68 H
following sciatic nerve CCI galanin expression in DRG manner from below at the center of the plantar surface of
neurons is more strongly upregulated in non-allodynic rats hindpaw ipsi-lateral to the nerve injury. Each filament was
when compared to the allodynic group. In the present study delivered three times with approximately 2-s intervals. The
we used the same model to examine the effect of i.t. lowest force at which each of the three applications of the
galanin and a putative galanin antagonist, M35 [1], in both filament elicited a paw withdrawal was taken as the
allodynic and non-allodynic rats. mechanical response threshold.
2.4. Experimental design
2. Materials and methods
The measurement of the mechanical threshold started 14 Adult male Sprague–Dawley rats 240–260 g; ALAB,
days after the nerve injury. The basal threshold was usually Stockholm, Sweden were used. The rats were housed in
between 2.85 and 13.5 g or higher, and then the rats were cages at room temperature 20–258C under a 12 12 h
divided into two groups according to the threshold, non- light dark cycle with free access to food and water. The
allodynic 8.8 g and allodynic 5.2 g rats. experiments were conducted according to the Ethical
In the first experiment, galanin was administered i.t. in Guidelines for Investigation of Experimental Pain in
four allodynic rats in a cumulative dose regime of 1, 9, and Conscious Animals [50] and were approved by the local
20 mg, totally 30 mg. In non-allodynic rats, M35 was ethics committee for animal research.
administered in a similar manner in doses of 0.1, 0.2, and 0.4 mg, totally 0.7 mg. The same volume of saline was
2.1. Unilateral sciatic nerve injury injected in two allodynic and two non-allodynic rats.
To further confirm the effects of galanin in allodynic, Unilateral sciatic nerve injury was produced under deep
and of M35 in non-allodynic rats, a second series of anesthesia with intraperitoneal i.p. injection of sodium
experiments was performed blindly six allodynic rats
pentobarbital Mebumal ; 60 mg kg, as described by received galanin, and six saline; six non-allodynic rats
Bennett and Xie [2]. The common sciatic nerve was received M35 and five saline. All testing procedures were
exposed and freed for about 10 mm at mid thigh level. the same as in the first series, except that the dose of M35
Four ligatures Ethicon ; 4.0 plain gut were tied loosely of the last injection was increased to 0.7 mg, to give a total
around the nerve with about 1-mm spacing. Great care was cumulative dose of 1 mg.
taken to tie the ligatures such that the nerve was barely Additional control tests were carried out to check the
constricted. The incision was closed in layers with 3-0 silk effect of galanin 1, 9, 20 mg, bradykinin 0.047, 0.095,
sutures. 0.33 mg, bradykinin fragment2–9 0.040, 0.080, 0.28
mg in non-allodynic rats, and a high dose M35 equimolar 2.2. Intrathecal catheterization and injection
to the galanin, that is 0.7, 6.34, 14.1 mg in allodynic rats in an alternate non-blind design. The experiments with
Seven days after sciatic nerve loose ligation, a chronic bradykinin and the bradykinin fragment were applied,
i.t. catheter was implanted according to a modification since bradykinin2–9 represents the C-terminal part of
described by Storkson et al. [31]. After anesthesia with i.p. M35 [1].
sodium pentobarbital, a catheter PE 10, o.d. 0.61 mm was inserted into the subarachnoid space through a guide-
2.5. Drugs
cannula Sterican ; 20 G, 0.90340 mm at the level of the gap between the L5 and L6 vertebrae. It was then carefully
Rat galanin was purchased from Peninsula Belmont, implanted rostrally with its tip at the lumbar enlargement.
CA, USA and M35 [galanin1–13-bradykinin2–9 I.t. injection was given 7 days later. The proper location of
amide] from Bachem Bubendorf, Switzerland. Brady- the catheter was tested 24 h before the pharmacological
kinin and bradykinin fragment 2–9 were purchased from experiments by assessing sensory and motor blockade after
ICN Biomedicals, Aurora, OH, USA. All the peptides were ¨
i.t. injection of 7 ml lidocaine 50 mg ml; Astra, Soder- dissolved in distilled water and diluted with sterile saline
talje, Sweden. and stored in aliquots at 2208C until use.
2.3. Measurement of mechanical allodynia 2.6. Statistics
Behavioral testing was performed during daytime 9.00– The results were presented as median6median-derived
18.00 14 days after sciatic nerve constriction. Rats were absolute deviation MAD. Friedman ANOVA one-way
placed in transparent plastic domes 838318 cm on a repeated measures ANOVA on ranks was used to analyze
metal mesh floor with a hole size of 333 mm. After 15 the data in each group with the time course. Kruskal–
min of adaptation, a series of von Frey filaments 0.5, 0.88, Wallis ANOVA test one-way ANOVA on ranks was used
1.28, 2.85, 5.2, 7.5, 8.8, 13.5 g were applied in ascending for the comparison of data among groups at the same time
H .-X. Liu, T. Hokfelt Brain Research 886 2000 67 –72
69
point. A P value less than 0.05 was chosen as the significant level.
3. Results
