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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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Microvascular Alterations Post Radiation.Immunohistochemical co-staining of CD31 and TUNEL confirms endothelial cell apoptosis occurs as early as 1 hour post radiation at site of cranial radiation. (A) Increasing radiation doses from 2Gy to 15Gy increases endothelial cell apoptosis as seen in immunohistochemistry sections (Brown: CD31, Pink: TUNEL), 40× magnification. (B) Graphical representation of endothelial apoptosis, CD31+ TUNEL+, illustrates that 15Gy significantly induces the most endothelial apoptosis when compared to 2Gy and 6Gy at 1 hour post radiation, (p = 0.0001***) and that endothelial cell apoptosis levels at 15Gy 1 hour post radiation are significantly increased compared to 1 day post radiation, (p = 0.0004**). (C) Quantification of apoptosis of total parenchymal cells shows that 15Gy significantly induces most apoptosis (p = 0.0075**) at 1 day post radiation, however significantly more apoptosis is induced 1 day post radiation than at 1 hour post radiation (p = 0.0224*). Compared with what is seen with endothelial cell apoptosis it is clear that maximal endothelial cell apoptosis occurs earlier than overall parenchymal cell apoptosis. (D) Further analysis of vessels structure at the site of radiation, 7 days post 3*2Gy, demonstrates significant increases in both the density, p<0.0001***, and diameter of vessels, p<0.0001***, when compared to non-irradiated controls. (E) CD31 immunohistochemistry sections confirm the change in vessel density and diameter 7 days following 3*2Gy radiation when compared to non-irradiated control tissue, 10× magnification.
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pone-0038366-g004: Microvascular Alterations Post Radiation.Immunohistochemical co-staining of CD31 and TUNEL confirms endothelial cell apoptosis occurs as early as 1 hour post radiation at site of cranial radiation. (A) Increasing radiation doses from 2Gy to 15Gy increases endothelial cell apoptosis as seen in immunohistochemistry sections (Brown: CD31, Pink: TUNEL), 40× magnification. (B) Graphical representation of endothelial apoptosis, CD31+ TUNEL+, illustrates that 15Gy significantly induces the most endothelial apoptosis when compared to 2Gy and 6Gy at 1 hour post radiation, (p = 0.0001***) and that endothelial cell apoptosis levels at 15Gy 1 hour post radiation are significantly increased compared to 1 day post radiation, (p = 0.0004**). (C) Quantification of apoptosis of total parenchymal cells shows that 15Gy significantly induces most apoptosis (p = 0.0075**) at 1 day post radiation, however significantly more apoptosis is induced 1 day post radiation than at 1 hour post radiation (p = 0.0224*). Compared with what is seen with endothelial cell apoptosis it is clear that maximal endothelial cell apoptosis occurs earlier than overall parenchymal cell apoptosis. (D) Further analysis of vessels structure at the site of radiation, 7 days post 3*2Gy, demonstrates significant increases in both the density, p<0.0001***, and diameter of vessels, p<0.0001***, when compared to non-irradiated controls. (E) CD31 immunohistochemistry sections confirm the change in vessel density and diameter 7 days following 3*2Gy radiation when compared to non-irradiated control tissue, 10× magnification.

Mentions: Microvascular injury, specifically endothelial cell (EC) apoptosis, is considered to play a pivotal role in radiation-induced cranial injury. We assessed the extent of EC apoptosis in a longitudinal and radiation dose dependent manner and in relation to the extent of BMDC recruitment, through double immunohistochemical staining for EC (CD31) and apoptosis (TUNEL) according to previous published protocols by Fuks et al [21], [22]. A statistically significant (p<0.0001) increase in EC apoptosis (CD31+ TUNEL+) was observed with each increasing radiation dose, from 2Gy to 15Gy (Figure 4A,B). The highest degree of EC apoptosis occurred at 1 hour post-15Gy RT and diminished thereafter in a radiation dose and time dependent manner, decreasing significantly by 1 day post-RT, (p = 0.0001). At radiation doses less than 6Gy minimal EC apoptosis was observed. We also quantified overall apoptotic cells in the normal brain parenchyma (TUNEL+). The peak of apoptosis in the normal brain parenchyma occurred significantly later than EC specific apoptosis, with the highest level seen 1 day post-RT (p = 0.0224). The highest degree of parenchymal TUNEL+ cells was seen in response to 15Gy, which was significantly higher than that seen with the other radiation doses (p = 0.0075) (Figure 4C). We analyzed alterations to vascular structure in response to CR by measuring microvascular density, vessel diameter and vessel leakiness. There was a statistically significant increase in vessel diameter by 7 days post-RT together with an increase in microvascular density (p<0.0001) (Figure 4D,E). Increased vessel leakiness was detected at 7 days following CR using Evans Blue perfusion fixation (Figure S1A). We investigated whether there was an associated change in cerebral blood flow (CBF) with ultrastructural changes observed in the vasculature at the site of CR. We used MRI flow-alternating-inversion-recovery techniques to measure CBF, comparing irradiated (R) hemisphere to non-irradiated (L) hemisphere. We found that at site of CR associated with the area of increased MVD and dilated vessels there was no significant change in CBF (Figure S2).


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

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

Microvascular Alterations Post Radiation.Immunohistochemical co-staining of CD31 and TUNEL confirms endothelial cell apoptosis occurs as early as 1 hour post radiation at site of cranial radiation. (A) Increasing radiation doses from 2Gy to 15Gy increases endothelial cell apoptosis as seen in immunohistochemistry sections (Brown: CD31, Pink: TUNEL), 40× magnification. (B) Graphical representation of endothelial apoptosis, CD31+ TUNEL+, illustrates that 15Gy significantly induces the most endothelial apoptosis when compared to 2Gy and 6Gy at 1 hour post radiation, (p = 0.0001***) and that endothelial cell apoptosis levels at 15Gy 1 hour post radiation are significantly increased compared to 1 day post radiation, (p = 0.0004**). (C) Quantification of apoptosis of total parenchymal cells shows that 15Gy significantly induces most apoptosis (p = 0.0075**) at 1 day post radiation, however significantly more apoptosis is induced 1 day post radiation than at 1 hour post radiation (p = 0.0224*). Compared with what is seen with endothelial cell apoptosis it is clear that maximal endothelial cell apoptosis occurs earlier than overall parenchymal cell apoptosis. (D) Further analysis of vessels structure at the site of radiation, 7 days post 3*2Gy, demonstrates significant increases in both the density, p<0.0001***, and diameter of vessels, p<0.0001***, when compared to non-irradiated controls. (E) CD31 immunohistochemistry sections confirm the change in vessel density and diameter 7 days following 3*2Gy radiation when compared to non-irradiated control tissue, 10× magnification.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3366930&req=5

pone-0038366-g004: Microvascular Alterations Post Radiation.Immunohistochemical co-staining of CD31 and TUNEL confirms endothelial cell apoptosis occurs as early as 1 hour post radiation at site of cranial radiation. (A) Increasing radiation doses from 2Gy to 15Gy increases endothelial cell apoptosis as seen in immunohistochemistry sections (Brown: CD31, Pink: TUNEL), 40× magnification. (B) Graphical representation of endothelial apoptosis, CD31+ TUNEL+, illustrates that 15Gy significantly induces the most endothelial apoptosis when compared to 2Gy and 6Gy at 1 hour post radiation, (p = 0.0001***) and that endothelial cell apoptosis levels at 15Gy 1 hour post radiation are significantly increased compared to 1 day post radiation, (p = 0.0004**). (C) Quantification of apoptosis of total parenchymal cells shows that 15Gy significantly induces most apoptosis (p = 0.0075**) at 1 day post radiation, however significantly more apoptosis is induced 1 day post radiation than at 1 hour post radiation (p = 0.0224*). Compared with what is seen with endothelial cell apoptosis it is clear that maximal endothelial cell apoptosis occurs earlier than overall parenchymal cell apoptosis. (D) Further analysis of vessels structure at the site of radiation, 7 days post 3*2Gy, demonstrates significant increases in both the density, p<0.0001***, and diameter of vessels, p<0.0001***, when compared to non-irradiated controls. (E) CD31 immunohistochemistry sections confirm the change in vessel density and diameter 7 days following 3*2Gy radiation when compared to non-irradiated control tissue, 10× magnification.
Mentions: Microvascular injury, specifically endothelial cell (EC) apoptosis, is considered to play a pivotal role in radiation-induced cranial injury. We assessed the extent of EC apoptosis in a longitudinal and radiation dose dependent manner and in relation to the extent of BMDC recruitment, through double immunohistochemical staining for EC (CD31) and apoptosis (TUNEL) according to previous published protocols by Fuks et al [21], [22]. A statistically significant (p<0.0001) increase in EC apoptosis (CD31+ TUNEL+) was observed with each increasing radiation dose, from 2Gy to 15Gy (Figure 4A,B). The highest degree of EC apoptosis occurred at 1 hour post-15Gy RT and diminished thereafter in a radiation dose and time dependent manner, decreasing significantly by 1 day post-RT, (p = 0.0001). At radiation doses less than 6Gy minimal EC apoptosis was observed. We also quantified overall apoptotic cells in the normal brain parenchyma (TUNEL+). The peak of apoptosis in the normal brain parenchyma occurred significantly later than EC specific apoptosis, with the highest level seen 1 day post-RT (p = 0.0224). The highest degree of parenchymal TUNEL+ cells was seen in response to 15Gy, which was significantly higher than that seen with the other radiation doses (p = 0.0075) (Figure 4C). We analyzed alterations to vascular structure in response to CR by measuring microvascular density, vessel diameter and vessel leakiness. There was a statistically significant increase in vessel diameter by 7 days post-RT together with an increase in microvascular density (p<0.0001) (Figure 4D,E). Increased vessel leakiness was detected at 7 days following CR using Evans Blue perfusion fixation (Figure S1A). We investigated whether there was an associated change in cerebral blood flow (CBF) with ultrastructural changes observed in the vasculature at the site of CR. We used MRI flow-alternating-inversion-recovery techniques to measure CBF, comparing irradiated (R) hemisphere to non-irradiated (L) hemisphere. We found that at site of CR associated with the area of increased MVD and dilated vessels there was no significant change in CBF (Figure S2).

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