Ethics access to food and water ad libitum.

Ethics and Animals

The protocol were used in
this study was approved by Ethics Committee of the School of Veterinary Medicine, Shiraz
University, Shiraz, Iran.

forty-nine adult male Sprague-Dawley rats (280 ±
30 g) were purchased.  They were
maintained a week for adaptation in an animal room of the School of
Veterinary,shiraz university on a  22 ±
2°C and 12/12 h light/dark cycle with free access to food and water ad libitum.

Study design

The animals were divided into seven groups
(n=7): group 1 (Sham control 1 group) 
recived  normal saline (1 ?L,
Intra ARC) and (50 ?L formalin 2.5% SC in the left hind paw) ; group 2 (Sham chontrol
2 group) recived  normal saline (5 ?L, I.C.V)
and (50 ?L formalin 2.5% SC in the left hind paw); group 3 (Negative control group)  No
injection intra arcuate nucleus or I.C.V but they recived  50 µl formalin
2.5% SC in
the left hind paw. group 4 (Test 1 group) recived ghrelin ( 5?L ,I.C.V) and (50
?L formalin 2.5%, SC in the left hind paw); group 5 (Test 2 group) recived
ghrelin (1 ?L, Intra ARC ) and (50 ?L formalin 2.5%, SC in the left hind paw) ;
group 6 (Test 3) recived ghrelin antagonist (2.5 µl, I.C.V) and 10 min later
ghrelin (5 ?L, I.C.V) and (50 ?L formalin 2.5%, SC in the left hind paw); group
7 (Test 4) resived ghrelin antagonist (0.5 µl, intra ARC) and 10 min later
ghrelin (1 ?L, intra ARC) and (50 ?L formalin 2.5%, SC in the left hind paw) .

Implantation of probe and
guide cannula and injection drugs

The rats were anesthetized
by sodium pentobarbital (50 mg/kg i.p). By stereotactic The guide cannulas were then implanted in the lateral ventricle (AP= -0.8 mm, L= +1.5 mm, and DV= -3.6 mm)  and at angle 15° from vertical in
the arcuate nucleus of  hypotalamus (AP= -2.3 mm, L= +0.5 mm, and DV= -10 mm) (Paxinos, 1977) .After implantation of guide cannulas microdialysis’s prob were implanted
in the periacueductal gray matter (AP=
-7.6 mm, L= +0.6 mm, and DV= -5.8 mm). 24 hr after surgery, the microdialysis experiment and behavioral test were done. Ghrelin and d-lys were injected ICV and intra ARC in the test groups;  and in the sham
control groups equal volumes
of normal saline was used.

 Microdialysis and sample collection

Microdialysis study was started 24
hours after putting of the microdialysis probe. Animals were put down into
small chamber for 15 min to conformity. Focal injection fared by the Hamilton syringe 5µl during one
min. Formalin test was do after the optimal time for the outcome of the drugs(20
min). during the Microdialysis, ACSF
(Artificial cerebrospinal fluid) (NaCl 114, CaCl2 1, KCl 3, MgSO4 2, NaH2PO4 1.25, NaHCO3
26, NaOH 1, glucose 10, and pH=7.4) was perfused through micro injection pump into
microdialysis probe with a flow rate of 2.0 ?l/min (WPI, SP 210, syringe pump). After 20 Min, 40?l of the perfused
ACSF were collected in each 1.5 ml sterile Eppendorf tubes located in dry ice. Eight samples were collected
and immediately stored in freezer at -80°C until Hplc analysis (16, 17). S1 was base sample without ghrelin effect, S2
was base sample without formalin effect but with ghrelin effect, S3, S4, S5 and
S6 were samples associated to the formalin test. S7 and S8 were samples after
windup formalin test.


For induction of pain, in all groups after 10 min of the ICV or
Intra ARC injections, 50 ?L of 2.5% formalin were injected subcutaneously into
the dorsal surface of the left hind paw with a 27-gauge needle. The level of
nociception was recorded every 15s for one hour following injection of formalin
(Dubbuisson, 1977). If the animal does not display any certain sign, number
zero, if the animal’s feet collect on the ground and it does not put pressure
on it, number one, if the animal dap the ground with its feet or collect its
legs in the abdomen, number two, and finally if the animal licked the injected
paw, number three was record (19mR). Injection of formalin
in the hind paw subcutaneously generates a biphasic response. Acute phase is the
first 5 min after injection. Chronic phase is from the firs of 20 min until the
end of 60 min. Also, between the 5 until 20 min is the middle phase (20MR).


HPLC analysis

Microdialysis samples were
measured by high performance liquid chromatography (HPLC) system. Beta
endorphin and Met-enkephalin were separeted 
in samples on reversed phace column (C18) with a buffer KH2PO4(68) and
acetonitril (32) 0.1 M, PH: 2.3 and mobile phace of acetonitril/buffer and
acetonitril/water to detection at 250 nm. The  mobile phase was acetonitril/buffer in the
first 4min and acetonitril/water between 4-6 minute and from 6-12 it was again
acetonitril/buffer. The flow rate was set on 0.2 ml/min and tempreture was 40 °C. Plotting graphs and data
analyses were done by Autochro data module software. Concentrations of each
sample were calculated by interpolating the areas obtained after fraction
injection with those obtained from 3 known standard concentrations being
injected regularly to the HPLC to monitor the linearity of the system.
Integration of chromatographic data was performed by Perkin-Elmer 1020 software
program (21).????? 2


 For verification of correct location of guide
cannula into the lateral ventricle and into the arcuate nucleus of hypothalamus
and microdialysis prob into the PAG, at the end of each test, rats were
euthanized with an overdose of diethyl ether, then their brains were exited and
supplied in formalin and after regular slice of the brain to confirm the
correct position of cannula and prob tracing ,they were compared with the rat
brain atlas (Paxinos, 1977).????

Data analysis

The data statistical analysis was done by SPSS® ver 22.0 for Windows. One-way analysis of variance  (ANOVA) were used for analysis among the
groups and Dunkan multiple range
test was used as the post hoc test. Results are expressed as mean±SEM. The significant
level was considered P<0.05