Posttransplant oxygen inhalation improves the outcome of subcutaneous islet transplantation: A promising clinical alternative to the conventional intrahepatic site.

Subcutaneous tissue is a promising site for islet transplantation, due to its large area and accessibility, which allows minimally invasive procedures for transplantation, graft monitoring, and removal of malignancies as needed. However, relative to the conventional intrahepatic transplantation site, the subcutaneous site requires a large number of islets to achieve engraftment success and diabetes reversal, due to hypoxia and low vascularity. We report that the efficiency of subcutaneous islet transplantation in a Lewis rat model is significantly improved by treating recipients with inhaled 50% oxygen, in conjunction with prevascularization of the graft bed by agarose-basic fibroblast growth factor. Administration of 50% oxygen increased oxygen tension in the subcutaneous site to 140 mm Hg, compared to 45 mm Hg under ambient air. In vitro, islets cultured under 140 mm Hg oxygen showed reduced central necrosis and increased insulin release, compared to those maintained in 45 mm Hg oxygen. Six hundred syngeneic islets subcutaneously transplanted into the prevascularized graft bed reversed diabetes when combined with postoperative 50% oxygen inhalation for 3 days, a number comparable to that required for intrahepatic transplantation; in the absence of oxygen treatment, diabetes was not reversed. Thus, we show oxygen inhalation to be a simple and promising approach to successfully establishing subcutaneous islet transplantation.

Pharmacological strategies for protection of extrahepatic islet transplantation.

The safety and effectiveness of islet transplantation has been proven through world-wide trials. However, acute and chronic islet loss has hindered the ultimate objective of becoming a widely used treatment option for type 1 diabetes. A large islet loss is attributed, in part, to the liver being a less-than-optimal site for transplantation. Over half of the transplanted islets are destroyed shortly after transplantation due to direct exposure to blood and non-specific inflammation. Successfully engrafted islets are continuously exposed to the liver micro-environment, a unique immune system, low oxygen tension, toxins and high glucose, which is toxic to islets, leading to premature islet dysfunction/death. Investigations have continued to search for alternate sites to transplant islets that provide a better environment for prolonged function and survival. This article gathers courses and conditions that lead to islet loss, from organ procurement through islet transplantation, with special emphasis on hypoxia, oxidative stress, and antigen non-specific inflammation, and reviews strategies using pharmacological agents that have shown effectiveness in protecting islets, including a new treatment approach utilizing siRNA. Pharmacological agents that support islet survival and promote ß-cell proliferation are also included. Treatment of donor pancreata and/or islets with these agents should increase the effectiveness of islets transplanted into extrahepatic sites. Furthermore, the development of methods designed to release these agents over an extended period, will further increase their efficacy. This requires the combined efforts of both islet transplant biologists and bioengineers.

Implementation of a personalized workplace smoking cessation programme.

BACKGROUND: Providing smoking cessation programmes through workplaces is an effective method of assisting employees to quit smoking; however, few employers provide such services, and achieving long-term success remains challenging. AIMS: To evaluate the effectiveness of a workplace-based tailored smoking cessation programme that combined telephone-based counselling with group behaviour therapy sessions in helping employees to quit. METHODS: A smoking cessation programme was offered to employees of a large corporation that is respons ible for the passenger rail network in New South Wales (NSW), Australia. Two hundred and thirty participants enrolled in the programme, which offered telephone-based coaching and group sessions designed around cognitive behavioural therapy principles. One hundred and eight participants (47%) completed the 6 month follow-up assessment. RESULTS: Of the estimated 2850 smokers in the organization, 8% (230) registered for the smoking cessation programme, with 77% (176) participating in telephone-based coaching and/or group sessions. Intention-to-treat analysis indicated 22% of participants achieved 7 day point prevalence abstinence and 10% achieved 3 month prolonged abstinence at the 6 month follow-up. Over 75% of those still smoking at follow-up reported intentions to quit in the next 6 months. Psychological distress was also significantly lower at 6 month follow-up. Participants reported high levels of satisfaction with the programme. CONCLUSIONS: The smoking cessation programme successfully assisted employees to quit smoking. Unique aspects of the programme such as continuity of care were valued by participants and may have contributed to the programme's success.

Promoter methylation of Wnt5a is associated with microsatellite instability and BRAF V600E mutation in two large populations of colorectal cancer patients.

BACKGROUND: In colorectal cancer (CRC), tumour microsatellite instability (MSI) status and CpG island methylator phenotype (CIMP) status are indicators of patient outcome, but the molecular events that give rise to these outcomes remain largely unknown. Wnt5a is a critical regulator of non-canonical Wnt activity and promoter hypermethylation of this gene has emerging prognostic roles in CRC; however the frequency and prognostic significance of this epigenetic event have not been explored in the context of colorectal tumour subtype. Consequently, we investigated the frequency and prognostic significance of Wnt5a methylation in a large cohort of MSI-stratified CRCs. METHODS: Methylation was quantified in a large cohort of 1232 colorectal carcinomas from two clinically distinct populations from Canada. Associations were examined between methylation status and clinicopathlogical features, including tumour MSI status, BRAF V600E mutation, and patient survival. RESULTS: In Ontario, Wnt5a methylation was strongly associated with MSI tumours after adjustment for age, sex, and tumour location (odds ratio (OR)=4.2, 95% confidence interval (CI)=2.4-7.4, P<10(-6)) and with BRAF V600E mutation, a marker of CIMP (OR=12.3, 95% CI=6.9-21.7, P<10(-17)), but was not associated with patient survival. Concordant results were obtained in Newfoundland. CONCLUSION: Methylation of Wnt5a is associated with distinct tumour subtypes, strengthening the evidence of an epigenetic-mediated Wnt bias in CRC.

mRNA of the pro-apoptotic gene BBC3 serves as a molecular marker for TNF-α-induced islet damage in humans.

AIMS/HYPOTHESIS: TNF-α plays important roles in the pathogenesis of type 1 and type 2 diabetes mellitus. In light of this, we examined the involvement of a pro-apoptotic gene, BBC3 (also known as PUMA), in TNF-α-mediated beta cell dysfunction and destruction in human islets. METHODS: Human islets were exposed in vitro to TNF-α alone or in combination with IFN-γ. Gene expression was assessed by RT-PCR using a set of single islets. Protein abundance and cellular localisation of BBC3 were assessed by immunoblot and immunohistochemistry. A marginal number of islets were transplanted into diabetic NODscid mice to correlate in vivo islet function with BBC3 expression. RESULTS: BBC3 and IL8 mRNA were upregulated in TNF-α-stimulated islets in a dose-dependent manner and enhanced through addition of IFN-γ, but not upregulated by IFN-γ alone. Immunohistochemistry revealed that TNF-α in combination with IFN-γ upregulated basal BBC3 abundance in the cytoplasm of beta cells along with the perinuclear clustering of mitochondria partially co-localised with BBC3. TNF-α alone did not induce beta cell death, but did abrogate preproinsulin precursor mRNA synthesis in response to high glucose stimulation, which was inversely associated with upregulation of BBC3 mRNA expression by TNF-α. Higher BBC3 mRNA expression in islets correlated with decreased graft function in vivo. CONCLUSIONS/INTERPRETATION: These results suggest that BBC3 mRNA can serve as a molecular marker to detect early TNF-α-induced beta cell stress and may help identify islet-protective compounds for the treatment of diabetes.

Estradiol-17beta-responsive A1 and A2 noradrenergic cells of the brain stem project to the bed nucleus of the stria terminalis in the ewe brain: a possible route for regulation of gonadotropin releasing hormone cells.

We have studied brain stem cells in the ewe brain that project to the bed nucleus of the stria terminalis (BNST) and determined if these cells are activated by estradiol-17beta. This would predicate an indirect role in the estradiol-17beta regulation of gonadotropin releasing hormone (GnRH) cells, since these receive input from the BNST. Ovariectomized ewes received 50 mug estradiol-17beta benzoate (i.m.) 1 h prior to brain collection, so that activated cells could be identified by Fos immunohistochemistry. Retrograde tracer (FluoroGold; FG), was injected into the three divisions of the BNST and labeled cells were mapped to the A1 and A2 regions and the parabrachial nucleus (PBN) of the brain stem. With FG injection into the dorsal and lateral BNST, all FG-containing cells in the caudal A1 and 45% of those in A2 stained for dopamine-beta-hydroxylase (DBH), indicating noradrenergic type. No FG-labelled cells in the PBN were DBH-positive. In A1 and A2 respectively, 42% and 46% of FG-labelled cells were Fos-positive, with no double-labeling in cells of the PBN. In ewes receiving FG injections into the ventral BNST, estrogen receptor (ER)alpha-immunoreactive nuclei were found in 82% of A1-FG labeled and 38% of A2-FG labeled cells. No FG-labelled cells of the PBN were ERalpha-positive. Anterograde tracing from A1 with microruby injection identified projections to the PBN, BNST and preoptic area (POA). Thus, A1 and A2 noradrenergic neurons project to the BNST in the ewe brain, express ERalpha and are activated by estradiol-17beta. These noradrenergic, estrogen-responsive cells may provide indirect input to GnRH cells, via the BNST.

Mesenchymal stem cells facilitate the induction of mixed hematopoietic chimerism and islet allograft tolerance without GVHD in the rat.

Induction of hematopoietic chimerism and subsequent donor-specific immune tolerance via bone marrow transplantation is an ideal approach for islet transplantation to treat type-1 diabetes. We examined the potential of mesenchymal stem cells (MSCs) in the induction of chimerism and islet allograft tolerance without the incidence of graft-versus-host disease (GVHD). Streptozotocin-diabetic rats received a conditioning regimen consisting of antilymphocyte serum and 5 Gy total body irradiation, followed by an intraportal co-infusion of allogeneic MSCs, bone marrow cells (BMCs) and islets. Although all the recipients rejected the islets initially, half of them developed stable mixed chimerism and donor-specific immune tolerance, shown by the engraftment of donor skin and second-set islet transplants and acute rejection of a third-party skin. The engraftment of the primary islet allografts with stable chimerism was achieved by the addition of a 2-week peritransplant administration of 15-deoxyspergualin (DSG). Without MSCs, none of the recipients treated with DSG developed chimerism or reversal of diabetes. GVHD was not observed in any of the recipients infused with MSCs (0/15), whereas it occurred in 4/11 recipients without MSCs. These results indicate a potential use of MSCs for induction of hematopoietic chimerism and subsequent immune tolerance in clinical islet transplantation.

Multipotent progenitor cells isolated from adult human pancreatic tissue.

The supply of islet cells is a limiting factor for the widespread application of islet transplantation of type-1 diabetes. Islets constitute 1% to 2% of pancreatic tissue, leaving approximately 98% as discard after islet isolation and purification. In this report we present our data on the isolation of multipotent progenitor cells from discarded adult human pancreatic tissue. The collected cells from discarded nonislet fractions, after enzymatic digestion and gradient purification of islets, were dissociated for suspension culture in a serum-free medium. The cell clusters grown to a size of 100 to 150 mum contained cells staining for stage-specific embryonic antigens, but not insulin or C-peptide. To direct cell differentiation toward islets, clusters were recultured in a pancreatic differentiation medium. Insulin and C-peptide-positive cells by immunocytochemistry appeared within a week, reaching over 10% of the cell population. Glucagon and somatostatin-positive cells were also detected. The cell clusters were found to secrete insulin in response to glucose stimulation. Cells from the same clusters also had the capacity for differentiation into neural cells, as documented by staining for neural and glial cell markers when cultured as monolayers in media containing neurotrophic factors. These data suggest that multipotent pancreatic progenitor cells exist within the human pancreatic tissue that is typically discarded during islet isolation procedures. These adult progenitor cells can be successfully differentiated into insulin-producing cells, and thus they have the potential for treatment of type-1 diabetes mellitus.

Effect of sleep deprivation on the performance of simulated laparoscopic surgical skill.

BACKGROUND: Resident work hours may impact patient care. We hypothesized that "call-associated" acute sleep deprivation has no effect on technical dexterity as measured on a minimally invasive surgery trainer, virtual reality (MIST VR) surgical simulator. METHODS: Thirty-five surgical residents were prospectively evaluated pre-call (rested), on-call (rested), and post-call (acutely sleep deprived). Participants completed questionnaires regarding sleep hours and level of fatigue. Technical skill was assessed using the MIST VR. Speed, errors, and economy of motion were automatically recorded by the MIST VR computer simulator. Data were analyzed by paired Student t test and analysis of variance. RESULTS: Estimated hours of sleep and subjective indicators of fatigue were different between rested and sleep-deprived residents. The number of errors and time to complete all tasks increased at the post-call assessment. CONCLUSIONS: Resident work schedules lead to sleep deprivation and fatigue. Call-associated sleep deprivation and fatigue are associated with increased technical errors in the performance of simulated laparoscopic surgical skills.

Cells of the arcuate nucleus and ventromedial nucleus of the ovariectomized ewe that respond to oestrogen: a study using Fos immunohistochemistry.

Oestrogen produces a positive feedback effect on the secretion of gonadotropin releasing hormone (GnRH) and luteinizing hormone (LH) when implanted into the ventromedial/arcuate nucleus of the ovariectomized (OVX) ewe. This has led to the belief that it is in this area of the hypothalamus that oestrogen causes the preovulatory surge in GnRH/LH. To date, however, the cell types that are integral to this response have not been identified. The present study aimed to examine cellular responsiveness to oestrogen in this region of the brain using Fos immunohistochemistry and further aimed to determine the cell type that shows an acute response to oestrogen. OVX ewes (n = 4-6 per group) were given i.m. injections of oestradiol benzoate or oil (vehicle) and were killed 1-6 h later. Brains were perfused for immunohistochemistry. The number of cells in the arcuate nucleus which were immunopositive for Fos was greater (two- to fourfold) in the oestradiol benzoate-treated OVX ewes (n = 5) 1 h after injection. The number of Fos-positive cells in the ventromedial hypothalamic nucleus was 10-fold greater in the oestradiol benzoate-treated ewes 1 h after injection. Because there were high levels of Fos-immunoreactive cells in oil-treated ewes, we repeated the experiment with i.v. injection of 50 microg oestrogen or vehicle (n = 5). With this latter procedure, we found that oestrogen injection caused a significant increase in the number of Fos immunoreactive cells in the arcuate nucleus within 1 h, but there was no response in the ventromedial hypothalamus. To further characterize the types of cells that might respond to oestrogen, we double-labelled cells for Fos and either adrenocorticotropin hormone, neuropeptide Y or tyrosine hydroxylase (a marker for dopaminergic cells). These cell types could account for less than 30% of the total number of cells that were Fos-positive and oestrogen treatment did not cause an increase in the Fos labelling of any of these types of cell. These data show that oestrogen activates cells of the arcuate/ventromedial hypothalamus within 1 h of injection and that this response could relate to the feedback effects of this gonadal hormone. The majority of cells that produce Fos following oestrogen injection are of unknown phenotype. The data further suggest that induction of cells of the ventromedial hypothalamic nucleus require more prolonged oestrogen stimulus than cells of the arcuate nucleus.

Projections from the arcuate/ventromedial region of the hypothalamus to the preoptic area and bed nucleus of stria terminalis in the brain of the ewe; lack of direct input to gonadotropin-releasing hormone neurons.

This study aimed to determine whether cells in the region of the arcuate and ventromedial hypothalamic nuclei (ARC/VMH) project to the gonadotropin-releasing hormone (GnRH) cells in the preoptic area (POA) and diagonal band of Broca (dbB) of the female sheep brain. An anterograde tracer, biotinylated dextran amine (BDA), was injected (70 nl) into the ARC/VMH (n=7) and the brains were perfused 3 weeks later. BDA terminals were mainly found in the dbB, POA and bed nucleus of stria terminalis (BNST). In order to determine the extent of input to GnRH neurons, we performed immunocytochemistry on the same sections with a GnRH antibody and examined close association of GnRH-immunoreactive (GnRH-IR) neurons (cell bodies and proximal dendrites) with BDA terminals. Of 223 GnRH-IR neurons that were examined, only three (1.3%) had BDA terminals in close proximity. Neither was close proximity observed between BDA terminals and GnRH-IR fibres. Injection of BDA into the BNST (n=6) showed terminals in POA, but only one of 273 GnRH-IR cells examined had BDA terminals in close proximity and no GnRH-IR fibres had BDA terminals in close proximity. Our results suggest that (1) although there are projections from the VMH/ARC to the dbB, POA and BNST, an interneuron or chain of interneurons is required for input to the GnRH neurones; (2) any input to GnRH neurons from the BNST involves at least one interneuron. The identity of these interneurons remains to be determined. Thus, input to the GnRH neurons from the estrogen receptor-rich area of ARC/VMH and from the BNST is not direct.

Changes in preoptic and hypothalamic levels of progesterone receptor mRNA across the oestrous cycle of the ewe.

We measured the levels of progesterone receptor (PR) mRNA in the hypothalamus and preoptic area (POA) of the ewe across the oestrous cycle. Perfusion-fixed hypothalamic tissue was collected from sheep killed during the luteal and follicular phases and during behavioural oestrus. Blood samples taken at the time of tissue collection verified that the oestrous ewes were undergoing a preovulatory luteinizing hormone (LH) surge. Matched sections were taken from the POA, periventricular nucleus (PeVN), ventromedial nucleus (VMN) and arcuate nucleus of each animal. In situ hybridization was performed using a sheep specific, (35)S-labelled riboprobe for PR and semiquantitative image analysis was conducted on emulsion-dipped slides. The number of silver grains per cell was greater in the VMN and arcuate nucleus of oestrous ewes than in luteal and follicular phase ewes; there was no cyclic variation in the level of PR mRNA expression in the cells of the POA and PeVN. The number of labelled cells per mm2 in the VMN was higher in the oestrous ewes than in luteal phase and follicular phase ewes. The number of labelled cells in the PeVN was also higher in the oestrous ewes than in follicular phase ewes, but there was no cyclic variation in the POA and arcuate nucleus. In the ewe, the onset of behavioural oestrus corresponds to the onset of the preovulatory LH surge and increased PR mRNA expression at this time is likely to be due to the high concentrations of circulating oestrogen that precede this period.

Noradrenergic projections from the A1 field to the preoptic area in the brain of the ewe and Fos responses to oestrogen in the A1 cells.

Previous studies have shown that there is a population of noradrenergic cells in the caudal A1 field of the brainstem of the ewe that contain oestrogen receptors and project to the preoptic area, where gonadotrophin releasing hormone (GnRH) neurones are located. There are some discrepancies in the literature regarding the extent of this projection and the location of the cells in the A1 region. The issue has been a focus of attention because the positive feedback response to oestrogen that causes the ovulatory GnRH/luteinizing hormone surge may originate from this brainstem region. The aim of the present study was to determine the extent of the projections to the preoptic area and to determine whether the caudal A1 cells are activated by oestrogen. Eleven ovariectomized ewes received an injection of the retrograde tracer FluoroGold into the preoptic hypothalamus and four of these also received an i.m. injection of oestrogen 2 h before tissue collection. A further three sheep received i.m. oil injections to act as controls for those receiving oestrogen. Dopamine-beta-hydroxylase (DBH)-positive, retrogradely labelled cells were found within the A1 field in sheep that received preoptic FluoroGold injections. Cells in the vicinity of the A2 and A6 fields, that were retrogradely labelled with FluoroGold, were not DBH-positive. Thus, cells in the A1 field provide a direct noradrenergic projection to the preoptic area and may be involved in the control of the secretion of GnRH in this species. Cells that project to the preoptic hypothalamus from more rostrally located areas of the brainstem are not noradrenergic. In the animals that received oestrogen, double-labelling immunohistochemistry was performed throughout the A1 field for FluoroGold, DBH and Fos. DBH cells of the A1 field expressed Fos only in the oestrogen-treated animals and not in the oil-treated animals. There was a decline in the number of DBH cells that were retrogradely labelled from the caudal region of A1 towards obex. There was a similar gradient in the number of cells that were double-labelled for Fos and FluoroGold. We conclude that there is a population of noradrenergic cells in the caudal A1 field that project to the preoptic area; this is a larger group of cells than previously reported. Oestrogen elicits an acute Fos response in these cells, which may be involved in the time-delayed positive feedback response on GnRH cells. The caudal-to-rostral gradient in the labelling with FluoroGold and Fos in DBH-positive cells is similar to that seen previously for oestrogen receptor in DBH-positive cells in the A1 field.

The distribution of cells containing estrogen receptor-alpha (ERalpha) and ERbeta messenger ribonucleic acid in the preoptic area and hypothalamus of the sheep: comparison of males and females.

We have used in situ hybridization to compare the distributions of estrogen receptor alpha (ERalpha) and ERbeta messenger RNA (mRNA)-containing cells in the preoptic area and hypothalamus of ewes and rams. Perfusion-fixed brain tissue was collected from luteal phase ewes and intact rams (n = 4) during the breeding season. Matched pairs of sections were hybridized with sheep-specific, 35S-labeled riboprobes, and semiquantitative image analysis was performed on emulsion-dipped slides. A number of sex differences were observed, with females having a greater density of labeled cells than males (P < 0.001) and a greater number of silver grains per cell (P < 0.01) in the ventromedial nucleus for both ER subtypes. In addition, in the retrochiasmatic area, males had a greater (P < 0.05) cell density for ERalpha mRNA-containing cells than females, whereas in the paraventricular nucleus, females had a greater density (P < 0.05) of ERalpha mRNA-containing cells than males. There was a trend (P = 0.068) in the arcuate nucleus for males to have a greater number of silver grains per cell labeled for ERalpha mRNA. In both sexes, there was considerable overlap in the distributions of ERalpha and ERbeta mRNA-containing cells, but the density of labeled cells within each nucleus differed in a number of instances. Nuclei that contained a higher (P < 0.001) density of ERalpha than ERbeta mRNA-containing cells included the preoptic area, bed nucleus of the stria terminalis, and ventromedial nucleus, whereas the subfornical organ (P < 0.001), paraventricular nucleus (males only, P < 0.05), and retrochiasmatic nucleus (females only, P < 0.05) had a greater density of ERalpha than ERbeta mRNA-containing cells. The anterior hypothalamic area and supraoptic nucleus had similar densities of cells containing both ER subtypes. The lateral septum and arcuate nucleus contained only ERalpha, whereas only ERbeta mRNA-containing cells were seen in the zona incerta. The sex differences in the populations of ER mRNA-containing cells in the ventromedial and arcuate nuclei may explain in part the sex differences in the neuroendocrine and behavioral responses to localized estrogen treatment in these nuclei. Within sexes, the differences between the distributions of ERalpha and ERbeta mRNA-containing cells may reflect differential regulation of the actions of estrogen in the sheep hypothalamus. Low levels of ERbeta mRNA in the preoptic area and ventromedial and arcuate nuclei, regions known to be important for the regulation of reproduction, suggest that ERbeta may not be involved in these functions.

The distribution of progesterone receptor immunoreactivity and mRNA in the preoptic area and hypothalamus of the ewe: upregulation of progesterone receptor mRNA in the mediobasal hypothalamus by oestrogen.

The distribution of progesterone receptors (PR) was mapped in the hypothalamus of the ewe using immunocytochemistry. These results were confirmed using in situ hybridization with a sheep-specific 35S-labelled riboprobe. In addition, the effect of oestrogen on the level of PR mRNA in the hypothalamus was examined in ovariectomized (OVX) ewes following treatment with an oestrogen implant or without treatment. PR immunoreactive (-ir) cells were readily detected in OVX animals. Labelled cells were observed in four main hypothalamic regions: the preoptic area (POA), including the organum vasculosum of the lamina terminalis, periventricular nucleus (PeVN), ventromedial nucleus (VMN) and the arcuate nucleus (ARC) (including the region ventral to the mamillary recess). In addition, lightly stained PR-ir cells were observed in the supraoptic nucleus and a few PR-ir cells were also found in the diagonal band of Broca. No PR-ir cells were found in the brainstem. PR mRNA-containing cells were found in the same hypothalamic regions as the PR-ir cells. Image analysis of emulsion-dipped slides following in situ hybridization indicated that oestrogen treatment increased (P<0.01) the mean number of silver grains/cell and the density of labelled cells in the VMN and ARC but had no effect on the level of PR mRNA expression in the POA or PeN. The distribution of PR-containing cells in the hypothalamus is similar to that described in other species and all cells were located in nuclei that contain large populations of oestrogen receptor-containing cells. These include regions implicated in the regulation of reproductive neuroendocrine function, and reproductive behaviour. Oestrogen and progesterone synergize to inhibit GnRH secretion and the present results suggest that these functions may involve cells of the VMN and ARC, with oestrogen acting to upregulate PR.

Gonadal steroid receptors in the regulation of GnRH secretion in farm animals.

The sites of action and mechanisms by which gonadal steroids regulate gonadotrophin-releasing hormone (GnRH) in domestic animals remain largely unknown. This review summarises information gained from sheep regarding the distribution of the gonadal steroid receptors in the brain, the neurochemical identity and the projections of these steroid receptor-containing neurones. The cells in the hypothalamus that contain each of the gonadal steroid receptors (oestrogen receptor alpha (ERalpha), oestrogen receptor beta (ERbeta), progesterone receptor (PR) and androgen receptor (AR)) show a remarkably similar distribution, although the PR and AR-containing cells are less widespread than oestrogen receptors (ERs). There is considerable overlap in the distribution of ERalpha- and ERbeta-containing cells but also some unique sites for each subtype. This suggests differential regulation of the actions of oestrogen. There appears to be little sexual dimorphism in the distribution of the gonadal steroid receptors in the hypothalamus, with the notable exception of the ventromedial nucleus where females appear to have greater numbers of both ERalpha- and ERbeta-containing cells. Neuronal tracing studies have identified projections of some of the ERalpha-containing cells to sites that may allow interaction with the GnRH system. The receptor mapping, neuronal tracing and microimplantation studies suggest that the ventromedial nucleus is likely to be a key hypothalamic nucleus in the steroid regulation of GnRH secretion in sheep.

Oestrogen receptors in the brainstem of the female sheep: relationship to noradrenergic cells and cells projecting to the medial preoptic area.

Oestrogen regulates the secretion of gonadotropin releasing hormone (GnRH) and this could be mediated by noradrenergic systems originating in the brainstem. Whilst it is known that noradrenergic cells possess oestrogen receptors (ER), it is not known whether ER-immunoreactive (-ir) cells in the brainstem project to the regions of the hypothalamus in which GnRH neurons are found. We have used dual-label immunocytochemistry to determine the extent to which ER-alpha is found in noradrenergic cells in the brainstem of the ovariectomized (OVX) ewe. Noradrenergic/adrenergic cells were identified by immunostaining for dopamine beta-hydroxylase (DBH). Cells that stained for both DBH and ER were found in both the A1 and A2 cell groups, with the highest levels found in the most caudal regions. In the A1 group, at the most caudal extent, 73% of ER-ir cells were DBH-positive and 19% of DBH-ir cells were ER-positive. The degree of co-localization decreased in a linear manner towards the rostral brainstem. In the caudal half of A2, 9-14% of ER-ir cells were DBH-positive and 20-25% of DBH cells were ER-positive. Less than 2% of DBH-ir cells in the A5 group were dual-labelled and none of the cells in the A6 and A7 groups were ER-positive. The retrograde tracer FluoroGold was injected into the preoptic area of nine OVX ewes and labelled cells were examined in the brainstem to determine the extent of co-localization of ER. Only injections in the rostroventral part of the medial preoptic area near to the organum vasculosum of the lamina terminalis resulted in the labelling of cells in the brainstem. One ewe with very strong labelling of the brainstem was selected for detailed mapping. In the ventrolateral medulla, half the ER-ir cells in the most caudal regions were retrogradely labelled. Almost all the ER-ir cells in the mid-region of the ventrolateral medulla were retrogradely labelled but no co-localization of retrograde tracer and ER was observed rostral to obex. There were many ER-ir cells and retrogradely-labelled cells in the nucleus of the solitary tract but only a few double-labelled cells. Similarly, numerous ER-ir cells and retrogradely labelled cells were observed around the lateral edges of the caudal fourth ventricle and across to the lateral parabrachial nucleus but there were few double-labelled cells. These results suggest differential regulation of noradrenergic cells by oestrogen, with a direct action of the hormone confined to the cells in the most caudal region of the A1 and A2 cell groups. The cells of the caudal ventrolateral medulla which contain ER-ir cells that project to the preoptic area may be important in the mediation by noradrenaline of the actions of oestrogen on GnRH secretion in the ewe.

Levels of dopamine beta hydroxylase immunoreactivity in the preoptic hypothalamus of the ovariectomised ewe following injection of oestrogen: evidence for increased noradrenaline release around the time of the oestrogen-induced surge in luteinizing hormone.

We have measured dopamine beta hydroxylase (DBH) immunoreactivity in the preoptic hypothalamus as an index of release of noradrenaline in the ovariectomised (OVX) ewe at the time of an oestrogen-induced surge in luteinizing hormone (LH) release. OVX ewes (n=5) were given an injection of 50 microg oestradiol benzoate (or oil), which causes a surge in the secretion of LH. Blood samples were taken and sheep were killed 16 h later. Other groups (n=3) were given oil or oestrogen and killed 6 h later. Brains were collected for immunohistochemistry and image analysis. The number of DBH-stained cells and the integrated optical density of the cells was measured throughout the A1 field of the brainstem. The DBH staining was measured in the terminal beds of the hypothalamus. There was no difference between the controls and the EB-treated OVX ewes in the number of DBH positive cells or the optical density of DBH-staining cells in the A1 field. Within the preoptic area, there was reduced (P<0.02) DBH staining in the 16 h EB-treated ewes. There was no change in the DBH staining in the paraventricular nucleus or the arcuate nucleus of the hypothalamus. These data suggest that there is release of noradrenaline in the preoptic area at the time of the E-induced GnRH/LH surge in the OVX ewe.

The distribution of estrogen receptors in the brainstem of female sheep.

Estrogen receptors (ER) have been mapped in the brainstem of the female sheep with immunocytochemistry. A small group of ER-positive cells was found in the lateral and ventrolateral medulla and a larger number in the nucleus of the solitary tract. ER-positive cells were abundant in the area postrema. In the rostral brainstem a small number of ER-positive cells were found in the lateral and dorsal regions of the periaqueductal gray area, and some immediately lateral to the fourth ventricle. Many ER-positive cells were also present in the region around the superior cerebellar peduncle, particularly in the lateral parabrachial nucleus. These results describe for the first time the distribution of ER in the brainstem of the sheep and indicate that the majority of estrogen-containing cells are located in structures involved in the regulation of cardiovascular parameters and fluid balance.

Prevalence of endometriosis in asymptomatic women.

Numerous studies have identified the presence of endometriosis by its "classic" morphologic characteristics in high-risk patient populations. Recent histologic documentation of "subtle" laparoscopic appearances of this condition suggests that many of the earlier studies may have underestimated its prevalence in the general population. We evaluated the frequency of endometriosis in 86 women who denied having the symptoms typical of the condition and who underwent laparoscopy for other indications (acute pain, sterilization, etc.). Of those patients, 45.3% were found to have definitive evidence of pelvic endometriosis (stage I, 32.5%; II, 9.3%, III, 1.1%; IV, 2.3%). The exclusion of nine patients whose only laparoscopic indication was infertility or tubal occlusion resulted in a 44% finding of endometriosis, while 39% of the patients with documented fertility exhibited endometriosis. Forty-four percent of a subset of asymptomatic women undergoing laparoscopic evaluation prior to vaginal hysterectomy were found to have evidence of endometriosis; all but one had stage I, judged amendable to laser excision. The data suggest that endometriosis may be a more common peritoneal finding in asymptomatic, fertile women than previously believed.