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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.

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Bone Marrow Derived Cells Migrate Specifically to the Site of Cranial irradiation.(A) H&E staining of a normal brain 7 days following 3*2Gy radiation illustrates the location from where immunofluorescence images in (B) were obtained, 2× magnification. Black arrowhead shows the positioning of the intracranial window (ICW) and so the radiation path. (B) The right irradiated hemisphere demonstrates a trajectory of bone marrow derived cell (BMDC) recruitment which follows a cranial-caudal direction, with maximal recruitment at the cortex gradually decreasing beneath the cortical surface, compared to the contralateral-left, non-irradiated hemisphere which shows no BMDC recruitment (Green: BMDC, blue: nuclei), 10× magnification. (C) 2 photon laser microscopy in-vivo images 7 days post 3*2Gy radiation, illustrate distinct BMDC recruitment to the site of cranial radiation, whilst non-irradiated control brains demonstrate minimal incorporation of BMDCs (Green: BMDC, red: blank, blue: CD31-APC), 5× magnification. (D) To confirm that recruitment of BMDCs visualized on 2 photon laser microscopy is not as a consequence of green auto-fluorescent signals, red fluorescent chimeric mice were used to demonstrate a similar pattern of BMDC recruitment following 3*2Gy radiation but demonstrate a negative green channel (Green: blank, red: BMDC, blue: CD31-APC), 10× magnification. (E) Quantification of BMDCs visualized per field of tissue volume for 2PLM, blue bars, and traditional histology, black line. Note, the 10-fold difference in the scale bars highlighting the statistically significant difference in sensitivity of the 2 methods. This figure highlights the distinct advantage of 2 photon laser microscopy in-vivo imaging over traditional 5 µm cross-sectional histological analysis, clearly allowing visualization of higher levels of BMDCs in the same region of the brain, comparing (B) to (C) which when quantified in context of tissue volume (E) shows 2PLM is 10-fold more sensitive than traditional histology.
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pone-0038366-g001: Bone Marrow Derived Cells Migrate Specifically to the Site of Cranial irradiation.(A) H&E staining of a normal brain 7 days following 3*2Gy radiation illustrates the location from where immunofluorescence images in (B) were obtained, 2× magnification. Black arrowhead shows the positioning of the intracranial window (ICW) and so the radiation path. (B) The right irradiated hemisphere demonstrates a trajectory of bone marrow derived cell (BMDC) recruitment which follows a cranial-caudal direction, with maximal recruitment at the cortex gradually decreasing beneath the cortical surface, compared to the contralateral-left, non-irradiated hemisphere which shows no BMDC recruitment (Green: BMDC, blue: nuclei), 10× magnification. (C) 2 photon laser microscopy in-vivo images 7 days post 3*2Gy radiation, illustrate distinct BMDC recruitment to the site of cranial radiation, whilst non-irradiated control brains demonstrate minimal incorporation of BMDCs (Green: BMDC, red: blank, blue: CD31-APC), 5× magnification. (D) To confirm that recruitment of BMDCs visualized on 2 photon laser microscopy is not as a consequence of green auto-fluorescent signals, red fluorescent chimeric mice were used to demonstrate a similar pattern of BMDC recruitment following 3*2Gy radiation but demonstrate a negative green channel (Green: blank, red: BMDC, blue: CD31-APC), 10× magnification. (E) Quantification of BMDCs visualized per field of tissue volume for 2PLM, blue bars, and traditional histology, black line. Note, the 10-fold difference in the scale bars highlighting the statistically significant difference in sensitivity of the 2 methods. This figure highlights the distinct advantage of 2 photon laser microscopy in-vivo imaging over traditional 5 µm cross-sectional histological analysis, clearly allowing visualization of higher levels of BMDCs in the same region of the brain, comparing (B) to (C) which when quantified in context of tissue volume (E) shows 2PLM is 10-fold more sensitive than traditional histology.

Mentions: Using 2PLM imaging we observed a distinctive pattern of recruitment of BMDCs to the site of CR, as evidenced by the presence of Green Fluorescent Protein (GFP)+BMDCs as early as 1 hour post-RT, in a trajectory that parallels the radiation path in a cranial-caudal direction as seen with both immunofluorescence analysis and 2PLM imaging at the site of the Intra-Cranial Window (ICW) (Figure 1A,B,C). In order to confirm that the green fluorescent signal is not due to auto—fluorescence we used red fluorescent protein (RFP+BMDC) chimeric mice and see the same pattern of recruitment of BMDC to site of CR with no GFP+ signal evident (Figure 1D). The advantages of using a 2PLM in-vivo approach over traditional histological studies is significant, as illustrated by quantification of BMDCs recruited to the site of CR per volume of tissue (Figure 1E). There is a ten–fold increase in the number of detectable BMDCs in 2PLM, through the examination of multiple z-stacked images, when compared with traditional histology. Infiltration of GFP+BMDCs is very specific to the site of CR with minimal GFP+BMDCs present outside of the direct radiation beam. Furthermore, immunofluorescence examination of the contra-lateral non-irradiated hemispheres (Figure 1B), plus 2PLM images of control brains of non-irradiated mice (Figure 1C) showed minimal GFP+BMDC recruitment when compared to irradiated normal brain. To confirm that our observations were not dependant on mouse strain we demonstrate a similar pattern of recruitment in many strains of mice (ICR, Balb/C5 and NODSCID). We also saw the same pattern of recruitment in both our immediate (7 day post bone marrow transplant) and chronic (60+day post bone marrow transplant) chimeric models demonstrating no difference between BMDC recruitment between immediate circulating BMDC and complete reconstitution of BMDC. Furthermore, to verify that total body irradiation (TBI) does not bias our results of BM recruited to site of CR we used lead cranial shielding during TBI for BM reconstitution.


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

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

Bone Marrow Derived Cells Migrate Specifically to the Site of Cranial irradiation.(A) H&E staining of a normal brain 7 days following 3*2Gy radiation illustrates the location from where immunofluorescence images in (B) were obtained, 2× magnification. Black arrowhead shows the positioning of the intracranial window (ICW) and so the radiation path. (B) The right irradiated hemisphere demonstrates a trajectory of bone marrow derived cell (BMDC) recruitment which follows a cranial-caudal direction, with maximal recruitment at the cortex gradually decreasing beneath the cortical surface, compared to the contralateral-left, non-irradiated hemisphere which shows no BMDC recruitment (Green: BMDC, blue: nuclei), 10× magnification. (C) 2 photon laser microscopy in-vivo images 7 days post 3*2Gy radiation, illustrate distinct BMDC recruitment to the site of cranial radiation, whilst non-irradiated control brains demonstrate minimal incorporation of BMDCs (Green: BMDC, red: blank, blue: CD31-APC), 5× magnification. (D) To confirm that recruitment of BMDCs visualized on 2 photon laser microscopy is not as a consequence of green auto-fluorescent signals, red fluorescent chimeric mice were used to demonstrate a similar pattern of BMDC recruitment following 3*2Gy radiation but demonstrate a negative green channel (Green: blank, red: BMDC, blue: CD31-APC), 10× magnification. (E) Quantification of BMDCs visualized per field of tissue volume for 2PLM, blue bars, and traditional histology, black line. Note, the 10-fold difference in the scale bars highlighting the statistically significant difference in sensitivity of the 2 methods. This figure highlights the distinct advantage of 2 photon laser microscopy in-vivo imaging over traditional 5 µm cross-sectional histological analysis, clearly allowing visualization of higher levels of BMDCs in the same region of the brain, comparing (B) to (C) which when quantified in context of tissue volume (E) shows 2PLM is 10-fold more sensitive than traditional histology.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0038366-g001: Bone Marrow Derived Cells Migrate Specifically to the Site of Cranial irradiation.(A) H&E staining of a normal brain 7 days following 3*2Gy radiation illustrates the location from where immunofluorescence images in (B) were obtained, 2× magnification. Black arrowhead shows the positioning of the intracranial window (ICW) and so the radiation path. (B) The right irradiated hemisphere demonstrates a trajectory of bone marrow derived cell (BMDC) recruitment which follows a cranial-caudal direction, with maximal recruitment at the cortex gradually decreasing beneath the cortical surface, compared to the contralateral-left, non-irradiated hemisphere which shows no BMDC recruitment (Green: BMDC, blue: nuclei), 10× magnification. (C) 2 photon laser microscopy in-vivo images 7 days post 3*2Gy radiation, illustrate distinct BMDC recruitment to the site of cranial radiation, whilst non-irradiated control brains demonstrate minimal incorporation of BMDCs (Green: BMDC, red: blank, blue: CD31-APC), 5× magnification. (D) To confirm that recruitment of BMDCs visualized on 2 photon laser microscopy is not as a consequence of green auto-fluorescent signals, red fluorescent chimeric mice were used to demonstrate a similar pattern of BMDC recruitment following 3*2Gy radiation but demonstrate a negative green channel (Green: blank, red: BMDC, blue: CD31-APC), 10× magnification. (E) Quantification of BMDCs visualized per field of tissue volume for 2PLM, blue bars, and traditional histology, black line. Note, the 10-fold difference in the scale bars highlighting the statistically significant difference in sensitivity of the 2 methods. This figure highlights the distinct advantage of 2 photon laser microscopy in-vivo imaging over traditional 5 µm cross-sectional histological analysis, clearly allowing visualization of higher levels of BMDCs in the same region of the brain, comparing (B) to (C) which when quantified in context of tissue volume (E) shows 2PLM is 10-fold more sensitive than traditional histology.
Mentions: Using 2PLM imaging we observed a distinctive pattern of recruitment of BMDCs to the site of CR, as evidenced by the presence of Green Fluorescent Protein (GFP)+BMDCs as early as 1 hour post-RT, in a trajectory that parallels the radiation path in a cranial-caudal direction as seen with both immunofluorescence analysis and 2PLM imaging at the site of the Intra-Cranial Window (ICW) (Figure 1A,B,C). In order to confirm that the green fluorescent signal is not due to auto—fluorescence we used red fluorescent protein (RFP+BMDC) chimeric mice and see the same pattern of recruitment of BMDC to site of CR with no GFP+ signal evident (Figure 1D). The advantages of using a 2PLM in-vivo approach over traditional histological studies is significant, as illustrated by quantification of BMDCs recruited to the site of CR per volume of tissue (Figure 1E). There is a ten–fold increase in the number of detectable BMDCs in 2PLM, through the examination of multiple z-stacked images, when compared with traditional histology. Infiltration of GFP+BMDCs is very specific to the site of CR with minimal GFP+BMDCs present outside of the direct radiation beam. Furthermore, immunofluorescence examination of the contra-lateral non-irradiated hemispheres (Figure 1B), plus 2PLM images of control brains of non-irradiated mice (Figure 1C) showed minimal GFP+BMDC recruitment when compared to irradiated normal brain. To confirm that our observations were not dependant on mouse strain we demonstrate a similar pattern of recruitment in many strains of mice (ICR, Balb/C5 and NODSCID). We also saw the same pattern of recruitment in both our immediate (7 day post bone marrow transplant) and chronic (60+day post bone marrow transplant) chimeric models demonstrating no difference between BMDC recruitment between immediate circulating BMDC and complete reconstitution of BMDC. Furthermore, to verify that total body irradiation (TBI) does not bias our results of BM recruited to site of CR we used lead cranial shielding during TBI for BM reconstitution.

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