Crucial role of CB(2) cannabinoid receptor in the regulation of central immune responses during neuropathic pain

Abstract

Neuropathic pain is a clinical manifestation of nerve injury difficult to treat even with potent analgesic compounds. Here, we used different lines of genetically modified mice to clarify the role played by CB(2) cannabinoid receptors in the regulation of the central immune responses leading to the development of neuropathic pain. CB(2) knock-out mice and wild-type littermates were exposed to sciatic nerve injury, and both genotypes developed a similar hyperalgesia and allodynia in the ipsilateral paw. Most strikingly, knock-outs also developed a contralateral mirror image pain, associated with an enhanced microglial and astrocytic expression in the contralateral spinal horn. In agreement, hyperalgesia, allodynia, and microglial and astrocytic activation induced by sciatic nerve injury were attenuated in transgenic mice overexpressing CB(2) receptors. These results demonstrate the crucial role of CB(2) cannabinoid receptor in modulating glial activation in response to nerve injury. The enhanced manifestations of neuropathic pain were replicated in irradiated wild-type mice reconstituted with bone marrow cells from CB(2) knock-outs, thus demonstrating the implication of the CB(2) receptor expressed in hematopoietic cells in the development of neuropathic pain at the spinal cord.

Figure 1.

Development of neuropathic pain is enhanced in male CB₂ ^−/− after sciatic nerve injury when compared with CB₂ ^+/+. A single tight ligature around one-third or one-half of sciatic nerve was made to induce the neuropathic pain in CB₂ ^−/− (n = 16) and CB₂ ^+/+ mice (n = 16). Mice were tested in the ipsilateral and contralateral paw for evaluating mechanical allodynia (von Frey model; percentage of the basal CB₂ ^+/+ sham-operated values) (A), thermal hyperalgesia (plantar test; withdrawal latency in seconds) (B), and cold allodynia (cold-plate test; number of paw elevations) (C) on days 3, 6, 8, 10, and 15 after surgery. The black stars represent comparison between sciatic nerve injury and sham-operated animals. The white stars represent comparison between genotypes. One star, p < 0.05; two stars, p < 0.01. Error bars indicate SEM.

Figure 2.

Enhanced glial activation in spinal cord of CB₂ ^−/− after sciatic nerve injury when compared with CB₂ ^+/+ mice. A, Increase in iba-1 (microglia) (n = 5) and GFAP (astrocyte) (n = 5) staining in dorsal horn of lumbar spinal cord after sciatic nerve injury in CB₂ ^−/− and in CB₂ ^+/+ mice. White bars, Ipsilateral paw; black bars, contralateral paw. Iba-1 and GFAP visualized with CY3 anti-rabbit (red). Data are expressed as percentage of stained area. The black stars represent comparisons between sham-operated and nerve injury between ipsilateral and contralateral paw. The white stars represent comparisons between genotypes. One star, p < 0.05; two stars, p < 0.01. Error bars indicate SEM. B, Representative confocal images of the immunostaining of iba-1 and GFAP recorded with 40× objective in lumbar dorsal horn of CB₂ ^+/+ and CB₂ ^−/−. C, Representative low-magnification images of iba-1 and GFAP immunostaining recorded with 10× objective in the lumbar dorsal horn of sciatic nerve injury CB₂ ^+/+ and CB₂ ^−/− mice.

Figure 3.

CB₂ expression and colocalization in male transgenic mice overexpressing CB₂ receptors. A, Relative abundance of endogenous and transgenic CB₂ RNA in PAG, thalamus, cervical spinal cord, and thoracic spinal cord of transgenic mice was determined by real-time PCR quantification. After being analyzed using the ΔCt method, data were normalized to GAPDH expression. The group with the lower value was assigned a value of 1. The stars represent differences in relative transcript abundance between endogenous and transgenic CB₂ receptor (one-way repeated-measures ANOVA). Error bars indicate SEM. B, Negative immunostaining for CB₂ receptor in the lumbar spinal cord of CB₂ ^−/− mice that serves as a control for CB₂ staining in transgenic mice overexpressing CB₂ receptors. C, Double-fluorescence immunostaining for CB₂ receptor, GFAP (astrocyte), Neu-N (neuron), and iba-1 (microglia) in lumbar spinal cord sections from transgenic mice overexpressing CB₂ receptors. First plot, Immunostaining for CB₂ receptor (red; CY3 anti-rabbit) and for astrocyte marker GFAP (green; FITC anti-rabbit). Second plot, Same staining for CB₂ receptor (red; CY3 anti-rabbit) and for neuron (fluorescein-conjugated Neu-N). Third plot, Same staining for CB₂ receptor (red; CY3 anti-rabbit) and for microglial marker iba-1 (green; FITC anti-rabbit).

Figure 4.

Behavioral manifestations of neuropathic pain in male transgenic mice overexpressing CB₂ receptors. Development of neuropathic pain in male transgenic mice overexpressing CB₂ receptors (n = 12) and wild-type mice (n = 12). A single tight ligature around one-third or one-half of sciatic nerve was made to induce the neuropathic pain. Mice were tested in the ipsilateral and contralateral paw for evaluating mechanical allodynia (von Frey model; percentage of the basal CB₂ ^+/+ sham-operated values) (A), thermal hyperalgesia (plantar test; withdrawal latency in seconds) (B), and cold allodynia (cold-plate test; number of paw elevations) (C) on days 3, 6, 8, 10, and 15 after surgery. The black stars represent comparison between sciatic nerve injury and sham-operated animals. The white stars represent comparison between genotypes. One star, p < 0.05; two stars, p < 0.01. Error bars indicate SEM.

Figure 5.

Expression of glial activation in spinal cord of CB₂ ^+/+ and transgenic mice overexpressing CB₂ receptors. A, The increase of microglia and astrocytic response in dorsal horn of lumbar spinal cord after sciatic nerve injury is only observed in CB₂ ^+/+ mice. Iba-1 staining (microglia) (n = 4 per group) and GFAP staining (astrocytes) (n = 4 per group) were visualized with CY3 anti-rabbit (red). Ipsilateral dorsal horns were represented with white bars, and contralateral dorsal horns were represented with black bars. Data are expressed as percentage of stained area. Error bars indicate SEM. The black stars represent comparisons between sham-operated and nerve injury or between ipsilateral and contralateral paw. The white stars represent comparisons between genotypes. One star, p < 0.05; two stars, p < 0.01. B, Representative confocal images of the immunostaining of iba-1 (microglia) and GFAP (astrocyte) in lumbar dorsal horn of CB₂ ^+/+ and transgenic mice overexpressing CB₂ receptors recorded with the 40× objective.

Figure 6.

Reconstitution efficiency of BM chimeric mice and behavioral manifestations of neuropathic pain after transplantation of BM cells lacking CB₂ receptors in irradiated CB₂ ^+/+ mice. A, Lethally irradiated recipient mice (CD45.1) were reconstituted with BM from CB₂ ^−/− (left) or WT (right) animals expressing the congenic marker CD45.2. Flow cytometry of peripheral blood lymphocytes of BM chimeric mice was performed 8 weeks later by staining with anti-CD45.1 and -CD45.2 antibodies. Frequencies of CD45.2 donor-derived and CD45.1 recipient-derived cells are indicated in each quadrant. B, Presence of CB₂-derived microglia in the spinal cord of BM-chimeric mice 14 d after surgery. Representative flow cytometry demonstrating CD45.2⁺CD11b⁺ cells in the spinal cord of CB₂ ^−/− (CD45.2) control mice (left) and lethally irradiated CD45.1 congenic recipients after engraftment with CB₂ ^−/− BM cells (chimera-CB₂) (right). CD11b⁺ cells in the spinal cord of CD45.1 control mice (middle) are negative for the congenic marker CD45.2. Representative dot blots are shown. C, The following groups were studied: CB₂ ^−/− and CB₂ ^+/+ control animals, chimera-CB₂ (CB₂ ^+/+ mice receiving bone marrow from CB₂ ^−/− animals), and chimera-WT (CB₂ ^+/+ mice receiving bone marrow from CB₂ ^+/+ mice). Development of neuropathic pain in CB₂ ^+/+ (n = 10), CB₂ ^−/− (n = 10), chimera-WT (n = 8), and chimera-CB₂ (n = 12). Mechanical allodynia after partial sciatic nerve ligation was tested in the ipsilateral and contralateral paws on day 3, 6, 8, 10, and 15 after the surgery. The withdrawal thresholds are presented as percentage of the basal CB₂ ^+/+ values. Error bars indicate SEM. The black stars represent comparison between sciatic nerve injury and sham-operated animals. The white stars represent comparison between genotypes. One star, p < 0.05; two stars, p < 0.01.

Figure 7.

Histological changes in neuropathic pain after transplantation of BM cells lacking CB₂ receptors into irradiated CB₂ ^+/+ mice. A, Increase of microglia activation and astrocytic response in ipsilateral and contralateral dorsal horn of lumbar spinal cord after sciatic nerve injury in CB₂ ^−/− BM-chimeric mice (black bars) (n = 6) compared with sham-operated BM-chimeric mice (white bars) (n = 3). Iba-1 was used to stain microglia cells and GFAP antibody for astrocyte staining. Iba-1 staining and GFAP staining visualized with CY3 anti-rabbit (red). Data are expressed as percentage of stained area. Error bars indicate SEM. Black stars represent comparisons between sham-operated and nerve injury. One star, p < 0.05; two stars, p < 0.01. B, Representative confocal images of the immunostaining of iba-1 (microglia) and GFAP (astrocyte) recorded with 40× objective in lumbar dorsal horn of sham-operated and nerve injured CB₂ ^−/− BM-chimeric mice.