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Minocycline Treatment Inhibits Microglial Activation and Alters Spinal Levels of Endocannabinoids in a Rat Model of Neuropathic Pain


Minocycline Treatment Inhibits Microglial Activation and Alters Spinal Levels of Endocannabinoids in a Rat Model of Neuropathic Pain

Leonardo Guasti et al. Mol Pain.


Activation of spinal microglia contributes to aberrant pain responses associated with neuropathic pain states. Endocannabinoids (ECs) are present in the spinal cord, and inhibit nociceptive processing; levels of ECs may be altered by microglia which modulate the turnover of endocannabinoids in vitro. Here, we investigate the effect of minocycline, an inhibitor of activated microglia, on levels of the endocannabinoids anandamide and 2-arachidonoylglycerol (2-AG), and the related compound N-palmitoylethanolamine (PEA), in neuropathic spinal cord. Selective spinal nerve ligation (SNL) in rats resulted in mechanical allodynia and the presence of activated microglia in the ipsilateral spinal cord. Chronic daily treatment with minocycline (30 mg/kg, ip for 14 days) significantly reduced the development of mechanical allodynia at days 5, 10 and 14 post-SNL surgery, compared to vehicle-treated SNL rats (P < 0.001). Minocycline treatment also significantly attenuated OX-42 immunoreactivity, a marker of activated microglia, in the ipsilateral (P < 0.001) and contralateral (P < 0.01) spinal cord of SNL rats, compared to vehicle controls. Minocycline treatment significantly (P < 0.01) decreased levels of 2-AG and significantly (P < 0.01) increased levels of PEA in the ipsilateral spinal cord of SNL rats, compared to the contralateral spinal cord. Thus, activation of microglia affects spinal levels of endocannabinoids and related compounds in neuropathic pain states.


Figure 1
Figure 1
Chronic treatment with minocycline significantly attenuates spinal nerve ligation-induced reduction in mechanical paw withdrawal threshold. Minocycline (Mino, 30 mg/kg, ip) or vehicle (Veh, sterile water) was injected daily for 14 days and paw withdrawal threshold of the ipsilateral (Ipsi) and contralateral (Contra) hindpaw were measured using von Frey filaments (sham data not shown for clarity of figure). Data were analysed using 2-way ANOVA followed by Bonferroni's post-hoc tests, and are expressed as mean (± SEM, n = 6 rats/group). ** P < 0.01, *** P < 0.001 vs contralateral paw; δ P < 0.05, δδ P < 0.01, δδδ P < 0.001 vs vehicle-ipsilateral paw.
Figure 2
Figure 2
Chronic minocycline treatment attenuates the spinal nerve ligation-induced increase in OX-42 immunoreactivity (ir) in the lumbar spinal cord. Vehicle (A, B, C, sterile water) or minocycline (D, E, F, 30 mg/kg, ip) was injected daily for 14 days and spinal cords dissected and processed for immunohistochemical detection of OX-42ir at the level of L4 (A-a" and D-d"), L5 (B-b" and E-e") and L6 (C-c" and F-f"). High magnification images of the dorsal area used for OX-42ir signal quantification are also shown. Histograms (G-I) showing effects of minocycline or vehicle in L4-L6 spinal cord OX-42ir (white bars: ipsilateral side; black bars: contralateral side) are shown at the bottom. Data are expressed as mean ± SEM (n = 12 sections from 4 rats per group). Data were analysed using Kruskal Wallis non-parametric test followed by Dunn's multiple comparison posthoc analysis, * P < 0.05, ** P < 0.01, *** P < 0.001 vs ipsilateral-veh, # P < 0.05, ## P < 0.01 vs contralateral-vehicle. Scale bars: F = 1 mm (applies to A-F); f' = 100 μm (applies to a'-f' and a"-f").
Figure 3
Figure 3
Effects of chronic intraperitoneal dosing of minocycline (Mino, 30 mg/kg) or vehicle (Veh, sterile water) on the levels of endocannabinoids (A, B) and related fatty acid ethanolamides (C, D) in the ipsilateral spinal cord of spinal nerve ligated (SNL) or sham-operated rats. Data were analysed using Mann-Whitney nonparametric test and are expressed as a mean percentage of contralateral levels ± SEM (n = 5 – 6 rats/group). * P < 0.05, ** P < 0.01 vs contralateral paw. AEA, anandamide; 2-AG, 2-arachidonoylglycerol; PEA, N-palmitoylethanolamine; OEA, N-oleoylethanolamine.
Figure 4
Figure 4
Time course of hydrolysis of AEA (anandamide) and PEA (N-palmitoylethanolamine) in BV2 microglial cells. Data are means ± SEM of six independent experiments conducted in triplicate.

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