Impact of chronic nicotine on the development and maintenance of neuropathic hypersensitivity in the rat.

RATIONALE: Clinical data support a correlation between smoking and the incidence and severity of some chronic pain conditions. However, the impact of nicotine on neuropathic pain has been largely ignored in the laboratory setting. OBJECTIVES: The purpose of these studies was to determine if chronic nicotine would alter mechanical hypersensitivity after spinal nerve ligation. MATERIALS AND METHODS: Rats were implanted with osmotic mini pumps to administer either saline or nicotine (4, 10, or 24 mg/kg/day) for 7 or 21 days. On day 7 of saline/nicotine administration, rats receiving 24 mg/kg/day nicotine underwent spinal nerve ligation. Mechanical thresholds to pressure were measured across nicotine exposure and spinal cords were collected on days 7 or 21. Spinal cord slices were immunostained for phosphorylation of cAMP response element binding protein (pCREB), to determine general neuronal activity, and for cleaved caspase-3, as a marker for apoptosis. RESULTS: Chronic nicotine produced a dose-dependent and stable mechanical hypersensitivity, which could be blocked with the alpha4beta2-selective antagonist, dihydro-beta-erythroidine (DHbetaE). Spinal nerve ligation also produced a stable mechanical hypersensitivity, which was exacerbated in the presence of chronic nicotine. Differences in mechanical sensitivity were reflected in spinal pCREB, which was highly correlated with the degree of mechanical hypersensitivity. Chronic nicotine also altered the number of pro-apoptotic cells in the spinal cord as measured by cleaved caspase-3. CONCLUSIONS: These findings demonstrate that chronic nicotine produces a stable, long-lasting, mechanical hypersensitivity that exacerbates mechanical sensitivity resulting from peripheral nerve injury. The mechanism of this may involve an increase in spinal neuronal activity and apoptosis.

Spinal cord dynorphin expression increases, but does not drive microglial prostaglandin production or mechanical hypersensitivity after incisional surgery in rats.

Spinally released dynorphin contributes to hypersensitivity from nerve injury, inflammation, and sustained morphine treatment, but its role in post-operative pain has not been tested. Intrathecal injection of dynorphin activates cyclooxygenase (COX)-1 and -2 to induce hypersensitivity. Spinal COX-1 expression and activity increase following incisional paw surgery in rats, although the stimulus for this increase is not known. In the current study we tested whether spinal dynorphin expression increases after incisional surgery and induces hypersensitivity in this setting, and whether dynorphin stimulates COX-1 activity in spinal cord microglia. Paw incision resulted in increased prodynorphin immunoreactivity in laminae I, IIo, and V in the L4-L6 spinal cord dorsal horn ipsilateral to surgery. Change in prodynorphin expression did not parallel that of mechanical hypersensitivity. Repeated intrathecal dynorphin A antiserum injection failed to alter mechanical hypersensitivity after incisional surgery, although it was effective against mechanical hypersensitivity following spinal nerve ligation. Paw incision increased COX-1 immunoreactivity in the L4-L6 ipsilateral spinal cord, and these cells were confirmed to be microglia by co-localization with OX-42. Spinal cord microglia in culture expressed COX-1 immunoreactivity and released PGE2, but dynorphin A failed to increase release of PGE2 in these cultures. These results suggest that increased COX-1 expression occurs in spinal cord microglia following incisional surgery. Although prodynorphin immunoreactivity also increases, it likely does not drive COX-1 expression or mechanical hypersensitivity in this setting.

Knock down of the alpha 5 nicotinic acetylcholine receptor in spinal nerve-ligated rats alleviates mechanical allodynia.

Nicotinic acetylcholine receptor (nAChR) agonists are known to alleviate neuropathic and inflammatory pain via activation of a heterogeneous population of receptors. However, the function of nAChRs in the maintenance of neuropathic pain is not known. Spinal nerve ligation (SNL) increases the spinal expression of the alpha5 nAChR subunit ipsilateral to injury. The alpha5 subunit is unique because it modifies numerous characteristics of existing functional nAChRs, but it does not form functional nAChRs when expressed alone or with beta nicotinic subunits. Because there are no alpha5 subunit selective ligands, we used antisense oligonucleotides (ODNs) to assess the contribution of the alpha5 subunit to the maintenance of mechanical allodynia following SNL. Intrathecal antisense oligonucleotides were administered to SNL rats after the development of mechanical allodynia (10-12 days post-SNL). I.t. antisense specifically reduced alpha5 immunoreactivity (alpha5-IR) by 50-70% in the outer laminae of the dorsal horn and moderately alleviated mechanical allodynia. Furthermore, using the phosphorylation of cAMP response element-binding protein (pCREB) as a general marker of neuronal activation, a significant increase in pCREB immunoreactivity was observed in SNL rats. Treatment of SNL rats with alpha5-antisense significantly reduced pCREB immunoreactivity. These results suggest that the increased expression of the alpha5 nAChR subunit following SNL contributes to spinal CREB phosphorylation and the maintenance of mechanical allodynia.

Immunocytochemical localization of the alpha3, alpha4, alpha5, alpha7, beta2, beta3 and beta4 nicotinic acetylcholine receptor subunits in the locus coeruleus of the rat.

The presence of nicotinic acetylcholine receptors (nAChRs) within the locus coeruleus (LC) has been examined using a wide range of techniques. However, the expression pattern of individual nicotinic receptor subunits has not been described. Using immunocytochemistry, we demonstrate the distribution of the alpha3, alpha4, alpha5, alpha7, beta2, beta3 and beta4 nAChR subunits within the LC. Most nAChR subunits were expressed on neuronal perikarya within the LC nucleus. The alpha3, alpha4, alpha7 and beta3 immunoreactive neurons were evenly distributed in the dorsal and ventral LC whereas the alpha5, beta2 and beta4 nAChR subunits were preferentially confined to the upper dorsal section. In addition to neuronal perikarya, alpha4, alpha5 and beta2 immunoreactive fibers were observed. With the exception of the alpha3 subunit, punctate labeling was observed within and immediately surrounding the LC. These data are consistent with the presence of multiple nAChRs within the LC and extend these findings to show the distribution pattern of each nAChR subunit throughout the LC nucleus.