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Cell death of spinal cord ED1(+) cells in a rat model of multiple sclerosis.

Trifunović D, Djedović N, Lavrnja I, Wendrich KS, Paquet-Durand F, Miljković D - PeerJ (2015)

Bottom Line: Our findings suggest that activated macrophages/microglia of gray matter are less susceptible to cell death induction.Thus, further research on the gray matter macrophages/microglia cell death during EAE is warranted.They should be aimed at identification of the reasons for the observed differences and finding suitable ways to stimulate gray matter activated macrophages/microglia death.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute for Ophthalmic Research, University of Tuebingen , Tuebingen , Germany.

ABSTRACT
Infiltration of macrophages into the central nervous system and activation of microglia are hallmarks of multiple sclerosis and its animal model-experimental autoimmune encephalomyelitis (EAE). Cell death in EAE has been demonstrated as an essential mechanism in the local regulation of the inflammatory reaction, but also as one of the major factors contributing to the destruction of the nervous tissue. The focus of this study was on detection of cell death among ED1(+) cells (macrophages/activated microglia) in the spinal cord of Dark Agouti rats at the peak of EAE. Cell death was assessed using the TUNEL assay and immunostaining for cleaved caspase 3, as markers for cell death in general and "classical" apoptosis, respectively. Major infiltrates of immune cells were detected both in white matter and gray matter of spinal cords in rats at the disease peak. ED1, TUNEL, and caspase 3 positive cells were detected within, but also outside the infiltrates. There were more dying ED1(+) cells in white matter than in gray matter, both in the general population and in infiltrated regions. The observed discrepancy in the proportion of dying ED1(+) cells in spinal cord gray and white matter indicated that in EAE rat macrophages/microglia within gray matter are less prone to cell death induction. This is of special interest in the context of the increasingly appreciated contribution of spinal cord gray matter inflammation to multiple sclerosis pathogenesis. Our findings suggest that activated macrophages/microglia of gray matter are less susceptible to cell death induction. Alternatively, it can be assumed that intrinsic cell death-inductive mechanisms of nervous tissue differ in white and gray matter. Thus, further research on the gray matter macrophages/microglia cell death during EAE is warranted. They should be aimed at identification of the reasons for the observed differences and finding suitable ways to stimulate gray matter activated macrophages/microglia death.

No MeSH data available.


Related in: MedlinePlus

Cell death in EAE rats at the disease peak.(A) Representative spinal cord sections of immunized and control animals stained for nuclei with DAPI (blue), cleaved caspase 3 (green) and cell death with TUNEL (red) are presented. TUNEL cells were present both in infiltrates and non-infiltrated regions in white matter, while caspase 3 positive cells were more prominent in infiltrates. The merged pictures showed that vast majority of caspase 3 positive cells were TUNEL positive, while there was a population of TUNEL cells which were caspase 3 negative. Inserts in merged pictures are shown at higher magnification in the upper right corner. (B) Higher magnification of cleaved caspase 3 and TUNEL co-labeling, where caspase 3 staining (green) is visible in cytoplasm, while TUNEL staining (shown in red) is confined to nucleus. (C) The percentage of TUNEL and (D) caspase 3 positive cells within white matter and gray matter cells throughout spinal cord (all) or within infiltrates or in non-infiltrated regions is presented as mean + SEM. WM, white matter; GM, gray matter. *p < 0.05 represents statistically significant difference between infiltrates vs. non-infiltrated regions or white matter vs. gray matter. Scale bars are 20 µm.
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fig-3: Cell death in EAE rats at the disease peak.(A) Representative spinal cord sections of immunized and control animals stained for nuclei with DAPI (blue), cleaved caspase 3 (green) and cell death with TUNEL (red) are presented. TUNEL cells were present both in infiltrates and non-infiltrated regions in white matter, while caspase 3 positive cells were more prominent in infiltrates. The merged pictures showed that vast majority of caspase 3 positive cells were TUNEL positive, while there was a population of TUNEL cells which were caspase 3 negative. Inserts in merged pictures are shown at higher magnification in the upper right corner. (B) Higher magnification of cleaved caspase 3 and TUNEL co-labeling, where caspase 3 staining (green) is visible in cytoplasm, while TUNEL staining (shown in red) is confined to nucleus. (C) The percentage of TUNEL and (D) caspase 3 positive cells within white matter and gray matter cells throughout spinal cord (all) or within infiltrates or in non-infiltrated regions is presented as mean + SEM. WM, white matter; GM, gray matter. *p < 0.05 represents statistically significant difference between infiltrates vs. non-infiltrated regions or white matter vs. gray matter. Scale bars are 20 µm.

Mentions: To assess the rate of cell death in EAE rats, TUNEL staining was performed on both immunized and control animals. While TUNEL positive cells were rarely detected in control sections, immunization led to extensive cell death in both white and gray matter (Figs. 3A, 3C; Table 2). There were significantly more TUNEL cells in white matter than in gray matter. At the same time there were more dying cells in infiltrated vs. non-infiltrated regions in the white matter. On the other hand, there was no difference in the percentage of dying cells in infiltrated and non-infiltrated regions within the gray matter (11.6 ± 1.7 vs. 11.4 ± 2.3). To determine if the observed cell death followed the “classical” apoptotic pathway characterized by caspase 3 activation, we looked for cleaved and activated caspase 3 positive cells (Figs. 3A and 3D). Caspase 3 positive cells essentially followed the pattern of TUNEL cells, with majority of caspase 3 positive cell also being positive for TUNEL (Fig. 3A). In most of the analyzed cells, caspase 3 staining was observed in the cytoplasm, while the TUNEL assay marked the nucleus (Fig. 3B). In some cells, the TUNEL signal traced the morphology of the cell and was morphologically co-labeled with cleaved caspase-3, as can be seen in Fig. 3A. This can be explained by the known fact that TUNEL labels DNA nick-ends, which in very late stages of cell death after the nucleus has disintegrated can also be found in the cytoplasm (Labat-Moleur et al., 1998). Of special interest is the observation that only about 70% of TUNEL positive cells were caspase 3 positive (Table 2), suggesting that a certain cell population was dying using a caspase 3-independent pathway.


Cell death of spinal cord ED1(+) cells in a rat model of multiple sclerosis.

Trifunović D, Djedović N, Lavrnja I, Wendrich KS, Paquet-Durand F, Miljković D - PeerJ (2015)

Cell death in EAE rats at the disease peak.(A) Representative spinal cord sections of immunized and control animals stained for nuclei with DAPI (blue), cleaved caspase 3 (green) and cell death with TUNEL (red) are presented. TUNEL cells were present both in infiltrates and non-infiltrated regions in white matter, while caspase 3 positive cells were more prominent in infiltrates. The merged pictures showed that vast majority of caspase 3 positive cells were TUNEL positive, while there was a population of TUNEL cells which were caspase 3 negative. Inserts in merged pictures are shown at higher magnification in the upper right corner. (B) Higher magnification of cleaved caspase 3 and TUNEL co-labeling, where caspase 3 staining (green) is visible in cytoplasm, while TUNEL staining (shown in red) is confined to nucleus. (C) The percentage of TUNEL and (D) caspase 3 positive cells within white matter and gray matter cells throughout spinal cord (all) or within infiltrates or in non-infiltrated regions is presented as mean + SEM. WM, white matter; GM, gray matter. *p < 0.05 represents statistically significant difference between infiltrates vs. non-infiltrated regions or white matter vs. gray matter. Scale bars are 20 µm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig-3: Cell death in EAE rats at the disease peak.(A) Representative spinal cord sections of immunized and control animals stained for nuclei with DAPI (blue), cleaved caspase 3 (green) and cell death with TUNEL (red) are presented. TUNEL cells were present both in infiltrates and non-infiltrated regions in white matter, while caspase 3 positive cells were more prominent in infiltrates. The merged pictures showed that vast majority of caspase 3 positive cells were TUNEL positive, while there was a population of TUNEL cells which were caspase 3 negative. Inserts in merged pictures are shown at higher magnification in the upper right corner. (B) Higher magnification of cleaved caspase 3 and TUNEL co-labeling, where caspase 3 staining (green) is visible in cytoplasm, while TUNEL staining (shown in red) is confined to nucleus. (C) The percentage of TUNEL and (D) caspase 3 positive cells within white matter and gray matter cells throughout spinal cord (all) or within infiltrates or in non-infiltrated regions is presented as mean + SEM. WM, white matter; GM, gray matter. *p < 0.05 represents statistically significant difference between infiltrates vs. non-infiltrated regions or white matter vs. gray matter. Scale bars are 20 µm.
Mentions: To assess the rate of cell death in EAE rats, TUNEL staining was performed on both immunized and control animals. While TUNEL positive cells were rarely detected in control sections, immunization led to extensive cell death in both white and gray matter (Figs. 3A, 3C; Table 2). There were significantly more TUNEL cells in white matter than in gray matter. At the same time there were more dying cells in infiltrated vs. non-infiltrated regions in the white matter. On the other hand, there was no difference in the percentage of dying cells in infiltrated and non-infiltrated regions within the gray matter (11.6 ± 1.7 vs. 11.4 ± 2.3). To determine if the observed cell death followed the “classical” apoptotic pathway characterized by caspase 3 activation, we looked for cleaved and activated caspase 3 positive cells (Figs. 3A and 3D). Caspase 3 positive cells essentially followed the pattern of TUNEL cells, with majority of caspase 3 positive cell also being positive for TUNEL (Fig. 3A). In most of the analyzed cells, caspase 3 staining was observed in the cytoplasm, while the TUNEL assay marked the nucleus (Fig. 3B). In some cells, the TUNEL signal traced the morphology of the cell and was morphologically co-labeled with cleaved caspase-3, as can be seen in Fig. 3A. This can be explained by the known fact that TUNEL labels DNA nick-ends, which in very late stages of cell death after the nucleus has disintegrated can also be found in the cytoplasm (Labat-Moleur et al., 1998). Of special interest is the observation that only about 70% of TUNEL positive cells were caspase 3 positive (Table 2), suggesting that a certain cell population was dying using a caspase 3-independent pathway.

Bottom Line: Our findings suggest that activated macrophages/microglia of gray matter are less susceptible to cell death induction.Thus, further research on the gray matter macrophages/microglia cell death during EAE is warranted.They should be aimed at identification of the reasons for the observed differences and finding suitable ways to stimulate gray matter activated macrophages/microglia death.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute for Ophthalmic Research, University of Tuebingen , Tuebingen , Germany.

ABSTRACT
Infiltration of macrophages into the central nervous system and activation of microglia are hallmarks of multiple sclerosis and its animal model-experimental autoimmune encephalomyelitis (EAE). Cell death in EAE has been demonstrated as an essential mechanism in the local regulation of the inflammatory reaction, but also as one of the major factors contributing to the destruction of the nervous tissue. The focus of this study was on detection of cell death among ED1(+) cells (macrophages/activated microglia) in the spinal cord of Dark Agouti rats at the peak of EAE. Cell death was assessed using the TUNEL assay and immunostaining for cleaved caspase 3, as markers for cell death in general and "classical" apoptosis, respectively. Major infiltrates of immune cells were detected both in white matter and gray matter of spinal cords in rats at the disease peak. ED1, TUNEL, and caspase 3 positive cells were detected within, but also outside the infiltrates. There were more dying ED1(+) cells in white matter than in gray matter, both in the general population and in infiltrated regions. The observed discrepancy in the proportion of dying ED1(+) cells in spinal cord gray and white matter indicated that in EAE rat macrophages/microglia within gray matter are less prone to cell death induction. This is of special interest in the context of the increasingly appreciated contribution of spinal cord gray matter inflammation to multiple sclerosis pathogenesis. Our findings suggest that activated macrophages/microglia of gray matter are less susceptible to cell death induction. Alternatively, it can be assumed that intrinsic cell death-inductive mechanisms of nervous tissue differ in white and gray matter. Thus, further research on the gray matter macrophages/microglia cell death during EAE is warranted. They should be aimed at identification of the reasons for the observed differences and finding suitable ways to stimulate gray matter activated macrophages/microglia death.

No MeSH data available.


Related in: MedlinePlus