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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

Spectral versus standard Image J immunofluorescent intensity quantification comparison. (A) Utilizing Image J software for immunofluorescent quantification, no significant IL-1β IR differences between CCI-induced neuropathy or nonneuropathic sham-treated rats in either the ipsilateral or contralateral dorsal horn of the spinal cord. IL-1β IR was observed by FITC-labeled secondary antibody. (B) Following a second exposure for image capture, fluorophore fading was virtually absent thereby lacking potential artificial IR intensity differences between experimental conditions. (C) A comparison of sham- and CCI-treated rats with i.t. vehicle using Image J immunofluorescent quantification resulted in no significant fluorescent intensity differences of labeled IL-1β IR between groups, in either the ipsilateral or contralateral dorsal horn. (D) Utilizing spectral immunofluorescent quantification, significant differences of fluorescent intensity from FITC-labeled IL-1β between sham- and CCI-treated rats given i.t. vehicle was observed in the ipsilateral, but not contralateral dorsal horn spinal cord. (E) An examination of fluorescent intensity between groups with a flourophore of a different spectral signature, Rhodamine Red (600 nm), using standard Image J immunofluorescent quantification revealed no significant group differences between sham- and CCI-treated rats despite a trend of increased IL-1β IR in CCI-treated rats with i.t. vehicle in either the ipsilateral or contralateral dorsal horn. (F) Spectral immunofluorescent quantification of Rhodamine Red labeled IL-1β yielded significant group differences between nonneuropathic sham rats and CCI-treated rats with vehicle in the ipsilateral dorsal spinal cord. IL-1β IR increases were absent in the contralateral dorsal horn. (G, H) Representative fluorescent images analyzed with Image J at 20× magnification of IL-1β fluorescent labeling (red). (I, J) Representative spectrally unmixed images at 20× magnification of IL-1β fluorescent labeling (red) with DAPI nuclear stain (blue). In all images, the scale bar is equal to 50 μm. All sections were 7-μm thick, and collected 1.5 h after i.t. vehicle administration 10 days after CCI or sham surgery.
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fig03: Spectral versus standard Image J immunofluorescent intensity quantification comparison. (A) Utilizing Image J software for immunofluorescent quantification, no significant IL-1β IR differences between CCI-induced neuropathy or nonneuropathic sham-treated rats in either the ipsilateral or contralateral dorsal horn of the spinal cord. IL-1β IR was observed by FITC-labeled secondary antibody. (B) Following a second exposure for image capture, fluorophore fading was virtually absent thereby lacking potential artificial IR intensity differences between experimental conditions. (C) A comparison of sham- and CCI-treated rats with i.t. vehicle using Image J immunofluorescent quantification resulted in no significant fluorescent intensity differences of labeled IL-1β IR between groups, in either the ipsilateral or contralateral dorsal horn. (D) Utilizing spectral immunofluorescent quantification, significant differences of fluorescent intensity from FITC-labeled IL-1β between sham- and CCI-treated rats given i.t. vehicle was observed in the ipsilateral, but not contralateral dorsal horn spinal cord. (E) An examination of fluorescent intensity between groups with a flourophore of a different spectral signature, Rhodamine Red (600 nm), using standard Image J immunofluorescent quantification revealed no significant group differences between sham- and CCI-treated rats despite a trend of increased IL-1β IR in CCI-treated rats with i.t. vehicle in either the ipsilateral or contralateral dorsal horn. (F) Spectral immunofluorescent quantification of Rhodamine Red labeled IL-1β yielded significant group differences between nonneuropathic sham rats and CCI-treated rats with vehicle in the ipsilateral dorsal spinal cord. IL-1β IR increases were absent in the contralateral dorsal horn. (G, H) Representative fluorescent images analyzed with Image J at 20× magnification of IL-1β fluorescent labeling (red). (I, J) Representative spectrally unmixed images at 20× magnification of IL-1β fluorescent labeling (red) with DAPI nuclear stain (blue). In all images, the scale bar is equal to 50 μm. All sections were 7-μm thick, and collected 1.5 h after i.t. vehicle administration 10 days after CCI or sham surgery.

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)

Spectral versus standard Image J immunofluorescent intensity quantification comparison. (A) Utilizing Image J software for immunofluorescent quantification, no significant IL-1β IR differences between CCI-induced neuropathy or nonneuropathic sham-treated rats in either the ipsilateral or contralateral dorsal horn of the spinal cord. IL-1β IR was observed by FITC-labeled secondary antibody. (B) Following a second exposure for image capture, fluorophore fading was virtually absent thereby lacking potential artificial IR intensity differences between experimental conditions. (C) A comparison of sham- and CCI-treated rats with i.t. vehicle using Image J immunofluorescent quantification resulted in no significant fluorescent intensity differences of labeled IL-1β IR between groups, in either the ipsilateral or contralateral dorsal horn. (D) Utilizing spectral immunofluorescent quantification, significant differences of fluorescent intensity from FITC-labeled IL-1β between sham- and CCI-treated rats given i.t. vehicle was observed in the ipsilateral, but not contralateral dorsal horn spinal cord. (E) An examination of fluorescent intensity between groups with a flourophore of a different spectral signature, Rhodamine Red (600 nm), using standard Image J immunofluorescent quantification revealed no significant group differences between sham- and CCI-treated rats despite a trend of increased IL-1β IR in CCI-treated rats with i.t. vehicle in either the ipsilateral or contralateral dorsal horn. (F) Spectral immunofluorescent quantification of Rhodamine Red labeled IL-1β yielded significant group differences between nonneuropathic sham rats and CCI-treated rats with vehicle in the ipsilateral dorsal spinal cord. IL-1β IR increases were absent in the contralateral dorsal horn. (G, H) Representative fluorescent images analyzed with Image J at 20× magnification of IL-1β fluorescent labeling (red). (I, J) Representative spectrally unmixed images at 20× magnification of IL-1β fluorescent labeling (red) with DAPI nuclear stain (blue). In all images, the scale bar is equal to 50 μm. All sections were 7-μm thick, and collected 1.5 h after i.t. vehicle administration 10 days after CCI or sham surgery.
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fig03: Spectral versus standard Image J immunofluorescent intensity quantification comparison. (A) Utilizing Image J software for immunofluorescent quantification, no significant IL-1β IR differences between CCI-induced neuropathy or nonneuropathic sham-treated rats in either the ipsilateral or contralateral dorsal horn of the spinal cord. IL-1β IR was observed by FITC-labeled secondary antibody. (B) Following a second exposure for image capture, fluorophore fading was virtually absent thereby lacking potential artificial IR intensity differences between experimental conditions. (C) A comparison of sham- and CCI-treated rats with i.t. vehicle using Image J immunofluorescent quantification resulted in no significant fluorescent intensity differences of labeled IL-1β IR between groups, in either the ipsilateral or contralateral dorsal horn. (D) Utilizing spectral immunofluorescent quantification, significant differences of fluorescent intensity from FITC-labeled IL-1β between sham- and CCI-treated rats given i.t. vehicle was observed in the ipsilateral, but not contralateral dorsal horn spinal cord. (E) An examination of fluorescent intensity between groups with a flourophore of a different spectral signature, Rhodamine Red (600 nm), using standard Image J immunofluorescent quantification revealed no significant group differences between sham- and CCI-treated rats despite a trend of increased IL-1β IR in CCI-treated rats with i.t. vehicle in either the ipsilateral or contralateral dorsal horn. (F) Spectral immunofluorescent quantification of Rhodamine Red labeled IL-1β yielded significant group differences between nonneuropathic sham rats and CCI-treated rats with vehicle in the ipsilateral dorsal spinal cord. IL-1β IR increases were absent in the contralateral dorsal horn. (G, H) Representative fluorescent images analyzed with Image J at 20× magnification of IL-1β fluorescent labeling (red). (I, J) Representative spectrally unmixed images at 20× magnification of IL-1β fluorescent labeling (red) with DAPI nuclear stain (blue). In all images, the scale bar is equal to 50 μm. All sections were 7-μm thick, and collected 1.5 h after i.t. vehicle administration 10 days after CCI or sham surgery.
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