Region-Specific Effects of Fractionated Low-Dose Versus Single-Dose Radiation on Hippocampal Neurogenesis and Neuroinflammation

Background: Despite technical advances in hippocampus-sparing radiotherapy, radiationinduced injury to neural stem cell compartments may affect neurocognitive functions. In pre-clinical mouse models with fractionated low-dose radiation (FLDR) and single-dose radiation (SDR), the accurate response to radiation-induced injury was analyzed in different hippocampal subregions. Methods: Adult and juvenile C57BL/6NCrl mice were exposed to FLDR (20 × 0.1 Gy, daily exposure from Monday to Friday for 4 weeks) or SDR (1 × 2 Gy). In addition, 72 h after the last exposure, neuroglia (astrocytes and microglia) and neuroprogenitor cells were characterized and quantified in the hippocampal cornu ammonis (CA) and dentate gyrus (DG) by immunofluorescence studies. Results: After analyzing different hippocampal subregions, it was observed that radiation responses varied between non-neurogenic CA, with no detectable inflammatory alterations, and neurogenic DG, characterized by impaired neurogenesis and subsequent neuroinflammation. Age-dependent differences in radiosensitivity appeared to depend on the varying proliferative potential of neural stem cell niches. Using the same overall dose for FLDR and SDR (2 Gy), both the cumulative dose over time and also the single dose fraction have decisive impacts on hippocampal damage. Conclusion: Region-specific effects of radiation-induced hippocampal injury relies primarily on cell deaths of proliferating neuroprogenitors. Dose per fraction defines the extent of neuronal injury, and subsequently activated microglia and reactive astrocytes modulate dynamic processes of neuroinflammation. Thus, limiting both cumulative doses and dose fractions to hippocampal DG is an important issue of clinical radiotherapy to preserve neurocognitive functions

Einfluss fraktionierter Niedrig-Dosis-Bestrahlung auf die Neurogenese des adulten und juvenilen Hippocampus

Radiotherapie mit ionisierender Strahlung stellt eine wirkungsvolle Behandlungsmethode dar, um Tumorwachstum effektiv zu kontrollieren. Über die Anpassung des Bestrahlungsfeldes an die Tumorkontur durch verschiedene Einstrahlrichtungen und die fraktionierte Applikation von Strahlendosen wird eine optimale Tumorkontrolle mit gleichzeitiger Minimierung der Normalgewebstoxizitäten gewährleistet, da nur der Tumor hohen Strahlendosen und das umliegende gesunde Gewebe niedrigen Strahlendosen ausgesetzt ist. Die Effekte und Konsequenzen niedriger Strahlendosen wurden bisher nur unzureichend untersucht. Da jedoch bereits kognitive Beeinträchtigungen nach einer pädiatrischen Radiotherapie zu beobachten waren, sollten Auswirkungen niedriger Strahlendosen stärker in den Fokus aktueller Forschungen treten. Im Rahmen dieser Arbeit wurden die Auswirkungen fraktionierter Niedrig-Dosis-Bestrahlung mit 0.1 Gy auf den Hippocampus und die adulte Neurogenese charakterisiert. Hierbei wurden sowohl juvenile (11 Tage alte), als auch adulte (8 Wochen alte) gesunde C57BL6-Mäuse bestrahlt. Die Untersuchungen fanden während der Bestrahlung (72 Stunden) sowie lange nach Bestrahlungsende (1 Monat, 3 Monate und 6 Monate) statt, um auch mögliche Spätfolgen zu erfassen. Für die Analyse der DNA-Schadensantwort wurden persistierende 53BP1-Foci quantifiziert, wobei eine Foci-Akkumulation mit steigender kumulativer Dosis und ein erhöhtes Foci-Level bis zu 3 Monate nach Bestrahlung im Vergleich zur altersgerechten Kontrolle nachgewiesen werden konnten. Potenzielle Auswirkungen auf die adulte Neurogenese wurden durch Immunfluoreszenzmarkierungen von Stamm- (SOX2) und Vorläuferzellen (DCX) analysiert. Bereits drei Tage nach der niedrigsten applizierten Dosis (5x0.1 Gy) lag die proliferierende Vorläuferzellpopulation deutlich vermindert vor und auch ihre dendritische Arborisation war signifikant reduziert. Beide Abnahmen waren bis zu sechs Monate nach Bestrahlung eindeutig nachweisbar. Eingeschränkte Arborisation impliziert eine beeinträchtigte neuronale Verschaltung und Konnektivität der Vorläuferzellen. Die verminderte synaptische Plastizität setzte sich ebenfalls auf neuronaler Ebene durch die reduzierte Expression von PSD-95 fort. Im Gegensatz zu den Vorläuferzellen lag die Stammzellpopulation erst einen Monat nach Applikation der letzten Dosisfraktion deutlich dezimiert vor und blieb es, zumindest in den juvenil-bestrahlten Mäusen, bis zu sechs Monate nach Bestrahlung. Fraktionierte Niedrig-Dosis-Bestrahlung führt somit zu einer starken Beeinträchtigung der Neurogenese. Diese Annahme wurde durch eine umfassende Proteomanalyse juvenil-bestrahlter Hippocampi belegt, die unter anderem eine Deregulation des CREB-Signalweges zeigte. CREB ist sowohl für die Neurogenese als auch für die synaptische Plastizität entscheidend. Aufgrund der ebenfalls neuroprotektiven Funktion von CREB wurde weiterhin mithilfe verschiedener Immunfluoreszenzmarker der Einfluss auf die Neuroinflammation und Gliazellen eruiert. Fraktionierte Niedrig-Dosis-Bestrahlung nahm auf die verschiedenen Gliazellen Einfluss, indem sowohl die Mikroglia- (IBA1) als auch die Oligodendrozyten-Population (OLIG2) in den bestrahlten Proben stark erhöht vorlagen. Auch die Astrozyten (GFAP) zeigten zunächst (72h) eine deutliche Zunahme, bevor sie einen Monat nach Bestrahlung stark dezimiert vorlagen. Die Markierung mit BrdU offenbarte zudem eine Änderung des Zellschicksals von der Neurogenese hin zur Gliogenese, da markierte Stammzellen nach Bestrahlung eher in einen glialen Phänotyp aus-differenzierten. Um herauszufinden, ob die strahlungsinduzierten Veränderungen bereits durch nicht-invasive Methoden feststellbar sind, wurden zusätzlich verschiedene MRT-Untersuchungen durchgeführt. Diese zeigten einen erhöhten zerebralen Blutfluss, sowie er-höhte T1- und T2-Relaxationszeiten im Hippocampus nach Bestrahlung, was indikativ für eine Entzündung oder Zellatrophie steht, wie sie bereits durch die immunhistologischen Experimente gezeigt werden konnte. Die tierexperimentellen Untersuchungen der vorliegenden Arbeit offenbaren eine Beeinträchtigung der adulten Neurogenese durch repetitive Exposition mit niedrigen Strahlendosen. Die Entstehung neuer Neurone wurde stark eingeschränkt und auch das Verhältnis der Gliazellen lag nach Bestrahlung gestört vor, was beides eine verminderte synaptische Plastizität und damit kognitive Beeinträchtigung zur Folge haben könnte. Die vorliegende Arbeit verdeutlicht die Relevanz für Untersuchungen von Niedrig-Dosis exponiertem Gehirngewebe, um potenzielle Nebenwirkungen einer Strahlentherapie benennen und möglicherweise reduzieren zu können. Aufgrund der ausgeprägteren Effekte in jungen Hippocampi sollte dieser während einer Radiotherapie möglichst maximal geschont werden. Ionizing radiation is an effective treatment modality utilized in radiotherapy for tumor growth control. Optimized tumor control with minimal normal tissue toxicity is achieved through ad-justments of the irradiation field with multiple photon-beams from varying directions, as well as, fractionated application of radiation dose. Consequently, a high dose is delivered to the tumor while surrounding tissue is spared and exposed only to low radiation doses. However, consequences of such low-dose radiation (LDR) are, to date, unknown and as cognitive im-pairments following pediatric radiotherapy have been observed, an increased focus of current research is required. The scope of this work was to determine the effects of fractionated LDR with 0.1 Gy on the hippocampus, focusing specifically on adult neurogenesis. For this purpose both juvenile, 11 days old at beginning of irradiation, and adult, 8 weeks old, healthy C57BL6-mice were ana-lyzed. Analysis took place 72 hours after irradiation, and to study late effects, 1 month, 3 months and 6 months after irradiation. DNA damage response was analyzed by 53BP1-foci quantification in neurons which revealed foci accumulation with increasing cumulative doses and elevated foci levels in irradiated samples detectable as late 3 months following irradiation. Potential consequences for adult neurogenesis were determined by immunofluorescence staining of stem- (SOX2) and neuroprogenitor cells (DCX). Proliferating neuroprogenitors al-ready declined three days after application of the lowest dose (5x0.1 Gy) and, additionally, their dendritic arborisation was significantly reduced. These effects were present until 6 months after irradiation. Diminished arborisation may implicate impaired neuronal connectivity and less synaptic plasticity, which was also shown on a neuronal level by the reduced expres-sion of PSD-95. In contrast, stem cells did not decline until one month after irradiation and at least in the juvenile mice, remained low until 6 months after irradiation. Thus, fractionated LDR has a major impact on adult neurogenesis, which was further confirmed by proteomic assay. This showed, amongst others, deregulated CREB signaling. CREB signaling plays a crucial role in adult neurogenesis, synaptic plasticity and also neuroprotection. Due to its in-volvement in neuroprotection, the impact on neuroinflammation and glial cells was investigat-ed by immunofluorescence staining of different glia markers. Fractionated LDR strongly in-fluenced glial cells by increasing microglia (IBA1) and oligodendrocyte (OLIG2) populations. Additionally, astrocytes (GFAP) increased initially (72h), followed by a drastic decline. Label-ing mice with BrdU enabled observation of a cell fate shift from neurogenesis to gliogenesis induced by LDR. Functional MRI was performed to demonstrate that the ascertained changes could be determined by non-invasive methods. Higher cerebral blood flow and increased T1- and T2- relaxation time was detected which may indicate inflammation or cell atrophy. The animal experiments of this present work identified that repeated exposition to low radia-tion doses induced strong impairment in adult neurogenesis. Formation of new neurons was notably restricted and the proportion of glial cells was affected, both of which can lead to re-duced synaptic plasticity and thereby to cognitive impairment. These results illustrate the rele-vance of low dose exposed tissue analysis to determine potential side effects of radiotherapy and to possibly reduce them. As juvenile patients show more pronounced side effects and longer life expectancy, all efforts should be made to treat the hippocampus with maximum care.Ionizing radiation is an effective treatment modality utilized in radiotherapy for tumor growth control. Optimized tumor control with minimal normal tissue toxicity is achieved through adjust-ments of the irradiation field with multiple photon-beams from varying directions, as well as, fractionated application of radiation dose. Consequently, a high dose is delivered to the tumor while surrounding tissue is spared and exposed only to low radiation doses. However, conse-quences of such low-dose radiation (LDR) are, to date, unknown and as cognitive impairments following pediatric radiotherapy have been observed, an increased focus of current research is required. The scope of this work was to determine the effects of fractionated LDR with 0.1 Gy on the hippocampus, focusing specifically on adult neurogenesis. For this purpose both juvenile, 11 days old at beginning of irradiation, and adult, 8 weeks old, healthy C57BL6-mice were ana-lyzed. Analysis took place 72 hours after irradiation, and to study late effects, 1 month, 3 months and 6 months after irradiation. DNA damage response was analyzed by 53BP1-foci quantification in neurons which revealed foci accumulation with increasing cumulative doses and elevated foci levels in irradiated samples detectable as late 3 months following irradiation. Potential consequences for adult neurogenesis were determined by immunofluorescence staining of stem- (SOX2) and neuroprogenitor cells (DCX). Proliferating neuroprogenitors al-ready declined three days after application of the lowest dose (5x0.1 Gy) and, additionally, their dendritic arborisation was significantly reduced. These effects were present until 6 months after irradiation. Diminished arborisation may implicate impaired neuronal connectivity and less synaptic plasticity, which was also shown on a neuronal level by the reduced expression of PSD-95. In contrast, stem cells did not decline until one month after irradiation and at least in the juvenile mice, remained low until 6 months after irradiation. Thus, fractionated LDR has a major impact on adult neurogenesis, which was further confirmed by proteomic assay. This showed, amongst others, deregulated CREB signaling. CREB signaling plays a crucial role in adult neurogenesis, synaptic plasticity and also neuroprotection. Due to its involvement in neu-roprotection, the impact on neuroinflammation and glial cells was investigated by immunofluo-rescence staining of different glia markers. Fractionated LDR strongly influenced glial cells by increasing microglia (IBA1) and oligodendrocyte (OLIG2) populations. Additionally, astrocytes (GFAP) increased initially (72h), followed by a drastic decline. Labeling mice with BrdU ena-bled observation of a cell fate shift from neurogenesis to gliogenesis induced by LDR. Func-tional MRI was performed to demonstrate that the ascertained changes could be determined by non-invasive methods. Higher cerebral blood flow and increased T1- and T2- relaxation time was detected which may indicate inflammation or cell atrophy. The animal experiments of this present work identified that repeated exposition to low radiation doses induced strong impairment in adult neurogenesis. Formation of new neurons was nota-bly restricted and the proportion of glial cells was affected, both of which can lead to reduced synaptic plasticity and thereby to cognitive impairment. These results illustrate the relevance of low dose exposed tissue analysis to determine potential side effects of radiotherapy and to possibly reduce them. As juvenile patients show more pronounced side effects and longer life expectancy, all efforts should be made to treat the hippocampus with maximum care

Cultured Human Foreskin as a Model System for Evaluating Ionizing Radiation-Induced Skin Injury

Purpose: Precise molecular and cellular mechanisms of radiation-induced dermatitis are incompletely understood. Histone variant H2A.J is associated with cellular senescence and modulates senescence-associated secretory phenotype (SASP) after DNA-damaging insults, such as ionizing radiation (IR). Using ex vivo irradiated cultured foreskin, H2A.J was analyzed as a biomarker of radiation-induced senescence, potentially initiating the inflammatory cascade of radiation-induced skin injury. Methods: Human foreskin explants were collected from young donors, irradiated ex vivo with 10 Gy, and cultured in air-liquid interphase for up to 72 h. At different time-points after ex vivo IR exposure, the foreskin epidermis was analyzed for proliferation and senescence markers by immunofluorescence and immunohistochemical staining of sectioned tissue. Secretion of cytokines was measured in supernatants by ELISA. Using our mouse model with fractionated in vivo irradiation, H2A.J expression was analyzed in epidermal stem/progenitor cell populations localized in different regions of murine hair follicles (HF). Results: Non-vascularized foreskin explants preserved their tissue homeostasis up to 72 h (even after IR exposure), but already nonirradiated foreskin epithelium expressed high levels of H2A.J in all epidermal layers and secreted high amounts of cytokines. Unexpectedly, no further increase in H2A.J expression and no obvious upregulation of cytokine secretion was observed in the foreskin epidermis after ex vivo IR. Undifferentiated keratinocytes in murine HF regions, by contrast, revealed low H2A.J expression in non-irradiated skin and significant radiation-induced H2A.J upregulations at different time-points after IR exposure. Based on its staining characteristics, we presume that H2A.J may have previously underestimated the importance of the epigenetic regulation of keratinocyte maturation. Conclusions: Cultured foreskin characterized by highly keratinized epithelium and specific immunological features is not an appropriate model for studying H2A.J-associated tissue reactions during radiation-induced dermatitis

Human skin aging is associated with increased expression of the histone variant H2A.J in the epidermis

Cellular senescence is an irreversible growth arrest that occurs as a result of damaging stimuli, including DNA damage and/or telomere shortening. Here, we investigate histone variant H2A.J as a new biomarker to detect senescent cells during human skin aging. Skin biopsies from healthy volunteers of different ages (18–90 years) were analyzed for H2A.J expression and other parameters involved in triggering and/or maintaining cellular senescence. In the epidermis, the proportions of H2A.J-expressing keratinocytes increased from ≈20% in young to ≈60% in aged skin. Inverse correlations between Ki67- and H2A.J staining in germinative layers may reflect that H2A.J-expressing cells having lost their capacity to divide. As cellular senescence is triggered by DNA-damage signals, persistent 53BP1-foci, telomere lengths, and telomere-associated damage foci were analyzed in epidermal keratinocytes. Only slight age-related telomere attrition and few persistent nuclear 53BP1-foci, occasionally colocalizing with telomeres, suggest that unprotected telomeres are not a significant cause of senescence during skin aging. Quantification of integrin-α6+ basal cells suggests that the number and function of stem/progenitor cells decreased during aging and their altered proliferation capacities resulted in diminished tissue renewal with epidermal thinning. Collectively, our findings suggest that H2A.J is a sensitive marker of epidermal aging in human skin

Region-Specific Effects of Fractionated Low-Dose Versus Single-Dose Radiation on Hippocampal Neurogenesis and Neuroinflammation

Background: Despite technical advances in hippocampus-sparing radiotherapy, radiation-induced injury to neural stem cell compartments may affect neurocognitive functions. In pre-clinical mouse models with fractionated low-dose radiation (FLDR) and single-dose radiation (SDR), the accurate response to radiation-induced injury was analyzed in different hippocampal subregions. Methods: Adult and juvenile C57BL/6NCrl mice were exposed to FLDR (20 × 0.1 Gy, daily exposure from Monday to Friday for 4 weeks) or SDR (1 × 2 Gy). In addition, 72 h after the last exposure, neuroglia (astrocytes and microglia) and neuroprogenitor cells were characterized and quantified in the hippocampal cornu ammonis (CA) and dentate gyrus (DG) by immunofluorescence studies. Results: After analyzing different hippocampal subregions, it was observed that radiation responses varied between non-neurogenic CA, with no detectable inflammatory alterations, and neurogenic DG, characterized by impaired neurogenesis and subsequent neuroinflammation. Age-dependent differences in radiosensitivity appeared to depend on the varying proliferative potential of neural stem cell niches. Using the same overall dose for FLDR and SDR (2 Gy), both the cumulative dose over time and also the single dose fraction have decisive impacts on hippocampal damage. Conclusion: Region-specific effects of radiation-induced hippocampal injury relies primarily on cell deaths of proliferating neuroprogenitors. Dose per fraction defines the extent of neuronal injury, and subsequently activated microglia and reactive astrocytes modulate dynamic processes of neuroinflammation. Thus, limiting both cumulative doses and dose fractions to hippocampal DG is an important issue of clinical radiotherapy to preserve neurocognitive functions

Cultured Human Foreskin as a Model System for Evaluating Ionizing Radiation-Induced Skin Injury

Purpose: Precise molecular and cellular mechanisms of radiation-induced dermatitis are incompletely understood. Histone variant H2A.J is associated with cellular senescence and modulates senescence-associated secretory phenotype (SASP) after DNA-damaging insults, such as ionizing radiation (IR). Using ex vivo irradiated cultured foreskin, H2A.J was analyzed as a biomarker of radiation-induced senescence, potentially initiating the inflammatory cascade of radiation-induced skin injury. Methods: Human foreskin explants were collected from young donors, irradiated ex vivo with 10 Gy, and cultured in air-liquid interphase for up to 72 h. At different time-points after ex vivo IR exposure, the foreskin epidermis was analyzed for proliferation and senescence markers by immunofluorescence and immunohistochemical staining of sectioned tissue. Secretion of cytokines was measured in supernatants by ELISA. Using our mouse model with fractionated in vivo irradiation, H2A.J expression was analyzed in epidermal stem/progenitor cell populations localized in different regions of murine hair follicles (HF). Results: Non-vascularized foreskin explants preserved their tissue homeostasis up to 72 h (even after IR exposure), but already non-irradiated foreskin epithelium expressed high levels of H2A.J in all epidermal layers and secreted high amounts of cytokines. Unexpectedly, no further increase in H2A.J expression and no obvious upregulation of cytokine secretion was observed in the foreskin epidermis after ex vivo IR. Undifferentiated keratinocytes in murine HF regions, by contrast, revealed low H2A.J expression in non-irradiated skin and significant radiation-induced H2A.J upregulations at different time-points after IR exposure. Based on its staining characteristics, we presume that H2A.J may have previously underestimated the importance of the epigenetic regulation of keratinocyte maturation. Conclusions: Cultured foreskin characterized by highly keratinized epithelium and specific immunological features is not an appropriate model for studying H2A.J-associated tissue reactions during radiation-induced dermatitis