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Restoration of axon conduction and motor deficits by therapeutic treatment with glatiramer acetate.

Moore S, Khalaj AJ, Patel R, Yoon J, Ichwan D, Hayardeny L, Tiwari-Woodruff SK - J. Neurosci. Res. (2014)

Bottom Line: The underlying multifactorial anti-inflammatory, neuroprotective effect of GA is in the induction of reactive T cells that release immunomodulatory cytokines and neurotrophic factors at the injury site.These GA-induced cytokines and growth factors may have a direct effect on axon function.Finally, GA improved callosal axon conduction and nodal protein organization in EAE.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurology, UCLA School of Medicine, Los Angeles, California.

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Therapeutic GA reduces inflammation in spinal cords of EAE mice. Representative PLP_EGFP (green) thoracic spinal cord sections costained with DAPI (red) from normal, EAE + vehicle and EAE + GA groups described in Figure 1B, with treatment initiated on peak EAE day 21 (A; ×4 magnification). All animals were sacrificed at EAE post-induction day ∼40. White-dashed perimeters (DC, dorsal column) and boxes (VC, ventral column) denote regions of the spinal cord used for quantification. No inflammatory nuclei are observed in normal controls, whereas vehicle-treated EAE spinal cord shows multifocal to coalescing infiltrates in the leptomeninges. Treatment with 2 mg/day GA for 8 days resulted in fewer DAPI+ cell infiltrates in the lateral funiculus (LF), DC, and VC*. Consecutive PLP_EGFP (green) thoracic spinal cord sections co-immunostained for CD3 (B, red, ×10), GFAP (D, red, ×40), or CD45 (F, red, ×40) are shown from partial images (dashed boxes in B) of normal control, vehicle-treated EAE, and GA-treated EAE mice. Vehicle-treated EAE spinal cords had large areas of CD45+ and GFAP+ cell staining in the DC compared with normal controls, whereas GA-treated EAE mice showed a significant decrease in CD3 and CD45 positivity. Number of CD3+ (C), GFAP+ (E), and CD45+ (G) cells per 400 µm2 or cell intensity within the DC were quantified. Compared with normal controls, treatment with GA induced a decrease in CD3+ and CD45+ cell numbers, but not GFAP+ immunoreactivity. Data are representative of experiments repeated in their entirety on another set of EAE mice. *P < 0.05, **P < 0.001, 1 × 4 ANOVA; n = 6–8 mice/group. [Color figure can be viewed in the online issue, which is available at http://wileyonlinelibrary.com.]
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fig03: Therapeutic GA reduces inflammation in spinal cords of EAE mice. Representative PLP_EGFP (green) thoracic spinal cord sections costained with DAPI (red) from normal, EAE + vehicle and EAE + GA groups described in Figure 1B, with treatment initiated on peak EAE day 21 (A; ×4 magnification). All animals were sacrificed at EAE post-induction day ∼40. White-dashed perimeters (DC, dorsal column) and boxes (VC, ventral column) denote regions of the spinal cord used for quantification. No inflammatory nuclei are observed in normal controls, whereas vehicle-treated EAE spinal cord shows multifocal to coalescing infiltrates in the leptomeninges. Treatment with 2 mg/day GA for 8 days resulted in fewer DAPI+ cell infiltrates in the lateral funiculus (LF), DC, and VC*. Consecutive PLP_EGFP (green) thoracic spinal cord sections co-immunostained for CD3 (B, red, ×10), GFAP (D, red, ×40), or CD45 (F, red, ×40) are shown from partial images (dashed boxes in B) of normal control, vehicle-treated EAE, and GA-treated EAE mice. Vehicle-treated EAE spinal cords had large areas of CD45+ and GFAP+ cell staining in the DC compared with normal controls, whereas GA-treated EAE mice showed a significant decrease in CD3 and CD45 positivity. Number of CD3+ (C), GFAP+ (E), and CD45+ (G) cells per 400 µm2 or cell intensity within the DC were quantified. Compared with normal controls, treatment with GA induced a decrease in CD3+ and CD45+ cell numbers, but not GFAP+ immunoreactivity. Data are representative of experiments repeated in their entirety on another set of EAE mice. *P < 0.05, **P < 0.001, 1 × 4 ANOVA; n = 6–8 mice/group. [Color figure can be viewed in the online issue, which is available at http://wileyonlinelibrary.com.]

Mentions: Immunostaining was quantified using unbiased stereology. The dorsal column (DC) was delineated (Fig. 3A) using the drawing tool in ImageJ version 1.29 (Windows version of NIH Image; http://rsb.info/nih/gov/ij), and MBP, GFAP, CD3, and CD45 staining intensity was quantified within this region. NF200+ and MBP+ axons were counted in the ventral column (VC) of thoracic spinal cord (Fig. 3A). All images (RGB) were converted to gray scale, split, and separated by color channel in ImageJ. To avoid experimenter bias, autoadjustment of brightness, contrast, and threshold of staining signal were carried out in NIH software. A grid plug-in (ImageJ) was used for counting points per area of interest. Adenomatus polyposis coli (CC1)+ mature OLs, olig2+ OL lineage cells, and CD3+ T cells within the CC or spinal cord dorsal column were counted manually using ×10 or ×40 magnification images and compared blindly among normal, vehicle-treated EAE, and GA-treated EAE groups. Inflammatory cells were quantified by counting the number of CD45+ and CD3+ cells with DAPI+ nuclei in delineated thoracic spinal cord dorsal column (and/or delineated CC). Myelin (MBP+) and astrocytes (GFAP+) were calculated as percentage area intensity within the spinal cord dorsal column (Fig. 3) and delineated CC. Spinal cord axonal densities were calculated by counting the number of NF200+ cells in a ×40 image of ventral column of thoracic spinal cords, where coherent and similar diameter axons are present. Myelinated axon densities were calculated by counting axons (NF200+) with a clear ring of MBP+ myelin staining around them. Damaged axons were calculated by counting APP+ axons.


Restoration of axon conduction and motor deficits by therapeutic treatment with glatiramer acetate.

Moore S, Khalaj AJ, Patel R, Yoon J, Ichwan D, Hayardeny L, Tiwari-Woodruff SK - J. Neurosci. Res. (2014)

Therapeutic GA reduces inflammation in spinal cords of EAE mice. Representative PLP_EGFP (green) thoracic spinal cord sections costained with DAPI (red) from normal, EAE + vehicle and EAE + GA groups described in Figure 1B, with treatment initiated on peak EAE day 21 (A; ×4 magnification). All animals were sacrificed at EAE post-induction day ∼40. White-dashed perimeters (DC, dorsal column) and boxes (VC, ventral column) denote regions of the spinal cord used for quantification. No inflammatory nuclei are observed in normal controls, whereas vehicle-treated EAE spinal cord shows multifocal to coalescing infiltrates in the leptomeninges. Treatment with 2 mg/day GA for 8 days resulted in fewer DAPI+ cell infiltrates in the lateral funiculus (LF), DC, and VC*. Consecutive PLP_EGFP (green) thoracic spinal cord sections co-immunostained for CD3 (B, red, ×10), GFAP (D, red, ×40), or CD45 (F, red, ×40) are shown from partial images (dashed boxes in B) of normal control, vehicle-treated EAE, and GA-treated EAE mice. Vehicle-treated EAE spinal cords had large areas of CD45+ and GFAP+ cell staining in the DC compared with normal controls, whereas GA-treated EAE mice showed a significant decrease in CD3 and CD45 positivity. Number of CD3+ (C), GFAP+ (E), and CD45+ (G) cells per 400 µm2 or cell intensity within the DC were quantified. Compared with normal controls, treatment with GA induced a decrease in CD3+ and CD45+ cell numbers, but not GFAP+ immunoreactivity. Data are representative of experiments repeated in their entirety on another set of EAE mice. *P < 0.05, **P < 0.001, 1 × 4 ANOVA; n = 6–8 mice/group. [Color figure can be viewed in the online issue, which is available at http://wileyonlinelibrary.com.]
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fig03: Therapeutic GA reduces inflammation in spinal cords of EAE mice. Representative PLP_EGFP (green) thoracic spinal cord sections costained with DAPI (red) from normal, EAE + vehicle and EAE + GA groups described in Figure 1B, with treatment initiated on peak EAE day 21 (A; ×4 magnification). All animals were sacrificed at EAE post-induction day ∼40. White-dashed perimeters (DC, dorsal column) and boxes (VC, ventral column) denote regions of the spinal cord used for quantification. No inflammatory nuclei are observed in normal controls, whereas vehicle-treated EAE spinal cord shows multifocal to coalescing infiltrates in the leptomeninges. Treatment with 2 mg/day GA for 8 days resulted in fewer DAPI+ cell infiltrates in the lateral funiculus (LF), DC, and VC*. Consecutive PLP_EGFP (green) thoracic spinal cord sections co-immunostained for CD3 (B, red, ×10), GFAP (D, red, ×40), or CD45 (F, red, ×40) are shown from partial images (dashed boxes in B) of normal control, vehicle-treated EAE, and GA-treated EAE mice. Vehicle-treated EAE spinal cords had large areas of CD45+ and GFAP+ cell staining in the DC compared with normal controls, whereas GA-treated EAE mice showed a significant decrease in CD3 and CD45 positivity. Number of CD3+ (C), GFAP+ (E), and CD45+ (G) cells per 400 µm2 or cell intensity within the DC were quantified. Compared with normal controls, treatment with GA induced a decrease in CD3+ and CD45+ cell numbers, but not GFAP+ immunoreactivity. Data are representative of experiments repeated in their entirety on another set of EAE mice. *P < 0.05, **P < 0.001, 1 × 4 ANOVA; n = 6–8 mice/group. [Color figure can be viewed in the online issue, which is available at http://wileyonlinelibrary.com.]
Mentions: Immunostaining was quantified using unbiased stereology. The dorsal column (DC) was delineated (Fig. 3A) using the drawing tool in ImageJ version 1.29 (Windows version of NIH Image; http://rsb.info/nih/gov/ij), and MBP, GFAP, CD3, and CD45 staining intensity was quantified within this region. NF200+ and MBP+ axons were counted in the ventral column (VC) of thoracic spinal cord (Fig. 3A). All images (RGB) were converted to gray scale, split, and separated by color channel in ImageJ. To avoid experimenter bias, autoadjustment of brightness, contrast, and threshold of staining signal were carried out in NIH software. A grid plug-in (ImageJ) was used for counting points per area of interest. Adenomatus polyposis coli (CC1)+ mature OLs, olig2+ OL lineage cells, and CD3+ T cells within the CC or spinal cord dorsal column were counted manually using ×10 or ×40 magnification images and compared blindly among normal, vehicle-treated EAE, and GA-treated EAE groups. Inflammatory cells were quantified by counting the number of CD45+ and CD3+ cells with DAPI+ nuclei in delineated thoracic spinal cord dorsal column (and/or delineated CC). Myelin (MBP+) and astrocytes (GFAP+) were calculated as percentage area intensity within the spinal cord dorsal column (Fig. 3) and delineated CC. Spinal cord axonal densities were calculated by counting the number of NF200+ cells in a ×40 image of ventral column of thoracic spinal cords, where coherent and similar diameter axons are present. Myelinated axon densities were calculated by counting axons (NF200+) with a clear ring of MBP+ myelin staining around them. Damaged axons were calculated by counting APP+ axons.

Bottom Line: The underlying multifactorial anti-inflammatory, neuroprotective effect of GA is in the induction of reactive T cells that release immunomodulatory cytokines and neurotrophic factors at the injury site.These GA-induced cytokines and growth factors may have a direct effect on axon function.Finally, GA improved callosal axon conduction and nodal protein organization in EAE.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurology, UCLA School of Medicine, Los Angeles, California.

Show MeSH
Related in: MedlinePlus