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Context Daily variation in the thermic effect of food (TEF) is commonly reported and proposed as a contributing factor to weight gain with late eating. However, underlying circadian variability in resting metabolic rate (RMR) is an overlooked factor when calculating TEF associated with eating at different times of the day. Objective This work aimed to determine whether methodological approaches to calculating TEF contribute to the reported phenomena of daily variation in TEF. Methods Fourteen overweight to obese but otherwise healthy individuals had their resting and postprandial energy expenditure (EE) measured over 15.5 hours at a clinical research unit. TEF was calculated for breakfast, lunch, and dinner using standard methods (above a baseline and premeal RMR measure) and compared to a method incorporating a circadian RMR by which RMR was derived from a sinusoid curve model and TEF was calculated over and above the continuously changing RMR. Main outcome measures were TEF at breakfast, lunch, and dinner calculated by different methods. Results Standard methods of calculating TEF above a premeal measured RMR showed that morning TEF (60.8 kcal ± 5.6) (mean ± SEM) was 1.6 times greater than TEF at lunch (36.3 kcal ± 8.4) and 2.4 times greater than dinner TEF (25.2 kcal ± 9.6) (P = .022). However, adjusting for modeled circadian RMR nullified any differences between breakfast (54.1 kcal ± 30.8), lunch (49.5 kcal ± 29.4), and dinner (49.1 kcal ± 25.7) (P = .680). Conclusion Differences in TEF between morning and evening can be explained by the underlying circadian resting EE, which is independent of an acute effect of eating.

Objectives The grant of marketing authorisation (MA) for new medicines requires comprehensive assessment by regulatory authorities. This study sought to identify ophthalmic medicines granted United Kingdom MA and consider trends in licence approvals. Methods This retrospective study reviewed published lists of products granted MA by the UK Medicines & Healthcare products Regulatory Agency between January 2001 and December 2018, inclusive. Eye drops and medicinal products intended for ophthalmic use were identified. All regulatory data sources consulted are in the public domain. Data analyses were descriptive. Results A total of 295 MAs were issued for ophthalmic products between 2001 and 2018, inclusive. Of these 229 (78%) were for single-active substances and 66 (22%) fixed-dose combination (FDC). Approvals for products with single-active substance included ocular hypotensives (115; 50%), antibiotics (48; 22%), allergy medicines (30; 13%), lubricants (18; 8%) and anti-inflammatories (11; 5%). Approvals for FDCs were predominantly ocular hypotensives (56; 85%), with timolol combined with carbonic anhydrase inhibitors (27; 48%) and prostaglandin analogues (26; 46%) accounting for the majority of glaucoma FDCs. Other FDCs were approved for antibiotic/inflammatory (5; 7.5%), pupillary mydriasis (2; 3%), allergy (2; 3%) and ocular surface lubrication (1; 1.5%) products. A median of 16 licences were approved per year (range 7 [2005] to 35 [2011]). Conclusions The majority of MAs granted were for single-agent products, particularly ocular hypotensives and antibiotic preparations. Most products were generic versions of well-established active substances. A trend for increased approvals for FDCs is evident, particularly for the treatment of raised IOP.

Objectives:Regulatory approval of new medicines requires a thorough assessment of the potential clinical benefits and risks. Study end-points are expected to demonstrate a clinically relevant treatment effect that will translate into direct patient benefits. This study sought to review the ophthalmic medicines with European Union–wide approval granted via the Centralised Procedure and characterise the key efficacy end-points underpinning the demonstration of clinical benefit.Methods:This study was a retrospective review of published data pertaining to the European regulatory authorisation of centrally approved ophthalmic products between 1999 and 2017, inclusive. All sources and data consulted are in the public domain. European Public Assessment Reports published by the European Medicines Agency were consulted for data concerning the pivotal clinical efficacy studies supporting the applications. Data analyses were descriptive.Results:The European Medicines Agency have authorised 30 products via the Centralised Procedure between 1999 and 2017. For these products, a total of 24 additional approvals for line extensions or additional therapeutic indications were granted. Four products have been approved for orphan indications, including one approval ‘under exceptional circumstances’ and one ‘Conditional Marketing Authorisation’. Approvals for products in retina (36%) and glaucoma (28%) indications together accounted for the majority of authorisations, with trial end-points predominantly based on visual acuity and intraocular pressure parameters, respectively. Products were also approved for indications in ocular surface disease, inflammation, optic neuropathy and surgical adjuncts, with a range of functional and anatomical end-points.Conclusion:Approvals for ophthalmic medicines have been granted for a range of clinical indications, representing a considerable portion of available major therapeutics for practitioners. Benefit–risk assessments rely on clinical trial data demonstrating a clearly relevant patient benefit.

Circadian rhythms play a critical role in the physiological processes involved in energy metabolism and energy balance (EB). A large array of metabolic processes, including the expression of many energy-regulating endocrine hormones, display temporal rhythms that are driven by both the circadian clock and food intake. Mealtime has been shown to be a compelling zeitgeber in peripheral tissue rhythms. Inconsistent signalling to the periphery, because of mismatched input from the central clock vs time of eating, results in circadian disruption in which central and/or peripheral rhythms are asynchronously time shifted or their amplitudes reduced. A growing body of evidence supports the negative health effects of circadian disruption, with strong evidence in murine models that mealtime-induced circadian disruption results in various metabolic consequences, including energy imbalance and weight gain. Increased weight gain has been reported to occur even without differences in energy intake, indicating an effect of circadian disruption on energy expenditure. However, the translation of these findings to humans is not well established because the ability to undertake rigorously controlled dietary studies that explore the chronic effects on energy regulation is challenging. Establishing the neuroendocrine changes in response to both acute and chronic variations in mealtime, along with observations in populations with routinely abnormal mealtimes, may provide greater insight into underlying mechanisms that influence long-term weight management under different meal patterns. Human studies should explore mechanisms through relevant biomarkers; for example, cortisol, leptin, ghrelin and other energy-regulating neuroendocrine factors. Mistiming between aggregate hormonal signals, or between hormones with their receptors, may cause reduced signalling intensity and hormonal resistance. Understanding how mealtimes may impact on the coordination of endocrine factors is essential for untangling the complex regulation of EB. Here a review is provided on current evidence of the impacts of mealtime on energy metabolism and the underlying neuroendocrine mechanisms, with a specific focus on human research.

Thyroid hormone (TH) is essential for adult brain function and its actions include several key roles in the hypothalamus. Although TH controls gene expression via specific TH receptors of the nuclear receptor class, surprisingly few genes have been demonstrated to be directly regulated by TH in the hypothalamus, or the adult brain as a whole. This study explored the rapid induction by TH of retinaldehyde dehydrogenase 1 (Raldh1), encoding a retinoic acid (RA)-synthesizing enzyme, as a gene specifically expressed in hypothalamic tanycytes, cells that mediate a number of actions of TH in the hypothalamus. The resulting increase in RA may then regulate gene expression via the RA receptors, also of the nuclear receptor class. In vivo exposure of the rat to TH led to a significant and rapid increase in hypothalamic Raldh1 within 4 hours. That this may lead to an in vivo increase in RA is suggested by the later induction by TH of the RA-responsive gene Cyp26b1. To explore the actions of RA in the hypothalamus as a potential mediator of TH control of gene regulation, an ex vivo hypothalamic rat slice culture method was developed in which the Raldh1-expressing tanycytes were maintained. These slice cultures confirmed that TH did not act on genes regulating energy balance but could induce Raldh1. RA has the potential to upregulate expression of genes involved in growth and appetite, Ghrh and Agrp. This regulation is acutely sensitive to epigenetic changes, as has been shown for TH action in vivo. These results indicate that sequential triggering of two nuclear receptor signalling systems has the capability to mediate some of the functions of TH in the hypothalamus.

Animals have evolved diverse seasonal variations in physiology and reproduction to accommodate yearly changes in environmental and climatic conditions. These changes in physiology are initiated by changes in photoperiod (daylength) and are mediated through melatonin, which relays photoperiodic information to the pars tuberalis of the pituitary gland. Melatonin drives thyroid-stimulating hormone transcription and synthesis in the pars tuberalis, which, in turn, regulates thyroid hormone and retinoic acid synthesis in the tanycytes lining the third ventricle of the hypothalamus. Seasonal variation in central thyroid hormone signalling is conserved among photoperiodic animals. Despite this, different species adopt divergent phenotypes to cope with the same seasonal changes. A common response amongst different species is increased hypothalamic cell proliferation/neurogenesis in short photoperiod. That cell proliferation/neurogenesis may be important for seasonal timing is based on (i) the neurogenic potential of tanycytes; (ii) the fact that they are the locus of striking seasonal morphological changes; and (iii) the similarities to mechanisms involved in de novo neurogenesis of energy balance neurones. We propose that a decrease in hypothalamic thyroid hormone and retinoic acid signalling initiates localised neurodegeneration and apoptosis, which leads to a reduction in appetite and body weight. Neurodegeneration induces compensatory cell proliferation from the neurogenic niche in tanycytes and new cells are born under short photoperiod. Because these cells have the potential to differentiate into a number of different neuronal phenotypes, this could provide a mechanistic basis to explain the seasonal regulation of energy balance, as well as reproduction. This cycle can be achieved without changes in thyroid hormone/retinoic acid and explains recent data obtained from seasonal animals held in natural conditions. However, thyroid/retinoic acid signalling is required to synchronise the cycles of apoptosis, proliferation and differentiation. Thus, hypothalamic neurogenesis provides a framework to explain diverse photoperiodic responses.

Seasonal or photoperiodically sensitive animals respond to altered day length with changes in physiology (growth, food intake and reproductive status) and behaviour to adapt to predictable yearly changes in the climate. Typically, different species of hamsters, voles and sheep are the most studied animal models of photoperiodism. Although laboratory rats are generally considered nonphotoperiodic, one rat strain, the inbred Fischer 344 (F344) rat, has been shown to be sensitive to the length of daylight exposure by changing its physiological phenotype and reproductive status according to the season. The present study aimed to better understand the nature of the photoperiodic response in the F344 rat. We examined the effects of five different photoperiods on the physiological and neuroendocrine responses. Young male F344 rats were held under light schedules ranging from 8 h of light/day to 16 h of light/day, and then body weight, including fat and lean mass, food intake, testes weights and hypothalamic gene expression were compared. We found that rats held under photoperiods of ≥ 12 h of light/day showed increased growth and food intake relative to rats held under photoperiods of ≤ 10 h of light/day. Magnetic resonance imaging analysis confirmed that these changes were mainly the result of a change in lean body mass. The same pattern was evident for reproductive status, with higher paired testes weight in photoperiods of ≥ 12 h of light/day. Accompanying the changes in physiological status were major changes in hypothalamic thyroid hormone (Dio2 and Dio3), retinoic acid (Crabp1 and Stra6) and Wnt/β-Catenin signalling genes (sFrp2 and Mfrp). Our data demonstrate that a photoperiod schedule of 12 h of light/day is interpreted as a stimulatory photoperiod by the neuroendocrine system of young male F344 rats.

Purpose: To evaluate the efficacy of anti-VEGF agents for treating choroidal neovascularization (CNV) when delivered topically using novel cell-penetrating peptides (CPPs) compared with delivery by intravitreal (ivit) injection.Methods: CPP toxicity was investigated in cell cultures. Ivit concentrations of ranibizumab and bevacizumab after topical administration were measured using ELISA. The biological efficacy of topical anti-VEGF + CPP complexes was compared with ivit anti-VEGF injections using an established model of CNV.Results: CPPs were nontoxic in vitro. In vivo, after topical eye drop delivery, CPPs were present in the rat anterior chamber within 6 minutes. A single application of CPP + bevacizumab eye drop delivered clinically relevant concentrations of bevacizumab to the posterior chamber of the rat eye in vivo. Similarly, clinically relevant levels of CPP + ranibizumab and CPP + bevacizumab were detected in the porcine vitreous and retina ex vivo. In an established model of CNV, mice treated with either a single ivit injection of anti-VEGF, twice daily CPP + anti-VEGF eye drops or daily dexamethasone gavage for 10 days all had significantly reduced areas of CNV when compared with lasered eyes without treatment.Conclusions: CPPs are nontoxic to ocular cells and can be used to deliver therapeutically relevant doses of ranibizumab and bevacizumab by eye drop to the posterior segment of mouse, rat, and pig eyes. The CPP + anti-VEGF drug complexes were cleared from the retina within 24 hours, suggesting a daily eye drop dosing regimen. Daily, topically delivered anti-VEGF with CPP was as efficacious as a single ivit injection of anti-VEGF in reducing areas of CNV in vivo.

Eye injuries are common in warfare with an incidence of approximately 10%. They carry a high morbidity, as they can determine an injured person’s future independence and employability. The majority are a combination of primary and secondary blast mechanisms, though tertiary and quaternary types are common. There is some evidence of quinary types from toxic elements from the explosion. Eye protection significantly reduces the incidence and severity of ballistic eye injury but does not eliminate it. Thermal ocular burns are relatively common in warfare. The treatment goal is to minimise limbal stem cell damage. Human amniotic membrane can be used to promote this. Retinal and optic nerve injury following closed eye trauma are currently untreatable, but neuroprotective and neuroregenerative agents are being developed to improve outcomes. Sensory substitution of the sense of touch for sight can help orientate blinded individual in their surroundings. Ophthalmology has a major impact on the lives of the war wounded.

The retinoids are a family of compounds that in nature are derived from vitamin A or pro-vitamin A carotenoids. An essential part of the diet for mammals, vitamin A has long been known to be essential for many organ systems in the adult. More recently, however, they have been shown to be necessary for function of the brain and new discoveries point to a central role in processes ranging from neuroplasticity to neurogenesis. Acting in several regions of the central nervous system including the eye, hippocampus and hypothalamus, one common factor in its action is control of biological rhythms. This review summarizes the role of vitamin A in the brain; its action through the metabolite retinoic acid via specific nuclear receptors, and the regulation of its concentration through controlled synthesis and catabolism. The action of retinoic acid to regulate several rhythms in the brain and body, from circadian to seasonal, is then discussed to finish with the importance of retinoic acid in the regular pattern of sleep. We review the role of vitamin A and retinoic acid (RA) as mediators of rhythm in the brain. In the suprachiasmatic nucleus and hippocampus they control expression of circadian clock genes while in the cortex retinoic acid is required for delta oscillations of sleep. Retinoic acid is also central to a second rhythm that keeps pace with the seasons, regulating function in the hypothalamus and pineal gland.

In seasonal animals, photoperiod exerts profound effects on physiology, such as growth, energy balance and reproduction, via changes in the neuroendocrine axes. A key element of the photoperiodic response is the thyroid hormone level in the hypothalamus, which is controlled via retrograde transport of thyroid-stimulating hormone (TSH) from the pars tuberalis of the pituitary. TSH regulates type II deiodinase (Dio2) expression, which transforms inactive thyroid hormone to its active form, via TSH receptors expressed in the ependymal cells of the hypothalamus. In the present study, we hypothesised that a second peptide hormone, neuromedin U (NMU), may play a role in the photoperiodic response alongside TSH because the gene for NMU is also expressed in a strongly photoperiod-dependent manner in the pars tuberalis and its receptor NMU2 is expressed in the ependymal layer of the third ventricle in photoperiod-sensitive F344 rats. Consistent with other studies conducted in nonseasonal mammals, we found that acute i.c.v. injections of NMU into the hypothalamus negatively regulated food intake and body weight and increased core body temperature in F344 rats. At the same time, NMU increased Dio2 mRNA expression in the ependymal region of the hypothalamus similar to the effects of TSH. These data suggest that NMU may affect acute and photoperiodically controlled energy balance through distinct pathways. We also showed that TSH inhibits the expression of type III deiodinase (Dio3) in F344 rats, a response not mimicked by NMU. Furthermore, NMU also increased the expression of genes from the Wnt/β-catenin pathway within the ependymal layer of the third ventricle. This effect was not influenced by TSH. These data indicate that, although NMU acts with some similarities to TSH, it also has completely distinct signalling functions that do not overlap. In summary, the present study of NMU signalling reveals the potential for a new player in the control of seasonal biology.

Long-term and reversible changes in body weight are typical of seasonal animals. Thyroid hormone (TH) and retinoic acid (RA) within the tanycytes and ependymal cells of the hypothalamus have been implicated in the photoperiodic response. We investigated signalling downstream of RA and how this links to the control of body weight and food intake in photoperiodic F344 rats. Chemerin, an inflammatory chemokine, with a known role in energy metabolism, was identified as a target of RA. Gene expression of chemerin (Rarres2) and its receptors were localised within the tanycytes and ependymal cells, with higher expression under long (LD) versus short (SD) photoperiod, pointing to a physiological role. The SD to LD transition (increased food intake) was mimicked by 2 weeks of ICV infusion of chemerin into rats. Chemerin also increased expression of the cytoskeletal protein vimentin, implicating hypothalamic remodelling in this response. By contrast, acute ICV bolus injection of chemerin on a 12 h:12 h photoperiod inhibited food intake and decreased body weight with associated changes in hypothalamic neuropeptides involved in growth and feeding after 24 hr. We describe the hypothalamic ventricular zone as a key site of neuroendocrine regulation, where the inflammatory signal, chemerin, links TH and RA signaling to hypothalamic remodeling.

Retinoic acid (RA) has been found to regulate hypothalamic function, but precisely where it acts is unknown. This study shows expression of retinaldehyde dehydrogenase (RALDH) enzymes in tanycytes that line the third ventricle in an area overlapping with the site of hypothalamic neural stem cells. The influence of RA was examined on the proliferation of progenitors lining the third ventricle using organotypic slice cultures. As has been shown in other regions of neurogenesis, RA was found to inhibit proliferation. Investigations of the dynamics of RALDH1 expression in the rat hypothalamus have shown that this enzyme is in tanycytes under photoperiodic control with highest levels during long versus short days. In parallel to this shift in RA synthesis, cell proliferation in the third ventricle was found to be lowest during long days when RA was highest, implying that RALDH1 synthesized RA may regulate neural stem cell proliferation. A second RA synthesizing enzyme, RALDH2 was also present in tanycytes lining the third ventricle. In contrast to RALDH1, RALDH2 showed little change with photoperiodicity, but surprisingly the protein was present in the apparent absence of mRNA transcript and it is hypothesized that the endocytic tanycytes may take this enzyme up from the cerebrospinal fluid (CSF).

Hydrogel-based three-dimensional (3D) scaffolds are widely used in the field of regenerative medicine, translational medicine, and tissue engineering. Recently, we reported the effect of scaffold formation on the differentiation and survival of human neural progenitor cells (hNPCs) using PuraMatrix™ (RADA-16) scaffolds. Here, we were interested in the impact of PuraMatrix modified by the addition of short peptide sequences, based on a bone marrow homing factor and laminin. The culture and differentiation of the hNPCs in the modified matrices resulted in an approximately fivefold increase in neuronal cells. The examination of apoptotic and necrotic cells, as well as the level of the anti-apoptotic protein Bcl-2, indicates benefits for cells hosted in the modified formulations. In addition, we found a trend to lower proportions of apoptotic or necrotic neuronal cells in the modified matrices. Interestingly, the neural progenitor cell pool was increased in all the tested matrices in comparison to the standard 2D culture system, while no difference was found between the modified matrices. We conclude that a combination of elevated neuronal differentiation and a protective effect of the modified matrices underlies the increased proportion of neuronal cells.

Both retinoic acid (RA) and thyroid hormone (TH) regulate transcription via specific nuclear receptors. TH regulates hypothalamic homeostasis and active T3 is generated by deiodinase enzymes in tanycytes surrounding the third ventricle. However, RA has not been previously considered in such a role. Data presented here highlights novel parallels between the TH and RA synthetic pathways in the hypothalamus implying that RA also acts to regulate hypothalamic gene expression and function. Key elements of the RA cellular signaling pathway were shown to be regulated in the rodent hypothalamus. Retinoid synthetic enzymes and the retinol transport protein Stra6 were located in the cells lining the third ventricle allowing synthesis of RA from retinol present in the CNS to act via RA receptors and retinoid X receptors in the hypothalamus. Photoperiod manipulation was shown to alter the expression of synthetic enzymes and receptors with lengthening of photoperiod leading to enhanced RA signaling. In vitro RA can regulate the hypothalamic neuroendocrine peptide adrenocorticotrophic hormone. This work presents the new concept of controlled RA synthesis by hypothalamic tanycytes giving rise to possible involvement of this system in endocrine, and possibly vitamin A, homeostasis.

Hypothalamic genes involved in food intake and growth regulation were studied in F344 rats in response to photoperiod. Two sub-strains were identified: F344/NHsd (F344/N) and F344/NCrHsd (F344/NCr); sensitive and relatively insensitive to photoperiod respectively. In F344/N rats, marked, but opposite, changes in the genes for neuropeptide Y (NPY) (+97.5%) and agouti-related protein (AgRP) (-39.3%) expression in the arcuate nucleus were observed in response to short (8 : 16 h light/dark cycle, SD) relative to long (16 : 8 h light/dark cycle, LD) day photoperiods. Changes were associated with both reduced food intake and growth. Expression of the genes for cocaine and amphetamine-regulated transcript (CART) and pro-opiomelanocortin (POMC) in the arcuate nucleus was unchanged by photoperiod. POMC in the ependymal layer around the third ventricle was markedly inhibited by SD. Parallel decreases in the genes for growth hormone-releasing hormone (GHRH) and somatostatin (Somatostatin) mRNA in the arcuate nucleus and Somatostatin in the periventricular nucleus were observed in SD. Serum levels of insulin-like growth factor (IGF)-1 and insulin were lower in F344/N rats in SD, whereas neither leptin nor corticosterone levels were affected. By contrast, F344/NCr rats that show only minor food intake and growth rate changes showed minimal responses in these genes and hormones. Thus, NPY/AgRP neurones may be pivotal to the photoperiodic regulation of food intake and growth. Potentially, the SD increase in NPY expression may inhibit growth by decreasing GHRH and Somatostatin expression, whereas the decrease in AgRP expression probably leads to reduced food intake. The present study reveals an atypical and divergent regulation of NPY and AgRP, which may relate to their separate roles with respect to growth and food intake, respectively.

In the Siberian hamster, seasonal weight loss occurs gradually over many weeks during autumn and winter. This is driven by a regulatory mechanism that is able to integrate duration of exposure to short days (SDs) with the size of body energy reserves. After food restriction in SDs, followed by ad libitum refeeding, body weight of the hamster does not return to its former level; rather, it increases to a level defined by the length of time spent in SDs. In this report, we show that components of the thyroid hormone system that are involved in seasonal weight loss change expression in response to 48 h of starvation. Eight weeks in an SD photoperiod induced weight loss in the Siberian hamster. In the hypothalamus of these hamsters, type II deiodinase expression was decreased and type III deiodinase expression was induced, but there was no change in hypothalamic neuropeptide Y or thyrotropin-releasing hormone gene expression. For the first time, we show that the thyroid hormone transporter monocarboxylate transporter 8 is expressed in tanycytes and is increased in response to an SD photoperiod. Food restriction (48 h of starvation) reversed the direction of gene expression change for type II and III deiodinase and monocarboxylate transporter 8 induced by SD photoperiods. Furthermore, fasting increased neuropeptide Y expression and decreased thyrotropin-releasing hormone expression. VGF, a gene upregulated in SDs in the dorsal region of the medial posterior area of the arcuate nucleus, was not changed by starvation. These data point to a mechanism whereby energy deprivation can interact with an SD photoperiod on hypothalamic tanycytes to regulate components of the thyroid hormone system involved in photoperiodic regulation of seasonal physiology.

Photoperiod exerts profound influence on the physiology of mammals through the action of melatonin on the neuroendocrine system. Over the last 20 years, studies have moved away from a melatonin receptor-focused approach to understanding how photoperiod regulates neuroendocrine activity through studies of downstream effects on gene expression. This paper reviews the recent progress made in our understanding of the effects of photoperiod on gene expression in the hypothalamus, and considers how this new information can be reconciled with the species-specific location of melatonin receptors.

Hypothalamic energy balance genes have been examined in the context of seasonal body weight regulation in the Siberian hamster. Most of these long photoperiod (LD)/short photoperiod (SD) comparisons have been of tissues collected at a single point in the light-dark cycle. We examined the diurnal expression profile of hypothalamic genes in hamsters killed at 3-h intervals throughout the light-dark cycle after housing in LD or SD for 12 wk. Gene expression of neuropeptide Y, agouti-related peptide, proopiomelanocortin, cocaine- and amphetamine-regulated transcript, long-form leptin receptor, suppressor of cytokine signaling-3, melanocortin-3 receptor, melanocortin-4 receptor, and the clock gene Per1 as control were measured by in situ hybridization in hypothalamic nuclei. Effects of photoperiod on gene expression and leptin levels were generally consistent with previous reports. A clear diurnal variation was observed for Per1 in the suprachiasmatic nucleus in both photoperiods. Temporal effects on expression of energy balance genes were restricted to long-form leptin receptor in the arcuate nucleus and ventromedial nucleus, where similar diurnal expression profiles were observed, and melanocortin-4 receptor in the paraventricular nucleus; these effects were only observed in LD hamsters. There was no variation in serum leptin concentration. The 24-h profiles of hypothalamic energy balance gene expression broadly confirm photoperiodic differences that were observed previously, based on single time point comparisons, support the growing consensus that these genes have a limited role in seasonal body weight regulation, and further suggest limited involvement in daily rhythms of food intake.

In a previous study performed in mouse models of energetic challenge, there was evidence to suggest that the orphan G protein-coupled receptor GPCR101 may have a role in the regulation of energy balance. To further investigate this possibility, we utilised in situ hybridisation to determine the effect of energetic challenges experienced by pregnant and lactating rats on GPCR101 mRNA expression. In the rat hypothalamus, GPCR101 mRNA expression was detected in a number of hypothalamic nuclei. During pregnancy and lactation, GPCR101 mRNA level remained unchanged in most nuclei, but had increased in the supraoptic nucleus by the end of pregnancy and remained elevated during lactation. GPCR101 mRNA expression showed a similar pattern of expression in the rostral ventromedial parvocellular subdivision of the paraventricular nucleus. A common feature of these two nuclei is the production of the peptide oxytocin. Dual in situ hybridisation revealed GPCR101 and oxytocin mRNA co-expression in neurons of these two nuclei. In the supraoptic nucleus, in situ hybridisation revealed that the temporal regulation of oxytocin and GPCR101 mRNA expression were similar. In the paraventricular nucleus, although temporal changes in oxytocin mRNA expression were similar to GPCR101, the spatial expression of the two mRNA species was different; in contrast to GPCR101, oxytocin mRNA expression changed in both parvo- and magnocellular neurons during lactation. In conclusion, increased GPCR101 mRNA expression in supraoptic and paraventricular nuclei from late pregnancy to late lactation may reflect the functional importance of this receptor in the regulation of neurons of these nuclei during this period.

Previously, 40% food restriction of male Siberian hamsters over 21 days in short-day (SD) photoperiod induced characteristic changes in expression of hypothalamic arcuate nucleus energy balance genes; mRNAs for neuropeptide Y, agouti-related peptide, and leptin receptor were upregulated, and those of proopiomelanocortin and cocaine- and amphetamine-regulated transcript were depressed. The present study examined the effect of refeeding hamsters for 6 days (∼50% recovery of weight differential) or 19 days (resumption of appropriate weight trajectory). Hyperphagia continued throughout refeeding, but differences in fat pad weights and leptin levels had disappeared after 19 days. Cocaine- and amphetamine-regulated transcript gene expression was depressed by prior restriction in both refed groups. The depressive effect of prior restriction on proopiomelanocortin gene expression had disappeared after 19 days of refeeding. There was no effect of prior food restriction on neuropeptide Y or agouti-related peptide gene expression. Expression of the anorexigenic brain-derived neurotrophic factor was downregulated in the ventromedial nucleus after SD exposure for 12 wk. In the SD food restriction study, there were effects of photoperiod on brain-derived neurotrophic factor gene expression but not of prior food restriction. Hypothalamic energy balance genes in the hamster respond asynchronously to return to a seasonally appropriate body weight. The achievement of this weight rather than the weight at which caloric restriction was imposed is the critical factor. The differential responses of hypothalamic energy balance genes to food restriction and refeeding are poorly characterized in any species, a critical issue given their potential relevance to human weight loss strategies that involve caloric restriction.

The Siberian hamster survives winter by decreasing food intake and catabolizing abdominal fat reserves, resulting in a sustained, profound loss of body weight. VGF gene expression is photoperiodically regulated in the hypothalamus with significantly higher expression in lean Siberian hamsters. The aim of this study was to investigate the role of VGF in regulating these seasonal cycles by determining the effects of a VGF-derived peptide (TLQP-21) on food intake and body weight. Acute intracerebroventricular administration of TLQP-21 decreased food intake, and chronic treatment caused a sustained reduction in food intake and body weight and decreased abdominal fat depots. Behavioral analysis revealed that TLQP-21 reduced meal size but not the frequency of feeding bouts, suggesting a primary action on satiety. Hamsters treated with TLQP-21 lost a similar amount of weight as a pair-fed group in which food intake was matched to that of the TLQP-21-treated group. Central or peripheral treatment with TLQP-21 did not produce a significant effect on resting metabolic rate. We conclude that the primary action of TLQP-21 is to decrease food intake rather than increase energy expenditure. TLQP-21 treatment caused a decrease in UCP-1 mRNA in brown adipose tissue, but hypothalamic expression of orexigenic and anorexigenic neuropeptide genes remained unchanged after TLQP-21 treatment, although compensatory increases in NPY and AgRP mRNA were observed in the pair-fed hamsters. The effects of TLQP-21 administration are similar to those in hamsters in short days, suggesting that increased VGF activity may contribute to the hypophagia that underlies the seasonal catabolic state.

PURPOSE:\ud\udTo investigate whether Decorin, a matrikine that regulates extracellular matrix (ECM) deposition, can reverse established trabecular meshwork (TM) fibrosis, lower IOP, and reduce progressive retinal ganglion cell (RGC) death in a novel rodent model of TM fibrosis.\udMETHODS:\ud\udAdult rats had intracameral (IC) injections of human recombinant (hr) TGF-β over 30 days (30 d; to induce TM fibrosis, raise IOP, and initiate RGC death by 17 d) or PBS (controls) and visually evoked potentials (VEP) were measured at 30 d to evaluate resultant visual pathway dysfunction. In some animals TGF-β injections were stopped at 17 d when TM fibrosis and IOP were consistently raised and either hrDecorin or PBS IC injections were administered between 21 d and 30 d. Intraocular pressure was measured biweekly and eyes were processed for immunohistochemical analysis of ECM deposition to assess TM fibrosis and levels of matrix metalloproteinases (MMP) and tissue inhibitors of matrix metalloproteinases (TIMP) to assess fibrolysis. The effect of hrDecorin treatment on RGC survival was also assessed.\udRESULTS:\ud\udTransforming growth factor-β injections caused sustained increases in ECM deposition in the TM and raised IOP by 17 d, responses that were associated with 42% RGC loss and a significant decrease in VEP amplitude measured at 30 d. Decorin treatment from 17 d reduced TGF-β-induced TM fibrosis, increased levels of MMP2 and MMP9 and lowered TIMP2 levels, and lowered IOP, preventing progressive RGC loss.\udCONCLUSIONS:\ud\udHuman recombinant Decorin reversed established TM fibrosis and lowered IOP, thereby rescuing RGC from progressive death. These data provide evidence for the candidacy of hrDecorin as a treatment for open-angle glaucoma.

Peter J Morgan-Warren, Walter Andreatta, Amit K Patel Department of Ophthalmology, Solihull Hospital, Heart of England NHS Foundation Trust, Birmingham,UK Purpose: Opacification of hydrophilic acrylic intraocular lenses (IOLs) is an emerging complication following Descemet stripping automated endothelial keratoplasty (DSAEK). We report six cases and review the current literature.Methods: In this retrospective, noncomparative, observational case series, patients with IOL opacification after previous DSAEK surgery were identified from corneal clinic records. Case notes were reviewed for demographic details, indication for DSAEK, IOL model, incidence of rebubbling, and postoperative course.Results: Six patients developed IOL opacification after DSAEK. All patients had Fuchs’ endothelial dystrophy and had previously received hydrophilic acrylic IOL models. Central anterior IOL opacification was noted in all six cases. Five cases (83%) had required rebubbling due to dislocated graft tissue, and one had an early postoperative intraocular pressure (IOP) rise. Five cases (83%) were managed conservatively, and one case with a failed graft underwent redo DSAEK and IOL exchange.Conclusion: Repeated exposure to intracameral air, raised IOP, and other patient influences may be major etiological factors for IOL opacification after DSAEK. We advise avoiding hydrophilic acrylic IOL models in patients who may require future endothelial keratoplasty. Keywords: IOL, DSAEK, lamellar keratoplasty, endothelial corneal transplantation

Tanycytes in the ependymal layer of the third ventricle act both as a barrier and a communication gateway between the cerebrospinal fluid, brain and portal blood supply to the pituitary gland. However, the range, importance and mechanisms involved in the function of tanycytes remain to be explored. In this study, we have utilized a photoperiodic animal to examine the expression of three unrelated gene sequences in relation to photoperiod-induced changes in seasonal physiology and behaviour. We demonstrate that cellular retinoic acid-binding protein 1 (CRBP1), a retinoic acid transport protein, GPR50, an orphan G-protein-coupled receptor and nestin, an intermediate filament protein, are down-regulated in short-day photoperiods. The distribution of the three sequences is very similar, with expression located in cells with tanycyte morphology in the region of the ependymal layer where tanycytes are located. Furthermore, CRBP1 expression in the ependymal layer is shown to be independent of a circadian clock and altered testosterone levels associated with testicular regression in short photo-period. Pinealectomy of Siberian hamsters demonstrates CRBP1 expression is likely to be dependent on melatonin output from the pineal gland. This provides evidence that tanycytes are seasonally responsive cells and are likely to be an important part of the mechanism to facilitate seasonal physiology and behaviour in the Siberian hamster.

In anticipation of seasonal climate changes, Siberian hamsters display a strategy for survival that entails profound physiological adaptations driven by photoperiod. These include weight loss, reproductive quiescence, and pelage growth with shortening photoperiod and vice versa with lengthening photoperiod. This study reports gene expression changes in the hypothalamus of Siberian hamsters switched from short days (SD) to long days (LD), and also in photorefractory hamsters. Siberian hamsters were maintained in either LD or SD for 14 wk, conditions that generate physiological states of obesity under LD and leanness under SD. After 14 wk, SD lighting was switched to LD and gene expression investigated after 0, 2, 4, and 6 wk by in situ hybridization. Genes encoding nuclear receptors (RXR/RAR), retinoid binding proteins (CRBP1 and CRABP2), and histamine H3 receptor were photoperiodically regulated with significantly lower expression in SD, whereas VGF mRNA expression was significantly higher in SD, in the dorsomedial posterior arcuate nucleus. After a SD-to-LD switch, gene expression changes of CRABP2, RAR, H3R, and VGF occurred relatively rapidly toward LD control levels, ahead of body weight recovery and testicular recrudescence, whereas CRBP1 responded less robustly and rxrgamma did not respond at the mRNA level. In this brain nucleus in photorefractory animals, the CRABP2, RAR, H3R, and VGF mRNA returned toward LD levels, whereas CRBP1 and rxrgamma remained at the reduced SD level. Thus, genes described here are related to photoperiodic programming of the neuroendocrine hypothalamus through expression responses within a subdivision of the arcuate nucleus.

Melatonin regulates circadian and seasonal physiology via melatonin receptors expressed in the brain. However, little is known about the signal transduction mechanisms that mediate the action of melatonin in neuronal cells. To begin to address this issue, we expressed the human MT(1) receptor in the human neuroblastoma SH-SY5Y cell line. In this cell line, melatonin acutely stimulated cAMP synthesis through a calcium-calmodulin dependent pathway. This stimulatory effect was independent of an interaction with G(i) or G(s) G proteins and dependent upon internal calcium stores. Melatonin also potentiated forskolin-activated cAMP synthesis. Differentiation of the neuroblastoma cells with retinoic acid to the neuronal phenotype did not alter the ability of melatonin to acutely stimulate cAMP. These data may be relevant to the neuronal action of melatonin and highlight the importance of the cellular context of expression of melatonin and other G protein-coupled receptors.

Neuromedin-U (NMU) has been reported to drive several physiological or behavioural responses following i.c.v. injection of the peptide into the third ventricle of rodent brains. Many of these responses are mediated through a change in corticotrophin-releasing factor (CRF) output from the paraventricular nucleus (PVN). A number of the physiological or behavioural responses are regulated in a circadian manner, e.g. feeding. We have previously reported NMU gene expression in the suprachiasmatic nucleus (SCN) and NMU-2 receptor expression in the PVN, dorsal medial hypothalamus (DMH) and other regions of the mouse brain. We therefore hypothesized that NMU would be regulated by the circadian clock and may consequently drive a circadian rhythm of CRF expression in the PVN. Here we report that NMU is regulated in a circadian manner with peak expression during the light phase of a light-dark cycle. In C3H mice held in constant darkness, the NMU rhythm free runs with a period predicted by the free running period of locomotor activity in this mouse. The NMU mRNA transcript colocalizes with cells expressing AVP in the SCN and shows a coincident rhythm of expression with AVP. On the other hand, CRF did not express a circadian rhythm of expression in a light-dark cycle, although a rhythm was evident in constant darkness with a peak of expression prior to the rise of NMU in the same conditions. This would suggest that the circadian rhythm in NMU expression in the SCN does not drive a circadian rhythm in CRF in the PVN to be translated into physiological and behavioural responses mediated by NMU.

The melatonin receptor family is a small group of receptors within the G protein-coupled receptor (GPCR) superfamily. The group comprises of three subtypes which bind melatonin and one member, the melatonin related receptor (MRR), that shares >40% sequence identity with the other melatonin receptors but does not bind melatonin. Identification of two subtypes expressed in the mouse suprachiasmatic nucleus, one of which (MT1) inhibits neuronal firing and the other (MT2) mediating the phase advancing properties of melatonin has given renewed interest to the development of subtype specific compounds for each of the mammalian melatonin receptors. Towards this goal site-directed and chimaeric receptor mutagenesis studies have been performed which have provided some insight into the structure-function relationships of the melatonin receptors. Furthermore, these studies may lead to the identification of the ligand for the orphan MRR.

In Soay rams in which the pituitary gland has been surgically separated from the hypothalamus, blood prolactin concentrations vary in response to changes in photoperiod and the administration of melatonin, as in intact animals, providing evidence that melatonin acts within the pituitary gland to control prolactin secretion. In this study the presence of potentially functional melatonin receptors in the pars tuberalis and zona tuberalis (PT/ZT) of hypothalamo-pituitary disconnected (HPD) Soay rams is confirmed using both in vitro autoradiography with the ligand 2-(125I)-iodomelatonin and in situ hybridization for the melatonin receptor. There was no effect of the HPD operation on the pattern and quantity of 2-(125I)iodomelatonin binding in the brain demonstrating that this binding is independent of hypothalamic regulation. The possibility that melatonin may control prolactin secretion directly via specific receptors on lactotrophs was investigated using dual in situ hybridization with a (35S) labelled probe for the ovine melatonin receptor (Mel 1a(b)) and a Digoxigenin labelled probe for ovine prolactin. Melatonin receptor gene expression was observed in the PT/ZT in both intact and HPD rams, however, there was no colocalization with prolactin gene expression; only in the ZT was there a close association between cells expressing the melatonin receptor and lactotrophs. The results provide strong support for the view that melatonin acts via the PT/ZT to mediate the effects of photoperiod on the seasonal cycle in prolactin secretion.

Neuromedin U (NMU) has been associated with the regulation of food-intake and energy balance in rats. The objective of this study was to identify the sites of gene expression for NMU and the NMU receptor-2 (NMU2R) in the mouse and rat hypothalamus and ascertain the effects of nutritional status on the expression of these genes. In situ hybridization studies revealed that NMU is expressed in several regions of the mouse hypothalamus associated with the regulation of energy balance. Analysis of NMU expression in the obese ob/ob mouse revealed that NMU mRNA levels were elevated in the dorsomedial hypothalamic (DMH) nucleus of obese ob/ob mice compared to lean litter-mates. In addition, NMU mRNA levels were elevated in the DMH of mice fasted for 24 h relative to ad libitum fed controls. The pattern of expression of NMU and NMU2R were more widespread in the hypothalamus of mice than rats. These data provide the first detailed anatomical analysis of the NMU and NMU2R expression in the mouse and advance our knowledge of expression in the rat. The data from the obese rodent models supports the hypothesis that NMU is involved in the regulation of nutritional status.

The expression of precursor-protein convertase (PC)1, PC2 and paired basic amino acid cleaving enzyme four mRNA was studied by in situ hybridisation in regions of the hypothalamus involved in energy regulation in relation to obese (ob) gene mutation and energy deficit. PC1 gene was differentially expressed in hypothalamic nuclei of mice from different genetic backgrounds or energetic status, whereas no differences in expression were observed for either the PC2 or paired basic amino acid cleaving enzyme four genes. In obese ob/ob mice, PC1 mRNA levels were increased in the paraventricular nucleus, decreased in the lateral hypothalamus and unchanged in the ventromedial nucleus and arcuate nucleus relative to lean controls. In response to intraperitoneal injection of murine leptin, PC1 mRNA levels in obese ob/ob mice decreased in the arcuate nucleus, increased in the lateral hypothalamus and were unchanged in both the paraventricular nucleus and ventromedial nucleus. In mice deprived of food for 24 h, PC1 mRNA levels were reduced in the ventromedial nucleus, increased in the lateral hypothalamus and unchanged in the paraventricular nucleus and arcuate nucleus relative to ad libitum-fed controls. Overall, whilst the data show effects related to leptin and energetic status, they do not support a strong and consistent link between PC1 gene expression and energy balance. This suggests that if PC1 is important to the control of energy balance then protein expression and activity, rather than gene expression may be the more critical parameters of regulation. The relationship between PC1 and candidate energy balance-related genes in the lateral hypothalamus was investigated by dual in situ hybridisation. PC1 mRNA was localised in prepro-orexin mRNA expressing neurons in the lateral hypothalamus, which suggests a functional relationship.

Sensitization of adenylate cyclase is a recently discovered phenomenon. Melatonin can induce a sensitized response of adenylate cyclase in ovine pars tuberalis cells where the receptor for melatonin is endogenously expressed. Although the mechanism is not fully understood, sensitization of adenylate cyclase may be an important part of the mechanism by which melatonin encodes daylength in the pars tuberalis of sheep and other animals. We used this as a hypothesis to search for a natural ligand that would activate adenylate cyclase in ovine pars tuberalis cells. The approach revealed pituitary adenylate cyclase activating polypeptide to be an indirect activator of adenylate cyclase in the ovine pars tuberalis. We discuss this in relation to the mechanism and importance of sensitization to the function to the pars tuberalis.

Cocaine- and amphetamine-regulated transcript (CART) mRNA is expressed in a number of hypothalamic nuclei including the arcuate nucleus (ARC). An increase in CART gene expression in the ARC of juvenile female Siberian hamsters (Phodopus sungorus) 14 days after transfer to short photoperiod at weaning and prior to major divergence of body weight trajectory in this seasonal mammal implicates CART in the induction of programmed weight change. In the current series of experiments, elevated CART mRNA in short photoperiod juvenile female animals relative to long photoperiod controls was apparent throughout the caudal-rostral extent of the ARC after 14 days, but was not observed when short photoperiod exposure was limited to 4-7 days. Elevated CART gene expression was also observed in juvenile males 14 days after transfer to short photoperiod at weaning, in adult female hamsters 14 days after transfer to short photoperiod and in adult male hamsters 21 days after transfer to short photoperiod. There were no consistent trends in expression levels of other energy balance-related genes with these relatively short duration photoperiod manipulations, suggesting that CART may be involved in short photoperiod-programmed body weight regulation.

We investigated the role of the hypothalamic melanocortin system in the regulation of food intake in the Siberian hamster, which shows a profound seasonal decrease in food intake and body weight in short photoperiod (SP). In male hamsters maintained in long photoperiod (LP), intracerebroventricular injection of melanotan II (MTII) just before lights off significantly decreased food intake relative to vehicle treatment over the 6-h observation period. Similar effects were observed in age-matched hamsters after exposure to a short daylength for 9 wk, when body weight had significantly decreased. There was no clear difference in either the magnitude of response or the dose required for half-maximal inhibition of food intake in hamsters in SP compared with those in LP. MTII significantly increased grooming in both LP and SP. Our results indicate that the melanocortin system is a potent short-term regulator of food intake. However, the lack of differential response or sensitivity to MTII treatment in the obese (LP) vs. lean (SP) states does not support the hypothesis that changes in this melanocortin pathway underlie the long-term decrease in food intake that occurs in this seasonal model.

A 37-year-old man presented with swelling under the lateral aspect of the right upper eyelid. He had sustained an alkali ocular chemical injury 10 years before resulting in persistent lateral canthal webbing. Diagnosis was made of lacrimal duct cyst (dacryops) and webbing of the lateral canthus and the findings were confirmed on computed tomography. He underwent lacrimal duct cyst marsupialisation and lateral canthoplasty with good cosmetic result, and there was no recurrence of symptoms at 2 months post-operatively. Lacrimal duct cyst is a rare clinical entity and has been postulated to result from localized inflammation or trauma to conjunctiva. To the best of our knowledge, this is the first report of dacryops following a chemical injury.

This study demonstrates the presence of two prolactin-releasing (PR) factors in media conditioned by primary pars tuberalis cells prepared from dispersed pars tuberalis tissue. One factor was identified as thyrotropin-releasing hormone (TRH) on the basis of immunoreactivity and following purification by high-performance liquid chromatography and mass spectrometry. The origin of TRH in the pars tuberalis conditioned media was investigated by measuring the expression of glutaminyl-cyclase (QC) by in situ hybridization. QC expression was not detected in pars tuberalis-specific cells, but was relatively abundant in cells in the pars distalis and hypothalamic paraventricular nucleus. These data suggest that TRH is not synthesized by the ovine pars tuberalis and more likely originated from the hypothalamic neuronal processes from the paraventricular nucleus that terminate in the median eminence. The second component of the conditioned media PR bioactivity was insensitive to the TRH-antiserum, less than 1 kDa and was not retained by the C18 reverse-phase column. The biosynthesis of the PR bioactivity by pars tuberalis cells was investigated using cycloheximide, forskolin and melatonin. Cycloheximide reduced the level of PR bioactivity produced by the pars tuberalis cells. Melatonin inhibited the increased level of PR bioactivity stimulated by forskolin. Collectively, these data demonstrate the synthesis of at least one regulator of prolactin secretion by ovine pars tuberalis-specific cells.

The basic helix-loop-helix transcription factors, neurological basic-helix-loop-helix-2 (Nhlh-2), neurogenic differentiation-1 (NeuroD-1) and single minded-1 (Sim-1) could have roles in energy balance regulation, although supporting evidence is inconclusive. This study in mice provides further evidence that Nhlh-2 and NeuroD-1 are involved in energy balance regulation. In situ hybridization was used to study the expression of the genes in relation to physiological status and genetic background within hypothalamic nuclei that are involved in energy balance regulation. These studies show reduced expression of Nhlh-2 mRNA in the arcuate (ARC) nucleus and NeuroD-1 mRNA in the paraventricular (PVN) nucleus in obese ob/ob and 24 h food-deprived mice relative to respective controls, suggesting regulation by leptin. Interestingly, Nhlh-2 mRNA expression is reduced in obese db/db mice, whereas NeuroD-1 remains unchanged, suggesting different mechanisms of regulation by leptin of these two genes. To study the role of leptin in the regulation of these genes, leptin was injected intraperitoneally in obese ob/ob mice and mRNA expression evaluated after 1 h or 4 h, or after twice-daily injection for 7 days. None of these regimes restored Nhlh-2 or NeuroD-1 to wild-type mRNA levels. These latter data suggest either that the regulation of the Nhlh-2 and NeuroD-1 genes by leptin is indirect or that the apparent leptin insensitivity of the gene expression reflects a developmental deficit that is a consequence of the phenotype of the obese ob/ob mice. The relationship between Nhlh-2 and candidate energy balance-related genes was studied by dual in situ hybridization. Nhlh-2 mRNA was coexpressed in a subpopulation (30%) of ARC neurons expressing pro-opiomelanocortin (POMC) mRNA, suggesting a potential functional relationship.

Ocular blast injuries in the military setting are particularly associated with significant maxillofacial trauma and/or brain injury. The opportunity to perform a comprehensive ophthalmic evaluation is frequently limited in the acute multiple trauma scenario. We aim to describe the relationship between the clinical effects of acute ocular and orbital blast trauma with the findings on computerised tomography (CT). This was a retrospective consecutive case series of all soldiers with facial and/or suspected ocular injuries. A total of 80 eyes that had suffered blast injuries of varying severity were studied. Assessment of orbital and ocular CT images were performed by military consultant radiologists. A comparison was made with actual clinical findings. Statistical analysis was performed using Fisher's exact test. No pathological findings were described in 37 of the 80 eyes imaged by orbital and ocular CT scans. Clinically, these eyes and orbits were all found to be intact, or had minor trauma. All foreign bodies and penetrating eye injuries were successfully diagnosed by CT. Absence of an orbital fracture did not rule out a globe injury. However, a corneal or scleral defect was less likely when an orbital fracture was absent. The eye is a delicate structure prone to injury that requires urgent repair if breached. It is difficult to assess thoroughly in the unconscious or distressed patient. In this context, CT imaging is invaluable to be able to make a relatively confident prediction of clinical findings and decide upon the necessity for acute ophthalmic surgical intervention.

Male Siberian hamsters undergo physiological weight change in changing photoperiod. Weight loss was induced by food restriction in long days to mimic short-day weight loss, or by food restriction superimposed on short-day weight loss, to test the hypothesis that the hypothalamus differentiates between weight change induced by imposed negative energy balance (inappropriate body weight) and seasonal, appropriate, body weight change, even when these are of similar magnitude. Short-day weight loss was accompanied by reduced POMC and leptin receptor (OB-Rb) mRNA in the arcuate nucleus but elevated cocaine- and amphetamine-regulated transcript. Melanocortin 3-receptor gene expression was reduced in the arcuate nucleus but elevated in the ventromedial nucleus compared with ad libitum-fed long-day controls. Weight loss in long-day restricted animals generated a gene expression profile typical of negative energy balance with low cocaine- and amphetamine-regulated transcript mRNA and elevated OB-Rb. Melanocortin 3-receptor mRNA levels were indistinguishable in short-day and long-day food-restricted hamsters. The hypothalamic correlates of food restriction in short days included up-regulated anabolic neuropeptides and increased OB-Rb mRNA. Low plasma leptin is integrated differently in short-day and long-day restricted animals, and seasonally-inappropriate body weight in either photoperiod engages the compensatory neuropeptide systems involved in the defense of body weight.

Previous studies have provided a limited examination of the expression of the orphan melatonin-related receptor in the pituitary and hypothalamus of human and sheep and retinal tissue in the sheep. The present study reports evidence of conservation of expression in regions of the hypothalamus (dorsal medial hypothalamus, lateral hypothalamus, arcuate nucleus), the epithelial layer lining the third ventricle and the paraventricular thalamic nucleus of the mouse, rat and hamster. An extensive and detailed analysis of melatonin-related receptor mRNA expression in the mouse central nervous system and peripheral tissues is presented. Mapping the distribution throughout the entire mouse brain has revealed new sites of expression in a number of brain nuclei, including preoptic areas, parabrachial nuclei and widespread distribution in the olfactory bulb. Reverse transcriptase-polymerase chain reaction was performed with RNA isolated from peripheral tissues revealing expression of the melatonin-related receptor mRNA in the mouse kidney, adrenal gland, intestine, stomach, heart, lung, skin, testis and ovary. These results suggest a conserved function in neuroendocrine regulation and a potential role in coordinating physiological responses in the central nervous system and peripheral tissues.

The cocaine-amphetamine regulated transcript (CART) encodes for a protein which has an important role in the regulation of appetite and body weight. To date, no details of the molecular events and signal transduction pathways which regulate this gene are available. We report the identification of CART gene expression in the GH3 pituitary cell line. We have used activators of the cAMP or protein kinase C (PKC) signal transduction pathways to show that, in GH3 cells, CART is transcriptionally up-regulated by activators of the cAMP signal transduction pathway. We also identify CART gene expression in ovine pars tuberalis (PT) tissue and primary cell cultures. In PT cells in contrast to GH3 cells, CART gene expression is upregulated by activators of the PKC signal transduction pathway. Cultured cells have provided a valuable resource for the detailed analysis of specific regulatory mechanisms underlying transcriptional or translational regulation of genes, signal transduction events and many other cellular processes. GH3 and PT cells may therefore provide a resource for the further detailed molecular analysis of the events regulating CART gene expression and processing.

Syrian hamsters exhibit a marked seasonal variation in prolactin secretion. The aim of this study was to analyse the nature of the photoperiodic regulation of prolactin gene expression, and to define the role of melatonin and the pars tuberalis of the anterior pituitary in this process. Pituitary prolactin gene expression, restricted to the pars distalis, was increased in hamsters maintained in long daylengths (16 h : 8 h, light : dark) compared to hamsters exposed to short daylengths (8 h : 16 h, light : dark) for 8–12 weeks. Analysis of single cells by in situ hybridization showed that photoperiod had no effect on the percentage of pars distalis cells expressing prolactin mRNA, but shifted the frequency distribution of prolactin mRNA expression per cell, such that in long photoperiods a greater proportion of cells were recruited to a higher expressing population. In vitro coculture of hamster pars tuberalis fragments increased prolactin promoter-driven luciferase activity in stably transfected GH3 cells in a dose- and duration-dependent manner. Conditioned medium from hamster and ovine pars tuberalis also activated the prolactin promoter. Furthermore, basal and forskolin-stimulated conditioned medium from hamster pars tuberalis increased prolactin mRNA expression in primary cultures of pars distalis cells. Melatonin attenuated the activity of pars tuberalis-conditioned medium but had no direct effect on either prolactin mRNA expression or secretion in pars distalis cell cultures. Finally, pars tuberalis fragments from long photoperiod hamsters stimulated prolactin gene promoter activity to a greater extent than those from short photoperiod hamsters. In conclusion, this study provides the first evidence in a seasonal mammal that the synthesis of prolactin depends on photoperiodic modulation of a pars tuberalis-derived factor. Our data support further the hypothesis that seasonal modulation of prolactin gene expression depends upon a melatonin-dependent paracrine action of the pars tuberalis on pars distalis lactotrophic cells.

In seasonal mammals, both the growth and reproductive axes are regulated by photoperiod. Female Siberian hamsters were kept, for up to 12 weeks, in long-day (LD) or short-day (SD) photoperiod, from weaning at 3 weeks of age (Exp 1). LD hamsters had characteristically faster growth and higher asymptotic body weight, adiposity, and leptin gene expression in adipose tissue. Only LD females attained puberty. Gene expression in the hypothalamic arcuate nucleus for leptin receptor (OB-Rb), POMC, and melanocortin 3-receptor (MC3-R) was higher in LD but did not change from weaning levels in SD. In contrast, gene expression in the arcuate nucleus for cocaine and amphetamine-regulated transcript (CART) was higher in SD than LD, a difference that was apparent at 2 weeks post weaning. Transfer of SD females to LD at 15 weeks post weaning (Exp 2) increased body weight, leptin signal, and gene expression for POMC but failed to induce normal puberty onset or to increase gene expression for OB-Rb and MC3-R. Therefore, ph...

Leptin burst onto the scene in 1994 and transformed research into obesity and energy balance. Immediately, hopes were raised that this peptide, secreted by adipose tissue, might be causally related to human obesity, and that the regulatory mechanisms underlying its action might be manipulated for therapeutic benefit. In the last 6 years, the concentrated effort of the research community, both academic and commercial, has made enormous strides towards understanding the full range of physiological effects of leptin. In the mouse, leptin deficiency (ob/ob) induces hyperphagia, massive obesity and diabetes. Administration of leptin protein reverses this phenotype and results in rapid bodyweight loss, mainly in the form of fat. Leptin also modestly reduces food intake and bodyweight in normal lean rodents on systemic administration. Leptin is much more potent when administered directly into the cerebroventricular system of the brain. This observation, and the identification of leptin receptor expression in hypothalamic nuclei with an established regulatory function in energy homeostasis, confirms the CNS as a primary site of action of leptin. A number of leptin-sensitive, receptor-expressing hypothalamic neurons of different neurochemical phenotype have been identified, and leptin receptor mRNA and protein are widespread elsewhere in the mammalian brain. Leptin provides two forms of feedback to regulatory systems in the brain, a dynamic reflection of diurnal feeding pattern (where it may interact with satiety signals), and a basal level of secretion encoding body fat storage (a plausible lipostat). The absence of leptin is a potent signal of starvation, and in this state reduced leptin feedback regulates the activity of compensatory neuropeptide systems in the hypothalamus to drive the voluntary hyperphagia displayed by rodents when released back to ad libitum feeding. Despite the initial excitement over the therapeutic potential of leptin, these expectations have now begun to wane. Assessments of the value of leptin, and of potential therapies based on its activities, are now more realistic and may involve targeting of specific subpopulations of individuals, or particular weight loss strategies. This review focuses deliberately upon the possible exploitation strategies that are still patent.

Previous studies demonstrated that the clock gene Per1 and the transcription factor ICER are expressed rhythmically in the suprachiasmatic nucleus (SCN) and in the pars tuberalis (PT). In the Syrian hamster the duration of photoperiod affects the amplitude of gene expression in the PT, and melatonin administered before lights-on suppressed the peak of Per1/ICER expression; these effects were not seen in the SCN. It was speculated that the inefficacy of melatonin was due to the low density of melatonin receptors in the SCN of this species. The aim of the present study was to determine whether this phenomenon also occurs in the Siberian hamster, which expresses a higher density of melatonin receptors in the SCN. Male Siberian hamsters were housed in long days (16 h light : 8 h dark) or short days (8 h light : 16 h dark) and expression of Per1 and ICER mRNA was studied by in situ hybridization. The expression of Per1 and ICER mRNA in the PT peaked 3 h following lights-on (ZT3) under both photoperiods. The amplitudes of these peaks were greatly attenuated under short photoperiod. In the SCN, the duration of Per1 gene expression was proportional to the length of the light phase, but only a modest amplitude effect was observed. Injections of melatonin (25 microg) 1 h before lights-on significantly reduced the expression of both genes in the PT at ZT3, but had no effect in the SCN. These data demonstrate that photoperiod-dependent amplitude modulation of Per1 and ICER gene expression in the PT is conserved across species, and reinforce the argument that this phenomenon is driven by melatonin.

In the ovine pituitary the in vivo expression of the bFGF gene was studied using in situ hybridisation and Northern analysis. Expression of bFGF mRNA was highest in the pars tuberalis (PT) and zona tuberalis (ZT) and this expression was higher in the pituitaries of animals acclimited to long days than in those from short day housed animals. Expression in the pars intermedia (PI) was also detectable but the pars distalis (PD) showed negligible expression compared with the PT. Regulation of bFGF gene was investigated in primary cultured PT cells. Both forskolin and PMA increased bFGF mRNA expression significantly and melatonin was able to inhibit these effects partially, however there was no independent effect of melatonin on bFGF mRNA levels. Despite the inducibility of the bFGF gene, bFGF protein levels in PT culture media were insensitive to the same stimuli and detectable bFGF protein declined with time. Exogenous bFGF had no effect on c-fos mRNA levels and did not increase prolactin secretion from ovine lactotrophs. In contrast c-fos mRNA was induced in GH3 cells by bFGF. These data suggest that although basic fibroblast growth factor (bFGF) mRNA expression in the ovine PT is photoperiodically-sensitive, it is not directly involved in the seasonal regulation of lactotrophic activity.

The mammalian Per1 gene is expressed in the suprachiasmatic nucleus of the hypothalamus, where it is thought to play a critical role in the generation of circadian rhythms. Per1 mRNA also is expressed in other tissues. Its expression in the pars tuberalis (PT) of the pituitary is noteworthy because, like the suprachiasmatic nucleus, it is a known site of action of melatonin. The duration of the nocturnal melatonin signal encodes photoperiodic time, and many species use this to coordinate physiological adaptations with the yearly climatic cycle. This study reveals how the duration of photoperiodic time, conveyed through melatonin, is decoded as amplitude of Per1 and ICER (inducible cAMP early repressor) gene expression in the PT. Syrian hamsters display a robust and transient peak of Per1 and ICER gene expression 3 h after lights-on (Zeitgeber time 3) in the PT, under both long (16 h light/8 h dark) and short (8 h light/16 h dark) photoperiods. However, the amplitude of these peaks is greatly attenuated under a short photoperiod. The data show how amplitude of these genes may be important to the long-term measurement of photoperiodic time intervals.

Mammalian Per1 (or RIGUI) is a recently described putative clock gene that is expressed in the suprachiasmatic nucleus. It is also expressed in the pars tuberalis (PT) of the pituitary, where melatonin appears to drive its expression. This study examines the regulation of Per1 expression. In ovine PT cells, oPer1 is an early response gene transiently expressed after stimulation with forskolin, but melatonin has no independent effect on its expression. In sheep, PT tissue photoperiodic background influences the magnitude or timing of expression of oPer1 2 h after lights-on. These data demonstrate that oPer1 mRNA is elevated in the PT following the decline in night-time melatonin, and that the amplitude or timing of this elevation is dependent upon the duration of the nocturnal melatonin signal.