Calcium Signaling Mechanisms Mediate Clock-Controlled ATP Gliotransmission among Immortalized Rat SCN2.2 Cell Cultures

The hypothalamus is an integral part of the brain's regulation of mammalian physiology and behavior. Among many functions, this regulatory center activates the sympathetic nervous system, maintains appropriate body temperature, controls food intake, and controls release of hormones from the pituitary gland. Deep within the hypothalamus lie a paired cluster of cells, the suprachiasmatic nuclei (SCN), which function as the chief circadian pacemaker. The goal of the present thesis research was to study rhythmically controlled ATP gliotransmission. I used an immortalized SCN2.2 hypothalamic cell line to determine the mechanism by which ATP signaling is regulated in this context. Additionally, this research aimed to elucidate if clock-controlled ATP gliotransmission is fundamentally distinct from stimulus-evoked calcium-dependent mechanisms that regulate intercellular ATP signaling among astrocytes. In this thesis, I show that there are multiple ATP signaling mechanisms present among SCN2.2 cells. cAMP-dependent signaling mediates clock-controlled ATP accumulation but not stimulus-evoked ATP signaling. In addition, pharmacological studies suggest that disparate purinergic receptor-mediated mechanisms are involved in the regulation of clock-controlled versus stimulus-evoked ATP signaling. Rhythmic accumulation of ATP in SCN2.2 cultures is modulated by calcium-dependent processes. Peaks in ATP accumulation coincide with elevated mitochondrial calcium levels, while troughs in ATP accumulation coincide with periods of high cytosolic calcium levels, suggesting a possible mechanistic link between circadian shifts in intracellular calcium handling and ATP handling in SCN2.2 cells. Clock-controlled ATP accumulation in SCN2.2 cells is not a by-product of rhythmic cell cycle or rhythmic cell death. Overall, my research suggests that the ATP accumulation rhythm in SCN2.2 cells is likely an output of the biological clock, mediated by astrocytic calcium signaling processes, and not an output of cell division or cell death. Estimation of ATP accumulation in SCN2.2 cultures at peak time points suggests that clock-controlled ATP release is critical to the function of astrocytes in the mammalian brain, perhaps in the regulation of brain metabolism, the regulation of sleep/wake physiology, or the integration of both

Multi-domain neurocognitive classification of primary brain tumor patients prior to radiotherapy on a prospective clinical trial

IntroductionWe investigated multi-domain baseline neurocognition of primary brain tumor patients prior to radiotherapy (RT), including clinical predictors of function and association between pre-RT and post-RT impairment on a prospective trial.MethodsA multi-domain neuropsychological battery (memory, executive functioning, language, attention, processing) was performed on 37 patients, pre-RT and 3-(n = 21), 6-(n = 22) and 12-(n = 14) months post-RT. Impairment rate was the proportion of patients with standardized T-scores ≤ 1.5 standard deviations below normative means. Per-patient impairment across all domains was calculated using a global deficit score (GDS; higher value indicates more impairment). Associations between baseline GDS and clinical variables were tested. Global GDS impairment rate at each time point was the fraction of patients with GDS scores > 0.5.ResultsStatistically significant baseline neurocognitive impairments were identified on 4 memory (all p ≤ 0.03) and 2 out of 3 (p = 0.01, p = 0.027) executive functioning tests. Per-patient baseline GDS was significantly associated with tumor volume (p = 0.048), tumor type (p = 0.043), seizure history (p = 0.007), and use of anti-epileptics (p = 0.009). The percentage of patients with the same impairment status at 3-, 6-, and 12-months as at baseline were 88%, 85%, and 85% respectively.ConclusionsMemory and executive functioning impairment were the most common cognitive deficits prior to RT. Patients with larger tumors, more aggressive histology, and use of anti-epileptics had higher baseline GDS values. GDS is a promising tool to encompass multi-domain neurocognitive function, and baseline GDS can identify those at risk of cognitive impairment

Radiation Dose–Dependent Hippocampal Atrophy Detected With Longitudinal Volumetric Magnetic Resonance Imaging

PurposeAfter radiation therapy (RT) to the brain, patients often experience memory impairment, which may be partially mediated by damage to the hippocampus. Hippocampal sparing in RT planning is the subject of recent and ongoing clinical trials. Calculating appropriate hippocampal dose constraints would be improved by efficient in vivo measurements of hippocampal damage. In this study we sought to determine whether brain RT was associated with dose-dependent hippocampal atrophy.Methods and materialsHippocampal volume was measured with magnetic resonance imaging (MRI) in 52 patients who underwent fractionated, partial brain RT for primary brain tumors. Study patients had high-resolution, 3-dimensional volumetric MRI before and 1 year after RT. Images were processed using software with clearance from the US Food and Drug Administration and Conformité Européene marking for automated measurement of hippocampal volume. Automated results were inspected visually for accuracy. Tumor and surgical changes were censored. Mean hippocampal dose was tested for correlation with hippocampal atrophy 1 year after RT. Average hippocampal volume change was also calculated for hippocampi receiving high (>40 Gy) or low (<10 Gy) mean RT dose. A multivariate analysis was conducted with linear mixed-effects modeling to evaluate other potential predictors of hippocampal volume change, including patient (random effect), age, hemisphere, sex, seizure history, and baseline volume. Statistical significance was evaluated at α = 0.05.ResultsMean hippocampal dose was significantly correlated with hippocampal volume loss (r=-0.24, P=.03). Mean hippocampal volume was significantly reduced 1 year after high-dose RT (mean -6%, P=.009) but not after low-dose RT. In multivariate analysis, both RT dose and patient age were significant predictors of hippocampal atrophy (P<.01).ConclusionsThe hippocampus demonstrates radiation dose-dependent atrophy after treatment for brain tumors. Quantitative MRI is a noninvasive imaging technique capable of measuring radiation effects on intracranial structures. This technique could be investigated as a potential biomarker for development of reliable dose constraints for improved cognitive outcomes

Cerebral Cortex Regions Selectively Vulnerable to Radiation Dose-Dependent Atrophy.

Purpose and objectivesNeurologic deficits after brain radiation therapy (RT) typically involve decline in higher-order cognitive functions such as attention and memory rather than sensory defects or paralysis. We sought to determine whether areas of the cortex critical to cognition are selectively vulnerable to radiation dose-dependent atrophy.Methods and materialsWe measured change in cortical thickness in 54 primary brain tumor patients who underwent fractionated, partial brain RT. The study patients underwent high-resolution, volumetric magnetic resonance imaging (T1-weighted; T2 fluid-attenuated inversion recovery, FLAIR) before RT and 1 year afterward. Semiautomated software was used to segment anatomic regions of the cerebral cortex for each patient. Cortical thickness was measured for each region before RT and 1 year afterward. Two higher-order cortical regions of interest (ROIs) were tested for association between radiation dose and cortical thinning: entorhinal (memory) and inferior parietal (attention/memory). For comparison, 2 primary cortex ROIs were also tested: pericalcarine (vision) and paracentral lobule (somatosensory/motor). Linear mixed-effects analyses were used to test all other cortical regions for significant radiation dose-dependent thickness change. Statistical significance was set at α = 0.05 using 2-tailed tests.ResultsCortical atrophy was significantly associated with radiation dose in the entorhinal (P=.01) and inferior parietal ROIs (P=.02). By contrast, no significant radiation dose-dependent effect was found in the primary cortex ROIs (pericalcarine and paracentral lobule). In the whole-cortex analysis, 9 regions showed significant radiation dose-dependent atrophy, including areas responsible for memory, attention, and executive function (P≤.002).ConclusionsAreas of cerebral cortex important for higher-order cognition may be most vulnerable to radiation-related atrophy. This is consistent with clinical observations that brain radiation patients experience deficits in domains of memory, executive function, and attention. Correlations of regional cortical atrophy with domain-specific cognitive functioning in prospective trials are warranted

Cerebral Cortex Regions Selectively Vulnerable to Radiation Dose-Dependent Atrophy.

Purpose and objectivesNeurologic deficits after brain radiation therapy (RT) typically involve decline in higher-order cognitive functions such as attention and memory rather than sensory defects or paralysis. We sought to determine whether areas of the cortex critical to cognition are selectively vulnerable to radiation dose-dependent atrophy.Methods and materialsWe measured change in cortical thickness in 54 primary brain tumor patients who underwent fractionated, partial brain RT. The study patients underwent high-resolution, volumetric magnetic resonance imaging (T1-weighted; T2 fluid-attenuated inversion recovery, FLAIR) before RT and 1 year afterward. Semiautomated software was used to segment anatomic regions of the cerebral cortex for each patient. Cortical thickness was measured for each region before RT and 1 year afterward. Two higher-order cortical regions of interest (ROIs) were tested for association between radiation dose and cortical thinning: entorhinal (memory) and inferior parietal (attention/memory). For comparison, 2 primary cortex ROIs were also tested: pericalcarine (vision) and paracentral lobule (somatosensory/motor). Linear mixed-effects analyses were used to test all other cortical regions for significant radiation dose-dependent thickness change. Statistical significance was set at α = 0.05 using 2-tailed tests.ResultsCortical atrophy was significantly associated with radiation dose in the entorhinal (P=.01) and inferior parietal ROIs (P=.02). By contrast, no significant radiation dose-dependent effect was found in the primary cortex ROIs (pericalcarine and paracentral lobule). In the whole-cortex analysis, 9 regions showed significant radiation dose-dependent atrophy, including areas responsible for memory, attention, and executive function (P≤.002).ConclusionsAreas of cerebral cortex important for higher-order cognition may be most vulnerable to radiation-related atrophy. This is consistent with clinical observations that brain radiation patients experience deficits in domains of memory, executive function, and attention. Correlations of regional cortical atrophy with domain-specific cognitive functioning in prospective trials are warranted

Radiation Dose–Dependent Hippocampal Atrophy Detected With Longitudinal Volumetric Magnetic Resonance Imaging

PurposeAfter radiation therapy (RT) to the brain, patients often experience memory impairment, which may be partially mediated by damage to the hippocampus. Hippocampal sparing in RT planning is the subject of recent and ongoing clinical trials. Calculating appropriate hippocampal dose constraints would be improved by efficient in vivo measurements of hippocampal damage. In this study we sought to determine whether brain RT was associated with dose-dependent hippocampal atrophy.Methods and materialsHippocampal volume was measured with magnetic resonance imaging (MRI) in 52 patients who underwent fractionated, partial brain RT for primary brain tumors. Study patients had high-resolution, 3-dimensional volumetric MRI before and 1 year after RT. Images were processed using software with clearance from the US Food and Drug Administration and Conformité Européene marking for automated measurement of hippocampal volume. Automated results were inspected visually for accuracy. Tumor and surgical changes were censored. Mean hippocampal dose was tested for correlation with hippocampal atrophy 1 year after RT. Average hippocampal volume change was also calculated for hippocampi receiving high (>40 Gy) or low (<10 Gy) mean RT dose. A multivariate analysis was conducted with linear mixed-effects modeling to evaluate other potential predictors of hippocampal volume change, including patient (random effect), age, hemisphere, sex, seizure history, and baseline volume. Statistical significance was evaluated at α = 0.05.ResultsMean hippocampal dose was significantly correlated with hippocampal volume loss (r=-0.24, P=.03). Mean hippocampal volume was significantly reduced 1 year after high-dose RT (mean -6%, P=.009) but not after low-dose RT. In multivariate analysis, both RT dose and patient age were significant predictors of hippocampal atrophy (P<.01).ConclusionsThe hippocampus demonstrates radiation dose-dependent atrophy after treatment for brain tumors. Quantitative MRI is a noninvasive imaging technique capable of measuring radiation effects on intracranial structures. This technique could be investigated as a potential biomarker for development of reliable dose constraints for improved cognitive outcomes