Peptides32(2011)1262–1269
Contents lists available at ScienceDirect
Peptides
j o u r n a l h o m e p a g e:w w w.e l s e v i e r.c o m/l o c a t e/p e p t i d e
s
Anti-allodynic effects of intrathecally and intracerebroventricularly administered 26RFa,an intrinsic agonist for GRP103,in the rat partial sciatic nerve ligation model
Tatsuo Yamamoto?,Rika Miyazaki,Toshihiko Yamada,Tomonari Shinozaki
Department of Anesthesiology,Graduate School of Medical Science,Kumamoto University,1-1-1Honjo,Kumamoto-shi,Kumamoto860-8556,Japan
a r t i c l e i n f o
Article history:
Received20October2010
Received in revised form13March2011 Accepted14March2011
Available online23March2011
Keywords:
Dorsal root ganglion
GPR103
Neuropathic pain Immunohistochemistry a b s t r a c t
26RFa and QRFP are endogenous ligands of GPR103.26RFa binding sites are widely distributed in the brain and the spinal cord where they are involved in processing pain.In the present study,the effects of intrathecal and intracerebroventricular applications of26RFa on the level of mechanical allodynia induced by partial sciatic nerve ligation were examined in rats.The level of mechanical allodynia was measured using von Frey?laments.Intrathecal and intracerebroventricular injection of26RFa attenuated the level of mechanical allodynia.26RFa has been reported to activate not only GPR103but also neuropep-tide FF2receptor and the effect of intrathecally and intracerebroventricularly administered26RFa was not antagonized by BIBP3226,an antagonist of neuropeptide FF receptor.Immunohistochemical exami-nation revealed that QRFP-like immunoreactivity(QRFP-LI)was expressed mainly in the small to medium sized neurons in the L5dorsal root ganglion(DRG)and that partial sciatic nerve injury increased the per-centage of QRFP-LI positive neurons.7days after the nerve injury,QRFP-LI positive neurons in the L5DRG ipsilateral to the partial sciatic nerve injury were larger than those in the L5DRG ipsilateral to the sham operation.These data suggest that(1)exogenously applied26RFa modulates nociceptive transmission at the spinal and the supraspinal brain in the neuropathic pain model,(2)the mechanism26RFa uses to produce an anti-allodynic effect may be mediated by the activation of GPR103,and(3)partial sciatic nerve ligation affects the expression of QRFP-LI in the dorsal root ganglion.
?2011Published by Elsevier Inc.
1.Introduction
GPR103(also referred to as SP9155or AQ27)is known as an orphan G protein-coupled receptor.GPR103shows similarities with orexin,neuropeptide FF(NPFF),and cholecystokinin recep-tors.Recently,two endogenous ligands for GPR103were identi?ed, 26RFa(also referred to as P518)and QRFP(also referred to as 43RFa)[9].26RFa is a novel26-residue RFamide peptide,initially isolated from a frog brain extract[4].QRFP is an N-terminally elongated form of26RFa.Although the role of GPR103and its endogenous ligands,26RFa and QRFP,is not fully understood, intravenous administered26RFa has been reported to cause a biphasic change in blood pressure and an increase in heart rate in urethane-anaesthetized rats[8].It has been reported that intrac-erebroventricular(ICV)administration of26RFa stimulates feeding
Abbreviations:NPFF,neuropeptide FF;LI,like immunoreactivity;IT,intrathe-cal;ICV,intracerebroventricular;DRG,dorsal root ganglion;NPY,neuropeptide Y; ANOVA,analysis of variance;IP,intraperitoneal.
?Corresponding author.Tel.:+81963735275;fax:+81963639697.
E-mail address:yamamotot@fc.kuh.kumamoto-u.ac.jp(T.Yamamoto).behavior and locomotion activity and that QRFP plays an important role in energy homeostasis[12,14,18,27].
Recently,Bruzzone et al.[1]reported that,in rats,26RFa bind-ing sites are widely distributed in the brain and the spinal cord, whereas the expression of GPR103mRNA is more discrete,notably in the mid brain,the pons,the medulla oblongata,and the spinal cord.In the spinal cord,a very strong expression of GPR103mRNA was detected in the super?cial layers of the dorsal horn and a high density of26RFa binding sites was also observed in the super?cial layers of the dorsal horn[1].In addition,many26RFa binding sites have also been observed in various nuclei of the brain involved in processing pain such as the parafascicular thalamic nucleus,the locus coeruleus,the dorsal raphe nucleus,and the parabrachial nucleus and these data strongly suggest that26RFa may modu-late transmission of nociceptive stimuli[1,2].The authors have reported that intrathecal(IT)injection of26RFa inhibited the agi-tation behavior induced by paw formalin injection and attenuated the level of mechanical allodynia induced by paw carrageenan injection and that these effects of26RFa may be mediated by the activation of spinal GPR103[23].The authors also reported that ICV injection of26RFa attenuated the agitation behavior induced by paw formalin injection[22].However,IT or ICV injection of
0196-9781/$–see front matter?2011Published by Elsevier Inc. doi:10.1016/j.peptides.2011.03.008
T.Yamamoto et al./Peptides32(2011)1262–12691263
26RFa had no effect on thermal nociceptive pain[22,23].Formalin induced agitation behavior and carrageenan induced mechanical allodynia are known to be an in?ammatory pain model and these data strongly suggest that the GPR103–26RFa system is involved in nociceptive transmission from spinal cord to brain during in?am-mation.
Sensory nerve injury may induce neuropathic pain that is con-stant,intermittent,or paroxysmal,with a burning,sharp,or aching sensation.Neuropathic pain is intractable pain and neuropathic pain is thought to be refractory to standard analgesics such as non-steroidal anti-in?ammatory drugs and opioids.It has been suggested that both in?ammatory pain and neuropathic pain,but not thermal nociceptive pain,are mediated,at least partly,by the sensitized systems of the spinal cord[11,24,25].Thus,it is pos-sible that activation of GPR103in the brain or in the spinal cord may produce an analgesic effect in the neuropathic pain models. In the present study,the authors examined the effect of either IT or ICV injection of26RFa on mechanical allodynia in the partial sciatic nerve ligation model,a neuropathic pain model[17],in the rat.
The authors previously reported that QRFP-like immunore-activity(QRFP-LI)is expressed in the rat dorsal root ganglion (DRG)[23].It has been reported that,in DRG,peripheral nerve injury decreased the expression of substance P and calcitonin gene related peptide and increased the expression of cholecys-tokinin,galanin,neuropeptide Y(NPY)and vasoactive intentinal polypeptide[5,10,19,20].In the present study,the authors exam-ined whether partial sciatic nerve ligation affects the expression of QRFP-LI in the L5DRG or not.
2.Materials and methods
The following investigations were performed according to a protocol approved by the Institutional Animal Care Committee of Kumamoto University,Kumamoto,Japan.Male Sprague–Dawley rats weighing250–300g were?tted with long-term IT catheters and ICV cannulae and examined for the effects of the agents on the level of mechanical allodynia induced by partial sciatic nerve ligation.
In the present study,the effect of QRFP was not examined because QRFP is insoluble in water.The manufacturer recommends that100?g of QRFP is dissolved in50?l of60%acetonitrile in water with0.1%dimethyl sulfoxide and then the solution is diluted up with any desired buffer.When10?g of QRFP was injected IT or ICV, the animal showed abnormal behavior and could not be examined for the effect of QRFP.
2.1.IT catheters and ICV cannulae
Chronic IT catheters were inserted,during halothane anesthesia, by passing a PE-10catheter through an incision in the atlanto-occipital membrane to a position8cm caudal to the cisterna at the level of lumbar enlargement[21].The catheter was externalized on the top of the skull and sealed with a steel wire,and the wound was closed with3-0silk sutures.
Implantation of the injection cannula into the right lateral ven-tricle was performed stereotaxically under halothane anesthesia.
A stainless steel thin-wall guide cannula(24gauge,0.64mm outer diameter,15mm long)was stereotaxically placed through a burr hole(0.5mm caudal to coronal suture and1mm lateral to sagi-tal suture;3mm deep to the dura)and af?xed to the skull with stainless steel screws and cranioplastic cement.
All animals displayed normal feeding and drinking behaviors post-operatively.Rats showing neurological de?cits were not stud-ied.2.2.Partial sciatic nerve ligation
Anesthesia was induced by inhalation of5%halothane,main-tained at a concentration of2–3%,as needed.After a local incision, the biceps femoralis of the right leg was bluntly dissected at mid-thigh to expose the sciatic nerve.The right sciatic nerve was then carefully mobilized,with care taken to avoid undue stretching.Par-tial sciatic nerve injury was created by a tight ligation of1/3to1/2 of the right sciatic nerve[17].An8-0silicon-treated silk suture was inserted into the sciatic nerve just proximal to the sciatic trifurca-tion with a3/8curved,reversed-cutting mini-needle,and tightly ligated so that the dorsal1/3to1/2of the nerve thickness was trapped in the ligature.Incisions were closed,layer-to-layer,with 3-0silk sutures,and the rats were allowed to recover from the anesthetic.After surgery,the animals were individually maintained in plastic cages with solid?oors covered with3–6cm sawdust. All animals postoperatively displayed normal feeding and drinking behavior.
2.3.Assessments of mechanical allodynia
Mechanical thresholds were measured using von Frey?la-ments with logarithmically incremental stiffnesses(0.41,0.70, 1.20,2.00,3.63,5.50,8.50and15.1g)(Stoelting,Wood Dale,IL) to calculate the50%probability thresholds for mechanical paw withdrawal[3].Beginning with the2.00g probe,?laments were applied to the plantar surface of a hind paw for6–8s,in a step-wise ascending or descending order following negative or positive withdrawal responses,respectively,until six consecutive responses were noted.50%probability thresholds were calculated according to the method suggested by Dixon[6].In a case where continuous negative or positive responses were observed to the limits of the stimulus set,values of15.00g or0.25g were assigned,respectively.
2.4.Mechanical nociceptive test
The mechanical nociceptive threshold was evaluated by mea-suring the frequency of foot withdrawals in response to a 29g von Frey?lament.The rats were placed in a clear plas-tic cage(10cm×20cm×24cm)on an elevated mesh?oor(grid: 5mm×5mm).To initiate a test,a rat was placed in the box and allowed5–10min to habituate.The frequency of paw withdrawal to repetitive applications of a29g von Frey?lament to the center of the footpad was tested in an unrestrained rat standing on four legs. The authors chose a29g von Frey?lament because preliminary study revealed that ten applications of a29g von Frey?lament to the foot pad induced about three or four withdrawal responses in the untreated normal rat.Thus,both the analgesic and the hyper-algesic effects of drugs can be evaluated with this method.During one trial,the von Frey?lament was repetitively applied to the hind-paw10times with an inter-stimulus interval of5s.The number of paw withdrawal responses was counted.
2.5.Immunohistochemistry
An immnohistochemical study was performed in animals with partial sciatic nerve injury and in sham-operated animals whose sciatic nerves were only mobilized.After anesthetizing rats with 60mg/kg of sodium pentobarbital intraperitoneally,surgery pro-ceeded with sternotomy,transcardiac aortic needle cannulation, and perfusion with50ml of saline,followed by500ml of4% paraformaldehyde in0.1M phosphate buffer(pH=7.4).L5DRG ipsilateral to either the nerve injury or the sham operation was removed and post?xed in the same?xative solution overnight at 4?C.After storing in0.01M phosphate buffered saline(PBS)con-taining20%sucrose for6h at room temperature,the DRG was
1264T.Yamamoto et al./Peptides32(2011)1262–1269
sectioned at a thickness of10?m,for QRFP immunohistochemistry, on a cryostat.Sections were mounted onto gelatin-coated slides.
After immersion in PBS containing5%normal horse serum and0.3%Triton X-100for1h,the sections were incubated with rabbit antibody to QRFP(1:1000;Phoenix Pharmaceuticals Inc., Burlingame,CA)and mouse antibody to NeuN(1:1000,Chemi-con,Temecula,CA)diluted with PBS containing5%normal horse serum and0.3%Triton X-100for20h at4?C.In the present study, the authors examined the expression of QRFP-LI,but not26RFa-LI,because the speci?c antibody to26RFa,which is applicable to immunohistochemical study,could not be obtained.A neuron was de?ned as a NeuN-LI positive cell.The authors previously demonstrated that the antibody to QRFP,which was used in this experiment,was completely neutralized by QRFP,and this indi-cated that the QRFP antibody used in the present study is speci?c to QRFP[23].The sections were then incubated at room temperature for3h with Alexa488-conjugated donkey anti-rabbit IgG(1:100; Molecular Probes,Eugene,OR,USA)and Alexa546-conjugated goat anti-mouse IgG(1:200;Molecular Probes)in PBS containing5%nor-mal horse serum and0.3%Triton X-100,and cover slipped with PermaFluor Aqueous(Shandon,Pittsburgh,PA).
2.6.Behavioral analysis
The general behavior of each rat was carefully observed and tested.Motor functions were evaluated by the performance of two speci?c behavioral tasks,as follows.(1)The placing/stepping re?ex: this response was evoked by drawing the dorsum of either hind paw over the edge of a tabletop.In normal animals,this stimulus elicits an upward lifting of the paw onto the surface of the table, called stepping.Animals with any degree of hind limb?accidity will demonstrate an altered or absent re?ex.(2)The righting re?ex:an animal placed horizontally with its back on the table will normally show an immediate coordinated twisting of the body around its longitudinal axis to regain its normal position on its feet.Animals displaying ataxic behavior will show a decreased ability to right themselves.To quantify the evaluation of motor functions,both tasks were scored on a scale of0–2in which0=absence of function and2=normal motor functions.Animals that were able to perform the motor tasks but did so more slowly than normal animals were assigned a score of1.
2.7.Drugs
The IT administered drugs were delivered in a total volume of 10?l followed by10?l of saline to?ush the catheter.The ICV administered drugs were delivered in a total volume of2?l.The agents used in this study were dissolved in saline.26RFa(molec-ular weight=2820)was purchased from Phoenix Pharmaceuticals Inc.BIBP3226(molecular weight=474)was purchased from Sigma (St.Louis,MO).Although BIBP3226has been reported to be a selec-tive non-peptide NPY Y1receptor antagonist[16],BIBP3226has also been reported to have antagonistic effects on neuropeptide FF1(NPFF1)and NPFF2receptors expressed in CHO cells[13]. More recently,Fang et al.[7]reported that intravenous injection of BIBP3226antagonized the effect of NPFF on arterial blood pres-sure in rats.These data suggested that BIBP3226has the property of a mixed antagonist to NPY Y1and NPFF receptors.
2.8.Experimental protocol
2.8.1.Dose–response study
A preliminary study performed in the authors’laboratory revealed that the maximum level of mechanical allodynia was observed between7and14days after the nerve injury.Thus, drugs were administered7days after the nerve injury.IT catheters were inserted4days before the nerve injury.ICV cannulae were implanted3days after the nerve injury.ICV cannulae were some-times plugged7days after implantation.If ICV cannulae were implanted4days before the nerve injury,the authors sometimes could not inject drugs through the ICV cannulae.Before and7days after the nerve injury,the mechanical threshold of the right hind paw was measured as control data.Then26RFa was administered IT(30?g(n=5);10?g(n=5);3?g(n=5))or ICV(30?g(n=5); 10?g(n=6);3?g(n=5)),and the right hind paw was tested at5, 15,30,45,60and90min after the drug administration.To obtain control data,vehicle(saline)was injected IT(n=5)or ICV(n=5).
26RFa has been reported to activate not only GPR103but also NPFF2receptor[1].To test whether the effect of IT or ICV admin-istered26RFa on an anti-allodynic effect was produced by an interaction between26RFa and spinal NPFF2receptor or central NPFF2receptor,10?g of BIBP3226was administered IT or ICV 10min before the IT or ICV injection of30or10?g of26RFa(IT study:30?g:n=5;10?g:n=4;ICV study:30?g:n=5;10?g: n=4).10?g of BIBP3226was also injected IT(n=5)or ICV(n=5). The motor function was examined just after each measurement of mechanical threshold.
As mentioned above,QRFP could not be injected IT or ICV because of its insolubility in saline.To examine the role of intrinsic QRFP in the maintenance of mechanical allodynia induced by nerve injury,rabbit antibody to QRFP(0.25?l;Phoenix Pharmaceuticals Inc.,Burlingame,CA),which was also used for the immunohisto-chemical study,was administered IT or ICV,and the right hind paw was tested at5,15,30,45and60min after the drug administration.
2.8.2.Mechanical nociceptive test in the sham operated rats
Before the drug administration,baseline measurements of the response frequency were made.30?g of26RFa was administered IT(n=4)or ICV(n=4)and the response of the sham-operated paw to10applications of a29g von Frey?lament was measured at5,15, 30,45and60min after the drug administration.To obtain control data,saline was administered IT(n=5)or ICV(n=5).
2.8.
3.Immunohistochemical study
The sections were examined using a?uorescent microscope (Axioskop2,Carl Zeiss,Germany).For the quanti?cation of the immunohistochemical study,?ve sections from each rat were randomly selected.The investigator responsible for the immuno-histochemical study was blinded to the operation(partial sciatic nerve ligation or sham operation)that the animals received.
The number of neurons and the number of QRFP-LI positive neurons in the right L5DRG was counted7days after the par-tial sciatic nerve ligation(n=5)or the sham operation(n=5).The proportion of QRFP-LI positive neurons to the total neurons of L5 DRG was calculated.The immunohistochemical examination of the nerve-injured rats proceeded concurrently with that of the sham-operated rats.
The areas of the QRFP-LI positive neurons were measured7days after the partial sciatic nerve ligation(n=5)or the sham operation (n=5)using a computer-assisted imaging analysis system(AxioVi-sion,version2.05,Carl Zeiss,Germany).Only neurons with clearly visible nuclei were used for quanti?cation.
2.9.Statistical analysis
2.9.1.Dose–response study
To determine whether partial sciatic nerve ligation induced signi?cant mechanical allodynia,the50%probability threshold before the nerve injury was compared with that7days after the nerve injury with a Mann–Whitney rank sum test.To analyze the time–effect curves,one way repeated measured analysis of variance(repeated measured ANOVA)with the Dunnet’s multiple
T.Yamamoto et al./Peptides32(2011)1262–12691265
comparison test was used.To analyze the dose–response effect of drugs on a mechanical threshold,the area under the curve(AUC) was calculated by use of the trapezoidal rule over the entire time course of the time–effect curve.To analyze the dose-dependency, one-way ANOVA with Dunnet’s multiple comparison test was used. To analyze the antagonistic effect of BIBP3226on the effect of 26RFa,the AUC of the BIBP3226+26RFa time–effect curve was com-pared with the AUC of the26RFa time effect curve with a t-test. 2.9.2.Mechanical nociceptive test
To analyze the effects of drugs on the response frequency,the AUC of the entire time–effect curve was calculated by use of the trapezoidal rule over the entire time course of the response fre-quency.The effect of26RFa on the AUC was analyzed by t-test. 2.9.3.Immunohistochemical study
The immunohistochemical study data are arranged in2×2or 2×7contingency tables.The most common procedure for analyz-ing contingency table data is by using the chi-square statistic[26]. Thus,to compare the proportion of QRFP-LI positive neurons in the total neurons of L5DRG in the nerve injured rats with that in the sham-operated rats(2×2table)or to compare the distribution of areas of QRFP-LI positive neurons in the partial sciatic nerve ligation rats with that in the sham-operated rats(2×7table),the chi-square test was used.
Wherever appropriate,results are expressed as mean±SEM. Critical values that reached a p<0.05level of signi?cance were considered statistically signi?cant.
3.Results
3.1.Behavioral analysis
After IT or ICV injection of26RFa,all animals scored2(normal motor function)in the placing/stepping re?ex and righting re?ex tests.No scratching or biting behavior was observed after the IT or ICV injection of26RFa.
3.2.Anti-allodynic Effects of26RFa
The50%probability threshold7days after partial sciatic nerve ligation(IT study(n=44):1.56±0.15g;ICV study(n=43): 1.65±0.05g)is signi?cantly less than the pre-surgery50% probability threshold level(IT study:14.4±0.3g;ICV study: 15.0±0.00g)(p<0.001by Mann–Whitney Rank Sum test)and this suggested that partial sciatic nerve ligation induced signi?cant mechanical allodynia7days after nerve injury.7days after nerve injury,the50%probability threshold level in the IT study group was not different from that in the ICV study group(p>0.5by t-test). 3.2.1.Effects of IT injection of26RFa
There were no differences in mean threshold differences after grouping the animals(data not shown,p>0.5by ANOVA).
IT injection of30?g of26RFa increased the50%probability threshold5min after the drug administration and the effect lasted for45min(p<0.05by repeated measured ANOVA,Fig.1)and there was no statistically signi?cant differences between the50%proba-bility threshold60min after26RFa administration and the pre-drug 50%probability threshold(Fig.1).IT injection of26RFa signi?cantly increased AUC as compared with the saline-treated rats at doses of 10and30?g(p<0.001by ANOVA,Fig.2).The AUCs in the26RFa(10 or30?g)+BIBP3226(10?g)treated rats were not different from those in the26RFa(10or30?g)treated rats,respectively(p>0.4 by t test,Fig.2).BIBP322610?g itself had no effect on the level of AUC as compared with that in the saline treated rats(BIBP3226
5
%
p
r
o
b
a
b
i
l
i
t
y
t
h
r
e
s
h
o
l
d
(
g
)
μg
Time (min)
5
%
p
r
o
b
a
b
i
l
i
t
y
t
h
r
e
s
h
o
l
d
(
g
)
26RFa 30 μg
μg
Fig.1.Effects of IT injection of30?g of26RFa and saline(IT injection study,top) and effects of ICV administration of30?g of26RFa and saline(ICV injection study, bottom)on the time course of the50%probability threshold(g)of the right hind paw (sciatic nerve injured paw).The effect of coadministration of BIBP3226and26RFa is also presented.26RFa,BIBP3226or saline was administered IT or ICV7days after partial sciatic nerve ligation.Ordinate:50%probability threshold(g);abscissa:time after drug administration(min).Each line represents the group mean and SEM of ?ve rats.*p<0.05as compared with the pre-drug(time=0)level.
treated rats:95.8±12.3;saline treated rats:101±8.8,p>0.7by t test).
3.2.2.Effects of ICV injection of26RFa
There were no differences in mean threshold differences after grouping the animals(data not shown,p>0.4by ANOVA).
ICV injection of30?g of26RFa increased the50%probabil-ity threshold5min after the drug administration and their effects lasted for60min after the drug administration(p<0.05by repeated measured ANOVA,Fig.1),and there was no statistically signi?cant difference between50%probability threshold90min after26RFa ICV injection and pre-drug50%probability threshold(Fig.1).ICV injection of26RFa signi?cantly increased the AUC as compared with the saline-treated rats at doses of10?g and30?g(p<0.001 by ANOVA,Fig.2).There was no difference between AUCs in the 26RFa(10or30?g)+BIBP3226(10?g)treated rats and those in the26RFa(10or30?g)treated rats,respectively(p>0.4by t test, Fig.2).BIBP322610?g itself had no effect on the AUC as com-pared with that in the saline treated rats(BIBP3226treated rats: 119±17.4;saline treated rats:107±15.2,p>0.6).
3.2.3.Effects of an antibody to QRFP on mechanical allodynia
IT or ICV injection of an antibody to QRFP had no effect on the AUC as compared with IT or ICV saline-treated rats,respectively(IT antibody to QRFP(n=5):142±20.7; IT saline(n=5):101±8.8;ICV antibody to QRFP(n=5): 152±19.4;ICV saline(n=5):107±15.2,p>0.1by t test, Fig.3).
1266T.Yamamoto et al./Peptides 32(2011)1262–1269
A U C
Dose (μg)
A U C
Fig.2.Dose–response curves for IT injected 26RFa (IT injection study,top)and ICV injected 26RFa (ICV injection study,bottom).Ordinate:area under the curve (AUC);abscissa:dose of 26RFa (?g).AUC was calculated by use of trapezoidal rule over the entire time course of the time effect curve.26RFa or saline was administered,either IT or ICV,7days after the partial sciatic nerve ligation.26RFa increased the AUC in a dose-dependent manner in either the IT injection study or the ICV injection study.Pre-treatment with 10?g of BIBP3226had no effect on the anti-allodynic effect of either 30?g or 10?g of 26RFa in either IT injection study or ICV injection study.Each line represents the group mean and SEM of ?ve or six rats.*p <0.05as compared with the saline treated rats.
3.2.
4.Effects of 26RFa on the mechanical nociceptive test
IT or ICV injection of 30?g of 26RFa had no effect on the mechan-ical nociceptive test as compared with IT or ICV saline treated rats,respectively (AUC of IT study:26RFa treated rats:2440±240,saline treated rats:2160±142;AUC of ICV study:26RFa treated rats:2530±200,saline treated rats:2620±340,p >0.3,by t test,Fig.4).
3.3.Immunohistochemical Study
In the rats with partial sciatic nerve ligation,665neurons out of 3155neurons (21.1%)in the L5DRG ipsilateral to the nerve injury were immunoreactive for QRFP.In the rats with the sham operation,574neurons out of 3534neurons (16.2%)in the L5DRG ipsilateral to the sham operation were immunoreactive for QRFP.Proportion of QRFP-LI positive neurons in total neurons of L5DRG in the nerve injured rats was signi?cantly larger than that in the sham operated rats (Fig.5,p <0.001by chi-square test).
The QRFP-LI was observed mainly in small-to medium sized neurons in the rats with the sham operation (Figs.5and 6).7days after the nerve injury,QRFP-LI positive neurons in the L5DRG ipsi-
50 % p r o b a b i l i t y t h r e s h o l d (g )
12345
6
Time (min)
50 % p r o b a b i l i t y t h r e s h o l d (g )
123456
Fig.3.Effects of IT injection or ICV injection of an antibody to QRFP on the time course of the 50%probability threshold (g)of the right hind paw (sciatic nerve injured paw).Ordinate:50%probability threshold (g);abscissa:time after antibody to QRFP or saline administration (min).Each line represents the group mean and SEM of ?ve rats.
lateral to the partial sciatic nerve ligation were larger than those in the L5DRG ipsilateral to the sham operation (p <0.005by qui-square test,Figs.5and 6).4.Discussion
It has been clearly demonstrated that either IT or ICV injec-tion of 26RFa attenuated the level of mechanical allodynia induced by partial sciatic nerve ligation at doses of 10and 30?g.The anti-allodynic effect of IT administered 26RFa lasted 45min and the antiallodynic effect of ICV administered 26RFa lasted 60min.Either IT or ICV injection of 26RFa had no effect on the mechanical nociceptive test in the sham-operated rats.This suggested that IT or ICV injected 26RFa elevated the mechan-ical nociceptive threshold only when the sciatic nerve was injured.
Primeaux et al.[15]reported that ICV injection of 10and 15?g of 26RFa increased food intake in rats.Kampe et al.[12]also reported that centrally administered 10and 50?g of 26RFa increased food intake in rats.These data strongly suggest that 30?g of 26RFa is not a high dose in rats and that 30?g of 26RFa is needed to produce an effect when 26RFa is administered ICV.
In the present study,IT catheters were implanted 4days before the nerve injury and ICV cannulae were implanted 3days after the
T.Yamamoto et al./Peptides 32(2011)1262–12691267
R e s p o n s e F r e q u e n c y (%)
Time (min)
R e s p o n s e F r e q u e n c y (%)
Fig.4.Effects of IT or ICV injection of 30?g of 26RFa in the mechanical nociceptive study.Ordinate:response frequency to 10applications of a 29g von Frey ?lament (%);abscissa:time after antibody to 26RFa or saline administration (min).Each line represents the group mean and SEM.
nerve injury.ICV cannulation may affect the maintenance of neu-ropathic pain.The authors found that the 50%probability threshold level 7days after the nerve injury in the ICV group was not different from that in the IT group.Thus,it is believed that the ICV cannula-tion procedure itself did not affect the maintenance of neuropathic pain.
The mechanisms that 26RFa uses to attenuate mechanical allo-dynia induced by partial sciatic nerve ligation are not understood.It is not fully understood whether 26RFa interacts with recep-tors other than GPR103or not.Although Takayasu et al.[18]reported that the effect of ICV injected QRFP (10nmol)on feed-ing behavior was suppressed by pretreatment with 10nmol of BIBP3226,a mixed antagonist of NPY Y1and NPFF receptors,it has not been demonstrated that 26RFa directly activates the NPY Y1receptor.Bruzzone et
al.[1]reported that 26RFa inter-acts with another RFamide peptide receptor that might be the NPFF2receptor.Thus,it is possible that the effect of 26RFa is mediated by the interaction between 26RFa and NPFF2recep-tor.In the present study,the effect of either IT or ICV injected 26RFa (30?g (10nmol)or 10?g (3.3nmol))on the mechani-cal alldoynia induced by partial sciatic nerve ligation was not antagonized by pre-treatment of 10?g of BIBP3226(21nmol).As mentioned above,BIBP3226is a mixed antagonist of NPY Y1and NPFF receptors.Thus,these data indicate that the effect of either IT or ICV 26RFa on mechanical alldoynia induced by
par-tial sciatic nerve ligation was not mediated by the action of 26RFa on NPFF2receptor.As described in the introduction,the 26RFa
Fig.5.Fluorescent photomicrographs of QRFP in the L5dorsal root ganglion neurons ipsilateral to partial sciatic nerve ligation (top)or the sham operation (bottom).Scale bar,200?m.
% o f Q R F P p o s i t i v e n e u r o n s
μm
2
Fig.6.Size distribution of QRFP-LI positive neurons in the L5dorsal root ganglion ipsilateral to the nerve injury or the sham operation.7days after the nerve injury,QRFP-LI positive neurons in the rats with the partial sciatic nerve ligation were larger than those in the rats with the sham operation (p <0.005).
1268T.Yamamoto et al./Peptides32(2011)1262–1269
binding sites and the expression sites of GPR103mRNA were reported to be located in the spinal cord and in the brain nucleus where they are involved in processing of nociceptive stimuli[1,2]. These data strongly suggest that the GPR-103–26RFa system may modulate transmission of nociceptive stimuli.Although there is no speci?c antagonist of GPR103and the authors were not able to perform an antagonist study,the data may suggest that the activation of spinal and supra-spinal brain GPR103by26RFa pro-duces an anti-allodynic effect in the partial sciatic nerve ligation model.
In the present study,the authors examined the role of26RFa but were unable to examine the role of QRFP because of its insol-ubility in saline.It has been reported that26RFa and QRFP exert similar effects in a number of papers[14,27].IT or ICV injection of QRFP may produce a similar anti-allodynic effect in the par-tial sciatic nerve ligation model.On the other hand,IT or ICV injection of antibody to QRFP had no effect on the level of allo-dynia.This may suggest that intrinsic QRFP plays only a limited role in the maintenance of the level of allodynia induced by par-tial sciatic nerve injury.The impact of intrinsic QRFP on the level of allodynia may be different from that of exogenously applied QRFP.
Either IT or ICV injection of26RFa had no effect on the motor function and produced no behavioral arousal,such as scratching or biting.These data suggested that the anti-allodynic effect of either IT or ICV26RFa was not due to a motor effect,but was due to a speci?c anti-allodynic effect.
Regarding the formalin test,the authors previously reported that the effect of IT administered26RFa on both the phase1and the phase2response was not antagonized by BIBP3226[23],and that the effect of ICV injected26RFa on the phase1response,but not the phase2response,was antagonized by BIBP3226[22].These data suggest that IT injected26RFa exerts an analgesic effect that is mediated by the GPR103mediated mechanism,and that ICV injected26RFa elicited an analgesic effect by two mechanisms, the GPR103dependent mechanism and the GPR103independent mechanism.The mechanisms used by26RFa in the partial sciatic nerve ligation model are similar to those used by26RFa in the phase 2response of the formalin test.
The authors have previously reported that,in the immunohis-tochemial study,QRFP-LI was expressed in the DRG and GPR103-LI was predominantly observed in the super?cial laminae of spinal dorsal horn[23].In the present study,the authors found that QRFP-LI was predominantly expressed in the small to medium sized neurons of L5DRG ipsilateral to the nerve injury and this suggests that QRFP has a close relationship with C-?bers.This indicates that the GPR103–QRFP system is involved in the nociceptive transmis-sion from a peripheral nerve to the spinal cord.
Partial sciatic nerve injury increased the proportion of QRFP-LI positive neurons in total neurons of L5DRG ipsilateral to the nerve injury.As mentioned in the introduction,nerve injury increased the expression of inhibitory peptides,such as cholecys-tokinin,galanin,NPY and vasoactive intentinal polypeptide and nerve injury decreased the expression of excitatory peptides,such as substance P and calcitonin gene related peptide[5,10,19,20].IT injection of26RFa produced an analgesic effect and this suggested that either26RFa or QRFP may be inhibitory peptide.The authors think that nerve injury increased the expression of many inhibitory peptides including QRFP and that these up-regulated inhibitory peptides may suppress nociceptive transmission at the spinal cord. Unfortunately,as mentioned above,even though intrinsic QRFP increased after nerve injury,intrinsic QRFP may not be enough to alleviate the level of allodynia.7days after the nerve injury,QRFP-LI positive neurons in the L5DRG ipsilateral to the partial sciatic nerve ligation were larger than those in the L5DRG ipsilateral to the sham operation.Although the signi?cance of this change is not clear,nerve injury itself affects the expression of QRFP in many aspects.
5.Conclusions
Both IT and ICV injection of26RFa produced an anti-allodynic effect in the partial sciatic nerve ligation model.QRFP-LI was pre-dominantly expressed in the small to medium sized neurons and partial sciatic nerve ligation increased the proportion of QRFP-LI neurons in the L5DRG ipsilateral to the nerve injury.These data strongly suggest that the GPR103–26RFa and QRFP system plays some role in nociceptive transmission during neuropathic pain.
References
[1]Bruzzone F,Lectez B,Alexandre D,Jegou S,Mounien L,Tollemer H,et al.Distri-
bution of26RFa binding sites and GPR103mRNA in the central nervous system of the rat.J Comp Neurol2007;503:573–91.
[2]Bruzzone F,Lectez B,Tollemer H,Leprince J,Dujardin C,Rachidi W,et al.
Anatomical distribution and biochemical characterization of the novel RFamide peptide26RFa in the human hypothalamus and spinal cord.J Neurochem 2006;99:616–27.
[3]Chaplan SR,Bach FW,Pogrel JW,Chung JM,Yaksh TL.Quantitative assessment
of tactile allodynia in the rat paw.J Neurosci Methods1994;53:55–63.
[4]Chartrel N,Dujardin C,Anouar Y,Leprince J,Decker A,Clerens S,et al.Identi-
?cation of26RFa,a hypothalamic neuropeptide of the RFamide peptide family with orexigenic activity.Proc Natl Acad Sci USA2003;100:15247–52.
[5]Costigan M,Befort K,Karchewski L,Grif?n RS,D’Urso D,Allchorne A,et al.Repli-
cate high-density rat genome oligonucleotide microarrays reveal hundreds of regulated genes in the dorsal root ganglion after peripheral nerve injury.BMC Neurosci2002;3:16.
[6]Dixon WJ.Ef?cient analysis of experimental observations.Annu Rev Pharmacol
Toxicol1980;20:441–62.
[7]Fang Q,Guo J,He F,Peng Y-I,Chang M,Wang R.In vivo inhibition of neu-
ropeptide FF agonism by BIBP3226,an NPY Y1receptor antagonist.Peptide 2006;27:2207–13.
[8]Fang Q,Liu Q,Li N,Jiang TN,Li YL,Yan X,et al.Cardiovascular effects of intra-
venous administered26RFa,a novel RFamide peptide ligand for GPR103,in anaesthetised rats.Eur J Pharmacol2009;621:61–6.
[9]Fukusumi S,Yoshida H,Fujii R,Maruyama M,Komatsu H,Habata Y,et al.A new
peptidic ligand and its receptor regulating adrenal function in rats.J Biol Chem 2003;278:46387–95.
[10]H?kfelt T,Zhang X,Xu X,Wiesenfeld-Hallin Z.Central consequences of
peripheral nerve damage.In:McMahon SB,Koltzenburg M,editors.Wall and Melzack’s textbook of pain.5th ed.Philadelphia:Elsevier;2006.p.947–59. [11]Ji RR,Woolf CJ.Neuronal plasticity and signal transduction in nociceptive neu-
rons:implications for the initiation and maintenance of pathological pain.
Neurobiol Dis2001;8:1–10.
[12]Kampe J,Wiedmer P,P?uger PT,Castaneda TR,Burget L,Mondala H,et al.
Effect of central administration of QRFP(26)peptide on energy balance and characterization of a second QRFP receptor in rat.Brain Res2006;1119:133–49.
[13]Mollereau C,Mazarguil H,Marcus D,Quelven I,Kotani M,Lannoy V,et al.
Pharmacological characterization of human NPFF1and NPFF2receptors expressed in CHO cells by using NPY Y1receptor antagonists.Eur J Pharmacol 2002;451:245–56.
[14]Moriya R,Sano H,Umeda T,Ito M,Takahashi Y,Matsuda M,et al.RFamide
peptide QRFP43causes obesity with hyperphagia and reduced thermogenesis in mice.Endocrinology2006;147:2916–22.
[15]Primeaux SD,Blackmon C,Barnes MJ,Braymer HD,Bray GA.Central adminis-
tration of the RFamide peptides,QRFP-26and QRFP-43,increases high fat food intake in rats.Peptides2008;29:1994–2000.
[16]Rudolf K,Eberlein W,Engel W,Wieland HA,Willim KD,Entzeroth M,et al.
The?rst highly potent and selective non-peptide neuropeptide Y Y1receptor antagonist:BIBP3226.Eur J Pharmacol1994;271:R11–13.
[17]Seltzer Z,Dubner R,Shir Y.A novel behavioral model of neuropathic pain dis-
orders produced in rats by partial sciatic nerve injury.Pain1990;43:205–18.
[18]Takayasu S,Sakurai T,Iwasaki S,Teranishi H,Yamanaka A,Williams SC,et al.
A neuropeptide ligand of the G protein-coupled receptor GPR103regulates
feeding,behavioral arousal,and blood pressure in mice.Proc Natl Acad Sci USA 2006;103:7438–43.
[19]Wang H,Sun H,Della Penna K,Benz RJ,Xu J,Gerhold DL,et al.Chronic
neuropathic pain is accompanied by global changes in gene expression and shares pathobiology with neurodegenerative diseases.Neuroscience 2002;114:529–46.
[20]Xiao H-S,Huang Q-H,Zhang F-X,Bao L,Lu Y-J,Guo C,et al.Identi?cation of
gene expression pro?le of dorsal root ganglion in the rat peripheral axotomy model of neuropathic pain.Proc Natl Acad Sci USA2002;99:8360–5.
[21]Yaksh TL,Rudy TA.Chronic catheterization of the spinal subarachnoid space.
Physiol Behav1976;17:1031–6.
T.Yamamoto et al./Peptides32(2011)1262–12691269
[22]Yamamoto T,Miyazaki R,Yamada Y.Intracerebroventricular administration
of26RFa produces an analgesic effect in the rat formalin test.Peptides 2009;30:1683–8.
[23]Yamamoto T,Wada T,Miyazaki R.Analgesic Effects of intrathecally adminis-
tered26RFa,an intrinsic agonist for GPR103,on formalin test and carrageenan test in rats.Neuroscience2008;157:214–22.
[24]Yamamoto T,Yaksh https://www.doczj.com/doc/227620348.html,parison of the antinociceptive effects of pre-and
post-treatment with intrathecal morphine and MK801,an NMDA antagonist, on the formalin test in the rat.Anesthesiology1992;77:757–63.[25]Yamamoto T,Yaksh TL.Spinal pharmacology of thermal hyperesthesia induced
by constriction injury of sciatic nerve.Excitatory amino acid antagonists.Pain 1992;49:121–8.
[26]Zar JH.Biostatistical analysis.Second ed.Englewood Cliffs,NJ:Prentice-Hall
Inc.;1984.p.61–78.
[27]do Rego JC,Leprince J,Chartrel N,Vaudry H,Costentin J.Behav-
ioral effects of26RFamide and related peptides.Peptides2006;27:2715–
21.