Morphine tolerance attenuates the resolution of postoperative pain and enhances spinal microglial p38 and extracellular receptor kinase phosphorylation

Abstract

Persistent postoperative pain is a very common phenomenon which severely affects the lives of patients who develop it following common surgical procedures. Opioid analgesics are of limited efficacy in the treatment of persistent pain states because of side effects including antinociceptive tolerance. We have previously shown that surgical incision injury and morphine tolerance share similar mechanisms, including a CNS role of spinal cord glia. We therefore hypothesized that prior chronic morphine exposure would inhibit the resolution of postoperative allodynia through increased glial ionized calcium-binding adaptor protein 1 (Iba1) and glial fibrillary acidic protein (GFAP) protein expression and mitogen activated protein kinase (MAPK) activation. To test this hypothesis, rats were implanted with s.c. osmotic minipumps on day zero, releasing saline or morphine for 7 days preceding or 7 days preceding and following paw incision surgery, which was completed on day seven. Thermal hyperalgesia and mechanical allodynia were assessed postoperatively every 3 days. Chronic morphine attenuated the resolution of postoperative thermal hyperalgesia and mechanical allodynia through day 20. However, no changes in Iba1 or GFAP expression were observed in the spinal cord dorsal horn between groups. Assessment of MAPK protein phosphorylation revealed that chronic morphine administration enhanced both p38 and extracellular receptor kinase (pERK) phosphorylation compared to saline on day 20. p-p38 and pERK immunofluorescence were only observed to colocalize with a marker of microglial cells and not with markers of astrocytes or neurons. Together, these data demonstrate that chronic morphine administration attenuates the resolution of postoperative allodynia in association with microglial p38 and extracellular receptor kinase (ERK) phosphorylation, independent of changes in Iba1 and GFAP expression.

Figure 1

Chronic morphine administration attenuates the resolution of allodynia following paw incision surgery. (a) Thermal hyperalgesia measurement of acute analgesia and tolerance to continuous subcutaneous morphine administration. Thermal hyperalgesia (b) and mechanical allodynia (c) were measured in saline, morphine 7d and morphine 14d treatment groups following paw incision surgery on day 7. *p<0.05, **p<0.01, ***p<0.001 compared to saline. #p<0.05, ##p<0.01 compared to morphine 7d.

Figure 2

Chronic morphine administration does not affect contralateral hind paw sensitivity following paw incision surgery or upon withdrawal of morphine treatment. Thermal hyperalgesia (a) and mechanical allodynia (b) were measured in the contralateral hind paw of rats in saline, morphine 7d and morphine 14d treatment groups following paw incision surgery on day 7. Thermal (c) and mechanical (d) allodynia was also measured in the hindpaw of non-injured rats from day 7 (last day of morphine 7d pump output) to day 14 (last day of morphine 14d pump output).

Figure 3

Chronic morphine administration does not increase behavioral hypersensitivity following L5 nerve transection surgery. (a) Thermal hyperalgesia measurement of acute analgesia and tolerance to continuous subcutaneous morphine administration. Thermal hyperalgesia (b) and mechanical allodynia (c) were measured in saline, morphine 7d and morphine 14d treatment groups following L5 nerve transection surgery on day 7. **p<0.01, ***p<0.001 compared to saline.

Figure 4

Iba1 and GFAP expression is unaltered in morphine treated groups following paw incision injury. Representative western blot membranes of day 20 lumbar spinal cord protein from rats receiving saline or chronic subcutaneous morphine for 7 or 14 days probed for Iba1 (a) or GFAP (c) and β-actin protein expression. Quantification of fold changes of Iba1 (b) or GFAP (d) expression normalized to β-actin loading control ± SEM (n=6).

Figure 5

Chronic morphine treatment does not alter Iba1 and GFAP immunofluorescence in ipsilateral spinal cord dorsal horn regions following paw incision surgery on day 20. (a) Representative Iba1 immunofluorescence images of ipsilateral spinal cord dorsal horn sections from naïve, saline and morphine 14d treated animals on day 20. Quantification of Iba1 immunofluorescence in the whole dorsal horn (b), superficial dorsal horn (c) and deep dorsal horn (d) represented as number of pixels (n=6, 4 images per rat). (e) Representative GFAP immunofluorescence images of ipsilateral spinal cord dorsal horn sections from naïve, saline and morphine 14d treated animals on day 20. Quantification of GFAP immunofluorescence in the whole dorsal horn (b), superficial dorsal horn (c) and deep dorsal horn (d) represented as number of pixels (n=6, 4 images per rat).

Figure 6

p-p38 expression is enhanced in the morphine 7d and 14d treatment groups following paw incision injury. (a) Representative western blot membranes of day 20 lumbar spinal cord protein from rats treated with saline or chronic subcutaneous morphine for 7 or 14 days probed for p-p38, p38 and β-actin protein expression. (b) Quantification of fold changes of p-p38 expression normalized to p38 expression and β-actin loading control ± SEM (n=6). *p<0.05 compared to saline.

Figure 7

pERK expression is enhanced in the morphine 7d and 14d treatment group following paw incision injury. (a) Representative western blot membranes of day 20 lumbar spinal cord protein from rats treated with saline or chronic subcutaneous morphine for 7 or 14 days probed for pERK, ERK and β-actin protein expression. Quantification of fold changes of pERK 44 (b) and 42 (c) expression normalized to ERK expression and β-actin loading control ± SEM (n=6). *p<0.05 compared to saline.

Figure 8

Microglial p-p38 expression is enhanced in the morphine 7d and 14d treatment groups. Confocal images of p-p38 (red) and Iba1 (green) immunofluorescence colocalization in saline (a), morphine 7d (b) and morphine 14d (c) treatment groups on day 20 in the superficial dorsal horn of the spinal cord ipsilateral to paw incision injury. Higher magnification images of single cells are found in boxes. (d) Confocal images of p-p38 (red) and GFAP (green) immunofluorescence showing no colocalization in the morphine 14d treatment group on day 20. (e) Confocal images of p-p38 (red) and NeuN (green) immunofluorescence showing no colocalization in the morphine 14d treatment group on day 20. Scale bar equals 20 µm, magnified box width equals 20 µm.

Figure 9

Microglial pERK expression is enhanced in the morphine 7d and 14d treatment groups. Confocal images of pERK (red) and Iba1 (green) immunofluorescence colocalization in saline (a), morphine 7d (b) and morphine 14d (c) treatment groups on day 20 in the superficial dorsal horn of the spinal cord ipsilateral to paw incision injury. Higher magnification images of single cells are found in boxes. (d) Confocal images of pERK (red) and GFAP (green) immunofluorescence showing no colocalization in the morphine 14d treatment group on day 20. (e) Confocal images of pERK (red) and NeuN (green) immunofluorescence showing no colocalization in the morphine 14d treatment group on day 20. Scale bar equals 20 µm, magnified box width equals 20 µm.