Radiation-induced bystander effect on the brain after fractionated spinal cord irradiation of aging rats.

We investigated the influence of the so-called bystander effect on metabolic and histopathological changes in the rat brain after fractionated spinal cord irradiation. The study was initiated with adult Wistar male rats (n = 20) at the age of 9 months. The group designated to irradiation (n = 10) and the age-matched control animals (n = 10) were subjected to an initial measurement using in vivo proton magnetic resonance spectroscopy (1H MRS) and magnetic resonance imaging (MRI). After allowing the animals to survive until 12 months, they received fractionated spinal cord irradiation with a total dose of 24 Gy administered in 3 fractions (8 Gy per fraction) once a week on the same day for 3 consecutive weeks. 1H MRS and MRI of brain metabolites were performed in the hippocampus, corpus striatum, and olfactory bulb (OB) before irradiation (9-month-old rats) and subsequently 48 h (12-month-old) and 2 months (14-month-old) after the completion of irradiation. After the animals were sacrificed at the age of 14 months, brain tissue changes were investigated in two neurogenic regions: the hippocampal dentate gyrus (DG) and the rostral migratory stream (RMS). By comparing the group of 9-month-old rats and individuals measured 48 h (at the age of 12 months) after irradiation, we found a significant decrease in the ratio of total N-acetyl aspartate to total creatine (tNAA/tCr) and gamma-aminobutyric acid to tCr (GABA/tCr) in OB and hippocampus. A significant increase in myoinositol to tCr (mIns/tCr) in the OB persisted up to 14 months of age. Proton nuclear magnetic resonance (1H NMR)-based plasma metabolomics showed a significant increase in keto acids and decreased tyrosine and tricarboxylic cycle enzymes. Morphometric analysis of neurogenic regions of 14-month-old rats showed well-preserved stem cells, neuroblasts, and increased neurodegeneration. The radiation-induced bystander effect more significantly affected metabolite concentration than the distribution of selected cell types.

Anatomic and metabolic alterations in the rodent frontal cortex caused by clinically relevant fractionated whole-brain irradiation.

Radiation-induced brain injury (RII) is a harmful side-effect occurring after conventional radiation therapy (usually fractionated whole-brain irradiation/fWBI) of patients with cerebral tumors and metastases. An important role in the quality of patients' lives plays cognitive, executive, and emotional functions, regulation on which are involved in frontal cortices pathways. This study assessed the morphologic and metabolic alterations in the rodent frontal cortex caused by fWBI with the total dose of 32 Gy in 4 fractions performed by linear accelerator Clinac iX. Nine male Wistar rats underwent radiation procedures, whereas the other nine rats were investigated as a sham-irradiated group. All eighteen animals were examined using magnetic resonance (MR) in three intervals - before, on 2nd, and 70th day after sham/irradiation. After ten weeks of surviving, all rats underwent histopathological analysis determined by image analysis of immunofluorescent stained sections in the frontal cortex. MR examination was performed on 7T MR scanner Bruker BioSpec 70/20 and consisted of MR-volumetry, T2 relaxometry, and single-voxel proton-1 MR spectroscopy localized in the frontal cortex. Both tissue volume and T2 relaxation time of the frontal cortex were significantly lower in animals after 2 and 70 days of exposure than in controls; however, there were no differences between irradiated groups. Similarly, in animals' frontal cortex after fWBI, increased levels of myoinositol and glutamate/glutamine ratios were observed. Ratios of N-acetyl-aspartate, choline, and peaks of lactate and lipids did not change between groups. The histopathological analysis of the frontal cortex showed increased signs of neurodegeneration and a slight increase in astrocytes and microglia in exposed animals. Early (2 days, 10 weeks) after clinically relevant fWBI were in the frontal cortices of exposed rodents confirmed morphologic and metabolic changes indicating neurodegenerative changes, initializing cerebral atrophy, and evident signs of endothelial disruption and dysregulated neurotransmission that may cause a wide range of functional as well as cognitive deficits.

Effect of fractionated whole-brain irradiation on brain and plasma in a rat model: Metabolic, volumetric and histopathological changes.

In the present study, we investigated the correlation between histopathological, metabolic, and volumetric changes in the brain and plasma under experimental conditions. Adult male Wistar rats received fractionated whole-brain irradiation (fWBI) with a total dose of 32 Gy delivered in 4 fractions (dose 8 Gy per fraction) once a week on the same day for 4 consecutive weeks. Proton magnetic resonance spectroscopy (1H MRS) and imaging were used to detect metabolic and volumetric changes in the brain and plasma. Histopathological changes in the brain were determined by image analysis of immunofluorescent stained sections. Metabolic changes in the brain measured by 1H MRS before, 48 h, and 9 weeks after the end of fWBI showed a significant decrease in the ratio of total N-acetylaspartate to total creatine (tNAA/tCr) in the corpus striatum. We found a significant decrease in glutamine + glutamate/tCr (Glx/tCr) and, conversely, an increase in gamma-aminobutyric acid to tCr (GABA/tCr) in olfactory bulb (OB). The ratio of astrocyte marker myoinositol/tCr (mIns/tCr) significantly increased in almost all evaluated areas. Magnetic resonance imaging (MRI)-based brain volumetry showed a significant increase in volume, and a concomitant increase in the T2 relaxation time of the hippocampus. Proton nuclear magnetic resonance (1H NMR) plasma metabolomics displayed a significant decrease in the level of glucose and glycolytic intermediates and an increase in ketone bodies. The histomorphological analysis showed a decrease to elimination of neuroblasts, increased astrocyte proliferation, and a mild microglia response. The results of the study clearly reflect early subacute changes 9-11 weeks after fWBI with strong manifestations of brain edema, astrogliosis, and ongoing ketosis.

Model-based calculation of thyroid gland normal tissue complication probability in head and neck cancer patients after radiation therapy.

AIM: Primary hypothyroidism is one of the late complications that can occur after radiation therapy for malignant tumors in the head and neck region. The aim of this retrospective study was to show the validity of the Lyman-Kutcher-Burman (LKB) normal tissue complication model for thyroid gland based on clinical results. METHODS: Thyroid function was evaluated by measuring thyroid-stimulating hormone and free thyroxine serum levels before radiation therapy, 3 months after the beginning of radiation therapy, and afterwards at each follow-up visit. Cumulative incidence was calculated using the Kaplan-Meier method. Dose-volume histogram, total dose, fractionation schedule, total duration of the treatment, and other parameters were used for normal tissue complication probability calculation based on the LKB model. The model was evaluated after fitting with the three sets of parameters for grade 2 hypothyroidism: 1) "Emami," where n = 0.22; m = 0.26, and D50 = 80 Gy; 2) "mean dose," where n = 1; m = 0.27, and D50 = 60 Gy; and 3) "Lyman EUD," where n = 0.49; m = 0.24, and D50 = 60 Gy. A value 3.0 Gy was used for α/ß ratio RESULTS: Eighty-three patients treated with volumetric modulated arc therapy for head and neck cancers at the University Hospital Martin, Slovakia, from January 2014 to July 2017, were included in the retrospective study. Median follow-up was 1.2 years. Cumulative incidence of hypothyroidism grade 2 or higher after 12 and 24 months was 9.6 and 22.0%, respectively. Normal tissue complication probability values calculated with mean dose and Lyman EUD parameters showed the best correlation with our clinical findings. CONCLUSION: Empirically based modelling of normal tissue complication probability was valid for our cohort of patients. With carefully chosen parameters, the LKB model can be used for predicting the normal tissue complication probability value.

Metabolic and histopathological changes in the brain and plasma of rats exposed to fractionated whole-brain irradiation.

In the present study we investigated the correlation between radiation-induced metabolic and histopathological changes in the brain under experimental conditions. Adult male Wistar rats received fractionated whole-brain irradiation (fWBI) with a total dose of 40 Gy administered in 5 fractions (dose 8 Gy per fraction) once a week on the same day for 5 consecutive weeks. Radiation-induced alteration in plasma and brain metabolites were measured by proton nuclear magnetic resonance (1H NMR)-based metabolomics and proton magnetic resonance spectroscopy (1HMRS). Histopathological changes in the brain were evaluated to determine alteration of neurogenesis and glial cell responses in 2 neurogenic regions: the hippocampal dentate gyrus (DG) and the subventricular zone-olfactory bulb axis (SVZ-OB axis). Evaluation of brain metabolites 15 weeks after irradiation performed with 1H MRS showed a significant decrease in the total N-acetylaspartate to total creatine (tNAA/tCr) ratio in the striatum, hippocampus, and OB, while gamma-aminobutyric acid to tCr (GABA/tCr) ratio in the hippocampus as well as OB and total choline to tCr (tCho/tCr) in striatum and OB. Magnetic resonance imaging (MRI) volumetric analysis showed a significant reduction in total brain volume and atrophy of dorsal hippocampus and OB. 1H NMR in plasma of irradiated animals displayed decreased citrate and increased bile acids. Image analysis of the brain sections 16 weeks after fWBI showed an increase in neurodegeneration and inhibition of neurogenesis. Results showed that fWBI led to metabolic alterations associated with histopathological findings, suggesting a subacute and development of late radiation-induced changes.

Effects of fractionated whole-brain irradiation on cellular composition and cognitive function in the rat brain.

PURPOSE: The aim of this study was investigate whether histopathological changes in the neurogenic region correlate with appropriate cognitive impairment in the experimental model of radiation-induced brain injury. MATERIALS AND METHODS: Adult male Wistar rats randomized into sham (0 Gy) and two experimental groups (survived 30 and 100 days after treatment) received fractionated whole-brain irradiation (one 5 Gy fraction/week for four weeks) with a total dose of 20 Gy of gamma rays. Morris water maze cognitive testing, histochemistry, immunohistochemistry and confocal microscopy were used to determine whether the cognitive changes are associated with the alteration of neurogenesis, astrocytic response and activation of microglia along and/or adjacent to well-defined pathway, subventricular zone-olfactory bulb axis (SVZ-OB axis). RESULTS: Irradiation revealed altered cognitive functions usually at 100 days after treatment. Neurodegenerative changes were characterized by a significant increase of Fluoro-Jade-positive cells 30 days after irradiation accompanied by a steep decline of neurogenesis 100 days after treatment. A strong astrocytic response and upregulation of the activated microglia were seen in both of experimental groups. CONCLUSIONS: Results shows that fractionated irradiation led to cognitive impairment closely associated with accerelation of neuronal cell death, inhibition of neurogenesis, activation of astrocytes and microglia indicate early delayed radiation-induced changes.

Effect of whole-brain irradiation on the specific brain regions in a rat model: Metabolic and histopathological changes.

Effect of ionizing radiation on the brain affects neuronal, glial, and endothelial cell population and lead to significant morphological, metabolic, and functional deficits. In the present study we investigated a dose- and time-dependent correlation between radiation-induced metabolic and histopathological changes. Adult male Wistar rats received a total dose of 35Gy delivered in 7 fractions (dose 5Gy per fraction) once per week in the same weekday during 7 consecutive weeks. Proton magnetic resonance spectroscopy (1H MRS), histochemistry, immunohistochemistry and confocal microscopy were used to determine whether radiation-induced alteration of the brain metabolites correlates with appropriate histopathological changes of neurogenesis and glial cell response in 2 neurogenic regions: the hippocampal dentate gyrus (DG) and the subventricular zone-olfactory bulb axis (SVZ-OB axis). Evaluation of the brain metabolites 18-19 weeks after irradiation performed by 1H MRS revealed a significant decrease in the total N-acetylaspartate to total creatine (tNAA/tCr) ratio in the striatum and OB. A significant decline of gamma-aminobutyric acid to tCr (GABA/tCr) ratio was seen in the OB and hippocampus. MR revealed absence of gross inflammatory or necrotic lesions in these regions. Image analysis of the brain sections 18-21 weeks after the exposure showed a radiation-induced increase of neurodegeneration, inhibition of neurogenesis and strong resemblance to the reactive astrogliosis. Results showed that fractionated whole-brain irradiation led to the changes in neurotransmission and to the loss of neuronal viability in vivo. Metabolic changes were closely associated with histopathological findings, i.e. initiation of neuronal cell death, inhibition of neurogenesis and strong response of astrocytes indicated development of late radiation-induced changes.

Differential expression of doublecortin and microglial markers in the rat brain following fractionated irradiation.

Ionizing radiation induces altered brain tissue homeostasis and can lead to morphological and functional deficits. In this study, adult male Wistar rats received whole-body exposure with fractionated doses of gamma rays (a total dose of 5 Gy) and were investigated 30 and 60 days later. Immunohistochemistry and confocal microscopy were used to determine proliferation rate of cells residing or derived from the forebrain anterior subventricular zone (SVZa) and microglia distributed along and/or adjacent to subventricular zone-olfactory bulb axis. Cell counting was performed in four anatomical parts along the well-defined pathway, known as the rostral migratory stream (RMS) represented by the SVZa, vertical arm, elbow and horizontal arm of the RMS. Different spatiotemporal distribution pattern of cell proliferation was seen up to 60 days after irradiation through the migratory pathway. A population of neuroblasts underwent less evident changes up to 60 days after treatment. Fractionated exposure led to decline or loss of resting as well as reactive forms of microglia until 60 days after irradiation. Results showed that altered expression of the SVZa derived cells and ultimative decrease of microglia may contribute to development of radiation-induced late effects.

Ionizing radiation induced long-term alterations in the adult rat rostral migratory stream.

Ionizing radiation can induce significant injury to normal brain structures. To assess radiation-induced late effects, adult male Wistar rats received whole-body exposure with fractionated doses of gamma rays (a total dose of 4Gy) and were investigated thirty, sixty and ninety days later. Immunohistochemistry and confocal microscopy were used to determine the density of neuroblasts derived from the anterior subventricular zone (SVZa) and brain resident microglia distributed along and/or adjacent to subventricular zone-olfactory bulb axis (SVZ-OB axis). Cell counting was performed in four anatomical parts along the well defined pathway, known as the rostral migratory stream (RMS) represented by the SVZa, vertical arm, elbow and horizontal arm of the RMS. Strong overdistribution of neuroblasts was seen in the SVZa thirty and sixty days after irradiation replaced by a steep decline in the following parts of the RMS and the highest decrease ninety days after radiation treatment along the entire SVZ-OB axis. Radiation treatment led to a decline or loss of microglia in almost all counted parts through the entire experiment. Results showed that ultimate decline of the SVZa descendants and loss of microglia suggests a contributory role of reduced neurogenesis in the development of radiation-induced late effects.

Long-term alterations of cell population in the adult rat forebrain following the exposure to fractionated doses of ionizing radiation.

We investigated radiation-induced delayed alterations of proliferating population, cells undergoing apoptosis and glial cells housed rat brain neurogenic region. Adult male Wistar rats were investigated 30, 60 or 90 days after whole-body irradiation with fractionated doses of gamma rays (the total dose of 4 Gy). Using immunohistochemistry for detection of cell proliferation marker Ki-67, caspase3 as apoptotic marker and GFAP for mature astrocytes we have been performed quantitative analysis in different forebrain's areas along the SVZ-OB axis, i.e. in the anterior subvetricular zone (SVZa), vertical arm, elbow and horizontal arm. In animals that survived thirty days after radiation treatment initial decrease of the Ki-67-positive cells was seen in regions along the SVZ-OB axis. The highest increase was observed in vertical arm on the 60th day followed by the most striking decline on the 90th day after irradiation. Cells undergoing apoptosis didn't showed expressive increase during entire experiment except of horizontal arm. The most striking changes of GFAP-positive cells were seen 30 and 60 days after irradiation in vertical arm and elbow. Results suggested that radiation response of proliferating cells and astrocytes resides the SVZa may play contributory role in development of more adverse radiation-induced late effects.

Fractionated irradiation-induced altered spatio-temporal cell distribution in the rat forebrain.

Ionizing radiation as one of the strongest cytogenetic factors can induce significant injury to the adult brain. In the present study, adult male Wistar rats were exposed to whole-body irradiation with fractionated doses of gamma rays (a total dose of 3Gy). Seven, 14 and 21 days after irradiation the cell types located in the neurogenic anterior subventricular zone (SVZa) were labeled using immunohistochemistry for SVZa-derived young neurons and astrocytes. Cell counting was performed in four anatomical parts along the pathway known as the rostral migratory stream (RMS) represented by the SVZa, vertical arm, elbow and horizontal arm of the RMS. A considerable increase was seen in the number of neuroblasts in the SVZa, vertical arm and elbow on day 7 after irradiation. Until days 14 and 21 there was a marked decline in the density of young neurons, mostly in the horizontal arm of the RMS. In contrast, the number of astrocytes gradually increased in the caudal parts of the RMS until day 14 after irradiation. Strong enhancement was replaced by a steep decline within the RMS up to 21 days after treatment. Our results showed that the radiation response of proliferating cells originating from the SVZa may play a contributory role in the development of more adverse late radiation-induced effects.