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Immunofluorescent spectral analysis reveals the intrathecal cannabinoid agonist, AM1241, produces spinal anti-inflammatory cytokine responses in neuropathic rats exhibiting relief from allodynia.

Wilkerson JL, Gentry KR, Dengler EC, Wallace JA, Kerwin AA, Kuhn MN, Zvonok AM, Thakur GA, Makriyannis A, Milligan ED - Brain Behav (2012)

Bottom Line: During pathological pain, the actions of the endocannabinoid system, including the cannabinoid 2 receptor (CB(2)R), leads to effective anti-allodynia and modifies a variety of spinal microglial and astrocyte responses.AM1241 produced profound anti-allodynia with corresponding immunoreactive levels of p38 mitogen-activated kinase, IL-1β, IL-10, the endocannabinoid enzyme monoacylglycerol lipase, and astrocyte activation markers that were similar to nonneuropathic controls.The differences in fluorescent markers were determined within discrete anatomical regions by applying spectral analysis methods, which virtually eliminated nonspecific signal during the quantification of specific immunofluorescent intensity.

View Article: PubMed Central - PubMed

ABSTRACT
During pathological pain, the actions of the endocannabinoid system, including the cannabinoid 2 receptor (CB(2)R), leads to effective anti-allodynia and modifies a variety of spinal microglial and astrocyte responses. Here, following spinal administration of the CB(2)R compound, AM1241, we examined immunoreactive alterations in markers for activated p38 mitogen-activated protein kinase, interleukin-1β (IL-1β), the anti-inflammatory cytokine, interleukin-10 (IL-10) as well as degradative endocannabinoid enzymes, and markers for altered glial responses in neuropathic rats. In these studies, the dorsal horn of the spinal cord and dorsal root ganglia were examined. AM1241 produced profound anti-allodynia with corresponding immunoreactive levels of p38 mitogen-activated kinase, IL-1β, IL-10, the endocannabinoid enzyme monoacylglycerol lipase, and astrocyte activation markers that were similar to nonneuropathic controls. In contrast, spinal AM1241 did not suppress the increased microglial responses observed in neuropathic rats. The differences in fluorescent markers were determined within discrete anatomical regions by applying spectral analysis methods, which virtually eliminated nonspecific signal during the quantification of specific immunofluorescent intensity. These data reveal expression profiles that support the actions of intrathecal AM1241 control pathological pain through anti-inflammatory mechanisms by modulating critical glial factors, and additionally decrease expression levels of endocannabinoid degradative enzymes.

No MeSH data available.


Intrathecal (i.t.) AM1241, a cannabinoid 2 receptor agonist reverses CCI-induced allodynia. (A and B) AM1241 reverses CCI-induced allodynia in a dose-dependent manner. Before surgical manipulation, all AM1241 BL values of experimental groups exhibited similar ipsilateral and contralateral BL thresholds; CCI surgery produced significant bilateral allodynia at Day 3 and 10 following injury compared to sham-treated animals. Responses from AM1241 (10 μg) maximally reversed CCI-induced allodynia, (black squares), at 1.5 h with allodynia fully returning by 3 h after i.t. administration.
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fig02: Intrathecal (i.t.) AM1241, a cannabinoid 2 receptor agonist reverses CCI-induced allodynia. (A and B) AM1241 reverses CCI-induced allodynia in a dose-dependent manner. Before surgical manipulation, all AM1241 BL values of experimental groups exhibited similar ipsilateral and contralateral BL thresholds; CCI surgery produced significant bilateral allodynia at Day 3 and 10 following injury compared to sham-treated animals. Responses from AM1241 (10 μg) maximally reversed CCI-induced allodynia, (black squares), at 1.5 h with allodynia fully returning by 3 h after i.t. administration.

Mentions: Limited evaluation exists for i.t. spinal application of CB2R agonists to control allodynia produced by CCI, a widely used and well-characterized rodent model of chronic peripheral neuropathy with related pain-like behaviors (Bennett and Xie 1988). We first examined if the putative CB2R agonist from the aminoalkylindole class, AM1241 (36-fold CB2R > CB1R) (Yao et al. 2006; Thakur et al. 2009), could reverse ongoing allodynia produced by CCI. Prior to surgical manipulation, all groups exhibited similar bilateral (ipsilateral and contralateral) BL thresholds (ANOVA, F(5,35) = 1.105; P = 0.3764 and ANOVA, F(5,35) = 2.632; P = 0.5884, respectively) (Fig. 2A and 2B). Following CCI, clear bilateral allodynia developed by Day 3 and continued chronically through Day 10 compared to sham-operated rats (ANOVA, F(10,56) = 73.23; P < 0.0001 and ANOVA, F(10,56) = 71.32; P < 0.0001, respectively). On Day 10, compared to i.t. control injected neuropathic rats, AM1241 produced a dose-dependent reversal of allodynia, with maximal reversal observed at 1.5 h following the highest injected dose (10 μg). However, allodynia fully returned by 3 h after i.t. AM1241 treatment, with allodynia remaining constant through 24 h. While 0.1 μg produced attenuated allodynia, 0.01 μg did not alter allodynia for either the ipsilateral (Fig. 2A) (ANOVA, F(15,84) = 138.8; P < 0.0001) or contralateral (Fig. 2B) hindpaw responses (ANOVA, F(15,84) = 131.6; P < 0.0001). Post hoc analysis revealed that 10 μg AM1241 yielded maximal reversal similar to pretreatment BL values at 1.5 h after injection (P > 0.05).


Immunofluorescent spectral analysis reveals the intrathecal cannabinoid agonist, AM1241, produces spinal anti-inflammatory cytokine responses in neuropathic rats exhibiting relief from allodynia.

Wilkerson JL, Gentry KR, Dengler EC, Wallace JA, Kerwin AA, Kuhn MN, Zvonok AM, Thakur GA, Makriyannis A, Milligan ED - Brain Behav (2012)

Intrathecal (i.t.) AM1241, a cannabinoid 2 receptor agonist reverses CCI-induced allodynia. (A and B) AM1241 reverses CCI-induced allodynia in a dose-dependent manner. Before surgical manipulation, all AM1241 BL values of experimental groups exhibited similar ipsilateral and contralateral BL thresholds; CCI surgery produced significant bilateral allodynia at Day 3 and 10 following injury compared to sham-treated animals. Responses from AM1241 (10 μg) maximally reversed CCI-induced allodynia, (black squares), at 1.5 h with allodynia fully returning by 3 h after i.t. administration.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3345359&req=5

fig02: Intrathecal (i.t.) AM1241, a cannabinoid 2 receptor agonist reverses CCI-induced allodynia. (A and B) AM1241 reverses CCI-induced allodynia in a dose-dependent manner. Before surgical manipulation, all AM1241 BL values of experimental groups exhibited similar ipsilateral and contralateral BL thresholds; CCI surgery produced significant bilateral allodynia at Day 3 and 10 following injury compared to sham-treated animals. Responses from AM1241 (10 μg) maximally reversed CCI-induced allodynia, (black squares), at 1.5 h with allodynia fully returning by 3 h after i.t. administration.
Mentions: Limited evaluation exists for i.t. spinal application of CB2R agonists to control allodynia produced by CCI, a widely used and well-characterized rodent model of chronic peripheral neuropathy with related pain-like behaviors (Bennett and Xie 1988). We first examined if the putative CB2R agonist from the aminoalkylindole class, AM1241 (36-fold CB2R > CB1R) (Yao et al. 2006; Thakur et al. 2009), could reverse ongoing allodynia produced by CCI. Prior to surgical manipulation, all groups exhibited similar bilateral (ipsilateral and contralateral) BL thresholds (ANOVA, F(5,35) = 1.105; P = 0.3764 and ANOVA, F(5,35) = 2.632; P = 0.5884, respectively) (Fig. 2A and 2B). Following CCI, clear bilateral allodynia developed by Day 3 and continued chronically through Day 10 compared to sham-operated rats (ANOVA, F(10,56) = 73.23; P < 0.0001 and ANOVA, F(10,56) = 71.32; P < 0.0001, respectively). On Day 10, compared to i.t. control injected neuropathic rats, AM1241 produced a dose-dependent reversal of allodynia, with maximal reversal observed at 1.5 h following the highest injected dose (10 μg). However, allodynia fully returned by 3 h after i.t. AM1241 treatment, with allodynia remaining constant through 24 h. While 0.1 μg produced attenuated allodynia, 0.01 μg did not alter allodynia for either the ipsilateral (Fig. 2A) (ANOVA, F(15,84) = 138.8; P < 0.0001) or contralateral (Fig. 2B) hindpaw responses (ANOVA, F(15,84) = 131.6; P < 0.0001). Post hoc analysis revealed that 10 μg AM1241 yielded maximal reversal similar to pretreatment BL values at 1.5 h after injection (P > 0.05).

Bottom Line: During pathological pain, the actions of the endocannabinoid system, including the cannabinoid 2 receptor (CB(2)R), leads to effective anti-allodynia and modifies a variety of spinal microglial and astrocyte responses.AM1241 produced profound anti-allodynia with corresponding immunoreactive levels of p38 mitogen-activated kinase, IL-1β, IL-10, the endocannabinoid enzyme monoacylglycerol lipase, and astrocyte activation markers that were similar to nonneuropathic controls.The differences in fluorescent markers were determined within discrete anatomical regions by applying spectral analysis methods, which virtually eliminated nonspecific signal during the quantification of specific immunofluorescent intensity.

View Article: PubMed Central - PubMed

ABSTRACT
During pathological pain, the actions of the endocannabinoid system, including the cannabinoid 2 receptor (CB(2)R), leads to effective anti-allodynia and modifies a variety of spinal microglial and astrocyte responses. Here, following spinal administration of the CB(2)R compound, AM1241, we examined immunoreactive alterations in markers for activated p38 mitogen-activated protein kinase, interleukin-1β (IL-1β), the anti-inflammatory cytokine, interleukin-10 (IL-10) as well as degradative endocannabinoid enzymes, and markers for altered glial responses in neuropathic rats. In these studies, the dorsal horn of the spinal cord and dorsal root ganglia were examined. AM1241 produced profound anti-allodynia with corresponding immunoreactive levels of p38 mitogen-activated kinase, IL-1β, IL-10, the endocannabinoid enzyme monoacylglycerol lipase, and astrocyte activation markers that were similar to nonneuropathic controls. In contrast, spinal AM1241 did not suppress the increased microglial responses observed in neuropathic rats. The differences in fluorescent markers were determined within discrete anatomical regions by applying spectral analysis methods, which virtually eliminated nonspecific signal during the quantification of specific immunofluorescent intensity. These data reveal expression profiles that support the actions of intrathecal AM1241 control pathological pain through anti-inflammatory mechanisms by modulating critical glial factors, and additionally decrease expression levels of endocannabinoid degradative enzymes.

No MeSH data available.