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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 7 μm in thick sections from the dorsal root ganglion reveals significant differences in satellite cell activation, phosphorylated p38MAPK, IL-1β, and IL-10 in i.t. AM1241-injected rats. (A, B) GFAP (satellite cell activation) expression was increased in animals with CCI compared to sham-treated rats, given i.t. vehicle ipsilaterally as well as contralaterally, and robust bilateral suppression of GFAP IR was observed in rats given i.t. AM1241. (C, D) A small but significant unilateral increase in Phospho-p38 (activated) IR was detected on the side ipsilateral to the CCI treatment with i.t. vehicle, while i.t. AM1241 attenuated increased p-p38MAPK IR in CCI-treated rats. (E, F) Unilateral IL-1β IR was increased in DRG on the side ipsilateral to the CCI manipulation compared to sham-treated controls, while IL-1β expression was substantially decreased in CCI-treated rats with i.t. AM1241. (G, H) Ipsilateral, but not contralateral, IL-10 IR was significantly decreased in CCI-treated neuropathic rats given i.t. vehicle of AM1241 compared to sham controls given either i.t. vehicle or AM1241. However, a robust increase in IL-10 IR in the DRG of CCI-neuropathic rats given i.t. AM1241 was detected.
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fig07: Immunofluorescent intensity quantification of 7 μm in thick sections from the dorsal root ganglion reveals significant differences in satellite cell activation, phosphorylated p38MAPK, IL-1β, and IL-10 in i.t. AM1241-injected rats. (A, B) GFAP (satellite cell activation) expression was increased in animals with CCI compared to sham-treated rats, given i.t. vehicle ipsilaterally as well as contralaterally, and robust bilateral suppression of GFAP IR was observed in rats given i.t. AM1241. (C, D) A small but significant unilateral increase in Phospho-p38 (activated) IR was detected on the side ipsilateral to the CCI treatment with i.t. vehicle, while i.t. AM1241 attenuated increased p-p38MAPK IR in CCI-treated rats. (E, F) Unilateral IL-1β IR was increased in DRG on the side ipsilateral to the CCI manipulation compared to sham-treated controls, while IL-1β expression was substantially decreased in CCI-treated rats with i.t. AM1241. (G, H) Ipsilateral, but not contralateral, IL-10 IR was significantly decreased in CCI-treated neuropathic rats given i.t. vehicle of AM1241 compared to sham controls given either i.t. vehicle or AM1241. However, a robust increase in IL-10 IR in the DRG of CCI-neuropathic rats given i.t. AM1241 was detected.

Mentions: Immunohistochemical detection of GFAP, IL-1β, p-p38MAPK, and anti-inflammatory IL-10 in L4–L5 DRG that correspond to the ipsilateral and contralateral spinal cord segments was quantified. Results revealed that compared to non-neuropathic control rats, CCI-induced neuropathic rats displayed a robust bilateral increase in GFAP IR in DRG (ipsilateral ANOVA, F(1,8) = 9.133; P = 0.0165; contralateral ANOVA, F(1,8) = 8.443; P = 0.0197, respectively) (Fig. 7A and 7B). However, i.t. AM1241 injection robustly blocked bilateral increases in GFAP IR (ipsilateral ANOVA, F(1,8) = 27.19; P = 0.0008; contralateral ANOVA, F(1,8) = 5.223; P = 0.0516, respectively) (Fig. 7A and 7B). Intriguingly, DRG changes in levels of p-p38MAPK IR occurred in the ipsilateral (ANOVA, F(1,8) = 6.885; P = 0.0305), but not the contralateral DRG (ANOVA, F(1,8) = 0.2013; P = 0.6656) to the sciatic nerve damage (Fig. 7C and 7D), and i.t. AM1241 injection revealed ipsilateral p-p38MAPK IR that was similar to controls (ANOVA, F(1,8) = 15.92; P = 0.0040). No change in p38MAPK IR was detected in the contralateral DRG (ANOVA, F(1,8) = 2.051; P = 0.1900). This unilateral change was also observed with IL-1β IR due to CCI surgery (ipsilateral ANOVA, F(1,8) = 6.414; P = 0.0351; contralateral ANOVA, F(1,8) = 0.3111; P = 0.5923), and AM1241 treatment resulted in levels similar to controls (ipsilateral ANOVA, F(1,8) = 52.03; P < 0.0001; contralateral ANOVA, F(1,8) = 0.2221; P = 0.6500) (Fig. 7E and 7F). An intriguing unilateral decrease in IL-10 IR was measured in CCI neuropathic rats only (ipsilateral ANOVA, F(1,8) = 17.42; P = 0.0031; contralateral ANOVA, F(1,8) = 1.583; P = 0.2438), while an i.t. AM1241 injection resulted in increased IL-10 IR levels that were similar to controls (ipsilateral ANOVA, F(1,8) = 22.83; P = 0.0014; contralateral ANOVA, F(1,8) = 1.327; P = 0.2826) (Fig. 7G and 7H). Collectively, these data show that while GFAP-positive satellite cells in bilateral DRG are a target of AM1241, only ipsilateral IL-1β, p-p38MAPK, and IL-10 IR levels are altered.


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 7 μm in thick sections from the dorsal root ganglion reveals significant differences in satellite cell activation, phosphorylated p38MAPK, IL-1β, and IL-10 in i.t. AM1241-injected rats. (A, B) GFAP (satellite cell activation) expression was increased in animals with CCI compared to sham-treated rats, given i.t. vehicle ipsilaterally as well as contralaterally, and robust bilateral suppression of GFAP IR was observed in rats given i.t. AM1241. (C, D) A small but significant unilateral increase in Phospho-p38 (activated) IR was detected on the side ipsilateral to the CCI treatment with i.t. vehicle, while i.t. AM1241 attenuated increased p-p38MAPK IR in CCI-treated rats. (E, F) Unilateral IL-1β IR was increased in DRG on the side ipsilateral to the CCI manipulation compared to sham-treated controls, while IL-1β expression was substantially decreased in CCI-treated rats with i.t. AM1241. (G, H) Ipsilateral, but not contralateral, IL-10 IR was significantly decreased in CCI-treated neuropathic rats given i.t. vehicle of AM1241 compared to sham controls given either i.t. vehicle or AM1241. However, a robust increase in IL-10 IR in the DRG of CCI-neuropathic rats given i.t. AM1241 was detected.
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Related In: Results  -  Collection

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fig07: Immunofluorescent intensity quantification of 7 μm in thick sections from the dorsal root ganglion reveals significant differences in satellite cell activation, phosphorylated p38MAPK, IL-1β, and IL-10 in i.t. AM1241-injected rats. (A, B) GFAP (satellite cell activation) expression was increased in animals with CCI compared to sham-treated rats, given i.t. vehicle ipsilaterally as well as contralaterally, and robust bilateral suppression of GFAP IR was observed in rats given i.t. AM1241. (C, D) A small but significant unilateral increase in Phospho-p38 (activated) IR was detected on the side ipsilateral to the CCI treatment with i.t. vehicle, while i.t. AM1241 attenuated increased p-p38MAPK IR in CCI-treated rats. (E, F) Unilateral IL-1β IR was increased in DRG on the side ipsilateral to the CCI manipulation compared to sham-treated controls, while IL-1β expression was substantially decreased in CCI-treated rats with i.t. AM1241. (G, H) Ipsilateral, but not contralateral, IL-10 IR was significantly decreased in CCI-treated neuropathic rats given i.t. vehicle of AM1241 compared to sham controls given either i.t. vehicle or AM1241. However, a robust increase in IL-10 IR in the DRG of CCI-neuropathic rats given i.t. AM1241 was detected.
Mentions: Immunohistochemical detection of GFAP, IL-1β, p-p38MAPK, and anti-inflammatory IL-10 in L4–L5 DRG that correspond to the ipsilateral and contralateral spinal cord segments was quantified. Results revealed that compared to non-neuropathic control rats, CCI-induced neuropathic rats displayed a robust bilateral increase in GFAP IR in DRG (ipsilateral ANOVA, F(1,8) = 9.133; P = 0.0165; contralateral ANOVA, F(1,8) = 8.443; P = 0.0197, respectively) (Fig. 7A and 7B). However, i.t. AM1241 injection robustly blocked bilateral increases in GFAP IR (ipsilateral ANOVA, F(1,8) = 27.19; P = 0.0008; contralateral ANOVA, F(1,8) = 5.223; P = 0.0516, respectively) (Fig. 7A and 7B). Intriguingly, DRG changes in levels of p-p38MAPK IR occurred in the ipsilateral (ANOVA, F(1,8) = 6.885; P = 0.0305), but not the contralateral DRG (ANOVA, F(1,8) = 0.2013; P = 0.6656) to the sciatic nerve damage (Fig. 7C and 7D), and i.t. AM1241 injection revealed ipsilateral p-p38MAPK IR that was similar to controls (ANOVA, F(1,8) = 15.92; P = 0.0040). No change in p38MAPK IR was detected in the contralateral DRG (ANOVA, F(1,8) = 2.051; P = 0.1900). This unilateral change was also observed with IL-1β IR due to CCI surgery (ipsilateral ANOVA, F(1,8) = 6.414; P = 0.0351; contralateral ANOVA, F(1,8) = 0.3111; P = 0.5923), and AM1241 treatment resulted in levels similar to controls (ipsilateral ANOVA, F(1,8) = 52.03; P < 0.0001; contralateral ANOVA, F(1,8) = 0.2221; P = 0.6500) (Fig. 7E and 7F). An intriguing unilateral decrease in IL-10 IR was measured in CCI neuropathic rats only (ipsilateral ANOVA, F(1,8) = 17.42; P = 0.0031; contralateral ANOVA, F(1,8) = 1.583; P = 0.2438), while an i.t. AM1241 injection resulted in increased IL-10 IR levels that were similar to controls (ipsilateral ANOVA, F(1,8) = 22.83; P = 0.0014; contralateral ANOVA, F(1,8) = 1.327; P = 0.2826) (Fig. 7G and 7H). Collectively, these data show that while GFAP-positive satellite cells in bilateral DRG are a target of AM1241, only ipsilateral IL-1β, p-p38MAPK, and IL-10 IR levels are altered.

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