Spinal ceramide and neuronal apoptosis in morphine antinociceptive tolerance

Abstract

Opiates, like morphine, are the most effective analgesics for treating acute and chronic severe pain, but their use is limited by the development of analgesic tolerance and hypersensitivity to innocuous and noxious stimuli. Because opioids are a mainstay of pain management, restoring their efficacy has great clinical importance. We have recently demonstrated that spinal ceramide, a sphingolipid signaling molecule plays a central role in the development of morphine antinociceptive tolerance. We now report that ceramide upregulation in dorsal horn tissues in response to chronic morphine administration is associated with significant neuronal apoptosis. Inhibition of ceramide biosynthesis attenuated both the increase in neuronal apoptosis and the development of antinociceptive tolerance. These findings indicate that spinal ceramide upregulation is a key pro-apoptotic event that occurs upstream of the development of morphine antinociceptive tolerance and support the rationale for development of inhibitors of ceramide biosynthesis as adjuncts to opiates for the management of chronic pain.

Fig. 1. Schematic of the ceramide's mechanism of action in antinociceptive tolerance

Chronic administration of morphine leads to increased formation of ceramide via the de novo and SMase pathways leading to antinociceptive tolerance; this is blocked by inhibitors of ceramide biosynthesis (this study and [28]). Ceramide fosters the development of tolerance at least in part by neuroimmune activation, formation of PN, PN-mediated inactivation of MnSOD [28] and neuronal apopotosis.

Fig. 2. The development of morphine antinociceptive tolerance and activation of ceramide synthase in dorsal horn tissues are attenuated by FB1

Co-administration of morphine with the ceramide synthesis inhibitor FB1 (1 mg/kg/day, n=10) inhibited both the ceramide synthase activity (bars) and the development of morphine antinociceptive tolerance (closed circle line) in a dose-dependent manner (0.25-1 mg/kg/day, n=10). Results are expressed as mean ± SEM for 10 animals. *P<0.001 for FB1-Mor vs Veh-Mor.

Fig. 3. Ceramide synthesis was necessary for the development of oxidative DNA damage and PARP activation

When compared to Veh-Sal, chronic administration of morphine (Veh-Mor) led to oxidative DNA damage as evidenced by a significant increase in 8OHdG (A) and a substantial activation of PARP (B). These events were blocked in a dose-dependent manner by co-administration of morphine with FB1 (0.25-1 mg/kg/d, n=10) (A, B). Results are expressed as mean ± SEM for n=10 animals. * P<0.01 for Veh-Mor versus Veh-Sal; †P<0.01 and ††P<0.001 for FB1-Mor versus Veh-Mor.

Fig. 4. Biochemical indices of spinal cord apoptosis were dependent on ceramide synthesis

When compared to Veh-Sal, chronic administration of morphine (Veh-Mor) increased the activity of caspase-3 (A) in dorsal horn tissues which was inhibited in a dose-dependent manner by FB1 (n=10). Furthermore, chronic administration of morphine increased Bax (B,B1) and concomitantly decreased Bcl-2 (C,C1) as determined by Western blot in dorsal horn tissues. These events were blocked by FB1 (1 mg/kg/d; B,B1,C,C1). Results are expressed as mean ± SEM for n=5 (B1,C1) and n=10 (A) animals. * P<0.01 for Veh-Mor versus Veh-Sal; †P<0.01 and ††P<0.001 for FB1-Mor versus Veh-Mor.

Fig. 5. Visualization of ceramide-dependent spinal cord apoptosis

No apoptotic cells were detectable using Annexin-V FITC coloration in the spinal cord tissue of non-tolerant mice (A,A1). In tolerant animals a marked appearance of stain positive for Annexin-V FITC was observed (B). In addition, several cells showed positive staining with PI (B1). Spinal cord section from FB1-treated mice demonstrated a marked reduction in the number of apoptotic cells (C,C1). Figs A2, B2 and C2 represent the overlap images of panels A-A1, B-B1 and C-C1 respectively, superimposed on transmission light images of identical fields, depicting a significant number of cells captured in each field. Figure is representative of at least 3 independent experiments.