Radiotherapy of the Hepatocellular Carcinoma in Mice Has a Time-Of-Day-Dependent Impact on the Mouse Hippocampus.

Chronic liver diseases including hepatocellular carcinoma (HCC) create a state of chronic inflammation that affects the brain via the liver-brain axis leading to an alteration of neurotransmission and cognition. However, little is known about the effects of HCC on the hippocampus, the key brain region for learning and memory. Moreover, radiotherapy used to treat HCC has severe side effects that impair patients' life quality. Thus, designing optimal strategies, such as chronotherapy, to enhance the efficacy and reduce the side effects of HCC treatment is critically important. We addressed the effects of HCC and the timed administration of radiotherapy in mice on the expression of pro-inflammatory cytokines, clock genes, markers for glial activation, oxidative stress, neuronal activity and proliferation in the hippocampal neurogenic niche. Our data showed that HCC induced the upregulation of genes encoding for pro-inflammatory cytokines, altered clock gene expressions and reduced proliferation in the hippocampus. Radiotherapy, in particular when applied during the light/inactive phase enhanced all these effects in addition to glial activation, increased oxidative stress, decreased neuronal activity and increased levels of phospho(p)-ERK. Our results suggested an interaction of the circadian molecular clockwork and the brain's innate immune system as key players in liver-brain crosstalk in HCC and that radiotherapy when applied during the light/inactive phase induced the most profound alterations in the hippocampus.

Does timing matter in radiotherapy of hepatocellular carcinoma? An experimental study in mice.

This study investigates whether a chronotherapeutic treatment of hepatocellular carcinoma (HCC) may improve treatment efficacy and mitigate side effects on non-tumoral liver (NTL). HCC was induced in Per2::luc mice which were irradiated at four time points of the day. Proliferation and DNA-double strand breaks were analyzed in irradiated and nonirradiated animals by detection of Ki67 and γ-H2AX. Prior to whole animal experiments, organotypic slice cultures were investigated to determine the dosage to be used in whole animal experiments. Irradiation was most effective at the proliferation peaks in HCC at ZT02 (early inactivity phase) and ZT20 (late activity phase). Irradiation effects on NTL were minimal at ZT20. As compared with NTL, nonirradiated HCC revealed disruption in daily variation and downregulation of all investigated clock genes except Per1. Irradiation affected rhythmic clock gene expression in NTL and HCC at all ZTs except at ZT20 (late activity phase). Irradiation at ZT20 had no effect on total leukocyte numbers. Our results indicate ZT20 as the optimal time point for irradiation of HCC in mice at which the ratio between efficacy of tumor treatment and toxic side effects was maximal. Translational studies are now needed to evaluate whether the late activity phase is the optimal time point for irradiation of HCC in man.

Arcuate nucleus, median eminence, and hypophysial pars tuberalis.

The arcuate nucleus (ARC) is located in the mediobasal hypothalamus and forms a morphological and functional entity with the median eminence (ME), the ARC-ME. The ARC comprises several distinct types of neurons controlling prolactin release, food intake, and metabolism as well as reproduction and onset of puberty. The ME lacks a blood-brain barrier and provides an entry for peripheral signals (nutrients, leptin, ghrelin). ARC neurons are adjacent to the wall of the third ventricle. This facilitates the exchange of signals from and to the cerebrospinal fluid. The ventricular wall is composed of tanycytes that serve different functions. Axons of ARC neurons contribute to the tuberoinfundibular tract terminating in the ME on the hypophysial portal vessels (HPV) and establish one of the neurohumoral links between the hypothalamus and the pituitary. ARC neurons are reciprocally connected with several other hypothalamic nuclei, the brainstem, and reward pathways. The hypophysial pars tuberalis (PT) is attached to the ME and the HPV. The PT, an important interface of the neuroendocrine system, is mandatory for the control of seasonal functions. This contribution provides an update of our knowledge about the ARC-ME complex and the PT which, inter alia, is needed to understand the pathophysiology of metabolic diseases and reproduction.

Relationship between locomotor activity rhythm and corticosterone levels during HCC development, progression, and treatment in a mouse model.

Cancer-related fatigue (CRF) and stress are common symptoms in cancer patients and represent early side effects of cancer treatment which affect the life quality of the patients. CRF may partly depend on disruption of the circadian rhythm. Locomotor activity and corticosterone rhythms are two important circadian outputs which can be used to analyze possible effects on the circadian function during cancer development and treatment. The present study analyzes the relationship between locomotor activity rhythm, corticosterone levels, hepatocellular carcinoma (HCC) development, and radiotherapy treatment in a mouse model. HCC was induced in mice by single injection of diethylnitrosamine (DEN) and chronic treatment of phenobarbital in drinking water. Another group received chronic phenobarbital treatment only. Tumor bearing animals were divided randomly into four groups irradiated at four different Zeitgeber time points. Spontaneous locomotor activity was recorded continuously; serum corticosterone levels and p-ERK immunoreaction in the suprachiasmatic nucleus (SCN) were investigated. Phenobarbital treated mice showed damped corticosterone levels and a less stable 24 hours activity rhythm as well as an increase in activity during the light phase, reminiscent of sleep disruption. The tumor mice showed an increase in corticosterone level during the inactive phase and decreased activity during the dark phase, reminiscent of CRF. After irradiation, corticosterone levels were further increased and locomotor activity rhythms were disrupted. Lowest corticosterone levels were observed after irradiation during the early light phase; thus, this time might be the best to apply radiotherapy in order to minimize side effects.

Time-dependent changes in proliferation, DNA damage and clock gene expression in hepatocellular carcinoma and healthy liver of a transgenic mouse model.

Hepatocellular carcinoma (HCC) is highly resistant to anticancer therapy and novel therapeutic strategies are needed. Chronotherapy may become a promising approach because it may improve the efficacy of antimitotic radiation and chemotherapy by considering timing of treatment. To date little is known about time-of-day dependent changes of proliferation and DNA damage in HCC. Using transgenic c-myc/transforming growth factor (TGFα) mice as HCC animal model, we immunohistochemically demonstrated Ki67 as marker for proliferation and γ-H2AX as marker for DNA damage in HCC and surrounding healthy liver (HL). Core clock genes (Per1, Per2, Cry1, Cry2, Bmal 1, Rev-erbα and Clock) were examined by qPCR. Data were obtained from samples collected ex vivo at four different time points and from organotypic slice cultures (OSC). Significant differences were found between HCC and HL. In HCC, the number of Ki67 immunoreactive cells showed two peaks (ex vivo: ZT06 middle of day and ZT18 middle of night; OSC: CT04 and CT16). In ex vivo samples, the number of γ-H2AX positive cells in HCC peaked at ZT18 (middle of the night), while in OSC their number remained high during subjective day and night. In both HCC and HL, clock gene expression showed a time-of-day dependent expression ex vivo but no changes in OSC. The expression of Per2 and Cry1 was significantly lower in HCC than in HL. Our data support the concept of chronotherapy of HCC. OSC may become useful to test novel cancer therapies.

Synchronizing effects of melatonin on diurnal and circadian rhythms.

In mammals, the rhythmic secretion of melatonin from the pineal gland is driven by the circadian clock in the suprachiasmatic nucleus (SCN) of the hypothalamus. The robust nightly peak of melatonin secretion is an output signal of the circadian clock and is supposed to deliver the circadian message to the whole of the organism. Since the circadian system regulates many behavioral and physiological processes, its disruption by external (shift-work, jet-lag) or internal desynchronization (blindness, aging) causes many different health problems. Externally applied melatonin is used in humans as a chronobiotic drug to treat desynchronization and circadian disorders, and the success of these treatments does, at first glance, underline the supposed pivotal role of melatonin in the synchronization of the circadian system. On the other hand, pinealectomy in experimental animals and humans does not abolish their rhythms of rest and activity. Furthermore, mice with deficient melatoninergic systems neither display overt defects in their rhythmic behavior nor do they show obvious signs of disease susceptibility, let alone premature mortality. During the last years, our laboratory has investigated several mouse stains with intact or compromised internal melatonin signaling systems in order to better understand the physiological role of the melatoninergic system. These and other investigations which will be reviewed in the present contribution confirm the synchronizing effect of endogenous melatonin and the melatoninergic system. However, these effects are subtle. Thus melatonin does not appear as the master of internal synchronization, but as one component in a cocktail of synchronizing agents.

Signaling pathways to and from the hypophysial pars tuberalis, an important center for the control of seasonal rhythms.

Seasonal (circannual) rhythms play an important role for the control of body functions (reproduction, metabolism, immune responses) in nearly all living organisms. Also humans are affected by the seasons with regard to immune responses and mental functions, the seasonal affective disorder being one of the most prominent examples. The hypophysial pars tuberalis (PT), an important interface between the hypophysial pars distalis and neuroendocrine centers in the brain, plays an essential role in the regulation of seasonal functions and may even be the seat of the circannual clock. Photoperiodic signals provide a major input to the PT. While the perception of these signals involves extraocular photoreceptors in non-mammalian species (birds, fish), mammals perceive photoperiodic signals exclusively in the retina. A multisynaptic pathway connects the retina with the pineal organ where photoperiodic signals are translated into the neurohormone melatonin that is rhythmically produced night by night and encodes the length of the night. Melatonin controls the functional activity of the mammalian PT by acting upon MT1 melatonin receptors. The PT sends its output signals via retrograde and anterograde pathways. The retrograde pathway targetting the hypothalamus employs TSH as messenger and controls a local hypothalamic T3 system. As discovered in Japanese quail, TSH triggers molecular cascades mediating thyroid hormone conversion in the ependymal cell layer of the infundibular recess of the third ventricle. The local accumulation of T3 in the mediobasal hypothalamus (MBH) appears to activate the gonadal axis by affecting the neuro-glial interaction between GnRH terminals and tanycytes in the median eminence. This retrograde pathway is conserved in photoperiodic mammals (sheep and hamsters), and even in non-photoperiodic laboratory mice provided that they are capable to synthesize melatonin. The anterograde pathway is implicated in the control of prolactin secretion, targets cells in the PD and supposedly employs small molecules as signal substances collectively denominated as "tuberalins". Several "tuberalin" candidates have been proposed, such as tachykinins, the secretory protein TAFA and endocannabinoids (EC). The PT-intrinsic EC system was first demonstrated in Syrian hamsters and shown to respond to photoperiodic changes. Subsequently, the EC system was also demonstrated in the PT of mice, rats and humans. To date, 2-arachidonoylglycerol (2-AG) appears as the most important endocannabinoid from the PT. Likely targets for the EC are folliculo-stellate cells that contain the CB1 receptor and appear to contact lactotroph cells. The CB1 receptor was also found on corticotroph cells which appear as a further target of the EC. Recently, the CB1 receptor was also localized to CRF-containing nerve fibers running in the outer zone of the median eminence. This finding suggests that the EC system of the PT contributes not only to the anterograde, but also to the retrograde pathway. Taken together, the results support the concept that the PT transmits its signals via a "cocktail" of messenger molecules which operate also in other brain areas and systems rather than through PT-specific "tuberalins". Furthermore, they may attribute a novel function to the PT, namely the modulation of the stress response and immune functions.

Heat Shock Factor 1 Deficiency Affects Systemic Body Temperature Regulation.

INTRODUCTION: Heat shock factor 1 (HSF1) is a ubiquitous heat-sensitive transcription factor that mediates heat shock protein transcription in response to cellular stress, such as increased temperature, in order to protect the organism against misfolded proteins. In this study, we analysed the effect of HSF1 deficiency on core body temperature regulation. MATERIALS AND METHODS: Body temperature, locomotor activity, and food consumption of wild-type mice and HSF1-deficient mice were recorded. Prolactin and thyroid-stimulating hormone levels were measured by ELISA. Gene expression in brown adipose tissue was analysed by quantitative real-time PCR. Hypothalamic HSF1 and its co-localisation with tyrosine hydroxylase was analysed using confocal laser scanning microscopy. RESULTS: HSF1-deficient mice showed an increase in core body temperature (hyperthermia), decreased overall locomotor activity, and decreased levels of prolactin in pituitary and blood plasma reminiscent of cold adaptation. HSF1 could be detected in various hypothalamic regions involved in temperature regulation, suggesting a potential role of HSF1 in hypothalamic thermoregulation. Moreover, HSF1 co-localises with tyrosine hydroxylase, the rate-limiting enzyme in dopamine synthesis, suggesting a potential role of HSF1 in the hypothalamic control of prolactin release. In brown adipose tissue, levels of prolactin receptor and uncoupled protein 1 were increased in HSF1-deficient mice, consistent with an up-regulation of heat production. CONCLUSION: Our data suggest a role of HSF1 in systemic thermoregulation.

Hierarchical Distribution of the Tau Cytoskeletal Pathology in the Thalamus of Alzheimer's Disease Patients.

In spite of considerable progress in neuropathological research on Alzheimer's disease (AD), knowledge regarding the exact pathoanatomical distribution of the tau cytoskeletal pathology in the thalamus of AD patients in the advanced Braak and Braak AD stages V or VI of the cortical cytoskeletal pathology is still fragmentary. Investigation of serial 100 µm-thick brain tissue sections through the thalamus of clinically diagnosed AD patients with Braak and Braak AD stage V or VI cytoskeletal pathologies immunostained with the anti-tau AT8 antibody, along with the affection of the extraterritorial reticular nucleus of the thalamus, reveals a consistent and severe tau immunoreactive cytoskeletal pathology in the limbic nuclei of the thalamus (e.g., paraventricular, anterodorsal and laterodorsal nuclei, limitans-suprageniculate complex). The thalamic nuclei integrated into the associative networks of the human brain (e.g., ventral anterior and mediodorsal nuclei) are only mildly affected, while its motor precerebellar (ventral lateral nucleus) and sensory nuclei (e.g., lateral and medial geniculate bodies, ventral posterior medial and lateral nuclei, parvocellular part of the ventral posterior medial nucleus) are more or less spared. The highly stereotypical and characteristic thalamic distribution pattern of the AD-related tau cytoskeletal pathology represents an anatomical mirror of the hierarchical topographic distribution of the cytoskeletal pathology in the interconnected regions of the cerebral cortex of AD patients. These pathoanatomical parallels support the pathophysiological concept of a transneuronal spread of the disease process of AD along anatomical pathways. The AD-related tau cytoskeletal pathology in the thalamus most likely contributes substantially to the neuropsychiatric disease symptoms (e.g., dementia), attention deficits, oculomotor dysfunctions, altered non-discriminative aspects of pain experience of AD patients, and the disruption of their waking and sleeping patterns.

Improving Drug Penetrability with iRGD Leverages the Therapeutic Response to Sorafenib and Doxorubicin in Hepatocellular Carcinoma.

iRGD is a derivative of the integrin-binding peptide RGD, which selectively increases the penetrability of tumor tissue to various coadministered substances in several preclinical models. In this study, we investigated the ability of iRGD to improve the delivery of sorafenib and doxorubicin therapy in hepatocellular carcinoma (HCC) using established mouse models of the disease. A contrast-enhanced MRI method was developed in parallel to assess the in vivo effects of iRGD in this setting. We found that iRGD improved the delivery of marker substances to the tumors of HCC-bearing mice about three-fold without a parallel increase in normal tissues. Control peptides lacking the critical CendR motif had no effect. Similarly, iRGD also selectively increased the signal intensity from tumors in Gd-DTPA-enhanced MRI. In terms of antitumor efficacy, iRGD coadministration significantly augmented the individual inhibitory effects of sorafenib and doxorubicin without increasing systemic toxicity. Overall, our results offered a preclinical proof of concept for the use of iRGD coadministration as a strategy to widen the therapeutic window for HCC chemotherapy, as monitored by Gd-DTPA-enhanced MRI as a noninvasive, clinically applicable method to identify iRGD-reactive tumors.

Melatonin-induced changes in the expression of thyroid hormone-converting enzymes in hypothalamus depend on the timing of melatonin injections and genetic background in mice.

Recent studies have identified TSHB, Dio2, and Dio3 as key genes for the photoperiodic regulation of gonads. In mammals, the expression of these genes is controlled by melatonin. Surprisingly, this effect of melatonin was shown to be conserved in several reproductively non-photoperiodic laboratory mouse strains that have thus become a valuable model to decipher the mechanisms through which melatonin controls the expression of TSHB, Dio2, and Dio3. In this study, we assessed the effects of intraperitoneal melatonin injections and of their timing on the expression of TSHB, TSHR, Dio2, and Dio3 in the hypothalamo-hypophysial systems of melatonin-proficient CBA/N and melatonin-deficient C57BL/6J mice kept under long-day conditions. In CBA/N mice, Dio3 expression was induced by a daily melatonin injection at ZT14 only, whereas in C57BL/6J mice, a daily melatonin injection induced Dio3 expression at all time points investigated (ZT8, 14, and 20) without changes in TSHB expression in both strains. Dio2 expression was suppressed by a daily melatonin injection only in C57BL/6J mice and only at ZT8. Effect of a daily melatonin injection on TSHR expression was strain- and region- specific. Melatonin levels elevated in plasma and hypothalamus after intraperitoneal injections of melatonin at ZT8 for 7days in C57BL/6J returned to basal levels within 1h after the final injection, while in CBA/N mice melatonin levels in hypothalamus remained high for at least 1h. These data suggest that Dio2 and Dio3 expression in the hypothalamus is differentially regulated by the timing of melatonin injections through strain-specific mechanisms.

Disturbed sleep/wake rhythms and neuronal cell loss in lateral hypothalamus and retina of mice with a spontaneous deletion in the ubiquitin carboxyl-terminal hydrolase L1 gene.

Many neurodegenerative disorders including Parkinson's disease (PD) and Alzheimer's disease (AD) are associated with sleep disturbances with presumably multifactorial etiology. Ubiquitin C-terminal hydrolase L1 (UCH-L1) is involved in the pathophysiology of PD and AD. In the present study, we analyzed locomotor rhythms, orexin A-immunoreaction (Ir) in the lateral hypothalamus (LH) and melanopsin-Ir in the retina of gracile axonal dystrophy (gad) mice with a spontaneous deletion in the Uch-l1 gene. In constant darkness, gad mice showed circadian rhythms in locomotor activity, indicating the integrity of the endogenous circadian rhythm generator. However, gad mice showed an increased activity during subjective day and a decreased number of orexin A-immunoreactive neurons in the LH compared with the wild type (WT). In addition, gad mice showed increased locomotor activity in the light period when kept in a standard photoperiod and entrainment to phase shifts was significantly slower than in WT. Moreover, melanopsin-Ir was significantly reduced in the retina of gad mice, suggesting an impairment of circadian light perception in gad mice.

Cocultures of rat sensorimotor cortex and spinal cord slices to investigate corticospinal tract sprouting.

STUDY DESIGN: Experimental study of corticospinal axonal sprouting in an organotypic slice culture model. OBJECTIVE: To develop an in vitro model that simplifies the study of various factors regulating neuronal regeneration. SUMMARY OF BACKGROUND DATA: Spinal cord injury leads to permanent neurologic damage, mainly due to the inability of the adult central nervous system to regenerate. Much attention has been focused on promoting axonal regeneration and sprouting, either by exogenous administration of various neurotrophic factors or by the antagonization of factors inhibiting regeneration. METHODS: An in vitro system that allows coculture of slices from rat sensorimotor cortex and spinal cord (p4) was established. Two groups of cultures were investigated: In the first group, intact spinal cord slices were cultured adjacent to sensorimotor cortex slices, while in the second group the spinal cord slices were sagittally cut into halves, with the sectioned interface placed directly adjacent to the sensorimotor cortex, to prevent the spinal white matter from interference. Each group was further divided into 2 subgroups: The neurotrophin-3 (NT-3) group, where the culture medium contained 50 ng/mL NT-3 and the control group treated with normal culture medium. Sensorimotor cortex pyramidal neurons were anterogradely labeled with Mini-Ruby, a 10 kD biotinylated dextran amine. RESULTS: Cocultures of cortical and spinal cord tissue were propagated in vitro, and axonal sprouting occurred. The group of cocultures treated with NT-3 showed an improved cortical cytoarchitecture, and sprouting axons were more frequently observed. In NT-3-treated cocultures where spinal cord gray matter was directly opposed to cortical slices sprouting axons entered the adjacent spinal cord tissue. This phenomenon was not observed if spinal cord pia mater and white matter were opposed to the cortical slices, or if NT-3 was absent. CONCLUSION: Our data suggest that the absence of repellent factors such as white matter and the presence of neurotrophic factors promote axonal sprouting. Cocultures of sensorimotor cortex and spinal cord slices combined with anterograde axonal labeling could provide a valuable in vitro model for the simplified screening of factors influencing corticospinal tract regeneration.

The rhythm and blues of gene expression in the rodent pineal gland.

In all vertebrates, melatonin is rhythmically synthesized in the pineal gland and functions as a hormonal message, encoding for the duration of night. In rodents, the nocturnal rise and fall of the arylalkylamine N-ace-tyltransferase (AA-NAT) activity controls the rhythmic synthesis of melatonin. This rhythm is centered around the transcriptional regulation of the AA-NAT by two norepinephrine-inducible transcription factors, the activator CREB (Ca2+/cAMP-response element binding protein) and the inhibitor ICER (inducible cAMP early repressor). CREB is activated by phosphorylation, which is one of the fastest responses in pinealocytes upon adrenergic stimulation, occurring within minutes. ICER in turn accumulates only after several hours, a time gap resulting from the required de novo protein synthesis upon adrenergic stimulation. However, these molecular components of neuroendocrine signaling in the rodent pineal gland are supplemented by the impact of a variety of neurotransmitters and neuromodulators, and by translational and post-translational mechanisms. By molecular crosstalk, those different inputs on pinealocytes seem to fine-tune the shape of the melatonin signal, by interacting at various levels with the NE/cAMP/pCREB/ICER pathway. In addition, these alternate signaling routes may be important in acute "emergency" situations. Together, concerted signaling events in the rodent pineal gland help to generate a stable and reliable hormonal message of darkness for the body, that, however, can be altered rapidly upon sudden and unexpected "error" signals.