Mistimed restricted feeding disrupts circadian rhythms of male mating behavior and female preovulatory LH surges in mice.

In rodents, eating at atypical circadian times, such as during the biological rest phase when feeding is normally minimal, reduces fertility. Prior findings suggest this fertility impairment is due, at least in part, to reduced mating success. However, the physiological and behavioral mechanisms underlying this reproductive suppression are not known. In the present study, we tested the hypothesis that mistimed feeding-induced infertility is due to a disruption in the normal circadian timing of mating behavior and/or the generation of pre-ovulatory luteinizing hormone (LH) surges (estrogen positive feedback). In the first experiment, male+female mouse pairs, acclimated to be food restricted to either the light (mistimed feeding) or dark (control feeding) phase, were scored for mounting frequency and ejaculations over 96 h. Male mounting behavior and ejaculations were distributed much more widely across the day in light-fed mice than in dark-fed controls and fewer light-fed males ejaculated. In the second experiment, the timing of the LH surge, a well characterized circadian event driven by estradiol (E2) and the SCN, was analyzed from serial blood samples taken from ovariectomized and E2-primed female mice that were light-, dark-, or ad-lib-fed. LH concentrations peaked 2 h after lights-off in both dark-fed and ad-lib control females, as expected, but not in light-fed females. Instead, the normally clustered LH surges were distributed widely with high inter-mouse variability in the light-fed group. These data indicate that mistimed feeding disrupts the temporal control of the neural processes underlying both ovulation and mating behavior, contributing to infertility.

Natural food intake patterns have little synchronizing effect on peripheral circadian clocks.

BACKGROUND: Circadian rhythms across mammalian tissues are coordinated by a master clock in the suprachiasmatic nucleus (SCN) that is principally entrained by light-dark cycles. Prior investigations have shown, however, that time-restricted feeding (TRF)-daily alternation of fasting and food availability-synchronizes peripheral clocks independent of the light-dark cycle and of the SCN. This has led to the idea that downstream peripheral clocks are entrained indirectly by food intake rhythms. However, TRF is not a normal eating pattern, and it imposes non-physiologic long fasts that rodents do not typically experience. Therefore, we tested whether normal feeding patterns can phase-shift or entrain peripheral tissues by measuring circadian rhythms of the liver, kidney, and submandibular gland in mPer2Luc mice under different food schedules. RESULTS: We employed home cage feeders to first measure ad libitum food intake and then to dispense 20-mg pellets on a schedule mimicking that pattern. In both conditions, PER2::LUC bioluminescence peaked during the night as expected. Surprisingly, shifting the scheduled feeding by 12 h advanced peripheral clocks by only 0-3 h, much less than predicted from TRF protocols. To isolate the effects of feeding from the light-dark cycle, clock phase was then measured in mice acclimated to scheduled feeding over the course of 3 months in constant darkness. In these conditions, peripheral clock phases were better predicted by the rest-activity cycle than by the food schedule, contrary to expectation based on TRF studies. At the end of both experiments, mice were exposed to a modified TRF with food provided in eight equally sized meals over 12 h. In the light-dark cycle, this advanced the phase of the liver and kidney, though less so than in TRF with ad libitum access; in darkness, this entrained the liver and kidney but had little effect on the submandibular gland or the rest-activity cycle. CONCLUSIONS: These data suggest that natural feeding patterns can only weakly affect circadian clocks. Instead, in normally feeding mice, the central pacemaker in the brain may set the phase of peripheral organs via pathways that are independent of feeding behavior.

Circadian disruption of food availability significantly reduces reproductive success in mice.

Circadian disruptions impair reproductive health in human populations and in animal models. We tested the hypothesis that mistimed food, a common disruptive feature of shift work, impairs reproductive success in mice. Male and female mPer2Luc mice on a C57BL/6 background were fed during the light or dark phase in two experiments. Food-induced internal misalignment of the liver clock was verified by in vivo bioluminescence in anesthetized mice in both experiments. In Experiment 1, food-restricted pairs were monitored for litters for 18 weeks. In the light-fed group, birth of the first litter was significantly delayed, and total reproductive output was significantly reduced by 38%. In Experiment 2, estrous cycling was monitored for 3 weeks, and then after pairing, copulatory plugs, pregnancy, litter sizes, and uterine implantation sites were measured. Fewer light-fed females birthed litters (25% versus 73%). This was attributable to a difference in behavior as mating success was significantly reduced in light-fed mice: 42% were observed with a copulatory plug compared to 82% for dark-fed mice. The proportion of mice displaying uterine implantation sites was the same as the proportion observed with copulatory plugs, suggesting no deficit in initiating pregnancy after mating. Estrous cycling and pregnancy maintenance did not differ between the groups. We conclude that mistimed feeding inhibits reproduction in mice by reducing successful mating behavior.

Rapid Response and Slow Recovery of the H3K4me3 Epigenomic Marker in the Liver after Light-mediated Phase Advances of the Circadian Clock.

Mammalian tissues display circadian rhythms in transcription, translation, and histone modifications. Here we asked how an advance of the light-dark cycle alters daily rhythms in the liver epigenome at the H3K4me3 (trimethylation of lysine 4 on histone 3) modification, which is found at active and poised gene promoters. H3K4me3 levels were first measured at 4 time points (zeitgeber time [ZT] 3, 8, 15, and 20) during a normal 12L:12D light-dark cycle. Peak levels were observed during the early dark phase at ZT15 and dropped to low levels around lights-on (ZT0) between ZT20 and ZT3. A 6-h phase advance at ZT18 (new lights-on after only 6 h of darkness) led to a transient extension of peak H3K4me3 levels. Although locomotor activity reentrained within a week after the phase advance, H3K4me3 rhythms failed to do so, with peak levels remaining in the light phase at the 1-week recovery time point. Eight weekly phase advances, with 1-week recovery times between each phase advance, further disrupted the H3K4me3 rhythms. Finally, we used the mPer2Luc knockin mouse to determine whether the phase advance also disrupted Per2 protein expression. Similar to the results from the histone work, we found both a rapid response to the phase advance and a delayed recovery, the latter in sync with H3K4me3 levels. A model to explain these results is offered.

High-dose estrogen treatment at reperfusion reduces lesion volume and accelerates recovery of sensorimotor function after experimental ischemic stroke.

Estrogens have previously been shown to protect the brain against acute ischemic insults, by potentially augmenting cerebrovascular function after ischemic stroke. The current study hypothesized that treatment with sustained release of high-dose 17ß-estradiol (E2) at the time of reperfusion from middle cerebral artery occlusion (MCAO) in rats would attenuate reperfusion injury, augment post-stroke angiogenesis and cerebral blood flow, and attenuate lesion volume. Female Wistar rats underwent ovariectomy, followed two weeks later by transient, two-hour right MCAO (tMCAO) and treatment with E2 (n=13) or placebo (P; n=12) pellets starting at reperfusion. E2 treatment resulted in significantly smaller total lesion volume, smaller lesions within striatal and cortical brain regions, and less atrophy of the ipsilateral hemisphere after six weeks of recovery. E2-treated animals exhibited accelerated recovery of contralateral forelimb sensorimotor function in the cylinder test. Magnetic resonance imaging (MRI) showed that E2 treatment reduced the formation of lesion cysts, decreased lesion volume, and increased lesional cerebral blood flow (CBF). K(trans), a measure of vascular permeability, was increased in the lesions. This finding, which represents lesion neovascularization, was not altered by E2 treatment. Ischemic stroke-related angiogenesis and vessel formation was confirmed with immunolabeling of brain tissue and was not altered with E2 treatment. In summary, E2 treatment administered immediately following reperfusion significantly reduced lesion size, cyst formation, and brain atrophy while improving lesional CBF and accelerating recovery of functional deficits in a rat model of ischemic stroke.

Synergistic Antivascular and Antitumor Efficacy with Combined Cediranib and SC6889 in Intracranial Mouse Glioma.

Prognosis remains extremely poor for malignant glioma. Targeted therapeutic approaches, including single agent anti-angiogenic and proteasome inhibition strategies, have not resulted in sustained anti-glioma clinical efficacy. We tested the anti-glioma efficacy of the anti-angiogenic receptor tyrosine kinase inhibitor cediranib and the novel proteasome inhibitor SC68896, in combination and as single agents. To assess anti-angiogenic effects and evaluate efficacy we employed 4C8 intracranial mouse glioma and a dual-bolus perfusion MRI approach to measure Ktrans, relative cerebral blood flow and volume (rCBF, rCBV), and relative mean transit time (rMTT) in combination with anatomical MRI measurements of tumor growth. While single agent cediranib or SC68896 treatment did not alter tumor growth or survival, combined cediranib/SC68896 significantly delayed tumor growth and increased median survival by 2-fold, compared to untreated. This was accompanied by substantially increased tumor necrosis in the cediranib/SC68896 group (p<0.01), not observed with single agent treatments. Mean vessel density was significantly lower, and mean vessel lumen area was significantly higher, for the combined cediranib/SC68896 group versus untreated. Consistent with our previous findings, cediranib alone did not significantly alter mean tumor rCBF, rCBV, rMTT, or Ktrans. In contrast, SC68896 reduced rCBF in comparison to untreated, but without concomitant reductions in rCBV, rMTT, or Ktrans. Importantly, combined cediranib/SC68896 substantially reduced rCBF, rCBV. rMTT, and Ktrans. A novel analysis of Ktrans/rCBV suggests that changes in Ktrans with time and/or treatment are related to altered total vascular surface area. The data suggest that combined cediranib/SC68896 induced potent anti-angiogenic effects, resulting in increased vascular efficiency and reduced extravasation, consistent with a process of vascular normalization. The study represents the first demonstration that the combination of cediranib with a proteasome inhibitor substantially increases the anti-angiogenic efficacy produced from either agent alone, and synergistically slows glioma tumor growth and extends survival, suggesting a promising treatment which warrants further investigation.

Neurobehavioral and imaging correlates of hippocampal atrophy in a mouse model of vascular cognitive impairment.

Vascular cognitive impairment (VCI) is the second most common cause of dementia. Reduced cerebral blood flow is thought to play a major role in the etiology of VCI. Therefore, chronic cerebral hypoperfusion has been used to model VCI in rodents. The goal of the current study was to determine the histopathological and neuroimaging substrates of neurocognitive impairments in a mouse model of chronic cerebral hypoperfusion induced by unilateral common carotid artery occlusion (UCCAO). Mice were subjected to sham or right UCCAO (VCI) surgeries. Three months later, neurocognitive function was evaluated using the novel object recognition task, Morris water maze, and contextual and cued fear-conditioning tests. Next, cerebral perfusion was evaluated with dynamic susceptibility contrast magnetic resonance imaging (MRI) using an ultra-high field (11.75 T) animal MRI system. Finally, brain pathology was evaluated using histology and T2-weighted MRI. VCI, but not sham, mice had significantly reduced cerebral blood flow in the right vs. left cerebral cortex. VCI mice showed deficits in object recognition. T2-weighted MRI of VCI brains revealed enlargement of lateral ventricles, which corresponded to areas of hippocampal atrophy upon histological analysis. In conclusion, our data demonstrate that the UCCAO model of chronic hypoperfusion induces hippocampal atrophy and ventricular enlargement, resulting in neurocognitive deficits characteristic of VCI.

Soluble Epoxide Hydrolase in Hydrocephalus, Cerebral Edema, and Vascular Inflammation After Subarachnoid Hemorrhage.

BACKGROUND AND PURPOSE: Acute communicating hydrocephalus and cerebral edema are common and serious complications of subarachnoid hemorrhage (SAH), whose causes are poorly understood. Using a mouse model of SAH, we determined whether soluble epoxide hydrolase (sEH) gene deletion protects against SAH-induced hydrocephalus and edema by increasing levels of vasoprotective eicosanoids and suppressing vascular inflammation. METHODS: SAH was induced via endovascular puncture in wild-type and sEH knockout mice. Hydrocephalus and tissue edema were assessed by T2-weighted magnetic resonance imaging. Endothelial activation was assessed in vivo using T2*-weighted magnetic resonance imaging after intravenous administration of iron oxide particles linked to anti-vascular cell adhesion molecule-1 antibody 24 hours after SAH. Behavioral outcome was assessed at 96 hours after SAH with the open field and accelerated rotarod tests. RESULTS: SAH induced an acute sustained communicating hydrocephalus within 6 hours of endovascular puncture in both wild-type and sEH knockout mice. This was followed by tissue edema, which peaked at 24 hours after SAH and was limited to white matter fiber tracts. sEH knockout mice had reduced edema, less vascular cell adhesion molecule-1 uptake, and improved outcome compared with wild-type mice. CONCLUSIONS: Genetic deletion of sEH reduces vascular inflammation and edema and improves outcome after SAH. sEH inhibition may serve as a novel therapy for SAH.