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, 15 (7), 669-75

Dynamic Temporal and Spatial Regulation of Mu Opioid Receptor Expression in Primary Afferent Neurons Following Spinal Nerve Injury

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Dynamic Temporal and Spatial Regulation of Mu Opioid Receptor Expression in Primary Afferent Neurons Following Spinal Nerve Injury

Chun-Yi Lee et al. Eur J Pain.

Abstract

Despite using prescribed pain medications, patients with neuropathic pain continue to experience moderate to severe pain. There is a growing recognition of a potent peripheral opioid analgesia in models of inflammatory and neuropathic pain. The goal of this study was to characterize the temporal and spatial expression of mu opioid receptor (mOR) mRNA and protein in primary afferent neurons in a rat L5 spinal nerve ligation model of persistent neuropathic pain. Bilateral L4 and L5 dorsal root ganglia (DRGs), L4 and L5 spinal cord segments, and hind paw plantar skins were collected on days 0 (naïve), 3, 7, 14, and 35 post-spinal nerve ligation or post-sham surgery. We found that expression of mOR mRNA and protein in primary afferent neurons changed dynamically and site-specifically following L5 spinal nerve ligation. Real-time RT-PCR, immunohistochemistry, and Western blot analysis demonstrated a down-regulation of mOR mRNA and protein in the injured L5 DRG. In contrast, in the uninjured L4 DRG, mOR mRNA transiently decreased on day 7 and then increased significantly on day 14. Western blot analysis revealed a persistent increase in mOR protein expression, although immunohistochemistry showed no change in number of mOR-positive neurons in the uninjured L4 DRG. Interestingly, mOR protein expression was reduced in the skin on days 14 and 35 post-nerve injury and in the L4 and L5 spinal cord on day 35 post-nerve injury. These temporal and anatomically specific changes in mOR expression following nerve injury are likely to have functional consequences on pain-associated behaviors and opioid analgesia.

Figures

Fig. 1
Fig. 1
Spinal nerve ligation (SNL)-induced mechanical allodynia. SNL led to a significant decrease in paw withdrawal threshold in response to mechanical stimuli on the ipsilateral (ipsi), but not contralateral (contra), side on days 3, 7, 14, and 35 after L5 SNL. Sham surgery did not produce a significant change in paw withdrawal threshold on either the ipsilateral or contralateral side during the observation period.
Fig. 2
Fig. 2
Dynamic changes in mOR mRNA expression in L4 and L5 DRGs after SNL. Relative expression levels of mOR mRNA in the L4 (Top) and L5 (bottom) DRGs on days 3, 7, 14, and 35 after L5 SNL or sham surgery. * p < 0.05 or ** p < 0.01 compared to the corresponding sham groups.
Fig. 3
Fig. 3
Specificity and selectivity of rabbit anti-mOR antibody. (A) Expression of mOR protein in HEK 293 cells transfected only with mOR, but not dOR, kOR, or un-tranfected controls. (B) Expression of mOR protein in skin and spinal cord tissues could be detected when the rabbit mOR antibody was preadsorbed with excess of mOR peptide.
Fig. 4
Fig. 4
Dynamic changes in the number of mOR-positive neurons in L4 and L5 DRGs after SNL. Top panels: representative mOR immunohistochemical staining in the ipsilateral (ipsi) and contralateral (contra) L5 DRGs on days 3, 7, 14, and 35 post-SNL. Bottom: statistical summary of the percentage of mOR-positive neurons in L4 and L5 DRGs on both ipsilateral and contralateral sides on days 3, 7, 14, and 35 post-SNL. Data are presented as mean ± SEM (n = 3/group). * p < 0.05 or ** p < 0.01 compared to the corresponding naïve rats.
Fig. 5
Fig. 5
Dynamic changes in mOR protein expression in L4 and L5 DRG and lumbar spinal cord after SNL. (A) Top panels: examples of Western blots that show the expression of mOR protein in the ipsilateral L4 and L5 DRGs in naïve rats (N) and on days 3, 7, 14, and 35 post-SNL. β-actin is used as a loading control. Bottom: statistical summary of mOR protein expression in ipsilateral and contralateral L4 and L5 DRGs on days 3, 7, 14, and 35 post-SNL. Data are presented as mean ± SEM (n = 4/group). * p < 0.05 or ** p < 0.01 compared to the corresponding naïve rats. (B) Top panels: examples of Western blots that show the expression of mOR protein in ipsilateral L4 and L5 spinal cord in naïve rats (N) and on days 3, 7, 14, and 35 post-SNL. Bottom: statistical summary of mOR protein expression in ipsilateral and contralateral L4 and L5 spinal cord on days 3, 7, 14, and 35 post-SNL. Data are presented as mean ± SEM (n = 4/group). * p < 0.05 compared to the corresponding naïve rats.
Fig. 6
Fig. 6
Dynamic changes in mOR protein expression in the hind paw plantar skin after SNL. Top panels: examples of Western blots that show the expression of mOR protein in the ipsilateral hind paw skin in naïve rats (N) and on days 3, 7, 14, and 35 post-SNL. β-actin is used as a loading control. Bottom: statistical summary of mOR protein expression in ipsilateral hind paw skin on days 3, 7, 14, and 35 post-SNL. Data are presented as mean ± SEM (n = 4/group).* p < 0.05 or ** p < 0.01 compared to the corresponding naïve rats. # p < 0.05 compared to day 14 post-SNL.
Fig. 7
Fig. 7
Decreased axonal transport of mOR on day 14 post-SNL. Injured animals have less mOR receptor in the nerve that is proximal to the double ligature as compared to normal uninjured animals demonstrating decreased axonal transport of mOR. Top panels: examples of Western blots that show the expression of mOR protein in the sciatic nerve proximal, intermediate, and distal to two ligations in normal and SNL rats. Bottom: statistical summary of mOR protein expression in the sciatic nerve proximal, intermediate, and distal to two ligations in normal and SNL rats. Data presented as average arbitrary density normalized to β-actin ± SEM (n=6 animals/group). ** p < 0.01 compared to the corresponding normal rats.

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