Restoring Spinal Noradrenergic Inhibitory Tone Attenuates Pain Hypersensitivity in a Rat Model of Parkinson's Disease

Abstract

In the present study, we investigated whether restoring descending noradrenergic inhibitory tone can attenuate pain in a PD rat model, which was established by stereotaxic infusion of 6-hydroxydopamine (6-OHDA) into the bilateral striatum (CPu). PD rats developed thermal and mechanical hypersensitivity at the 4th week after surgery. HPLC analysis showed that NE content, but not dopamine or 5-HT, significantly decreased in lumbar spinal cord in PD rats. Additional noradrenergic depletion by injection of N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4) aggravated pain hypersensitivity in PD rats. At the 5th week after injection of 6-OHDA, systemic treatment with pharmacological norepinephrine (NE) precursor droxidopa (L-DOPS) or α2 adrenoceptor agonist clonidine significantly attenuated thermal and mechanical pain hypersensitivity in PD rats. Furthermore, application of norepinephrine (NE) and 5-hydroxytryptamine (5-HT) reuptake inhibitors duloxetine, but not 5-HT selective reuptake inhibitors sertraline, significantly inhibited thermal and mechanical pain hypersensitivity in PD rats. Systemic administration of Madopar (L-DOPA) or the D2/D3 agonist pramipexole slightly inhibited the thermal, but not mechanical, hypersensitivity in PD rats. Thus, our study revealed that impairment of descending noradrenergic system may play a key role in PD-associated pain and restoring spinal noradrenergic inhibitory tone may serve as a novel strategy to manage PD-associated pain.

Figure 1

Parkinson's disease model induced by stereotaxic infusion of 6-OHDA into the bilateral striatum of rats. (a) Representative photomicrographs of coronal section showing tyrosine hydroxylase-immunoreactive neurons and fibers in the substantia nigra (SN). The right lanes of pictures are higher magnification of boxed area. Scale bar = 100 μm. (b) The quantification analysis showed the TH-immunostaining intensity in SNc plus SNr (substantia nigra reticular part) at the 5th week after microinjection. Compared with the sham group, the TH-immunoreactivity was significantly decreased in the 6-OHDA-treated group (^∗ P < 0.05, ^∗∗ P < 0.01 compared to the sham control, n = 4 for each group). (c) The expression level of TH in striatum (CPu) was revealed by western blotting. TH expression in striatum (CPu) was significantly reduced in 6-OHDA-treated rats at the 5th week after operation compared to the sham control. Bottom bar graph showed the relative density of TH/GAPDH between 6-OHDA-treated group (n = 4) and sham group (n = 4). ^∗∗ P < 0.01 compared to the sham control. (d) There was no significant difference for body weight between 6-OHDA-treated group (n = 10) and sham group (n = 10). (e) The rotarod test showed that comparing to the sham group (n = 5), the latency time to fall from the rod for 6-OHDA-treated rats (n = 8) was significantly decreased from the 2nd week after operation (^∗ P < 0.05; ^∗∗ P < 0.01 compared to the sham group).

Figure 2

The development of pain hypersensitivity in the 6-OHDA-lesioned rats. After testing the baseline response to heat and mechanical stimuli, rats received a bilateral dorsal CPu injection of 6-OHDA or vehicle. (a) Paw thermal withdrawal threshold was significantly decreased in the 6-OHDA-treated rats (n = 12) than in the sham control rats (n = 5) on the 4th and 5th weeks after operation. ((b)–(d)) 4 g, 8 g, and 15 g von Frey filament were used to evaluate mechanical hypersensitivity. The 6-OHDA-lesioned rats (n = 12) showed mechanical hypersensitivity from the 4th and 5th week after the 6-OHDA lesion compared with the sham rats (n = 5). ^∗ P < 0.05, ^∗∗ P < 0.01, and ^∗∗∗ P < 0.001 compared to the sham animals. ((e)-(f)) At the 5th week after 6-OHDA microinjection, we used the formalin test to determine the changes of chemical-induced pain. There was a significant increase in both phase 1 (0–10 min) and phase 2 (10–60 min) in 6-OHDA-treated rats (n = 8) compared to the sham animals (n = 5) (^∗ P < 0.05, ^∗∗ P < 0.01, and ^∗∗∗ P < 0.001 compared to the sham animals).

Figure 3

Change of NE, 5-HT, and dopamine (DA) content in the lumbar spinal cord in the 6-OHDA-lesioned rat model. Samples were collected at the 5th week after 6-OHDA-lesion and NE (a), 5-HT (b), and DA (c) content in the lumbar spinal cord were analyzed by HPLC. Compared to the sham group, the NE content significantly decreased in the 6-OHDA-lesioned animals (^∗ P < 0.05 compared to the sham group, n = 4 for each group). 5-HT and DA content did not significantly change between the 6-OHDA-treated group and sham control group (P > 0.05 compared to the sham group, n = 4 for each group).

Figure 4

Effects of DSP-4 or 5,7-DHT injection alone on thermal and mechanical sensitivity in rats. (a) Compared to the sham group, paw thermal withdrawal threshold was decreased on the 4th week after bilateral ICV injection of DSP-4 (100 μg/4 μL in each side), but not 5,7-DHT (100 μg/4 μL in each side). ((b)–(d)) Mechanical hypersensitivity was significantly induced from the 2nd week of the end of testing after ICV injection DSP-4, but not 5,7-DHT (^∗ P < 0.05, ^∗∗ P < 0.01, and ^∗∗∗ P < 0.001 compared to the sham group, n = 6-7 for each group).

Figure 5

Effects of DSP-4 and 5,7-DHT on thermal and mechanical hypersensitivity in 6-OHDA-lesioned rats. (a) Compared to 6-OHDA-treated group, paw thermal withdrawal thresholds were decreased from the 2nd week after 6-OHDA plus DSP-4 injection. ((b)–(d)) Mechanical hypersensitivity was increased from the 2nd week after 6-OHDA plus DSP-4 injection and even increased from the 1st week after injection under 15 g von Frey filament (d). However, compared to 6-OHDA-treated group, coinjection of 5,7-DHT and 6-OHDA simultaneous did not further increase the mechanical hypersensitivity in rats (^∗ P < 0.05, ^∗∗ P < 0.01, and ^∗∗∗ P < 0.001 compared to the sham control, n = 6-7 for each group).

Figure 6

Effects of droxidopa and clonidine on thermal and mechanical hypersensitivity in 6-OHDA-lesioned rats. Rats were i.p. injected with 10 mg/kg droxidopa and 20 mg/kg droxidopa or equal volume of saline on the 5th week after surgery. ((a)–(d)) Systemic application of droxidopa significantly inhibited thermal (a) and mechanical hypersensitivity ((b)–(d)) in 6-OHDA-lesioned rats (^∗ P < 0.05, ^∗∗ P < 0.01, and ^∗∗∗ P < 0.001 for 10 mg/kg droxidopa and ^# P < 0.05 and ^## P < 0.01 for 20 mg/kg droxidopa compared to the saline control, n = 5-6 for each group). ((e)–(h)) Systemic application of clonidine (200 µg/kg, i.p.) significantly inhibited thermal (e) and mechanical hypersensitivity ((f)–(h)) in 6-OHDA-lesioned rats (^∗ P < 0.05, ^∗∗ P < 0.01, and ^∗∗∗ P < 0.001 compared to the saline control, n = 5-6 for each group).

Figure 7

Effects of duloxetine and sertraline on thermal and mechanical hypersensitivity in 6-OHDA-lesioned rats. Rats were i.p. injected with 10 mg/kg duloxetine or equal volume of 10% dimethylsulfoxide on the 5th week after 6-OHDA microinjection. ((a)–(d)) Systemic application of duloxetine significantly inhibited thermal (a) and mechanical hypersensitivity ((b)–(d)) in the 6-OHDA-lesioned rats (^∗ P < 0.05, ^∗∗ P < 0.01, and ^∗∗∗ P < 0.001 for duloxetine compared to the vehicle control, n = 5–7 for each group). ((e)–(h)) Systemic application of sertraline (10 mg/kg, i.p.) did not significantly affect thermal (e) and mechanical hypersensitivity ((f)–(h)) in the 6-OHDA-lesioned rats (P > 0.05, n = 5 for each group).

Figure 8

Effects of L-DOPA and pramipexole on thermal and mechanical hypersensitivity in 6-OHDA-lesioned rats. On the 5th week after microinjection of 6-OHDA, (a) systemic application of L-DOPA (15 mg/kg, i.p.) did not significantly inhibit thermal (a) and mechanical hypersensitivity ((b)–(d)) in the 6-OHDA-lesioned rats, although there was a trend to increase the paw thermal withdrawal threshold (n = 5-6 for each group). ((e)–(h)) Systemic application of pramipexole (PRA; 1 mg/kg, i.p.) significantly increased the paw thermal withdrawal threshold (e) after injection 1.5 h, but it had no effects on mechanical hypersensitivity ((f)–(h)) in the 6-OHDA-lesioned rats (^∗ P < 0.05 compared to the saline control, n = 5–7 for each group).