the mechanisms involved in the development of diabetic neuropathic pain are poorly understood due to their complexity .
currently , much attention has been given to the role of immune factors in the development of neuropathic pain [ 24 ] , and it seems that the chemokines may play an important role in this process [ 5 , 6 ] .
we have shown that , during streptozotocin - induced diabetic neuropathy , the level of some pronociceptive interleukins and proteins from the tumor necrosis factor family is upregulated , which suggests their role in diabetic neuropathic pain .
. there are four defined classes of chemokines : cc , cxc , cx3c , and xc .
their main function as chemoattractants is to guide the migration of receptor cells to the place of chemokine secretion .
they are second line proinflammatory factors and are induced by primary proinflammatory mediators such as tumor necrosis factor ( tnf ) .
recent studies have shown that chemokines from the cxc subfamily are involved in pain processes .
cxcl1 , cxcl11 , and cxcl13 were upregulated in a snl pain model [ 9 , 10 ] . furthermore ,
spinal application of cxcl1 or cxcl12 elicited pain hypersensitivity [ 9 , 11 ] similar to intraplantar cxcl5 injection .
the chemokine receptors are expressed not only on immune cells but also on microglial cells or astrocytes [ 5 , 6 , 13 ] . the activated microglia cells , resident macrophages of the central nervous system , play an important role in diabetic neuropathy [ 7 , 14 ] .
therefore , it seems that the chemokines may be crucial in the development and maintenance of diabetic neuropathic pain .
the aim of our study was to verify how the expression and protein levels of chemokines from the cxc subfamily vary with diabetic neuropathy .
using qrt - pcr and the raybio antibody array technique , we examined the mrna and protein levels of cxcl1 ( kc ) , cxcl5 ( lix ) , cxcl9 ( mig ) , cxcl11 ( i - tac ) , cxcl12 ( sdf-1 ) , and cxcl13 ( blc ) in lumbar spinal cords of diabetic mice .
we verified also the influence of intrathecal administration of cxcl1 , cxcl5 , cxcl9 , and cxcl12 on nociceptive transmission in nave mice .
male albino - swiss mice ( 3035  g ) were housed in cages lined with sawdust under a standard 12/12  h light / dark cycle ( lights on at 08:00  h ) with food and water available ad libitum .
the experiments were carried out according to the institute 's animal research bioethics committee ( cracow , poland ) , in accordance with iasp rules   and the nih guide for care and use of laboratory animals .
injection of streptozotocin ( stz ; 200  mg / kg ; sigma aldrich , usa ) prepared in 0.9% nacl [ 7 , 16 , 17 ] .
the behavioral tests were conducted on day 7 after stz injection , to reflect changes in the early stage of diabetes development .
von frey filaments of increasing strengths were applied to the plantar surface of the hind paw of each mouse sequentially ( from 0.6 to 6  g ) until the hind paw was withdrawn [ 7 , 18 ] .
hyperalgesia was assessed using the cold plate test ( cold / hot plate analgesia meter , columbus instruments ) as described in our previous papers [ 7 , 19 ] .
the mice were placed on the cold plate ( at a temperature of 2c ) , and the time until the hind paw was lifted was recorded .
the cut - off latency was 30  s. the pain threshold to a thermal stimulus was assessed by tail - flick latency determined by a tail - flick analgesic meter ( analgesia meter ; ugo basile , comerio , italy ) . in this test , the beam of light was focused on the dorsal tail surface 1  cm from the tip of the tail .
the baseline was determined at 34.5  s. the cut - off time was 9  s [ 7 , 20 ] .
the lumbar ( l4l6 ) region of the spinal cord was dissected after decapitation of nave mice and mice after stz - induced neuropathy on day 7 following stz administration .
the rna concentration was measured using a nanodrop nd-1000 spectrometer ( nanodrop technologies ) , and rna quality was determined by chip - based capillary electrophoresis using an rna 6000 nano labchip kit and agilent bioanalyzer 2100 ( agilent ) .
reverse transcription was performed on 2  g of total rna using omniscript reverse transcriptase ( qiagen inc . ) at 37c for 60  min .
rt reactions were carried out in the presence of an rnase inhibitor ( rrnasin , promega ) and oligo ( dt16 ) primer ( qiagen inc . ) .
cdna was diluted 1  :  10 with h2o , and , for each reaction , ~50  ng of cdna synthesized from the total rna template obtained from individual animals was used for quantitative real - time pcr ( qrt - pcr ) reactions .
qrt - pcr was performed using assay - on - demand taqman probes ( applied biosystems ) and run on an icycler device ( biorad , hercules ) .
the following taqman primers were used : mm00446968_m1 ( hprt , mouse hypoxanthine guanine phosphoribosyl transferase ) ; mm04207460_m1 ( cxcl1 ) ; mm00436451_g1 ( cxcl5 ) ; mm00434946_m1 ( cxcl9 ) ; mm00444662_m1 ( cxcl11 ) ; mm00445553_m1 ( cxcl12 ) ; and mm04214185_s1 ( cxcl13 ) .
the expression of hprt was measured and was quantified to control for variations in cdna amounts .
hprt did not significantly change for stz - injected mice and therefore served as a housekeeping gene ( data not shown ) .
the cycle threshold values were calculated automatically by icycler iq 3.0 software using default parameters .
the lumbar ( l4l6 ) portion of the spinal cord was removed from nave and stz - induced neuropathic pain mice on day 7 following stz administration .
the tissue samples were homogenized in 1x cell lysis buffer ( raybio ) with protease inhibitor cocktail ( sigma ) and cleared by centrifugation ( 14000  g for 30  min ) .
the protein concentration in the supernatant was determined using the bca protein assay kit ( sigma ) .
the samples were diluted with 1x blocking buffer to a final concentration of 250  g of protein per sample .
the raybio membranes ( table 1 ) were blocked at room temperature and incubated with 1  ml of sample overnight at 4c .
after incubation , the samples were decanted , and the membranes were washed 3x with 2  ml of 1x wash buffer i ( raybio ) and 2x with 2  ml of 1x wash buffer ii ( raybio ) at room temperature .
to each membrane , 1  ml of diluted biotin - conjugated anti - cytokines antibodies ( raybio ) was added and incubated at room temperature for 90  min .
the membranes were incubated for 2 hours at room temperature with 2  ml of 1000-fold diluted hrp - conjugated streptavidin ( raybio ) .
immunocomplexes were detected using detection buffer ( raybio ) and visualized using a fujifilm las-4000 fluorimager system .
the intrathecal ( i.t . ) administration was performed according to hylden and wilcox . using a hamilton syringe with a thin needle ,
5  l of each chemokine was injected between the l5-l6 vertebrae in the lumbar portion of the spinal cord .
cxcl1/kc and cxcl5/lix were obtained from sigma aldrich and cxcl9/mig and cxcl12/sdf-1 were from r&d system .
reconstituted chemokines were injected in the following concentrations : 10  ng/5  l , 100  ng/5  l , and 500  ng/5  l . the behavioral tests were performed after 1 , 4 , and 24 hours following chemokine administration . the behavioral data for diabetic neuropathic pain development are presented as a percentage of the nave mice  s.e.m . of 416 animals per group .
p < 0.05 and p < 0.001 indicate a significant difference versus nave animals . for the chemokine administration ,
the data are presented as fold change of the control ( nave )  s.e.m .
the results were evaluated using a one - way analysis of variance ( anova ) .
the intergroup differences were analyzed by anova plus bonferroni 's multiple comparison test ; p < 0.05 , p < 0.01 , and p < 0.001 indicate a significant difference compared with nave animals . the data from the biochemical experiments are presented as fold change of the control ( nave )  s.e.m .
the qrt - pcr analysis represents normalized averages derived from the threshold cycle in qpcr from 410 samples for each group .
the protein analysis was performed using the antibody array method from 11 - 12 samples per group .
p < 0.05 , p < 0.01 , and p < 0.001 indicate a significant difference in comparison to the control ( nave animals ) .
seven days after stz ( 200  mg / kg , i.p . ) administration , an increase in the plasma glucose concentration in stz - injected mice was observed ( 552  12.0  mg / dl versus nave 186  13.6  mg / dl ) ( figure 1(a ) ) .
furthermore , at day 7 following stz administration the decrease in nociceptive threshold , evaluated by the tail - flick test , was detected in stz - injected mice ( 3.7  0.3  s versus nave 4.8  0.3  s ) ( figure 1(b ) ) .
neuropathic pain syndromes such as allodynia and hyperalgesia were also detected at day 7 after stz administration .
the mechanical allodynia ( 0.9  0.1  g ) was assessed by the von frey test ( figure 1(c ) ) and the thermal hyperalgesia ( 6.5  0.7  s ) was measured using the cold plate test ( figure 1(d ) ) in stz - injected mice in comparison to nave animals ( 5.5  0.3  g and 27.2  2.0  s , resp . ) .
stz administration , the levels of cxcl1 , cxcl5 , cxcl9 , cxcl11 , cxcl12 , and cxcl13 mrna were measured in the lumbar ( l4l6 ) portion of the spinal cord in mice using qrt - pcr ( figures 2(a ) , 2(b ) , 2(c ) , 2(d ) , 2(e ) , and 2(f ) ) . in stz - induced diabetic mice , a significant increase in cxcl1 ( figure 2(a ) ; 1.25-fold ) , cxcl9 ( figure 2(c ) ; 28.38-fold ) , and cxcl11 ( figure 2(e ) ; 3.32-fold ) mrna levels was detected .
simultaneously , a decrease in cxcl12 ( figure 2(d ) ; 0.48-fold ) mrna level and no changes in cxcl5 ( figure 2(b ) ) and cxcl13 ( figure 2(f ) ) levels in stz - injected mice were observed .
the analysis of cxcl1 , cxcl5 , cxcl9 , cxcl11 , cxcl12 , and cxcl13 protein levels in the lumbar ( l4l6 ) portion of the spinal cord was conducted at day 7 after i.p .
stz administration , using the raybio mouse inflammation antibody array ( figures 3(a ) , 3(b ) , 3(c ) , 3(d ) , 3(e ) , and 3(f ) ) . in stz - induced diabetic mice , an upregulation of cxcl1 ( figure 3(a ) ; 1.63-fold ) , cxcl5 ( figure 3(b ) ; 1.32-fold ) , cxcl9 ( figure 3(c ) ; 1.32-fold ) , and cxcl12 ( figure 3(d ) ; 1.25-fold ) protein levels was observed .
changes in cxcl11 and cxcl13 levels were not detected ( figures 3(e ) and 3(f ) ) .
administration of cxcl1 , cxcl5 , cxcl9 , or cxcl12 at each dose ( 10 , 100 , and 500  ng/5  l ) caused a pronociceptive reaction as measured by the tail - flick test ( figures 4(a ) , 4(b ) , 4(c ) , and 4(d ) ) .
after cxcl1 ( figure 4(a ) ) treatment , the response at all doses to the thermal stimuli occurred in an hour after treatment and then slowly diminished .
one day after injection , the values observed after the lowest dose ( 10  ng ) were not significant in comparison to the control , but the highest dose ( 500  ng ) still caused a pronociceptive reaction as measured by the tail - flick test ( figure 4(a ) ) . the cxcl5 ( figure 4(b ) ) injection resulted in a dose - dependent response one hour after injection . furthermore , at the later time points , 10  ng cxcl5 induced a reaction similar to that obtained with 500  ng of this chemokine ( figure 4(b ) ) , and after 24 hours following the treatment the values were not statistically significant in comparison to the control ( figure 4(b ) ) .
one hour after cxcl9 application ( figure 4(c ) ) , the 100  ng and 500  ng doses caused a similar response that was stronger than seen with the 10  ng dose .
furthermore , these effects were still detectable up to 24 hours later ( figure 4(c ) ) .
the cxcl12 ( figure 4(d ) ) injection resulted in a dose - dependent response one hour after injection .
these effects were still measurable in the fourth hour after treatment for all doses except the 10  ng dose of cxcl12 ( figure 4(d ) ) .
twenty - four hours following cxcl12 treatment , the pronociceptive properties of this chemokine were not observed in animals that received a dose of 10  ng ( figure 4(d ) ) . a single i.t .
administration of cxcl1 , cxcl5 , cxcl9 , or cxcl12 at each dose ( 10 , 100 , and 500  ng/5  l ) induced the development of mechanical allodynia as measured by the von frey test ( figures 5(a ) , 5(b ) , 5(c ) , and 5(d ) ) .
one hour after cxcl1 treatment , a strong response to the mechanical stimuli was detected ( figure 5(a ) ) .
these effects were intensified in the fourth hour after treatment and were similar for all doses .
furthermore , 24 hours following administration , the mechanical allodynia was still observed ( figure 5(a ) ) .
cxcl5 ( figure 5(b ) ) or cxcl9 ( figure 5(c ) ) treatment resulted in a dose - dependent response in the von frey test one hour following injection .
moreover , at the fourth hour after cxcl5 treatment , this correlation was still detected ( figure 5(b ) ) and persisted for up to 24 hours ( figure 5(b ) ) . in the case of cxcl9 treatment , after four hours , all doses resulted in a similar , but weaker , reaction to mechanical stimuli than at one hour , which disappeared after 24 hours ( figure 5(c ) ) .
the administration of cxcl12 in doses of 100  ng and 500  ng caused a similar response in the von frey test measured one hour after application ( figure 5(d ) ) .
those effects gradually diminished in the fourth hour to achieve control values , measured one day after injection ( figure 5(d ) ) . a single i.t .
administration of cxcl1 , cxcl5 , cxcl9 , or cxcl12 at each dose ( 10 , 100 , or 500  ng/5  l ) induced the development of thermal hyperalgesia as measured by the cold plate test ( figures 6(a ) , 6(b ) , 6(c ) , and 6(d ) ) .
one hour after cxcl1 treatment , a dose - dependent reaction to the thermal stimuli was evident for all doses ( figure 6(a ) ) ; by the fourth hour , those effects were potentiated .
one day following administration of a 500  ng dose , cxcl1 still had strong pronociceptive properties ( figure 6(a ) ) . in the case of cxcl5 ,
the 100  ng and 500  ng doses triggered a dose - dependent pain response in the cold plate test at one hour after treatment ( figure 6(b ) ) . in the fourth hour ,
the lowest dose ( 10  ng ) induced a response to the thermal stimuli , although it was not as strong as seen for the other doses .
the thermal hyperalgesia dose - dependent effect was still measured one day after treatment ( figure 6(b ) ) .
the results obtained after cxcl9 treatment showed that the reaction to the thermal stimuli at one hour was the same for the 10  ng and 500  ng doses ( figure 6(c ) ) . those effects , excluding 500  ng cxcl9 , gradually diminished , and after one day thermal hyperalgesia was detected in only the animals that received 10  ng ( figure 6(c ) ) .
the dose - dependent effects obtained after cxcl12 application indicated their pronociceptive properties ( figure 6(d ) ) .
furthermore , after four hours , the 10  ng dose induced the strongest reaction in comparison to the reaction measured after one hour .
this study verifies that , in streptozotocin - induced diabetic neuropathy , the spinal protein levels of cxcl1 , cxcl5 , cxcl9 , and cxcl12 , but not cxcl11 and cxcl13 , are upregulated .
furthermore , the results show that the intrathecal administration of the chemokines that are changed in the spinal cord in stz - induced diabetic mice ( cxcl1 , cxcl5 , cxcl9 , and cxcl12 ) are associated with the development of mechanical allodynia and thermal hyperalgesia in parallel to a decrease in the nociceptive threshold . studying the neuropathic pain of diabetes using experimental diabetic animal models
in the case of type 1 diabetes , the most common mouse model of chemoinduced pancreatic toxicity is streptozotocin - induced diabetes [ 16 , 17 , 25 ] . in our studies ,
a single dose of stz induced the development of diabetes , as demonstrated by a high blood glucose level , and the development of neuropathic pain syndromes such allodynia and hyperalgesia , in agreement with our earlier experiments in a diabetic neuropathic pain model .
furthermore , we observed that mice which developed diabetic neuropathy demonstrated a decrease in nociceptive threshold .
those results correlate with ohsawa et al . , who showed that up to four weeks the reaction to heat stimuli in the tail - flick test was quicker in diabetic mice than in nave controls ; however , after 6 weeks the tail - flick latency was prolonged in diabetic mice which can be related to the progressive degeneration of the nerves within the tail of the animal .
neuropathic pain induced by diabetes may be a neuroinflammatory disorder because it is mediated by a variety of inflammatory mediators [ 26 , 27 ] .
recent studies have shown that , in stz - induced diabetic neuropathy , some pronociceptive factors , such as ifn - gamma , il-1 beta , and il-6 , are upregulated [ 7 , 28 ] .
however the participation of chemokines from the cxc subfamily in stz - induced diabetic neuropathy remains unexplored .
currently , studies in many pain - related models suggest that chemokines from the cxc subfamily play an important role .
the results of our studies suggest an important role for cxcl1 , cxcl5 , cxcl9 , and cxcl12 in stz - induced diabetic neuropathy . in our experiments ,
the protein and mrna levels of cxcl1 , also known as keratinocyte - derived chemokine ( kc ) or growth - related oncogene ( gro ) , were increased in diabetic animals , and its intrathecal administration caused the development of neuropathic pain syndromes .
those results are in agreement with zhang et al . , who observed the upregulation of cxcl1 expression in the spinal cord in the snl model of neuropathic pain .
furthermore , the spinal injection of cxcl1 induces dose - dependent thermal hyperalgesia , while snl - induced neuropathic pain was diminished by cxcl1 neutralizing antibody .
it was also reported that , in patients with diabetic neuropathy , the serum level of cxcl1 is higher than in healthy people .
in addition , cxcl1 seems to be crucial not only in neuropathic pain but also in inflammatory pain   and bone cancer pain . in multiple sclerosis ,
the upregulation of cxcl1 was induced by proinflammatory cytokines such as il-1 beta and ifn - gamma   and those cytokines were also increased in a streptozotocin - induced diabetic neuropathy model [ 7 , 28 ] .
our studies also reveal an increase in the cxcl5 protein level ; however , changes in the mrna amount in the spinal cord were undetectable .
those discrepancies in mrna and protein level can be related to the fact that process of translation does not always take place after transcription , but for some factors it can be shifted in time . additionally , when the concentration of ligand is high and completely saturated its receptor the process of transcription is suspended ; however translation may be still in process . in 2013 , liu et al .
reported mechanical allodynia and parallel upregulation of cxcl5 mrna in spinal lumbar dorsal horns after intrathecal injection of lipopolysaccharide .
our results are in partial agreement with nunemaker et al . , who reported an increased serum level of cxcl5 in type 2 diabetics .
these reports confirm our experiments , in which the intrathecal application of cxcl5 induced the development of mechanical allodynia .
the changes observed in our study in the mrna and protein levels of cxcl9 during diabetic neuropathy suggest its very important role in the neuropathic pain that develops in diabetes .
cxcl9 , also known as monokine - induced by gamma interferon ( mig ) , is a member of the ifn - gamma inducible subset of cxc chemokines . in our previous studies
, we have shown that ifn - gamma is upregulated in the spinal cord 7 days after stz treatment , which may explain the strong parallel upregulation of cxcl9 .
a number of previous studies have suggested a role for the cytokine ifn - gamma within the dorsal horn in chronic pain states . therefore , cxcl9 as a target of ifn - gamma signaling may be involved .
we have observed that , after injection of this chemokine , the nociceptive threshold was lower and neuropathic pain syndromes developed . additionally , after stimulation of schwann cells with high glucose levels , cxcl9 mrna expression was elevated and high levels of cxcl9 protein were detected in the supernatant .
moreover , the expression of cxcl9 was increased in the joints of patients with rheumatoid arthritis   and it was found that activation of spinal cxcl9 mediated bone cancer pain in rats . a pronociceptive role for cxcl9 within the dorsal horn would provide further support for the use of chemokine antagonists in the treatment of diabetic neuropathic pain .
cxcl12 , formerly named stromal cell - derived factor 1 ( sdf-1 ) , is expressed in various kinds of cells in the central nervous system .
it has been shown that increased signaling by sdf-1/cxcr4 contributes to chronic pain behavior [ 27 , 39 ] .
therefore , the results obtained in our studies showing an upregulation of cxcl12 in the lumbar spinal cord and the decrease in its mrna content in diabetic animals correlate with this thesis .
as was mentioned previously , this inconsistency may be associated with a shift in the process of transcription and translation . in stz - induced diabetic neuropathy
, sdf-1 increased the velocity of sciatic nerve conduction , and in spinal cord injury the cxcl12 was induced in the dorsal horn after spinal cord lesioning .
moreover , some authors have observed an increase in cxcl12 in the spinal cord in bone cancer pain   and in antiretroviral toxic neuropathy .
furthermore , spinal administration of cxcl12 to nave mice caused the development of allodynia and hyperalgesia .
moreover , single spinal administration of cxcl12 neutralizing antibody reversed the pain hypersensitivity induced by tumor cell implantation .
cxcl11 is called interferon - inducible t - cell alpha chemoattractant ( i - tac ) and interferon - gamma - inducible protein 9 ( ip-9 ) . gene expression of cxcl11 is strongly induced by ifn- , and it is chemotactic for activated t cells .
these findings corresponded to mccoll et al .   who indicated the increase of cxcl11 mrna expression in experimental autoimmune encephalomyelitis ( eae ) in spinal cord
however , in our study , the spinal protein level of this chemokine in stz - diabetic mice was not changed which may be associated with a shift in the process of transcription and translation .
we also did not observe differences in spinal cxcl13 protein and mrna level between nave and stz - treated mice .
however , in animals with eae , but not in nave controls , the cxcl13 mrna was detected . furthermore , cxcl13-deficient mice exhibited a mild , self - limited form of this disease .
it has also been suggested that cxcl13 could be a diagnostic marker of lyme neuroborreliosis   and rheumatoid arthritis .
our results suggest that , at day 7 , cxcl11 and cxcl13 are not involved in stz - induced diabetic neuropathy . in conclusion ,
the results of our study suggest that increased spinal levels of cxcl1 , cxcl5 , cxcl9 , or cxcl12 protein in mice after stz treatment may participate in the development of diabetic neuropathic pain .
furthermore , we have shown that intrathecal administration of cxcl1 and cxcl5 to naive mice caused immediate development of allodynia and hyperalgesia , but those effects were still observed 24 hours following administration . because diabetic neuropathy is a neuroinflammatory disorder ,
investigations should focus on better understanding the role of chemokines in its development and maintenance .
a more accurate explanation of the participation of these chemokines in diabetic neuropathy has been planned for future studies .
 the mechanism involved in the development of diabetic neuropathy is complex . 
 currently , it is thought that chemokines play an important role in this process . 
 the aim of this study was to determine how the level of some chemokines from the cxc subfamily varies in diabetic neuropathy and how the chemokines affect nociceptive transmission . 
 a single intraperitoneal ( i.p . ) injection of streptozotocin ( stz ; 200  mg / kg ) resulted in an increased plasma glucose . 
 the development of allodynia and hyperalgesia was measured at day 7 after stz administration . using antibody array techniques , the increases in cxcl1 ( kc ) , cxcl5 ( lix ) , cxcl9 ( mig ) , and cxcl12 ( sdf-1 ) protein levels 
 were detected in stz - injected mice . 
 no changes in cxcl11 ( i - tac ) or cxcl13 ( blc ) protein levels were observed . 
 the single intrathecal ( i.t . ) administration of cxcl1 , cxcl5 , cxcl9 , and cxcl12 ( each in doses of 10 , 100 , and 500  ng/5  l ) shows their pronociceptive properties as measured 1 , 4 , and 24 hours after injection using the tail - flick , von frey , and cold plate tests . 
 these findings indicate that the chemokines cxcl1 , cxcl5 , cxcl9 , and cxcl12 are important in nociceptive transmission and may play a role in the development of diabetic neuropathy . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion