly lower than those for the lps treated animals ( p < 0.001 ) ( figures 3 and 4 ) .
lps + rvd1 animals also had the lowest number of c8 particles compared to the lps group ( figures 3 and 4 ) .
the immunohistochemistry also showed that rvd1 reduced the infiltration of pmn leukocytes marked within the uvea .
cd11b positive particles were mainly localized inside the blood vessels with no infiltration within the adjacent tissues ; figure 3 shows the effects of rvd1 100  ng / kg .
the actions of rvd1 were evident for the doses of 100 and 1000  ng / kg , while 10  ng / kg gave no significant results in term of reduction of number of inflammatory components within the uvea ( figures 3 and 4 ) .
the development of eiu was paralleled by increase in mpo activity ( figure 5 ) .
treatments of eiu rats with rvd1 1 hour after lps caused a dose - dependent decrease of mpo activity in the aqh .
the decreases significantly started from the dose of 100  ng / kg ( p < 0.05 versus vehicle ) and reached the maximum with the highest dose of 1000  ng / kg ( p < 0.01 versus vehicle ) ( figure 5 ) .
tissue homogenates from the eyes of vehicle - treated rats had slightly appreciable levels of tnf - alpha , cxcl8 , and rantes ( figure 6 ) .
in contrast , tissue homogenates from eiu lps - induced rats showed high levels of the tnf - alpha , cxcl8 , and rantes as 280  20  pg / mg , 560  38  pg / mg , and 373  27  pg / mg , respectively ( figure 6 ) .
resolvin d1 treatment , 1 hour after lps , dose - dependently produced a significant reduction in either the cytokine and the chemokine levels within eye tissues ( figure 6 ) .
here we report that stimulation of resolvin d1 pathway in rats undergoing experimental uveitis ameliorates the immuno - inflammatory profile of the external and median tunics of the eye , accounting for eye protection .  
inflamed eye is the result of the altered functions of endothelial cells , leukocytes , retinal pigment epithelium , retinal neurons , glial cells , and other types of cells locally present .
these cells are targets of signalling molecules such as lipid mediators and cytokines that favour the shifting of the tissue from a physiological shape to pathological one .
targeting one , or more than one , of these mediators with specific agents prevents inflammation or promotes resolution of it .
this latter response is limited at the site of the noxious stimulus , in order to restore the right homeostasis through specialized proresolving mediators with tissue - protective and resolution - stimulating functions .
they are biosynthesized from eicosapentaenoic acid and from docosahexaenoic acid and so are denoted as e series and d series , respectively .
the actions of the resolvins include the scavenging of cytokines and chemokines from the inflamed site , the inhibition of the de novo production of cytokines and chemokines , the inhibition of the leukocytes trafficking / infiltration to inflamed tissue , and the inhibition of the production of pmn - derived free radicals [ 58 ] . however , an action on the recruitment of nonphlogistic monocytes and phagocytosis is also accredited .   from the molecular point of view , the proresolving properties of resolvins are exerted through the share of g - protein - coupled seven - transmembrane receptors located on human leukocytes with the anti - inflammatory peptide annexin 1 and chemerin .
receptors are called alx / fpr2 ( lxa4 receptor ) and gpr32 ( g - protein - coupled receptor ) [ 1518 ] and are aimed to translate the rvd1 signal into leukocytes activation and movement impediment . according to this evidence here we report that the pro - resolving properties of resolvin d1 is exerted on the typical actors leading experimental uveitis , the white blood cells recruited into the eye specimens .
mpo , a sensible marker of leucocytes infiltration , and tissue immunoreactivity for the cd11b were remarkably reduced by the rvd1 .
pmn leukocytes activation and infiltration are the key events of inflamed eye , because pmn leukocytes adhere to , roll along , infiltrate the endothelial wall of blood vessels and release reactive oxygen species to the site attacked causing inflammation .
furthermore , this treatment also reduced the local generation of cytokines and chemokines which are known to promote leukocyte - endothelium interaction   and finally eye damage . indeed , a cytokine able to increase the adhesive properties of the endothelial wall [ 21 , 22 ] , and that it is implicated in the pathology associated with experimental uveitis , the tnf - alpha was drastically reduced following the treatment of the rats with rvd1 as well as the chemokine cxcl8 , a chemokine able to recruit neutrophils in rodent species during experimental inflammation [ 21 , 24 ] . as chemoattractants
, cxcl8 stimulates directional leukocyte migration and activates the expression of integrin on leukocytes such as cd11b , which increases leukocyte binding to the ligands icam-1 and vcam-1 on the endothelium .
taken together , the present results show that the persistent inflammation of the eye and tissue damage following uveitis could be controlled by means of rvd1 .
an intriguing hypothesis would be that the rvd1 , acts through its receptor alx / fpr2 , ubiquitously present within the eye structures .
consistent with this contention are the recent studies by odusanwo et al . , in other settings .  
the resolution of the inflammation seems not be the only target to reach during uveitis ; unfortunately , the immune response has a major responsibility in it and thus needs to be suppressed .
much experience on the immune - mediated damage has arisen from clinical and experimental models of uveitis [ 2729 ] .
however , the interplay between innate and adaptive immunity and the crossover between autoinflammatory and autoimmune conditions need relative exploration . here
we show that although the predominant infiltrating cell type in eiu is the pmn neutrophil , cd4 and cd8 t cells are also partners of this company .
these cells have been found markedly infiltrated within the uveal structures together with pmn leukocytes . over the last two decades , the understanding of immunopathogenetic mechanisms associated with eiu
is increased and supports a fundamental role of t cells in it , especially cd4 t cells .
this is in accordance with a number of studies done through the last two decades supporting the fundamental role for t cells , especially cd4 t cells .
other studies showed that that anti - cd4 antibody significantly reduced the severity of eiu in endotoxin - responsive strains of mice , while an anti - cd8 antibody had no influence on the disease . in 2002 , avunduk et al .   also showed cd4 and cd8 cells infiltration in the anterior uveal tract , paving the way to the later concept that activated lymphocytes can invade vascular endothelium by degrading subendothelial matrix , and so memory t lymphocytes may be the predominant introducers of the extra vascular tissues .
activated t lymphocytes selectively cross the blood - aqueous barrier , accumulate in the uveal tissue of eiu animals , and secrete a number of damaging elements .  
rvd1 quantitatively reduced the number of infiltrated cd4 and cd8 elements within the perivascular uveal tissue .
also , rvd1 associated a reduction of the t - chemokine rantes ( regulated on activation , normal t expressed and secreted ) within the uveal tissues .
rantes , also known as ccl5 , is an 8  kda protein member of the interleukin-8 superfamily of cytokines .
firstly identified after t - cells activation , it was subsequently determined to be a cc chemokine and expressed in more than 100 human diseases .
rantes expression in t lymphocytes is regulated by kruppel like factor 13 ( klf13 ) [ 3537 ] and is chemotactic for t cells , eosinophils , basophils , neutrophils , and macrophages [ 3840 ] , and through its receptor ccr5 plays an active role in recruiting leukocytes into inflammatory sites .
rantes , along with the related chemokines mip-1alpha and mip-1beta , has been identified as a natural factor secreted by activated cd8 t cells and other immune cells . with the help of particular cytokines ( i.e. , il-2 and ifn- ) that are released by t cells
, ccl5 also induces the proliferation and activation of certain natural - killer ( nk ) cells to form chak ( cc - chemokine - activated killer ) cells .
therefore , limiting the infiltration of immune cells into inflamed sites or reducing their products could be mandatory for return to eye homeostasis .   in conclusion
, our study shows that stimulation of the resolving phase of eiu through resolvin d1 pathway ameliorates the immuno - inflammatory profile of the rat eye .
 this study investigated whether the administration of resolvin d1 to rats with endotoxininduced uveitis ( eiu ) ameliorates the immuno - inflammatory profile of the eye . 
 24  h after the administration of 200  g lps into the footpad of sprague - dawley rats , severe changes of the structure of the eye occurred concomitantly with a severe inflammatory and immune response . 
 these latter included strong infiltration of pmn leukocytes cd11b+ t - lymphocytes cd4 + and cd8 + within the eye and a significant release of the cytokines / chemokines tnf - alpha , cxcl8 , and rantes too . 
 bolus of resolvin d1 ( rvd1 ; 101001000  ng / kg in 200  l of sterile saline via the tail vein ) significantly and dose - dependently ( i ) reduced the development of the ocular derangement caused by lps ; ( ii ) reduced the clinical score attributed to eiu ; ( iii ) reduced the protein concentration and myeloperoxidase activity ( mpo ) in aqueous humor ( aqh ) ; and 
 ( iv ) reduced neutrophils , t - lymphocytes , and cytokines within the eye . 
1. Introduction 
2. Materials and Methods
3. Results
4. Discussion