| PART 1γ-H2AX:precise index to analyze the formation and repair of DNA Double-strand breaks in vivoPurpose:Double-strand breaks(DSBs) are the most deleterious form of DNA damage after ionizing radiation,and deficiencies in repairing DSBs lead to pronounced radiosensitivity.The clinical radiation responses of different organs vary widely and likely depend on the intrinsic radiosensitivities of their different cell populations but only little is known about how DSBs are repaired differentially in various normal tissues.H2AX phosphorylation is an early step in the response to DNA damage and it has been demonstrated that enumeratingγ-H2AX foci(the phosphorylated histone) can be used to measure the induction and repair of radiation-induced DSBs in vitro.Organs and tissues show substantially varying levels of sensitivity during radiotherapy but only little is known about how DSBs are repaired differentially in various normal tissues.In the present study theγ-H2AX foci approach was established in normal tissues of normal tissue of DSB repair-proficient and repair-deficient mice to evaluate if this approach is a precise index for the DSB repair in vivo and if there are differences in the DSB repair kinetics between the different tissues which may account for their considerably varying intrinsic radiosensitivities.Methods For the DSB induction,normal tissues(brain,small intestine,lung,heart) of C57BL/6 mice was analyzed at 10 min after whole body irradiation with 0.1,0.5 and 1.0 Gy.For the DSB repair kinetics,normal tissue of repair-proficient(C57BL/6 mice) and repair-deficient mouse strains(BALB/c,A-T and SCID mice) were analyzed at 0.5,2.5,5, 24 and 48 h after whole body irradiation with 2Gy.Normal tissues of sham-irradiated mice of each strain served as controls.γ-H2AX immunohistochemistry andγ-H2AX immunofluorescence analysis was used to measure DSBs formation and repair in the normal tissue of the different mouse strains.Results For the DSB induction,identicalγ-H2AX foci levels with a clear linear dose correlation and very low backgrounds in the nuclei in the normal tissue were observed. Scoring the loss ofγ-H2AX foci allowed us to verify the different,genetically determined DSB repair deficiencies,including the minor impairment of BALB/c mice. Repair-proficient C57BL/6 mice exhibited the fastest decrease in foci number with time, and displayed only low levels of residual damage at 24 h and 48 h postirradiation.In contrast,SCID mice showed highly increasedγ-H2AX foci levels at all repair times(0.5 h to 48 h) while A-T mice exhibited a lesser defect which was most significant at later repair times(≥5h).Radiosensitive BALB/c mice exhibited slightly elevated foci numbers compared with C57BL/6 mice at 5 h and 24 h but not at 48 h postirradiation.The DSB repair kinetics measured in complex organs were nearly identical,although these organs reveal clearly different clinical radiosensitivities.Conclusion The results provide evidence that quantifyingγ-H2AX foci in various normal tissues represents a sensitive tool for the detection of induction and repair of radiation-induced DSBs at clinically relevant doses in vivo.Strikingly,the various analyzed tissues exhibited similar kinetics forγ-H2AX foci loss despite their clearly different clinical radiation responses.Hence,the distinct radiosensitivity of parenchymal cells does not rely on tissue-specific differences in DSB repair.Rather,it is suggested that differing down-stream events determine the characteristic radiation responses of different tissues.PART 2 The difference of DNA Double-strand break repair between neurons and microglia in brain tissuePurpose:Double-strand breaks(DSBs) are the most deleterious form of DNA damage after ionizing radiation.Tissues show substantially varying levels of sensitivity during radiotherapy but only little is known about how DSBs are repaired differentially in various cell types of brain.H2AX phosphorylation is an early step in the response to DNA damage and it has been demonstrated that enumeratingγ-H2AX foci can be used to measure the repair of radiation-induced DSBs in vivo.In the present study theγ-H2AX foci approach was established to measure if there are differences in the DSB repair kinetics between the neurons and microglias in the brain tissue which may account for their considerably varying intrinsic functional change.Methods:For the DSB induction,brain tissue of C57BL/6 mice was analyzed at 10 min after cranial irradiation with 0.1,0.5 and 1.0 Gy.For the DSB repair kinetics,brain tissue was analyzed at 0.5,2.5,5,24 and 48 h after cranial irradiation with 2 Gy.Brain tissue of sham-irradiated mice of each strain served as controls.γ-H2AX and NeuN or Iba-1 double immunofluorescence analysis was used to measure DSBs repair and cell morphological change in the different regions of brain between the neurons and microglias.Results:For the DSB induction,identicalγ-H2AX foci levels with a clear linear dose correlation and very low backgrounds in the neurons and microglias of different brain regions were observed.Scoring the loss ofγ-H2AX foci found that repair-proficient C57BL/6 mice exhibited the fastest decrease in foci number with time,and displayed only low levels of residual damage at 24 and 48 h postirradiation.The DSB repair kinetics measured in neurons and microglias of different brain regions were nearly identical. Interestingly,the morphological change of microglias was found which showed a more rounded appearance and their processes retract into the soma.These indicated that microglia was activated after irradiation.Conclusions:The results provide evidence thatγ-H2AX foci kinetics measured in the neurons were similar to kinetics in microglia demonstrating that after clinical dose irradiation the DSB formation and repair kinetics in neurons and microglias are identical in the brain tissue despite their clearly different regions.Strikingly,activated microglias were found after clinical irradiation,which may relate to the microglia's functional change. Activated microglia are intricately participated in the initiation and propagation of the inflammatory response within the brain,If we can find the level of sporadic DNA damage a mature neuron can tolerate as well as the microglia be activated,which could indicate many clues for therapy of many diseases,such as:irradiation-induced brain injury,therapy of Parkinson's disease. analyzed by Western Blot after 16Gy irradiation at 3,6,12,24 and 48 h.Results:Interestingly,the morphological change of microglias was found which showed a more rounded appearance and their processes retract into the soma after irradiation.The the expression of NF-κB(P65) protein was increased after irradiation.These indicated that microglia was activated after irradiation.Conclusions:The results provide evidence that irradiation could induced microglia activation,the possible signal pathway is via NF-κB pathway,which could indicate many clues for therapy of many diseases,such as:irradiation-induced brain injury,therapy of Parkinson's disease.
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