Limits...
High-resolution in-vivo analysis of normal brain response to cranial irradiation.

Burrell K, Hill RP, Zadeh G - PLoS ONE (2012)

Bottom Line: However, despite recognized therapeutic success, significant negative consequences are associated with cranial irradiation (CR), which manifests months to years post-RT.We establish that BMDCs do not form endothelial cells but rather they differentiate predominantly into inflammatory cells and microglia.These results have invaluable therapeutic implications as BMDCs may be a primary therapeutic target to block acute and long-term inflammatory response following CR.

View Article: PubMed Central - PubMed

Affiliation: Brain Tumor Research Centre, SickKids Research Institute, Toronto, Canada.

ABSTRACT
Radiation therapy (RT) is a widely accepted treatment strategy for many central nervous system (CNS) pathologies. However, despite recognized therapeutic success, significant negative consequences are associated with cranial irradiation (CR), which manifests months to years post-RT. The pathophysiology and molecular alterations that culminate in the long-term detrimental effects of CR are poorly understood, though it is thought that endothelial injury plays a pivotal role in triggering cranial injury. We therefore explored the contribution of bone marrow derived cells (BMDCs) in their capacity to repair and contribute to neo-vascularization following CR. Using high-resolution in vivo optical imaging we have studied, at single-cell resolution, the spatio-temporal response of BMDCs in normal brain following CR. We demonstrate that BMDCs are recruited specifically to the site of CR, in a radiation dose and temporal-spatial manner. We establish that BMDCs do not form endothelial cells but rather they differentiate predominantly into inflammatory cells and microglia. Most notably we provide evidence that more than 50% of the microglia in the irradiated region of the brain are not resident microglia but recruited from the bone marrow following CR. These results have invaluable therapeutic implications as BMDCs may be a primary therapeutic target to block acute and long-term inflammatory response following CR. Identifying the critical steps involved in the sustained recruitment and differentiation of BMDCs into microglia at the site of CR can provide new insights into the mechanisms of injury following CR offering potential therapeutic strategies to counteract the long-term adverse effects of CR.

Show MeSH

Related in: MedlinePlus

Characterization of Bone Marrow Derived Cells.(A–H) Immunofluorescence analysis of brain sections for characterization of cell types that BMDCs potentially differentiate to at the site of cranial radiation 7 days post 6Gy radiation, in the staining a red fluorescent chimeric mouse was used. (Green: stain, Red: BMDC, Blue: nuclei), 40× magnification (A,B) No differentiation of BMDCs to vessel endothelial cells is evident, confirming what was seen on the 2 photon laser microscopy in-vivo imaging. (A) BMDCs do not co-localize with the endothelial cell marker CD31, using confocal immunofluoroscence, or (B) immunohistochemical overlay of CD31 (Brown: CD31), 10× magnification. (C) Few BMDCs co-localize with the pericytic differentiation marker smooth muscle actin, shown by white arrowhead. (D,E) Approximately 40% of recruited BMDCs stain positively for inflammatory markers: (D) MAC3, co-localization shown by white arrowhead, (E) CD11b, co-localization shown by white arrowhead. (F) 50% of recruited BMDCs found in the parenchyma co-localize with the microglial differentiation marker IBA1 shown here by white arrowhead. (G) IBA1+ BMDCs are also found in the perivascular space around vessel wall, shown by white arrowhead. (H) There was no astrocytic differentiation or glial scaring, as determined by the lack of GFAP co-localization.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3366930&req=5

pone-0038366-g005: Characterization of Bone Marrow Derived Cells.(A–H) Immunofluorescence analysis of brain sections for characterization of cell types that BMDCs potentially differentiate to at the site of cranial radiation 7 days post 6Gy radiation, in the staining a red fluorescent chimeric mouse was used. (Green: stain, Red: BMDC, Blue: nuclei), 40× magnification (A,B) No differentiation of BMDCs to vessel endothelial cells is evident, confirming what was seen on the 2 photon laser microscopy in-vivo imaging. (A) BMDCs do not co-localize with the endothelial cell marker CD31, using confocal immunofluoroscence, or (B) immunohistochemical overlay of CD31 (Brown: CD31), 10× magnification. (C) Few BMDCs co-localize with the pericytic differentiation marker smooth muscle actin, shown by white arrowhead. (D,E) Approximately 40% of recruited BMDCs stain positively for inflammatory markers: (D) MAC3, co-localization shown by white arrowhead, (E) CD11b, co-localization shown by white arrowhead. (F) 50% of recruited BMDCs found in the parenchyma co-localize with the microglial differentiation marker IBA1 shown here by white arrowhead. (G) IBA1+ BMDCs are also found in the perivascular space around vessel wall, shown by white arrowhead. (H) There was no astrocytic differentiation or glial scaring, as determined by the lack of GFAP co-localization.

Mentions: We took advantage of our 2PLM in-vivo intra-vital imaging strategy to examine in-vivo differentiation of BMDCs intracranially following CR, with specific interest in whether BMDC differentiate to form EC. We used intravenous injection of CD31+ tagged Allophycocyanin (APC) antibody to allow specific in-vivo identification of vessel ECs. Additionally, the use of time-lapse footage (Video S1) and z-stack imaging (Video S2) provided detailed information on three-dimensional correlation of BMDCs with the vasculature and in depth analysis of their relationship to the vessel wall and vessel ECs (Video S2 & Figure S3). BMDCs did not differentiate to form EC at any point following CR or at any radiation dose. On 2PLM images we saw BMDC migrate to the site of CR, migrate outside of the vessel lumen, where a subpopulation went onto encircle the vessel, but did not form CD31+APC cells. Migration of BMDCs out of the vessel lumen was independent of vessel size, caliber or location along the course of the vessel. There was no difference between branching points in comparison to regions along the vessels between branching points. This is in contrast to what is reported for cancer cell metastases by Winkler et al where a majority of metastatic cancer cells migrate out of the tumor vasculature at sites of branching [23]. Immunofluorescence and immunohistochemical analysis both at site of CR and outside the radiation field did not show any co-localization of CD31+ cells with BMDCs (Figure 5A,B). Immunofluorescence staining for smooth muscle actin (SMA) confirmed a small, although not statistically significant, increase in the extent of SMA+ BMDC that surrounded the vasculature in the area of CR, compared to the contralateral control non-irradiated brain. However, the majority of peri-vascular BMDCs are not SMA+ (Figure 5C) rather they are inflammatory and microglial-like cells as described below.


High-resolution in-vivo analysis of normal brain response to cranial irradiation.

Burrell K, Hill RP, Zadeh G - PLoS ONE (2012)

Characterization of Bone Marrow Derived Cells.(A–H) Immunofluorescence analysis of brain sections for characterization of cell types that BMDCs potentially differentiate to at the site of cranial radiation 7 days post 6Gy radiation, in the staining a red fluorescent chimeric mouse was used. (Green: stain, Red: BMDC, Blue: nuclei), 40× magnification (A,B) No differentiation of BMDCs to vessel endothelial cells is evident, confirming what was seen on the 2 photon laser microscopy in-vivo imaging. (A) BMDCs do not co-localize with the endothelial cell marker CD31, using confocal immunofluoroscence, or (B) immunohistochemical overlay of CD31 (Brown: CD31), 10× magnification. (C) Few BMDCs co-localize with the pericytic differentiation marker smooth muscle actin, shown by white arrowhead. (D,E) Approximately 40% of recruited BMDCs stain positively for inflammatory markers: (D) MAC3, co-localization shown by white arrowhead, (E) CD11b, co-localization shown by white arrowhead. (F) 50% of recruited BMDCs found in the parenchyma co-localize with the microglial differentiation marker IBA1 shown here by white arrowhead. (G) IBA1+ BMDCs are also found in the perivascular space around vessel wall, shown by white arrowhead. (H) There was no astrocytic differentiation or glial scaring, as determined by the lack of GFAP co-localization.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3366930&req=5

pone-0038366-g005: Characterization of Bone Marrow Derived Cells.(A–H) Immunofluorescence analysis of brain sections for characterization of cell types that BMDCs potentially differentiate to at the site of cranial radiation 7 days post 6Gy radiation, in the staining a red fluorescent chimeric mouse was used. (Green: stain, Red: BMDC, Blue: nuclei), 40× magnification (A,B) No differentiation of BMDCs to vessel endothelial cells is evident, confirming what was seen on the 2 photon laser microscopy in-vivo imaging. (A) BMDCs do not co-localize with the endothelial cell marker CD31, using confocal immunofluoroscence, or (B) immunohistochemical overlay of CD31 (Brown: CD31), 10× magnification. (C) Few BMDCs co-localize with the pericytic differentiation marker smooth muscle actin, shown by white arrowhead. (D,E) Approximately 40% of recruited BMDCs stain positively for inflammatory markers: (D) MAC3, co-localization shown by white arrowhead, (E) CD11b, co-localization shown by white arrowhead. (F) 50% of recruited BMDCs found in the parenchyma co-localize with the microglial differentiation marker IBA1 shown here by white arrowhead. (G) IBA1+ BMDCs are also found in the perivascular space around vessel wall, shown by white arrowhead. (H) There was no astrocytic differentiation or glial scaring, as determined by the lack of GFAP co-localization.
Mentions: We took advantage of our 2PLM in-vivo intra-vital imaging strategy to examine in-vivo differentiation of BMDCs intracranially following CR, with specific interest in whether BMDC differentiate to form EC. We used intravenous injection of CD31+ tagged Allophycocyanin (APC) antibody to allow specific in-vivo identification of vessel ECs. Additionally, the use of time-lapse footage (Video S1) and z-stack imaging (Video S2) provided detailed information on three-dimensional correlation of BMDCs with the vasculature and in depth analysis of their relationship to the vessel wall and vessel ECs (Video S2 & Figure S3). BMDCs did not differentiate to form EC at any point following CR or at any radiation dose. On 2PLM images we saw BMDC migrate to the site of CR, migrate outside of the vessel lumen, where a subpopulation went onto encircle the vessel, but did not form CD31+APC cells. Migration of BMDCs out of the vessel lumen was independent of vessel size, caliber or location along the course of the vessel. There was no difference between branching points in comparison to regions along the vessels between branching points. This is in contrast to what is reported for cancer cell metastases by Winkler et al where a majority of metastatic cancer cells migrate out of the tumor vasculature at sites of branching [23]. Immunofluorescence and immunohistochemical analysis both at site of CR and outside the radiation field did not show any co-localization of CD31+ cells with BMDCs (Figure 5A,B). Immunofluorescence staining for smooth muscle actin (SMA) confirmed a small, although not statistically significant, increase in the extent of SMA+ BMDC that surrounded the vasculature in the area of CR, compared to the contralateral control non-irradiated brain. However, the majority of peri-vascular BMDCs are not SMA+ (Figure 5C) rather they are inflammatory and microglial-like cells as described below.

Bottom Line: However, despite recognized therapeutic success, significant negative consequences are associated with cranial irradiation (CR), which manifests months to years post-RT.We establish that BMDCs do not form endothelial cells but rather they differentiate predominantly into inflammatory cells and microglia.These results have invaluable therapeutic implications as BMDCs may be a primary therapeutic target to block acute and long-term inflammatory response following CR.

View Article: PubMed Central - PubMed

Affiliation: Brain Tumor Research Centre, SickKids Research Institute, Toronto, Canada.

ABSTRACT
Radiation therapy (RT) is a widely accepted treatment strategy for many central nervous system (CNS) pathologies. However, despite recognized therapeutic success, significant negative consequences are associated with cranial irradiation (CR), which manifests months to years post-RT. The pathophysiology and molecular alterations that culminate in the long-term detrimental effects of CR are poorly understood, though it is thought that endothelial injury plays a pivotal role in triggering cranial injury. We therefore explored the contribution of bone marrow derived cells (BMDCs) in their capacity to repair and contribute to neo-vascularization following CR. Using high-resolution in vivo optical imaging we have studied, at single-cell resolution, the spatio-temporal response of BMDCs in normal brain following CR. We demonstrate that BMDCs are recruited specifically to the site of CR, in a radiation dose and temporal-spatial manner. We establish that BMDCs do not form endothelial cells but rather they differentiate predominantly into inflammatory cells and microglia. Most notably we provide evidence that more than 50% of the microglia in the irradiated region of the brain are not resident microglia but recruited from the bone marrow following CR. These results have invaluable therapeutic implications as BMDCs may be a primary therapeutic target to block acute and long-term inflammatory response following CR. Identifying the critical steps involved in the sustained recruitment and differentiation of BMDCs into microglia at the site of CR can provide new insights into the mechanisms of injury following CR offering potential therapeutic strategies to counteract the long-term adverse effects of CR.

Show MeSH
Related in: MedlinePlus