INTRODUCTION:Endogenous biological timing mechanisms are fundamental aspects of living cells, tissues, and organisms. Virtually every aspect of physiology and behavior is mediated by self-sustaining circadian clocks, which depend on light to synchronize with the external daily environment. However, exposure to artificial light at night (ALAN) can impair temporal adaptations and affect health and disease. METHODS:During a study of the effects of long-term ALAN exposure on cardiovascular function, we serendipitously detected ultradian rhythms in muscarinic receptor dependent relaxation of isolated aortic tissue. RESULTS AND CONCLUSION:Mice exposed to dark nights displayed an ultradian pattern of maximum endothelial-dependent relaxation that was antiphase between the sexes. Rhythmic patterns of relaxation were abolished by ALAN exposure in both sexes suggesting that ALAN exposure can affect ultradian rhythms in physiology and behavior.

The central nervous system (CNS), comprising the brain and spinal cord, is fortified by complex barriers that protect the underlying organs and maintain homeostasis. The importance of proper fortification and homeostatic regulation provided by these systems has broad implications for many physiological processes and several pathological conditions are associated with their disruption. Recent studies support the notion that CNS barriers and fluids are regulated by circadian rhythms. Whereas reciprocal associations between the structural and functional integrity of neural barriers and disease states are well-established, the role of circadian rhythms in mediating these relationships remains unspecified. The goals of this review are to provide a general overview of three primary systems responsible for maintaining CNS homeostasis, namely the blood-brain barrier, blood-cerebrospinal fluid barrier, and glymphatic system, and to synthesize recent evidence highlighting the role of circadian rhythms as a critical regulator of CNS fluid and barrier function.

Chemotherapy agents in breast cancer are associated with chemotherapy-related cognitive impairments (CRCI). Mechanisms are not fully clear, but alterations of glucose and lipid metabolism, neuroinflammation and neurodegeneration may contribute to CRCI. The aim of this study was to investigate the combined effects of a high fat (HF) diet combined with doxorubicin-based chemotherapy on glucose and lipid metabolism, neuroinflammation, and neurodegeneration in mice. Additionally, we examined the therapeutic potential of dietary eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) to attenuate these effects. Female C57Bl/6 mice (n = 42) were fed HF, HFn-3 (2 % kcals as EPA + DHA) or Low Fat (LF) diets for seven weeks, with and without chemotherapy. In this study, two chemotherapy injections led to weight and body fat loss associated with a decrease in insulin resistance measured by HOMA-IR. HOMA-IR was significantly greater in HF versus LF groups; but HOMA-IR in HFn-3 group did not significantly differ from either HF or LF groups. Chemotherapy resulted in higher brain concentrations of the inflammatory chemokine KC/GRO. Compared to LF diet plus chemotherapy, HF diet plus chemotherapy upregulated multiple genes involved in neuroinflammation and neurodegeneration pathways. HFn-3 diet plus chemotherapy attenuated gene expression by downregulating multiple genes involved in neuroinflammation and blood brain barrier regulation, including Mapkapk2, Aqp4, and s100b, and upregulating Kcnb1 and Atxn3, genes involved in reduction of oxidative stress and anxiety, respectively. Overall, a HF diet combined with chemotherapy is associated with neuroinflammatory and neurodegenerative gene expression changes in this mouse model; dietary enrichment of EPA and DHA attenuated these effects. Further studies are needed to understand how diet impacts behavioral outcomes of CRCI.

Chemotherapy-related cognitive impairment (CRCI) and affective symptoms negatively impact quality of life in breast cancer survivors. The aim of this study was to determine the efficacy of high eicosapentaenoic acid + docosahexaenoic acid (EPA+DHA) and low sucrose diets to alleviate these symptoms in a mouse model of chemotherapy. Potential mechanisms involving insulin resistance were explored. We hypothesized that diets enriched in EPA+DHA and low amounts of sucrose would protect against the impact of chemotherapy on measures of CRCI. Female C57Bl/6 mice were randomized to 1 of 4 diets (2% kcal eicosapentaenoic acid + docosahexaenoic acid [EPA+DHA]/high or low sucrose, low omega-3/high or low sucrose) for 6 weeks and treated with two injections of doxorubicin-based chemotherapy or vehicle during week 2 and 4. Behavioral tests were performed 7 days after second injection. Chemotherapy increased serum insulin and decreased body weight, locomotion and exploratory behavior (all p < .05). Low sucrose consumption resulted in better long-term memory regardless of chemotherapy or vehicle injection (p < .05). 2% EPA+DHA consumption lessened insulin resistance (p < .05); however, controlling for body weight attenuated this effect (p = .08). There were no significant differences by diet or injection on liver lipid content; however, liver lipid content was positively correlated with insulin resistance scores (p < .05). Low sucrose diets may protect long-term memory during chemotherapy. The effect of EPA+DHA on insulin resistance and affective side effects during chemotherapy requires further investigation.

Circadian rhythms are endogenous biological cycles that regulate physiology and behavior and are set to precisely 24-h by light exposure. Light at night (LAN) dysregulates physiology and function including immune response; a critical component that contributes to stroke pathophysiological progression of neuronal injury and may impair recovery from injury. The goal of this study is to explore the effects of dim LAN (dLAN) in a murine model of ischemic stroke to assess how nighttime lighting from hospital settings can affect stroke outcome. Further, this study sought to identify mechanisms underlying pathophysiological changes to immune response after circadian disruption. Male and female adult Swiss Webster (CFW) mice were subjected to transient or permanent focal cerebral ischemia, then were subsequently placed into either dark night conditions (LD) or one night of dLAN (5 lx). 24 h post-stroke, sensorimotor impairments and infarct sizes were quantified. A single night of dLAN following MCAO increased infarct size and sensorimotor deficits across both sexes and reduced survival in males after 24 h. Flow cytometry was performed to assess microglial phenotypes after MCAO, and revealed that dLAN altered the percentage of microglia that express pro-inflammatory markers (MHC II+ and IL-6) and microglia that express CD206 and IL-10 that likely contributed to poor ischemic outcomes. Following these results, microglia were reduced in the brain using Plexxikon 5622 (PLX 5622) a CSFR1 inhibitor, then the mice received an MCAO and were exposed to LD or dLAN conditions for 24 h. Microglial depletion by PLX5622 resulted in infarct sizes that were comparable between lighting conditions. This study provides supporting evidence that environmental lighting exacerbates ischemic injury and post-stroke mortality by a biological mechanism that exposure to dLAN causes a fundamental shift of activated microglial phenotypes from beneficial to detrimental at an early time point after stroke, resulting in irreversible neuronal death.

Disrupted or atypical light-dark cycles disrupts synchronization of endogenous circadian clocks to the external environment; extensive circadian rhythm desynchrony promotes adverse health outcomes. Previous studies suggest that disrupted circadian rhythms promote neuroinflammation and neuronal damage post-ischemia in otherwise healthy mice, however, few studies to date have evaluated these health risks with aging. Because most strokes occur in aged individuals, we sought to identify whether, in addition to being a risk factor for poor ischemic outcome, circadian rhythm disruption can increase risk for vascular cognitive impairment and dementia (VCID). We hypothesized that repeated 6 h phase advances (chronic jet lag; CJL) for 8 weeks alters cerebrovascular architecture leading to increased cognitive impairments in aged mice. Female CJL mice displayed impaired spatial processing during a spontaneous alternation task and reduced acquisition during auditory-cued associative learning. Male CJL mice displayed impaired retention of the auditory-cued associative learning task 24 h following acquisition. CJL increased vascular tortuosity in the isocortex, associated with increased risk for vascular disease. These results demonstrate that CJL increased sex-specific cognitive impairments coinciding with structural changes to vasculature in the brain. We highlight that CJL may accelerate aged-related functional decline and could be a crucial target against disease progression.

Despite its demonstrated biological significance, time of day is a broadly overlooked biological variable in preclinical and clinical studies. How time of day affects the influence of peripheral tumors on central (brain) function remains unspecified. Thus, we tested the hypothesis that peripheral mammary cancer tumors alter the transcriptome of immune responses in the brain and that these responses vary based on time of day; we predicted that time of day sampling bias would alter the interpretation of the results. Brain tissues collected at mid dark and mid light from mammary tumor-bearing and vehicle injected mice were analyzed using the Nanostring nCounter immune panel. Peripheral mammary tumors significantly affected expression within the brain of over 100 unique genes of the 770 represented in the panel, and fewer than 25% of these genes were affected similarly across the day. Indeed, between 65 and 75% of GO biological processes represented by the differentially expressed genes were dependent upon time of day of sampling. The implications of time-of-day sampling bias in interpretation of research studies cannot be understated. We encourage considering time of day as a significant biological variable in studies and to appropriately control for it and clearly report time of day in findings.

Aging is associated with cognitive decline and is the main risk factor for a myriad of conditions including neurodegeneration and stroke. Concomitant with aging is the progressive accumulation of misfolded proteins and loss of proteostasis. Accumulation of misfolded proteins in the endoplasmic reticulum (ER) leads to ER stress and activation of the unfolded protein response (UPR). The UPR is mediated, in part, by the eukaryotic initiation factor 2α (eIF2α) kinase protein kinase R‐like ER kinase (PERK). Phosphorylation of eIF2α reduces protein translation as an adaptive mechanism but this also opposes synaptic plasticity. PERK, and other eIF2α kinases, have been widely studied in neurons where they modulate both cognitive function and response to injury. The impact of astrocytic PERK signaling in cognitive processes was previously unknown. To examine this, we deleted PERK from astrocytes (AstroPERK KO ) and examined the impact on cognitive functions in middle‐aged and old mice of both sexes. Additionally, we tested the outcome following experimental stroke using the transient middle cerebral artery occlusion (MCAO) model. Tests of short‐term and long‐term learning and memory as well as of cognitive flexibility in middle‐aged and old mice revealed that astrocytic PERK does not regulate these processes. Following MCAO, AstroPERK KO had increased morbidity and mortality. Collectively, our data demonstrate that astrocytic PERK has limited impact on cognitive function and has a more prominent role in the response to neural injury.

Neither norepinephrine nor recombinant CCL2 have proliferative effects on B16F10 cells.

Supplementary Table for qPCR sequences

Aging is a risk factor for the development of breast cancer. Foundational science studies have supported associations among neuroinflammation, breast cancer, and chemotherapy, but to date, these associations are based on studies using young adult rodents. The current study examined the neuroinflammatory effects of chemotherapy in aged, tumor-naïve and tumor-bearing mice with or without social enrichment. Mice received two intravenous injections of doxorubicin (A) and cyclophosphamide (C) at a two-week interval. Brain immune cells were enriched/assessed via flow cytometry, seven days following the second chemotherapy injection. Social enrichment enhanced peripheral immune cell trafficking in aged tumor-naive mice treated with AC. Group housed aged tumor bearing mice receiving AC had reduced percentage of IL-6+ monocytes and granulocytes relative to their singly housed counterparts. Notably, group housing aged experimental mice with young cage partners significantly reduced TNF + monocytes, tumor volume, and tumor mass. These data illustrate the importance of social enrichment in attenuating neuroinflammation and are the first to demonstrate that social support with young housing partners reduces tumor growth in aged mice.

Ghrelin is a peptide hormone, primarily produced in the stomach, best known for its role as a regulator of nutrient sensing, appetite, and meal initiation. However, increasing evidence supports a more complex and nuanced role for ghrelin in a diverse array of biological processes, including cancer. Ghrelin’s effects on breast cancer development and progression are not clear, as the literature detailing its effects is conflicting. Several studies in breast cancer survivors have concluded that higher expression of ghrelin is correlated with better recurrence-free and breast cancer-specific survival. However, additional clinical and foundational science studies have suggested greater complexity for ghrelin’s role in breast cancer development as a splice variant of ghrelin, namely In1-ghrelin, is associated with increased tumor proliferation and reduced disease-free survival of breast cancer patients. Thus, the current study sought to determine the effects of ghrelin deletion within mammary tumor cells (67NR) on subsequent tumor growth. Specifically, we hypothesized that ghrelin deletion would significantly alter tumor growth. Adult female mice received bilateral orthotopic injections of the nonmetastatic murine breast carcinoma cell line 67NR or one of two CRISPR generated ghrelin deleted cell lines 1G9 or 2G2 (derived from the 67NR cell line). Ghrelin deletion significantly reduced tumor volume and tumor mass by ~60% and ~55%, respectively. Additionally, ghrelin deletion significantly increased median survival duration by ~30%. When examining changes within the tumor microenvironment we observed a significant increase in macrophage colonization in ghrelin knockouts relative to the parental 67NR tumors. Notably, the percent F4/80 staining within the tumor positively correlated with length of survival. To determine the role of these F4/80 cells on tumor progression, we ablated these macrophages by feeding chow containing the colony stimulating factor receptor 1 inhibitor, PLX5622, for the entirety of the study. Ablating macrophages significantly increased tumor volume and tumor mass and rendered the previous beneficial effects of ghrelin deletion null, suggesting that the positive effects of ghrelin deletion may be due to actions on macrophages. Lastly, because of the increased macrophage infiltration within ghrelin knockout tumors, we sought to determine their repose to an immunotherapy targeting macrophages, namely anti-CD47. Notably, ghrelin deleted tumors displayed a positive response to the immunotherapy and further reduction in tumor growth (~80% reduction in tumor volume compared to 67NR tumors). Whereas, 67NR tumors were unresponsive to the immunotherapy. These data add to the literature detailing the complex effects of ghrelin on tumor growth and suggest that ghrelin may be a druggable target to slow tumor development. Citation Format: William H Walker II, Brittany D Elliott, Claire O Kisamore, Randy J Nelson, A. Courtney DeVries. Ghrelin deletion reduces mammary tumor growth and enhances response to immunotherapy [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A072.

Stress confers survival in a murine model of melanoma.

Neither norepinephrine nor recombinant CCL2 have toxic effects on B16F10 cells.

Restraint stress increases tumor CD3+/CD4+ T cell infiltration

Stress correlates with reduced tumor size and burden, VEGF and CCL2 levels, and infiltrating macrophages compared to unstressed mice.

Circadian rhythms are internal manifestations of the 24-h solar day that allow for synchronization of biological and behavioral processes to the external solar day. This precise regulation of physiology and behavior improves adaptive function and survival. Chronotherapy takes advantage of circadian rhythms in physiological processes to optimize the timing of drug administration to achieve maximal therapeutic efficacy and minimize negative side effects. Chronotherapy for cancer treatment was first demonstrated to be beneficial more than five decades ago and has favorable effects across diverse cancer types. However, implementation of chronotherapy in clinic remains limited. The present review examines the evidence for chronotherapeutic treatment for solid tumors. Specifically, studies examining chrono-chemotherapy, chrono-radiotherapy, and alternative chronotherapeutics (e.g., hormone therapy, TKIs, antiangiogenic therapy, immunotherapy) are discussed. In addition, we propose areas of needed research and identify challenges in the field that remain to be addressed.

Physiology and behavior are synchronized to the external environment by endogenous circadian rhythms that are set to precisely 24 h by exposure to bright light early in the day. Exposure to artificial light outside of the typical solar day, such as during the night, may impair aspects of physiology and behavior in human and non-human animals. Both the intensity and the wavelength of light are important in mediating these effects. The present report is the result of an unplanned change in our vivarium lighting conditions, which led to the observation that dim light during the daytime affects body mass similarly to dim nighttime light exposure in male Swiss Webster mice. Mice exposed to bright days (>= 125 lux) with dark nights (0 lux) gained significantly less weight than those exposed to bright days with dim light at night (5 lux) or dim days (<= 60 lux) with either dark nights or dim light at night. Notably, among the mice exposed to dim daytime light, no weight gain differences were observed between dark nights and dim light at night exposure; however dim light at night exposure shifted food intake to the inactive phase as previously reported. The mechanisms mediating these effects remain un-specified, but it appears that dimly illuminated days may have similar adverse metabolic effects as exposure to artificial light at night.

The structure and function of the cardiovascular system are modulated across the day by circadian rhythms, making this system susceptible to circadian rhythm disruption. Recent evidence demonstrated that short-term exposure to a pervasive circadian rhythm disruptor, artificial light at night (ALAN), increased inflammation and altered angiogenic transcripts in the hippocampi of mice. Here, we examined the effects of four nights of ALAN exposure on mouse hippocampal vascular networks. To do this, we analyzed 2D and 3D images of hippocampal vasculature and hippocampal transcriptomic profiles of mice exposed to ALAN. ALAN reduced vascular density in the CA1 and CA2/3 of female mice and the dentate gyrus of male mice. Network structure and connectivity were also impaired in the CA2/3 of female mice. These results demonstrate the rapid and potent effects of ALAN on cerebrovascular networks, highlighting the importance of ALAN mitigation in the context of health and cerebrovascular disease.