Immunofluorescent intensity quantification following AM1710 –induced reversal of allodynia. A total of 12 animals were used for both the behavioral experiment reported here and tissues from these animals were analyzed in the reported immunohistochemical experiments. A,B, Prior to CCI, all groups exhibited similar ipsilateral and contralateral BL thresholds (ANOVA, F_(3,11) =2.396; p=0.1438, and ANOVA, F_(3,11) =1.432; p=0.3036, respectively). CCI produced significant bilateral allodynia at Day 3 and continued to Day 10 compared to sham-treated animals (ANOVA, F_(1,8) =284.8; p< 0.0001, and ANOVA, F_(1,8)=222.9; p=0.0001, respectively). Behavioral responses following i.t. AM1710 (10 μg) produced maximal bilateral reversal of allodynia (ANOVA, F_(1,8)= 269.7; p<0.0001 and ANOVA, F_(1,8)=146.0; p<0.0001, respectively). At peak reversal, animals were sacrificed and spinal tissue was collected. C, D, Bilateral IL-10-immunoreactivity (IR) in the dorsal horn spinal cord was dramatically decreased in CCI-induced neuropathic rats compared to sham-treated rats. In stark contrast, treatment with AM1710 rescued IL-10 IR to basal levels in both the ipsilateral and contralateral dorsal spinal cord (ANOVA, F_(3,11)=12.36; p=0.0023; ANOVA, F_(3,11)=30.68; p<0.0001, respectively). Inset, C, No changes in expression of meningeal IL-10 IR between non-neuropathic sham and neuropathic CCI rats following i.t. AM1710 or equivolume vehicle were observed (ANOVA, F_(3,11)=1.109; p=0.4008). E, F, Compared to non-neuropathic sham-operated rats given i.t. AM1710 or equivolume vehicle, CCI-induced neuropathy produced a robust unilateral increase in IL-1β IR in i.t. vehicle injected animals. Conversely, i.t. administration of AM1710 reversed increased dorsal horn spinal IL-1β IR. IL-1β IR observed in the contralateral dorsal horn spinal cord was not substantially elevated when compared to non-neuropathic control animals. (ANOVA, F_(3,11)= 6.240; p=0.0172; ANOVA, F_(3,11)=3.354; p=0.0.760, respectively). G, H, Compared to non-neuropathic sham-operated rats given i.t. AM1710 or equivolume vehicle, CCI-induced neuropathy produced a robust p-p38MAPK bilateral IR increase in dorsal horn spinal cord tissues following i.t. vehicle injection. AM1710 administered i.t. did not reverse CCI-induced increases in p38MAPK IR. (ANOVA, F_(3,11)=4.221; p=0.0459; ANOVA, F_(3,11)=22.26; p=0.0003, respectively). I, J, No differences in DAPI nuclear stain fluorescent intensity were observed in AM1710 ipsilateral or contralateral dorsal horn (ANOVA, F_(3,11)=0.4571; p=0.7197, ANOVA, F_(3,11)=0.0006230; p=1.0, respectively). K, L, M, Representative spectrally unmixed images at 20x magnification of IL-10 fluorescent staining (green) with DAPI nuclear stain (blue). N, O, P, Representative spectrally unmixed images at 20x magnification of IL1β fluorescent staining (red) and DAPI nuclear stain (blue). Q, R, S, Representative spectrally unmixed images at 20x magnification of phospho-p38 fluorescent staining (green) with DAPI nuclear stain (blue). In all images the scale bar is equal to 50 μm.

Immunofluorescent intensity quantification of the spinal cord dorsal horn reveals i.t. AM1710 reduces the expression of the endocannabinoid degragative enzyme, MAGL. A, B, No changes in expression of FAAH IR between non-neuropathic sham and neuropathic CCI rats following i.t. AM1710 or equivolume vehicle were observed (ANOVA, F_(3,11)=1.967; p=0.1976; ANOVA, F_(3,11)=3.068; p=0.0910, respectively) C, D, Compared to non-neuropathic sham operated rats given i.t. AM1710 or equivolume vehicle, neuropathic rats given i.t. vehicle showed a robust bilateral increase in dorsal horn MAGL IR. In contrast, an i.t. AM1710 injection robustly suppressed bilateral increases in dorsal spinal MAGL IR (ANOVA, F_(3,11)=11.38; p=0.0029, ANOVA, F_(3,11)=5.444; p=0.00247, respectively). Inset, C, non-neuropathic sham rats given i.t. AM1710 as well as neuropathic rats given i.t. vehicle showed an increase in meningeal MAGL IR compared to non-neuropathic rats given i.t. vehicle (ANOVA, F_(3,11)=8.153; p=0.0081). E, F, G, H, Representative spectrally unmixed images at 20x magnification of MAGL fluorescent staining (green), and DAPI nuclear stain (blue). In all images the scale bar is equal to 50 μm.

Qualitative confocal images of cellular immunostaining of IL-10 in spinal cord. A–I, Spinal cord tissue from rats with CCI-AM1710 treatment. A, B, C, Immunostaining of IL-10 (green) in meninges and superficial laminae of the spinal cord dorsal horn is not co-labeled with GFAP (red) positive cells. DAPI nuclear labeling is blue. Arrows indicate IL-10 in the superficial laminae. D, E, F, Immunostaining of IL-10 (green) in the deeper laminae of the dorsal horn spinal cord is co-labeled yellow with GFAP (red) positive cells, with DAPI nuclear labeling (blue). Arrows indicate co-labeling of IL-10 and GFAP positive cells. G, H, I, Immunostaining of IL-10 (green) in the meninges and superficial laminae of the dorsal horn spinal cord is co-labeled (yellow) with Iba-1 (red) positive cells, with DAPI nuclear labeling (blue). Arrows indicate co-labeling of IL-10 and Iba-1 positive cells. J, K,L, Immunostaining of MAGL (green) in the deeper laminae of the dorsal horn is co-labeled yellow with Iba-1 (red) positive cells, with NF-H neuronal labeling (blue). An arrow indicates co-labeling of MAGL and an Iba-1 positive cell. In all images the scale bar is equal to 20 μm.

Selective i.t. cannabinoid 2 receptor agonist AM1710 reverses CCI-induced allodynia. A, B, AM1710 reverses CCI-induced allodynia in a dose-dependent manner. A total of 36 animals were used in this experiment. Prior to surgical manipulation, all groups exhibited similar bilateral (ipsilateral and contralateral) BL thresholds (ANOVA, F_(5,35) =1.982 ; p=0.1124, ANOVA, F_(5,35) =1.142; p=0.3616, respectively). Following CCI, clear bilateral allodynia developed by Day 3 and continued chronically through Day 10 compared to sham-operated rats. On Day 10, compared to i.t. control injected rats, AM1710 produced a dose-dependent reversal from allodynia, with maximal reversal observed at 3 hours following the highest injected dose (10 μg). However, allodynia fully returned by 5 hours after i.t. AM1710 treatment, with allodynia remaining stable through 24 hours (ipsilateral paw ANOVA, F_(15,84) = 187.6; p<0.0001; and contralateral paw, ANOVA, F_(15,84)=403.7; p<0.0001). While 1.0 μg produced attenuated allodynia, 0.1 μg did not alter allodynia for either the ipsilateral or contralateral hindpaws. Post hoc analysis revealed that 1μg AM1710 produced a robust reversal from allodynia at 2 hours following i.t. injection, while all AM1710 treated animals returned to allodynia by 4 hours (p<0.001).

Immunofluorescent intensity quantification of the spinal cord dorsal horn reveals differences in astrocyte but not microglial activation in neuropathic rats treated with AM1710. A, B, Compared to non-neuropathic sham-operated rats given i.t. AM1710 or equivolume vehicle, CCI-induced neuropathy produced a robust bilateral increase in spinal cord dorsal horn Iba-1 IR in rats given i.t. vehicle (ANOVA, F_(3,11)=58.94; p<0.0001, ANOVA, F_(3,11)=175.5; p<0.0001, respectively). Inset, A, significant changes in expression of meningeal Iba-1 IR between non-neuropathic sham and neuropathic CCI rats following i.t. AM1710 or equivolume vehicle were observed (ANOVA, F_(3,11)=22.55; p=0.0003). C, D, Compared to non-neuropathic sham-operated rats given i.t. AM1710 or equivolume vehicle, neuropathic rats demonstrated a robust bilateral increase in dorsal horn GFAP IR given i.t. vehicle (ANOVA, F_(3,11)=30.32; p=0.0001, ANOVA, F_(3,11)=31.57; p<0.001, respectively). E, F, G, H, Representative spectrally unmixed images at 20x magnification of GFAP fluorescent staining (red) and DAPI nuclear stain(blue). In all images the scale bar is equal to 50 μm.

Immunofluorescent intensity quantification of the dorsal root ganglion reveals differences in astrocyte activation levels, phosphorylated p38MAPK, IL1β and IL-10. A, B, Compared to non-neuropathic sham operated rats given i.t. AM1710 or equivolume vehicle, CCI-induced neuropathic rats given i.t. vehicle revealed a robust bilateral increase in GFAP IR. However, i.t. AM1710 injection robustly blocked bilateral increases in GFAP IR (ANOVA, F_(3,11)=28.56; p=0.0001, ANOVA, F_(3,11)=6.067; p=0.0186, respectively). C, D, DRG changes in levels of p-p38MAPK IR occurred in the ipsilateral(ANOVA, F_(3,11)=8.097; p=0.0083), but not the contralateral (ANOVA, F_(3,11)=0.01644; p=0.9969) spinal cord to the sciatic nerve damage. E, F, Unilateral change was also observed with IL-1β IR (ipsilateral ANOVA, F_(3,11)=9.291; p=0.0055, contralateral ANOVA, F_(3,11)=0.2395; p=0.8664). G, H, IL-10 IR was also unilaterally decreased in neuropathic animals following CCI surgery given i.t. vehicle treatment of AM1710 (ipsilateral ANOVA, F_(3,11)=12.01; p=0.0025, contralateral ANOVA, F_(3,11)=1.612; p=0.2618). I, J, K, Representative spectrally unmixed images of phospho-p38 at 20x magnification, phospho-p38 fluorescent staining (green) and DAPI nuclear stain (blue). L, M, N, IL-10 representative spectrally unmixed images at 20x, IL-10 fluorescent staining (green) and DAPI nuclear stain (blue). In all images the scale bar is equal to 50 μm.

AM1710 pre-treatment blocks gp120-induced allodynia and IL-1β cytokine production. A total of 24 animals were used for both the behavioral experiment reported here and tissues from these animals were analyzed in the reported ELISA experiments. A, B, Prior to surgical manipulation, all groups exhibited similar bilateral (ipsilateral and contralateral) BL thresholds (ANOVA, F_(3,23) =1.781; p=0.1833; F_(3,23) =2.311; p=0.1072, respectively). Following indwelling catheter implantation, there was no major effect of surgery on Day 6 after surgery thresholds when compared to BL thresholds or following the i.t. pretreatment of either AM1710 or vehicle (ANOVA, F_(6,60) = 0.9381; p=0.1311; ANOVA, F_(6,60)=2.415 ; p=0.1648, respectively). Animals given i.t. gp120 developed strong allodynia in both left and right paws at 20 min compared to pre-gp120 threshold values (ANOVA, F_(3,20)=61.72; p<0.0001 ANOVA, F_(3,20)=75.73; p<0.0001, respectively). C, Quantification of IL-1β protein by ELISA revealed IL-1β was significantly increased in left DRG of allodynic rats following i.t. gp120 given a pretreatment with i.t. vehicle of AM1710 (ANOVA, F_(3,13)= 7.785; p=0.0057). D, a trend in the levels of TNF-α protein, although not statistically significant (ANOVA, F_(3,14) = 2.977; p=0.0741).