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Abstract 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a persistent environmental contaminant that disrupts hepatic function leading to steatotic liver disease (SLD)-like pathologies, such as steatosis, steatohepatitis, and fibrosis. These effects are mediated by the aryl hydrocarbon receptor following changes in gene expression. Although diverse cell types are involved, initial cell-specific changes in gene expression have not been reported. In this study, differential gene expression in hepatic cell types was examined in male C57BL/6 mice gavaged with 30 µg/kg of TCDD using single-nuclei RNA-sequencing. Ten liver cell types were identified with the proportions of most cell types remaining unchanged, except for neutrophils which increased at 72 h. Gene expression suggests TCDD induced genes related to oxidative stress in hepatocytes as early as 2 h. Lipid homeostasis was disrupted in hepatocytes, macrophages, B cells, and T cells, characterized by the induction of genes associated with lipid transport, steroid hormone biosynthesis, and the suppression of β-oxidation, while linoleic acid metabolism was altered in hepatic stellate cells (HSCs), B cells, portal fibroblasts, and plasmacytoid dendritic cells. Pro-fibrogenic processes were also enriched, including the induction retinol metabolism genes in HSCs and the early induction of anti-fibrolysis genes in hepatocytes, endothelial cells, HSCs, and macrophages. Hepatocytes also had gene expression changes consistent with hepatocellular carcinoma. Collectively, these findings underscore the effects of TCDD in initiating SLD-like phenotypes and identified cell-specific gene expression changes related to oxidative stress, steatosis, fibrosis, cell proliferation and the development of HCC.

Metabolic reprogramming by the pyruvate kinase M2 isoform is associated with cell proliferation and reactive oxygen species (ROS) defenses. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), an environmental contaminant that induces ROS and hepatotoxicity, dose-dependently induces pyruvate kinase muscle isoform M2 (PKM2) in the liver. To further investigate its role in combating TCDD hepatotoxicity, a PkmΔDRE mouse was constructed lacking the dioxin response element mediating aryl hydrocarbon receptor (AHR) induction. TCDD failed to induce hepatic PKM2 in PkmΔDRE mice and in primary hepatocytes isolated from an AHR knockout model (AHRV375Afl/flAlb-CreERT2), demonstrating induction is AHR dependent. Both wild-type (WT) and PkmΔDRE mice exhibited dose-dependent increases in liver weight after treatment with TCDD every 4 days for 28 days. Glutathione (GSH) levels increased in WT mice while oxidized glutathione (GSSG) levels increased in both models with a 24-fold decrease in the GSH/GSSG ratio in PkmΔDRE mice suggesting lower antioxidant and recycling capacity. Moreover, TCDD-induced fibrosis was more severe in PkmΔDRE mice while PkmΔDRE hepatocytes exhibited greater cytotoxicity following co-treatment with TCDD and hydrogen peroxide. TCDD also induced PKM2 in human HepaRG™ cells with AHR enrichment at a conserved DRE core within the locus. These results suggest AHR-mediated PKM2 induction is a novel antioxidant response to TCDD.

Perivascular adipose tissue (PVAT) is known for being anti-contractile in healthy tissues. We discovered a new function of PVAT, the ability to stress relax and maintain a tone in response to a stretch. This is of note because stress relaxation has been attributed to smooth muscle, of which PVAT has none that is organized in a functional layer. We test the hypothesis the interactions of integrins with collagen play a role in stress relaxation. Our model is the thoracic aorta of the male Dahl SS rat. The PVAT and aorta were physically separated for most assays. Results from single nuclei RNA sequencing (snRNAseq) experiments, histochemistry and isometric contractility were also used. Masson Trichrome staining made evident the expression of collagen in PVAT. From snRNA seq experiments of the PVAT, mRNA for multiple collagen and integrin isoforms were detected: the α1 and β1 integrin were most highly expressed. Pharmacological inhibition of integrin/collagen interaction was effected by the specific α1β1 distintegrin obtustatin or general integrin inhibitor RGD peptide. RGD peptide but not obtustatin increased the stress relaxation. Cell-cell communication inference identified integrins αv and α5, two major RGD motif containing isoforms, as potential signaling partners of collagens. Collectively, these findings validate that stress relaxation can occur in a non-smooth muscle tissue, doing so in part through integrin-collagen interactions that may not include α1β1 heterodimers. The importance of this lies in considering PVAT as a vascular layer that possesses mechanical functions.

Abstract 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is associated with metabolic syndrome (MetS) in humans and elicits pathologies in rodents that resemble non-alcoholic fatty liver disease (NAFLD) in humans through activation of the aryl hydrocarbon receptor (AHR) pathway. Dysregulation of cholesterol homeostasis, an aspect of MetS, is linked to NAFLD pathogenesis. TCDD exposure is also linked to the suppression of genes that encode key cholesterol biosynthesis steps and changes in serum cholesterol levels. In a previous experiment, treating mice with TCDD in the presence of simvastatin, a 3-Hydroxy-3-Methylglutaryl-CoA Reductase competitive inhibitor, altered lipid and glycogen levels, AHR-battery gene expression, and liver injury in male mice compared to TCDD alone. The aim of this study was to deduce a possible mechanism(s) for the metabolic changes and increased injury using single-nuclei RNA sequencing in mouse liver. We demonstrated that co-treated mice experienced wasting and increased AHR activation compared to TCDD alone. Furthermore, relative proportions of cell (sub)types were different between TCDD alone and co-treated mice including important mediators of NAFLD progression like hepatocytes and immune cell populations. Analysis of non-overlapping differentially expressed genes identified several pathways where simvastatin co-treatment significantly impacted TCDD-induced changes, which may explain the differences between treatments. Overall, these results demonstrate a connection between dysregulation of cholesterol homeostasis and toxicant-induced metabolic changes.

Abstract 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a persistent environmental contaminant that induces the progression of steatosis to steatohepatitis with fibrosis in mice. Furthermore, TCDD reprograms hepatic metabolism by redirecting glycolytic intermediates while inhibiting lipid metabolism. Here, we examined the effect of TCDD on hepatic acetyl-coenzyme A (acetyl-CoA) and β-hydroxybutyrate levels as well as protein acetylation and β-hydroxybutyrylation. Acetyl-CoA is not only a central metabolite in multiple anabolic and catabolic pathways, but also a substrate used for posttranslational modification of proteins and a surrogate indicator of cellular energy status. Targeted metabolomic analysis revealed a dose-dependent decrease in hepatic acetyl-CoA levels coincident with the phosphorylation of pyruvate dehydrogenase (E1), and the induction of pyruvate dehydrogenase kinase 4 and pyruvate dehydrogenase phosphatase, while repressing ATP citrate lyase and short-chain acyl-CoA synthetase gene expression. In addition, TCDD dose-dependently reduced the levels of hepatic β-hydroxybutyrate and repressed ketone body biosynthesis gene expression. Moreover, levels of total hepatic protein acetylation and β-hydroxybutyrylation were reduced. AMPK phosphorylation was induced consistent with acetyl-CoA serving as a cellular energy status surrogate, yet subsequent targets associated with re-establishing energy homeostasis were not activated. Collectively, TCDD reduced hepatic acetyl-CoA and β-hydroxybutyrate levels eliciting starvation-like conditions despite normal levels of food intake.

Abstract 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), a persistent environmental contaminant, induces steatosis by increasing hepatic uptake of dietary and mobilized peripheral fats, inhibiting lipoprotein export, and repressing β-oxidation. In this study, the mechanism of β-oxidation inhibition was investigated by testing the hypothesis that TCDD dose-dependently repressed straight-chain fatty acid oxidation gene expression in mice following oral gavage every 4 days for 28 days. Untargeted metabolomic analysis revealed a dose-dependent decrease in hepatic acyl-CoA levels, while octenoyl-CoA and dicarboxylic acid levels increased. TCDD also dose-dependently repressed the hepatic gene expression associated with triacylglycerol and cholesterol ester hydrolysis, fatty acid binding proteins, fatty acid activation, and 3-ketoacyl-CoA thiolysis while inducing acyl-CoA hydrolysis. Moreover, octenoyl-CoA blocked the hydration of crotonyl-CoA suggesting short chain enoyl-CoA hydratase (ECHS1) activity was inhibited. Collectively, the integration of metabolomics and RNA-seq data suggested TCDD induced a futile cycle of fatty acid activation and acyl-CoA hydrolysis resulting in incomplete β-oxidation, and the accumulation octenoyl-CoA levels that inhibited the activity of short chain enoyl-CoA hydratase (ECHS1).

Background and Aims: Characterization of cell specific transcriptional responses to hepatotoxicants is lost in the averages of bulk RNA-sequencing (RNA-seq). Single-cell/nuclei RNA-seq technologies enable the transcriptomes of individual cell (sub)types to be assessed within the context of in vivo models. Methods: Single-nuclei RNA-sequencing (snSeq) of frozen liver samples from male C57BL/6 mice gavaged with sesame oil vehicle or 30 μg/kg 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) every 4 days for 28 days was used to demonstrate the application of snSeq for the evaluation of xenobiotics. Results: A total of 19,907 genes were detected across 16,015 nuclei from control and TCDD-treated livers. Eleven cell (sub)types reflected the expected cell diversity of the liver including distinct pericentral, midzonal, and periportal hepatocyte subpopulations. TCDD altered relative proportions of cell types and elicited cell-specific gene expression profiles. For example, macrophages increased from 0.5% to 24.7%, while neutrophils were only present in treated samples, consistent with histological evaluation. The number of differentially expressed genes (DEGs) in each cell type ranged from 122 (cholangiocytes) to 7625 (midcentral hepatocytes), and loosely correlated with the basal expression level of Ahr, the canonical mediator of TCDD and related compounds. In addition to the expected functions within each cell (sub)types, RAS signaling and related pathways were specifically enriched in nonparenchymal cells while metabolic process enrichment occurred primarily in hepatocytes. snSeq also identified the expansion of a Kupffer cell subtype highly expressing Gpnmb, as reported in a dietary NASH model. Conclusions: We show that snSeq of frozen liver samples can be used to assess cell-specific transcriptional changes and population shifts in models of hepatotoxicity when examining freshly isolated cells is not feasible.

Abstract Aryl hydrocarbon receptor (AhR) activation is reported to alter the hepatic expression of circadian clock regulators, however the impact on clock-controlled metabolism has not been thoroughly investigated. This study examines the effects of AhR activation on hepatic transcriptome and metabolome rhythmicity in male C57BL/6 mice orally gavaged with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) every 4 days for 28 days. TCDD diminished the rhythmicity of several core clock regulators (e.g. Arntl, Clock, Nr1d1, Per1, Cry1, Nfil3) in a dose-dependent manner, involving either a ≥ 3.3-fold suppression in amplitude or complete loss of oscillation. Accordingly, protein levels (ARNTL, REV-ERBα, NFIL3) and genomic binding (ARNTL) of select regulators were reduced and arrhythmic following treatment. As a result, the oscillating expression of 99.6% of 5,636 clock-controlled hepatic genes was abolished including genes associated with the metabolism of lipids, glucose/glycogen, and heme. For example, TCDD flattened expression of the rate-limiting enzymes in both gluconeogenesis (Pck1) and glycogenesis (Gys2), consistent with the depletion and loss of rhythmicity in hepatic glycogen levels. Examination of polar hepatic extracts by untargeted mass spectrometry revealed that virtually all oscillating metabolites lost rhythmicity following treatment. Collectively, these results suggest TCDD disrupted circadian regulation of hepatic metabolism, altering metabolic efficiency and energy storage.

Objectives: Previous research has largely focused on the lived experience either of those who have fibromyalgia syndrome (FMS) or their spousal carers. This study aimed to explore the lived experiences of both those with FMS and their spousal carers.Methods: Participants were aged between 38 and 65 years and all came from the south-west of England. Semi-structured interviews were conducted with four women with FMS and their spousal carers, who were interviewed separately. The resultant transcripts were analysed using interpretative phenomenological analysis.An overriding theme running throughout was loss of identity, which fed into a sense of isolation. Participants reported feeling isolated from: healthcare professionals, whom they felt they had to convince that they had something 'real', and from friends and family because the unpredictability of their symptoms meant that they were less able to plan ahead and often had to pull out of arranged outings. They also felt isolated from their identity because they no longer recognized the person that they once were, and struggled to recognize the person that they had become. As a consequence, the people with FMS and their carers were both engaged in a process of reassessing who they were, now that FMS had become such a large part of their lives. This sense of isolation was evidenced for the carers as well as the people with FMS and is documented in three sub-themes described in the paper: 'others' attitudes', 'invisible illness' and 'role'.Conclusion: This study has provided new information regarding the lifeworlds both of people living with FMS and their spousal carers. We identified a number of practical and attitudinal barriers that had led to the diminution of social networks for both members of the couple and have explored the related clinical and theoretical implications of this. Copyright (c) 2010 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Abstract 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is a persistent environmental contaminant which elicits hepatotoxicity through activation of the aryl hydrocarbon receptor (AhR). Male C57BL/6 mice orally gavaged with TCDD (0.01–30 µg/kg) every 4 days for 28 days exhibited bile duct proliferation and pericholangitis. Mass spectrometry analysis detected a 4.6-fold increase in total hepatic bile acid levels, despite the coordinated repression of genes involved in cholesterol and primary bile acid biosynthesis including Cyp7a1. Specifically, TCDD elicited a >200-fold increase in taurolithocholic acid (TLCA), a potent G protein-coupled bile acid receptor 1 (GPBAR1) agonist associated with bile duct proliferation. Increased levels of microbial bile acid metabolism loci (bsh, baiCD) are consistent with accumulation of TLCA and other secondary bile acids. Fecal bile acids decreased 2.8-fold, suggesting enhanced intestinal reabsorption due to induction of ileal transporters (Slc10a2, Slc51a) and increases in whole gut transit time and intestinal permeability. Moreover, serum bile acids were increased 45.4-fold, consistent with blood-to-hepatocyte transporter repression (Slco1a1, Slc10a1, Slco2b1, Slco1b2, Slco1a4) and hepatocyte-to-blood transporter induction (Abcc4, Abcc3). These results suggest that systemic alterations in enterohepatic circulation, as well as host and microbiota bile acid metabolism, favor bile acid accumulation that contributes to AhR-mediated hepatotoxicity.

C7 M/N typing, the determination of the complement component C7 M/N phenotypes, was successfully used in family studies to trace haplotypes bearing C7 deficiency genes. Furthermore, it was shown to be preferable to C7 allotyping based on isoelectric focusing (IEF) since it distinguishes two common alleles (C7* M and C7*N), whereas one common C7 IEF allele (C7*I) predominates in most populations. It is also the more sensitive method, as it enabled detection of very low amounts of abnormal C7 molecules in the third generation of a combined subtotal C6/C7-deficient subject and thus confirmed that this partial deficiency gene is not silent in heterozygotes. In this respect C7 M/N typing is even more informative than DNA restriction fragment length polymorphism typing which will assess the presence but not necessarily the functional status of a gene. C6 and C7 genes are tightly linked and therefore C7 M/N typing was also applied to tracing C6 deficiency genes in families. C6/ C7 haplotype analysis of South African C6-deficient (C6Q0) subjects revealed a strong allelic association of C6*Q0 and C7*M. [ABSTRACT FROM AUTHOR]

The aryl hydrocarbon receptor (AhR) is a highly conserved transcription factor that mediates a broad spectrum of species-, strain-, sex-, age-, tissue-, and cell-specific responses elicited by structurally diverse ligands including 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Dose-dependent effects on liver-specific and sexually dimorphic gene expression were examined in male and female mice gavaged with TCDD every 4 days for 28 or 92 days. RNA-seq data revealed the coordinated repression of 181 genes predominately expressed in the liver including albumin (3.7-fold), α-fibrinogen (14.5-fold), and β-fibrinogen (17.4-fold) in males with corresponding AhR enrichment at 2 hr. Liver-specific genes exhibiting sexually dimorphic expression also demonstrated diminished divergence between sexes. For example, male-biased Gstp1 was repressed 3.0-fold in males and induced 4.5-fold in females, which were confirmed at the protein level. Disrupted regulation is consistent with impaired GHR-JAK2-STAT5 signaling and inhibition of female specific CUX2-mediated transcription as well as the repression of other key transcriptional regulators including Ghr, Stat5b, Bcl6, Hnf4a, Hnf6, Foxa1/2/3, and Zhx2. Attenuated liver-specific and sexually dimorphic gene expression was concurrent with the induction of fetal genes such as alpha-fetoprotein. The results suggest AhR activation causes the loss of liver-specific and sexually dimorphic gene expression producing a functionally "de-differentiated" hepatic phenotype.

Background: The methods of estimating vascular access (VA) flow rates are usually based on the indicator dilution theory by measuring recirculation during dialysis sessions., Methods: This is an observational study comparing the VA flow rates measured by NIKKISO DBB-EXA™ and Transonic®. Sixty-five patients (38 M/27 F, mean age 72 ± 10 years) participated in the study. We measured the VA flow rates during dialysis twice with each method and repeated the procedure 7 days later., Results: In 130 double measurements for each method on the same day, mean flow with Transonic® was 1413±715 ml/min and with DBB-EXA™ 1297 ± 664 ml/min. In Bland-Altman analysis, the mean difference between the two methods was 159 ± 211 ml/min (limits of agreement: -274 and 572 ml/min). Eighty-one out of the 130 DBB-EXA™ measurements were within 25% of the Transonic® measurements (62% accuracy). Regarding reproducibility of each method on different days, mean difference in the Bland-Altman analysis was 29 ± 620 ml/min (limits of agreement: -1186 and 1244 ml/min) for the Transonic® measurements and 132 ± 625 ml/min (limits of agreement: -1092 and 1356 ml/min) for the DBB-EXA™ measurements. The measurements on two different days were within 25% of each other for 52 of the 65 patients (80%) with the Transonic® method, and for 35 of the 65 patients (54%) with the DBB-EXA™ method., Conclusions: In conclusion, the DBB-EXA™ method underestimates VA flow rates compared to the Transonic® technique, resulting in a limited accuracy of 62%. There was poor reproducibility for both methods in different day measurements with better performance of the Transonic® technique. The DBB-EXA™ method could be used as a simple tool for a rough estimate of VA flow rates but cannot replace the Transonic® reference method., Competing Interests: Declaration of conflicting interestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

The prototypical aryl hydrocarbon receptor (AHR) ligand, 2,3,7,8-tetrachlorodibenzo- p -dioxin (TCDD), has been a valuable model for investigating toxicant-associated fatty liver disease (TAFLD). TCDD induces dose-dependent hepatic lipid accumulation, followed by the development of inflammatory foci and eventual progression to fibrosis in mice. Previously, bulk approaches and in vitro examination of different cell types were relied upon to study the mechanisms underlying TCDD-induced liver pathologies. However, the advent of single-cell transcriptomic technologies, such as single-nuclei RNA sequencing (snRNAseq) and spatial transcriptomics (STx), has provided new insights into the responses of hepatic cell types to TCDD exposure. This review explores the application of these single-cell transcriptomic technologies and highlights their contributions towards unraveling the cell-specific mechanisms mediating the hepatic responses to TCDD., Competing Interests: Rance Nault reports financial support was provided by National Institute of Environmental Health Sciences. Rance Nault reports financial support was provided by National Human Genome Research Institute. Rance Nault reports financial support was provided by National Heart Lung and Blood Institute.Declaration of competing interest The authors declare no conflict of interest.

Background&aims: Patients with end-stage renal failure on chronic hemodialysis present an important risk of malnutrition, which is associated with a significant risk of morbidity and mortality. Meals during the dialysis session are important for maintaining the nutritional status of dialysis patients but represent a risk for intradialytic hypotension. During the COVID-19 pandemic, several dialysis centers stopped providing meals during the dialysis session as a protective measure. The aim of this retrospective, multicentric cross-over study was to study the evolution of the nutritional parameters of a cohort of hemodialysis patients for 12 months before, during and after the suspension of meals during dialysis due to the COVID-19 pandemics., Methods: We registered the evolution of dry weight, C Reactive Protein (CRP), serum Potassium and Phosphate before the dialysis session, serum albumin and prealbumin levels as well as normalized Protein Catabolic Ratio (nPCR)., Results: We studied 168 hemodialysis patients (113M, 55F, mean age at inclusion:68.45 ± 0.45 years). The results ares shown as mean values (±SEM). The supression of the intradialytic meals led to significant reduction of the patients' dry weight (in Kg) from 78.66 ± 0.72 to 76.50 ± 0.49, p = 0.013, serum albumin (in g/l) (from 40.72 ± 0.16 to 39.25 ± 0.12, p < 0.001) and prealbumin levels (in g/l) (from 33.82 ± 0.31 to 32.73 ± 0.22, p = 0.004) as well as the nPCR values (from 1.08 ± 0.08 to 1.05 ± 0.11, p = 0.021). Serum CRP as well as predialytic Potassium and Phosphate levels did not change significantly. The reinstitution of the intradialytic meals led to a complete correction of the studied nutritional parameters with Body weight values evolving from 76.50 ± 0.49 to 78.28 ± 1.01, p = 0.025, serum albumin from 39.25 ± 0.12 to 40.53 ± 1.04, p < 0.001, serum prealbumin levels from 32.73 ± 0.22 to 33.95 ± 0.64, p = 0.001 an nPCR from 1.05 ± 0.11 to 1.08 ± 0.08, p = 0.021., Conclusion: In conclusion, the suppression of intradialytic meals during the COVID-19 pandemic had deleterious effects on the nutritional parameters of patients on chronic hemodialysis., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest., (Copyright © 2023 European Society for Clinical Nutrition and Metabolism. Published by Elsevier Ltd. All rights reserved.)

Poor success rates and high levels of dropout are common features in the treatment of anorexia nervosa. Using semi-structured interviews, this study elicited the views of 12 women who were recovered, or in recovery, for anorexia nervosa and had received treatment. Results derived from a thematic analysis revealed the women's high degree of dissatisfaction with treatment and their perception that the treatment system is overly focused on, and driven by, food and weight. In contrast, what the women really wanted was to be seen and treated as a 'whole person' and to have a 'real' relationship with their therapist.

Neurokinin B (NKB) and kisspeptin receptor signaling are essential components of the reproductive axis. A population of neurons resides within the arcuate nucleus of the rat that expresses NKB, kisspeptin, dynorphin, NK3 receptors and estrogen receptor alpha (ERalpha). Here we investigate the projections of these neurons using NKB-immunocytochemistry as a marker. First, the loss of NKB-immunoreactive (ir) somata and fibers was characterized after ablation of the arcuate nucleus by neonatal injections of monosodium glutamate. Second, biotinylated dextran amine was injected into the arcuate nucleus and anterogradely labeled NKB-ir fibers were identified using dual-labeled immunofluorescence. Four major projection pathways are described: (1) local projections within the arcuate nucleus bilaterally, (2) projections to the median eminence including the lateral palisade zone, (3) projections to a periventricular pathway extending rostrally to multiple hypothalamic nuclei, the septal region and BNST and dorsally to the dorsomedial nucleus and (4) Projections to a ventral hypothalamic tract to the lateral hypothalamus and medial forebrain bundle. The diverse projections provide evidence that NKB/kisspeptin/dynorphin neurons could integrate the reproductive axis with multiple homeostatic, behavioral and neuroendocrine processes. Interestingly, anterograde tract-tracing revealed NKB-ir axons originating from arcuate neurons terminating on other NKB-ir somata within the arcuate nucleus. Combined with previous studies, these experiments reveal a bilateral interconnected network of sex-steroid responsive neurons in the arcuate nucleus of the rat that express NKB, kisspeptin, dynorphin, NK3 receptors and ERalpha and project to GnRH terminals in the median eminence. This circuitry provides a mechanism for bilateral synchronization of arcuate NKB/kisspeptin/dynorphin neurons to modulate the pulsatile secretion of GnRH., (Copyright (c) 2010 IBRO. Published by Elsevier Ltd. All rights reserved.)

Descending input from the rostral ventromedial medulla (RVM) provides positive and negative modulation of spinal nociceptive transmission and has been proposed to be critical for maintaining neuropathic pain. This study tests the hypothesis that neuropathic pain requires the activity of a subset of RVM neurons that are distinguished by co-expression of mu opioid receptor (MOR) and cholecystokinin type 2 receptor (CCK2). Using male Sprague-Dawley rats, we demonstrate that discrete RVM neurons express MOR and CCK2; over 80% of these cells co-express both receptors. Agonist-directed cell lesion in the RVM with the cytotoxin, saporin, using either CCK-saporin to target CCK receptor expressing cells, or dermorphin-saporin to target MOR expressing cells, resulted in concomitant loss of CCK2 and MOR expressing cells, did not alter the basal sensory thresholds but abolished the hyperalgesia induced by microinjection of CCK into the RVM. The findings suggest that these CCK2-MOR co-expressing RVM neurons facilitate pain and can be directly activated by CCK input to the RVM. Furthermore, lesion of these RVM neurons did not affect the initial development of neuropathic pain in the hind paw upon injury to the sciatic nerve, but the abnormal pain states were short lived such that by about day 9 the sensory thresholds had reverted to pre-injury baselines despite the existing neuropathy. These data support our hypothesis and identify CCK2-MOR co-expressing neurons in the RVM as potential therapeutic targets for neuropathic pain.

Aging in women is associated with dramatic changes in neuronal morphology and neuropeptide gene expression in the medial basal hypothalamus. There is hypertrophy of neurons expressing substance P and neurokinin B gene transcripts in the infundibular (arcuate) nucleus, accompanied by increased tachykinin gene expression. In addition, gonadotropin-releasing hormone (GnRH) gene expression is increased in a separate subpopulation of neurons within the medial basal hypothalamus. In contrast, the number of neurons expressing proopiomelanocortin (POMC) mRNA in the infundibular nucleus of older women is decreased. To determine whether neuronal degeneration contributes to these phenomena, unbiased stereologic methods were used to compare the total number of infundibular neurons between groups of young (premenopausal) and older (postmenopausal) women. There was no significant difference in the total number of infundibular neurons between young (520,000 +/- 42,000 neurons, mean +/- SEM) and older women (505,000 +/- 51,000 neurons, mean +/- SEM). The mean volume of neuronal somata, however, was increased by 40% in the older women (young, 1,860 +/- 180 microm(3) vs. older, 2,610 +/- 230 microm(3), mean +/- SEM, P < 0.05). These data demonstrate that neuronal hypertrophy in older women is not accompanied by degeneration of the infundibular nucleus. We conclude that the loss of menstrual cyclicity in middle-aged women cannot be explained by loss of neurons within the hypothalamic control center for reproduction., (Copyright 2000 Wiley-Liss, Inc.)

Previous studies have shown that estrogen withdrawal decreases the secretion of beta-endorphin from the monkey hypothalamus. In addition, there are consistent age-associated changes in beta-endorphin neurons in the rodent. Based on these findings, we hypothesized that the activity of hypothalamic beta-endorphin neurons would be decreased in the hypothalamus of postmenopausal women. In the present study, we examined the expression of proopiomelanocortin (POMC) mRNA, the precursor mRNA for beta-endorphin, in the medial basal hypothalamus of premenopausal and postmenopausal women. Every 20th sagittal section through the hypothalamus was hybridized with a synthetic [35S]labeled, 48-base oligonucleotide probe complementary to POMC mRNA. Labeled neurons were counted and their somatic profile areas were measured with an image-combining computer microscope system. The number of POMC mRNA-containing neurons/section in the infundibular nucleus was reduced by 65% in postmenopausal women. In contrast, there was no significant difference in the number of neurons expressing POMC gene transcripts in the retrochiasmatic region. The POMC neurons in the retrochiasmatic area were also distinct morphologically from those in the infundibular nucleus. The differences between the infundibular and retrochiasmatic regions suggest that functional subgroups of POMC neurons exist in the human hypothalamus. Our findings provide evidence that the activity of hypothalamic POMC neurons is decreased in the infundibular nucleus of postmenopausal women. Both aging and gonadal steroid withdrawal may contribute to the decline in POMC gene expression in postmenopausal women., (Copyright 1999 Published by Elsevier Science B.V.)