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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 from 7-μm thick sections of dorsal horn spinal cord from behaviorally verified rats following i.t. vehicle or AM1241. (A, B) Prior to CCI, all groups exhibited similar ipsilateral and contralateral BL thresholds. CCI produced significant bilateral allodynia at Day 3 and 10 following injury compared to sham-treated animals. Behavioral responses following AM1241 (10 μg) produced maximal bilateral reversal of allodynia followed by tissue collection of immunofluorescent intensity quantification. (C, D) IL-10 expression was bilaterally decreased in CCI-treated rats that received i.t. vehicle compared to control sham-treated rats given either vehicle or AM1241, while IL-10 IR recovered to sham levels in CCI neuropathic rats given i.t. AM1241. (E, F) Compared to sham controls, IL-1β expression was increased ipsilaterally, but not contralaterally in CCI-treated animals given i.t. vehicle of AM1241. However, i.t. AM1241 in CCI-treated rats robustly suppressed increases in IL-1β IR. (G, H) Phospho-p38 expression was bilaterally increased in CCI-induced neuropathic rats treated with i.t vehicle of AM1241. Increased bilateral p-p38MAPK was significantly suppressed in CCI-treated rats given i.t. AM1241. (I, J) No differences in DAPI nuclear stain fluorescent intensity were observed in either sham control or CCI-treated rats given either i.t. vehicle or AM1241. (K, L, and M) Representative spectrally unmixed images at 20× magnification of p-p38 MAPK fluorescent labeling (green) with DAPI nuclear stain (blue). In all images, the scale bar is equal to 50 μm.
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fig04: Immunofluorescent intensity quantification from 7-μm thick sections of dorsal horn spinal cord from behaviorally verified rats following i.t. vehicle or AM1241. (A, B) Prior to CCI, all groups exhibited similar ipsilateral and contralateral BL thresholds. CCI produced significant bilateral allodynia at Day 3 and 10 following injury compared to sham-treated animals. Behavioral responses following AM1241 (10 μg) produced maximal bilateral reversal of allodynia followed by tissue collection of immunofluorescent intensity quantification. (C, D) IL-10 expression was bilaterally decreased in CCI-treated rats that received i.t. vehicle compared to control sham-treated rats given either vehicle or AM1241, while IL-10 IR recovered to sham levels in CCI neuropathic rats given i.t. AM1241. (E, F) Compared to sham controls, IL-1β expression was increased ipsilaterally, but not contralaterally in CCI-treated animals given i.t. vehicle of AM1241. However, i.t. AM1241 in CCI-treated rats robustly suppressed increases in IL-1β IR. (G, H) Phospho-p38 expression was bilaterally increased in CCI-induced neuropathic rats treated with i.t vehicle of AM1241. Increased bilateral p-p38MAPK was significantly suppressed in CCI-treated rats given i.t. AM1241. (I, J) No differences in DAPI nuclear stain fluorescent intensity were observed in either sham control or CCI-treated rats given either i.t. vehicle or AM1241. (K, L, and M) Representative spectrally unmixed images at 20× magnification of p-p38 MAPK fluorescent labeling (green) with DAPI nuclear stain (blue). In all images, the scale bar is equal to 50 μm.

Mentions: Following behavioral assessment at indicated time points (Figs. 3, 4), animals were overdosed with an i.p. injection (0.8-1.3 cc) of sodium phenobarbital (Sleepaway, Fort Dodge Animal Health, Fort Dodge, IA) and perfused transcardially with saline followed by 4% paraformaldehyde. Whole vertebral columns with intact spinal cord (cervical 2 through sacral 1 spinal column segments) were removed, and underwent overnight fixation in 4% paraformaldehyde at 4°C. This tissue collection procedure ensured that all relevant anatomical components, including the spinal cord, DRG, and related meninges, were intact within the vertebral column, allowing important spatial relationships to be examined for corresponding functional interactions at individual and specific spinal cord levels. All specimens underwent EDTA (Sigma Aldrich, St. Louis, MO) decalcification for 30 days, and spinal cord sections were subsequently paraffin processed and embedded in Paraplast Plus Embedding Media (McCormick Scientific, St. Louis, MO) as previously described (Wallace et al. 1996). Four adjacent tissue sections (7 μm) were mounted on a vectabond-treated slide (Vector Labs, Burlingame, CA), and allowed to adhere to the slide overnight at 40°C.


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 from 7-μm thick sections of dorsal horn spinal cord from behaviorally verified rats following i.t. vehicle or AM1241. (A, B) Prior to CCI, all groups exhibited similar ipsilateral and contralateral BL thresholds. CCI produced significant bilateral allodynia at Day 3 and 10 following injury compared to sham-treated animals. Behavioral responses following AM1241 (10 μg) produced maximal bilateral reversal of allodynia followed by tissue collection of immunofluorescent intensity quantification. (C, D) IL-10 expression was bilaterally decreased in CCI-treated rats that received i.t. vehicle compared to control sham-treated rats given either vehicle or AM1241, while IL-10 IR recovered to sham levels in CCI neuropathic rats given i.t. AM1241. (E, F) Compared to sham controls, IL-1β expression was increased ipsilaterally, but not contralaterally in CCI-treated animals given i.t. vehicle of AM1241. However, i.t. AM1241 in CCI-treated rats robustly suppressed increases in IL-1β IR. (G, H) Phospho-p38 expression was bilaterally increased in CCI-induced neuropathic rats treated with i.t vehicle of AM1241. Increased bilateral p-p38MAPK was significantly suppressed in CCI-treated rats given i.t. AM1241. (I, J) No differences in DAPI nuclear stain fluorescent intensity were observed in either sham control or CCI-treated rats given either i.t. vehicle or AM1241. (K, L, and M) Representative spectrally unmixed images at 20× magnification of p-p38 MAPK fluorescent labeling (green) with DAPI nuclear stain (blue). In all images, the scale bar is equal to 50 μm.
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Related In: Results  -  Collection

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fig04: Immunofluorescent intensity quantification from 7-μm thick sections of dorsal horn spinal cord from behaviorally verified rats following i.t. vehicle or AM1241. (A, B) Prior to CCI, all groups exhibited similar ipsilateral and contralateral BL thresholds. CCI produced significant bilateral allodynia at Day 3 and 10 following injury compared to sham-treated animals. Behavioral responses following AM1241 (10 μg) produced maximal bilateral reversal of allodynia followed by tissue collection of immunofluorescent intensity quantification. (C, D) IL-10 expression was bilaterally decreased in CCI-treated rats that received i.t. vehicle compared to control sham-treated rats given either vehicle or AM1241, while IL-10 IR recovered to sham levels in CCI neuropathic rats given i.t. AM1241. (E, F) Compared to sham controls, IL-1β expression was increased ipsilaterally, but not contralaterally in CCI-treated animals given i.t. vehicle of AM1241. However, i.t. AM1241 in CCI-treated rats robustly suppressed increases in IL-1β IR. (G, H) Phospho-p38 expression was bilaterally increased in CCI-induced neuropathic rats treated with i.t vehicle of AM1241. Increased bilateral p-p38MAPK was significantly suppressed in CCI-treated rats given i.t. AM1241. (I, J) No differences in DAPI nuclear stain fluorescent intensity were observed in either sham control or CCI-treated rats given either i.t. vehicle or AM1241. (K, L, and M) Representative spectrally unmixed images at 20× magnification of p-p38 MAPK fluorescent labeling (green) with DAPI nuclear stain (blue). In all images, the scale bar is equal to 50 μm.
Mentions: Following behavioral assessment at indicated time points (Figs. 3, 4), animals were overdosed with an i.p. injection (0.8-1.3 cc) of sodium phenobarbital (Sleepaway, Fort Dodge Animal Health, Fort Dodge, IA) and perfused transcardially with saline followed by 4% paraformaldehyde. Whole vertebral columns with intact spinal cord (cervical 2 through sacral 1 spinal column segments) were removed, and underwent overnight fixation in 4% paraformaldehyde at 4°C. This tissue collection procedure ensured that all relevant anatomical components, including the spinal cord, DRG, and related meninges, were intact within the vertebral column, allowing important spatial relationships to be examined for corresponding functional interactions at individual and specific spinal cord levels. All specimens underwent EDTA (Sigma Aldrich, St. Louis, MO) decalcification for 30 days, and spinal cord sections were subsequently paraffin processed and embedded in Paraplast Plus Embedding Media (McCormick Scientific, St. Louis, MO) as previously described (Wallace et al. 1996). Four adjacent tissue sections (7 μm) were mounted on a vectabond-treated slide (Vector Labs, Burlingame, CA), and allowed to adhere to the slide overnight at 40°C.

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