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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.


Related in: MedlinePlus

Immunofluorescent intensity quantification of the spinal cord dorsal horn reveals that AM1241 reduces the expression of the endocannabinoid degradative enzyme, MAGL but does not alter FAAH. (A, B) Compared to control rats, MAGL IR expression was increased ipsilaterally, with strong trends contralaterally in CCI-treated neuropathic rats that received i.t vehicle of AM1241, while spinal MAGL IR in CCI-treated rats given i.t. AM1241 was substantially reduced. (C, D) No changes in FAAH IR expression in ipsilateral and contralateral dorsal horn of either sham- or CCI-neuropathic rats given either i.t. vehicle or AM1241 was observed. All sections were 7 μm in thickness.
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fig06: Immunofluorescent intensity quantification of the spinal cord dorsal horn reveals that AM1241 reduces the expression of the endocannabinoid degradative enzyme, MAGL but does not alter FAAH. (A, B) Compared to control rats, MAGL IR expression was increased ipsilaterally, with strong trends contralaterally in CCI-treated neuropathic rats that received i.t vehicle of AM1241, while spinal MAGL IR in CCI-treated rats given i.t. AM1241 was substantially reduced. (C, D) No changes in FAAH IR expression in ipsilateral and contralateral dorsal horn of either sham- or CCI-neuropathic rats given either i.t. vehicle or AM1241 was observed. All sections were 7 μm in thickness.

Mentions: Endocannabinoids known to produce anti-allodynic effects are metabolized via enzymatic hydrolysis by fatty acid amide hydrolase (FAAH) and/or MAGL (Basavarajappa 2007). Inhibition of FAAH or MAGL increases the bioavailablity of CNS endocannabinoids with a corresponding attenuation of neuropathic pain rats (Kinsey et al. 2009; Long et al. 2009). Whether FAAH and MAGL IR expression levels are altered in the dorsal horn following i.t. CB2R agonist injections in neuropathic rats, is unknown. Therefore, we examined potential changes in MAGL and FAAH IR in tissue sections from rats given i.t. AM1241. Compared to non-neuropathic control rats, neuropathic rats showed a robust ipsilateral (ANOVA, F(1,8) = 34.19; P = 0.0004) and contralateral (ANOVA, F(1,8) = 27.51; P = 0.0008) increase in dorsal horn MAGL IR (Fig. 6A and 6B). In contrast, spinal tissue collected from rats given an i.t. AM1241 injection revealed significantly lower bilateral MAGL IR (ipsilateral ANOVA, F(1,8) = 8.356; P = 0.0202; contralateral ANOVA, F(1,8) = 4.146; P = 0.0761, respectively) (Fig. 6A and 6B). Interestingly, no interpretable or meaningful change in FAAH IR between non-neuropathic and neuropathic CCI rats was observed following surgical manipulation (ipsilateral ANOVA, F(1,8) = 8.072; P = 0.0218; contralateral ANOVA, F(1,8) = 0.09666; P = 0.7638), or following i.t. AM1241 or vehicle treatment (ANOVA, F(1,8) = 0.5436; P = 0.4820 and ANOVA, F(1,8) = 2.174; P = 0.1786, respectively) (Fig. 6C and 6D).


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)

Immunofluorescent intensity quantification of the spinal cord dorsal horn reveals that AM1241 reduces the expression of the endocannabinoid degradative enzyme, MAGL but does not alter FAAH. (A, B) Compared to control rats, MAGL IR expression was increased ipsilaterally, with strong trends contralaterally in CCI-treated neuropathic rats that received i.t vehicle of AM1241, while spinal MAGL IR in CCI-treated rats given i.t. AM1241 was substantially reduced. (C, D) No changes in FAAH IR expression in ipsilateral and contralateral dorsal horn of either sham- or CCI-neuropathic rats given either i.t. vehicle or AM1241 was observed. All sections were 7 μm in thickness.
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Related In: Results  -  Collection

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fig06: Immunofluorescent intensity quantification of the spinal cord dorsal horn reveals that AM1241 reduces the expression of the endocannabinoid degradative enzyme, MAGL but does not alter FAAH. (A, B) Compared to control rats, MAGL IR expression was increased ipsilaterally, with strong trends contralaterally in CCI-treated neuropathic rats that received i.t vehicle of AM1241, while spinal MAGL IR in CCI-treated rats given i.t. AM1241 was substantially reduced. (C, D) No changes in FAAH IR expression in ipsilateral and contralateral dorsal horn of either sham- or CCI-neuropathic rats given either i.t. vehicle or AM1241 was observed. All sections were 7 μm in thickness.
Mentions: Endocannabinoids known to produce anti-allodynic effects are metabolized via enzymatic hydrolysis by fatty acid amide hydrolase (FAAH) and/or MAGL (Basavarajappa 2007). Inhibition of FAAH or MAGL increases the bioavailablity of CNS endocannabinoids with a corresponding attenuation of neuropathic pain rats (Kinsey et al. 2009; Long et al. 2009). Whether FAAH and MAGL IR expression levels are altered in the dorsal horn following i.t. CB2R agonist injections in neuropathic rats, is unknown. Therefore, we examined potential changes in MAGL and FAAH IR in tissue sections from rats given i.t. AM1241. Compared to non-neuropathic control rats, neuropathic rats showed a robust ipsilateral (ANOVA, F(1,8) = 34.19; P = 0.0004) and contralateral (ANOVA, F(1,8) = 27.51; P = 0.0008) increase in dorsal horn MAGL IR (Fig. 6A and 6B). In contrast, spinal tissue collected from rats given an i.t. AM1241 injection revealed significantly lower bilateral MAGL IR (ipsilateral ANOVA, F(1,8) = 8.356; P = 0.0202; contralateral ANOVA, F(1,8) = 4.146; P = 0.0761, respectively) (Fig. 6A and 6B). Interestingly, no interpretable or meaningful change in FAAH IR between non-neuropathic and neuropathic CCI rats was observed following surgical manipulation (ipsilateral ANOVA, F(1,8) = 8.072; P = 0.0218; contralateral ANOVA, F(1,8) = 0.09666; P = 0.7638), or following i.t. AM1241 or vehicle treatment (ANOVA, F(1,8) = 0.5436; P = 0.4820 and ANOVA, F(1,8) = 2.174; P = 0.1786, respectively) (Fig. 6C and 6D).

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.


Related in: MedlinePlus