Acute inflammation induces segmental, bilateral, supraspinally mediated opioid release in the rat spinal cord, as measured by μ-opioid receptor internalization

Abstract

The objective of this study was to measure opioid release in the spinal cord during acute and long-term inflammation using μ-opioid receptor (MOR) internalization. In particular, we determined whether opioid release occurs in the segments receiving the noxious signals or in the entire spinal cord, and whether it involves supraspinal signals. Internalization of neurokinin 1 receptors (NK1Rs) was measured to track the intensity of the noxious stimulus. Rats received peptidase inhibitors intrathecally to protect opioids from degradation. Acute inflammation of the hindpaw with formalin induced moderate MOR internalization in the L5 segment bilaterally, whereas NK1R internalization occurred only ipsilaterally. MOR internalization was restricted to the lumbar spinal cord, regardless of whether the peptidase inhibitors were injected in a lumbar or thoracic site. Formalin-induced MOR internalization was substantially reduced by isoflurane anesthesia. It was also markedly reduced by a lidocaine block of the cervical-thoracic spinal cord (which did not affect the evoked NK1R internalization) indicating that spinal opioid release is mediated supraspinally. In the absence of peptidase inhibitors, formalin and hindpaw clamp induced a small amount of MOR internalization, which was significantly higher than in controls. To study spinal opioid release during chronic inflammation, we injected Complete Freund's Adjuvant (CFA) in the hindpaw and peptidase inhibitors intrathecally. Two days later, no MOR or NK1R internalization was detected. Furthermore, CFA inflammation decreased MOR internalization induced by clamping the inflamed hindpaw. These results show that acute inflammation, but not chronic inflammation, induce segmental opioid release in the spinal cord that involves supraspinal signals.

Sustained Suppression of Hyperalgesia during Latent Sensitization by μ-, δ-, and κ-opioid receptors and α2AAdrenergic Receptors: Role of Constitutive Activity

“…Spinal cord sections were labeled for MORs as described previously (Song and Marvizó n, 2003b;Chen et al, 2007;Chen et al, 2008;Chen and Marvizó n, 2009). Rats were killed with pentobarbital (100 mg/ kg) and fixed immediately by aortic perfusion of 100 ml phosphate buffer (0.1 M sodium phosphate, pH 7.4) containing 0.01% heparin, followed by 400 ml of ice-cold fixative (4% paraformaldehyde, 0.18% picric acid in phosphate buffer).…”

“…MOR internalization was measured as described previously Marvizó n, 2003a, 2005;Song and Marvizón, 2003b;Chen and Marvizó n, 2009). MOR neurons with or without internalization were counted using an objective of 63ϫ (numerical aperture 1.40) and a Zeiss Axio-Imager A1 microscope.…”

“…We have shown previously that all endogenous opioids produce MOR internalization when their degradation is suppressed with peptidase inhibitors (Song and Marvizó n, 2003b) and that opioid release induces MOR internalization in slices (Song and Marvizó n, 2003a;Chen et al, 2008) and in vivo (Lao et al, 2008;Chen and Marvizó n, 2009). Therefore, the absence of MOR internalization in the presence of peptidase inhibitors argues against sustained opioid release in latent sensitization.…”

“…It is possible that this activation would be accompanied by MOR internalization either as the result of endogenous opioid release (Song and Marvizó n, 2003b) or of MOR constitutive activity leading to MOR internalization (Walwyn et al, 2007). Therefore, we measured MOR internalization during the remission phase of latent sensitization by immunofluorescence, an approach that we have used and described extensively (Song and Marvizó n, 2003a;Song and Marvizó n, 2003b;Chen et al, 2007;Chen et al, 2008;Chen and Marvizó n, 2009). To maximize the detection of MOR internalization due to the release of opioid peptides, we prevented peptide cleavage by an intrathecal injection of a mixture of peptidase inhibitors (Song and Marvizó n, 2003a;Song and Marvizó n, 2003b;Chen et al, 2007;Lao et al, 2008;Chen and Marvizó n, 2009).…”

Inflammation enhances Y1 receptor signaling, neuropeptide Y-mediated inhibition of hyperalgesia, and substance P release from primary afferent neurons

“…Substance P (SP) is released into the dorsal horn upon noxious stimulation or electrical stimulation of C-fibers (Yaksh et al, 1980, Allen et al, 1997a, Calcutt et al, 2000, Adelson et al, 2009, Chen and Marvizon, 2009a). SP then binds to neurokinin 1 receptors (NK1Rs) in lamina I and III neurons causing their internalization (Mantyh et al, 1989), which has been extensively used to indirectly estimate SP release (Mantyh et al, 1995, Abbadie et al, 1997b, Allen et al, 1997b, Honore et al, 1999b, Adelson et al, 2009, Chen and Marvizon, 2009a, Zhang et al, 2010b).…”