Multiple events case-control study in a prospective cohort to identify systemic, cellular, and molecular biomarkers of obesity-induced accelerated aging in 30-years-olds: the ObAGE study protocol.

BACKGROUND: Aging is characterized by a progressive loss of capacities linked to fundamental alterations/damage in multiple cellular and molecular pathways. It is the most significant risk factor for all non-communicable diseases (NCDs). Another contributing factor to the rise in NCDs is obesity. It has been suggested that obesity not only accelerates the onset of metabolic imbalances but also decreases lifespan and impacts cellular and molecular processes in a manner similar to aging. Obesity might accelerate the pace of aging. Guided by a lifecourse approach, we will explore how exposure to obesity in critical developmental stages disrupt homeostatic resilience mechanisms that preserve physiological integrity, inducing an early expression of aging phenotypes. Also, we will determine whether exposure to early psychosocial adversity influences vulnerability to obesity as a risk factor for accelerated aging. METHODS: Multiple events case-control study embedded in a prospective cohort of Chileans at 30-31y, 50% females, of low- to-middle socioeconomic status, who participated in nutrition research since birth. At 23y, 25% had obesity and cardiometabolic risk was high. We will use a multi-layer approach including: anthropometric assessment; DXA scan for body composition; abdominal ultrasound of the liver; stool samples collection and sequencing of the ribosomal RNA 16S gene to characterize the gut microbiome; determination of age-related pro-inflammatory cytokynes and anti-inflammatory miokynes. For the first time in Chile, we will address age-related epigenetic changes using the Horvath´s epigenetic clock. In a subset we will conduct a controlled physical challenge to characterize physical resilience (autophagy). DISCUSSION: ObAGE is in an excellent position to: approach aging as a process whose expression involves multiple factors from the early stages of a person's life; understand how longitudinal changes in health trajectories impact the biological mechanisms of aging; identify potential resilience mechanisms that help prevent unhealthy aging. Because SLS participants are still young, our research setting combined with advanced scientific techniques may identify individuals or groups at risk of early onset health issues. Results from ObAGE may pave the way to address the contribution of obesity to aging through lifespan from cells to systems and might be instrumental to developing interventions to improve health span in the Chilean population. TRIAL REGISTRATION: The proposed study does not consider any health care intervention on human participants.

Metformin protects from oxaliplatin induced peripheral neuropathy in rats.

Oxaliplatin is a commonly used drug to treat cancer, extending the rate of disease-free survival by 20% in colorectal cancer. However, oxaliplatin induces a disabling form of neuropathy resulting in more than 60% of patients having to reduce or discontinue oxaliplatin, negatively impacting their chance of survival. Oxaliplatin-induced neuropathies are accompanied by degeneration of sensory fibers in the epidermis and hyperexcitability of sensory neurons. These morphological and functional changes have been associated with sensory symptoms such as dysesthesia, paresthesia and mechanical and cold allodynia. Various strategies have been proposed to prevent or treat oxaliplatin-induced neuropathies without success. The anti-diabetic drug metformin has been recently shown to exert neuroprotection in other chemotherapy-induced neuropathies, so here we aimed to test if metformin can prevent the development of oxaliplatin-induced neuropathy in a rat model of this condition. Animals treated with oxaliplatin developed significant intraepidermal fiber degeneration, a mild gliosis in the spinal cord, and mechanical and cold hyperalgesia. The concomitant use of metformin prevented degeneration of intraepidermal fibers, gliosis, and the altered sensitivity. Our evidence further supports metformin as a new approach to prevent oxaliplatin-induced neuropathy with a potential important clinical impact.

c-Jun N-terminal kinase (JNK)-dependent internalization and Rab5-dependent endocytic sorting mediate long-distance retrograde neuronal death induced by axonal BDNF-p75 signaling.

During the development of the sympathetic nervous system, signals from tropomyosin-related kinase receptors (Trks) and p75 neurotrophin receptors (p75) compete to regulate survival and connectivity. During this process, nerve growth factor (NGF)- TrkA signaling in axons communicates NGF-mediated trophic responses in signaling endosomes. Whether axonal p75 signaling contributes to neuronal death and how signaling endosomes contribute to p75 signaling has not been established. Using compartmentalized sympathetic neuronal cultures (CSCGs) as a model, we observed that the addition of BDNF to axons increased the transport of p75 and induced death of sympathetic neurons in a dynein-dependent manner. In cell bodies, internalization of p75 required the activity of JNK, a downstream kinase mediating p75 death signaling in neurons. Additionally, the activity of Rab5, the key GTPase regulating early endosomes, was required for p75 death signaling. In axons, JNK and Rab5 were required for retrograde transport and death signaling mediated by axonal BDNF-p75 in CSCGs. JNK was also required for the proper axonal transport of p75-positive endosomes. Thus, our findings provide evidence that the activation of JNK by p75 in cell bodies and axons is required for internalization to a Rab5-positive signaling endosome and the further propagation of p75-dependent neuronal death signals.

Distinct promoter methylation and isoform-specific expression of RASFF1A in placental biopsies from complicated pregnancies.

INTRODUCTION: Epigenetic changes in the placenta have been postulated to act as mediators between environmental influences and poor fetal growth. We assessed if genes with a plausible influence on growth could be aberrantly methylated in placental samples from pregnancies complicated by intrauterine growth restriction (IUGR). METHODS: A candidate gene approach was undertaken using a custom Illumina Goldengate® array on a collection of placental samples from growth restricted pregnancies and normally grown controls with confirmation using bisulphite pyrosequencing. RESULTS: The custom array analysis revealed that the promoter of RASSF1A was the only region with significant methylation differences between IUGR placentas and those from pregnancies with appropriate growth for gestational age (AGA). The RASSF1A promoter had increased levels of DNA methylation in IUGR samples compared to controls. Interestingly, the methylation difference was also observed in preeclamptic samples. Higher methylation was associated with a concomitant decrease in expression of the RASSF1 transcript A, but not other isoforms that originate from an alternative, nearby promoter interval. DISCUSSION: Our results do not support the hypothesis that altered DNA-methylation in the placenta is a mechanism generally involved in fetal growth restriction. A specific region corresponding to the promoter of RASSF1A does display methylation changes in placenta that could be used to identify at-risk pregnancies.

Sprouting of axonal collaterals after spinal cord injury is prevented by delayed axonal degeneration.

After an incomplete spinal cord injury (SCI), partial recovery of locomotion is accomplished with time. Previous studies have established a functional link between extension of axon collaterals from spared spinal tracts and locomotor recovery after SCI, but the tissular signals triggering collateral sprouting have not been identified. Here, we investigated whether axonal degeneration after SCI contributes to the sprouting of collaterals from axons spared after injury. To this end, we evaluated collateral sprouting from BDA-labeled uninjured corticospinal axons after spinal cord hemisection (SCI(H)) in wild type (WT) mouse and Wld(S) mouse strains, which shows a significant delay in Wallerian degeneration after injury. After SCI(H), spared fibers of WT mice extend collateral sprouts to both intact and denervated sides of the spinal cord distant from the injury site. On the contrary, in the Wld(S) mice collateral sprouting from spared fibers was greatly reduced after SCI(H). Consistent with a role for collateral sprouting in functional recovery after SCI, locomotor recovery after SCI(H) was impaired in Wld(S) mice compared to WT animals. In conclusion, our results identify axonal degeneration as one of the triggers for collateral sprouting from the contralesional uninjured fibers after an SCI(H). These results open the path for identifying molecular signals associated with tissular changes after SCI that promotes collateral sprouting and functional recovery.

Measurement of autophagy flux in the nervous system in vivo.

Accurate methods to measure autophagic activity in vivo in neurons are not available, and most of the studies are based on correlative and static measurements of autophagy markers, leading to conflicting interpretations. Autophagy is an essential homeostatic process involved in the degradation of diverse cellular components including organelles and protein aggregates. Autophagy impairment is emerging as a relevant factor driving neurodegeneration in many diseases. Moreover, strategies to modulate autophagy have been shown to provide protection against neurodegeneration. Here we describe a novel and simple strategy to express an autophagy flux reporter in the nervous system of adult animals by the intraventricular delivery of adeno-associated viruses (AAV) into newborn mice. Using this approach we efficiently expressed a monomeric tandem mCherry-GFP-LC3 construct in neurons of the peripheral and central nervous system, allowing the measurement of autophagy activity in pharmacological and disease settings.

Activation of the unfolded protein response enhances motor recovery after spinal cord injury.

Spinal cord injury (SCI) is a major cause of paralysis, and involves multiple cellular and tissular responses including demyelination, inflammation, cell death and axonal degeneration. Recent evidence suggests that perturbation on the homeostasis of the endoplasmic reticulum (ER) is observed in different SCI models; however, the functional contribution of this pathway to this pathology is not known. Here we demonstrate that SCI triggers a fast ER stress reaction (1-3 h) involving the upregulation of key components of the unfolded protein response (UPR), a process that propagates through the spinal cord. Ablation of X-box-binding protein 1 (XBP1) or activating transcription factor 4 (ATF4) expression, two major UPR transcription factors, leads to a reduced locomotor recovery after experimental SCI. The effects of UPR inactivation were associated with a significant increase in the number of damaged axons and reduced amount of oligodendrocytes surrounding the injury zone. In addition, altered microglial activation and pro-inflammatory cytokine expression were observed in ATF4 deficient mice after SCI. Local expression of active XBP1 into the spinal cord using adeno-associated viruses enhanced locomotor recovery after SCI, and was associated with an increased number of oligodendrocytes. Altogether, our results demonstrate a functional role of the UPR in SCI, offering novel therapeutic targets to treat this invalidating condition.

Idefix insulator activity can be modulated by nearby regulatory elements.

Insulators play important roles in controlling gene activity and maintaining regulatory independence between neighbouring genes. In this article, we show that the enhancer-blocking activity of the insulator present within the LTR retrotransposon Idefix can be abolished if two copies of the region containing the insulator--specifically, the long terminal repeat (LTR)--are fused to the retrotransposon's 5' untranslated region (5' UTR). The presence of this combination of two [LTR-5' UTR] modules is a prerequisite for the loss of enhancer-blocking activity. We further show that the 5' UTR causes flanking genomic sequences to be displaced to the nuclear periphery, which is not observed when two insulators are present by themselves. This study thus provides a functional link between insulators and independent genomic modules, which may cooperate to allow the specific regulation of defined genomic loci via nuclear repositioning. It further illustrates the complexity of genomic regulation within a chromatic environment with multiple functional elements.

Postoperative death in sows as a result of gastric mucosal de-gloving at the pars oesophagea.

It is well documented that pigs frequently die from postoperative acute gastric dilatation, and proximal gastric 'stress' ulceration. Three cases of gastric mucosal 'de-gloving' are reported. This was secondary to acute gastric dilatation and resulted in death from acute haemorrhage. All animals had undergone major abdominal surgery. Histology confirmed that the proximal gastric mucosa had been 'de-gloved', or torn from the gastro-oesophageal junction, leaving exposed muscle fibres. This syndrome has not been reported previously. The postmortem appearances of this mechanical injury could easily be mistaken for extensive oesophago-gastric peptic ulceration. This has major implications for prevention.

Endoscopic perductal electrolytic ablation of the pancreas: experimental studies of morbidity and mortality.

BACKGROUND: Palliation of pancreatic cancer remains the only option for the majority of patients. Palliative techniques such as surgical bypass and endoscopic retrograde cholangiopancreatography (ERCP) with stenting are not ideal. The 'ideal' palliative technique would combine the efficacy of surgery with the minimal complications of an endoscopic procedure. Endoscopically delivered perductal electrolytic ablation of pancreatic lesions has the potential to meet these criteria. METHODS: Fifteen pigs were used. The pancreatic duct was cannulated with an electrolysis catheter. Animals were randomised to either: controls, treatment 2-week survivor or treatment 8-week survivor. An electrolytic dose was administered to the treatment animals. Post-operatively, serum amylase and leucocyte count were assessed. Pancreata were histologically examined to detect evidence of acute pancreatitis. RESULTS: Electrolysis was well tolerated. There was no difference in post-operative hyperamylasaemia and leucocyte count between the groups. Histological examination showed inflammation at the ablation site at 2 weeks, by 8 weeks this was replaced by scarring. CONCLUSION: The results of this study suggest that endoscopic perductal electrolytic ablation of the pancreas is feasible and safe. Biochemical and histological findings indicate self-limiting localised inflammation of the pancreas. This technique may have a role in the palliation of pancreatic cancer and warrants further investigation.

Electrolytic liver ablation is not associated with evidence of a systemic inflammatory response syndrome.

BACKGROUND: Local ablation has been proposed for treatment of liver tumours. Cryoshock, a variant of the systemic inflammatory response syndrome (SIRS), is a potentially fatal complication of cryoablation caused by systemic release of necrotic breakdown products from ablated liver. The proinflammatory cytokines tissue necrosis factor (TNF) alpha and interleukin (IL) 1 are important mediators of this response. This study assessed the risk of SIRS complicating electrolytic liver ablation by measuring circulating levels of inflammatory cytokines, other inflammatory markers and clinical markers of organ function. METHODS: Electrolytic liver ablation was performed in 16 pigs and four pigs served as controls. Platelet count, and serum levels of urea, creatinine, liver enzymes, C-reactive protein (CRP), TNF-alpha and IL-1beta were measured before treatment and for 72 h after the procedure. RESULTS: There were significant dose-related increases in CRP and alanine aminotransferase levels with liver electrolysis. There was no significant derangement in renal function or platelet count following ablation. A rise in serum TNF-alpha and IL-1beta levels was not associated with liver electrolysis. CONCLUSION: There was no evidence of organ failure or significantly raised levels of proinflammatory cytokines as a result of liver electrolysis, suggesting that this is a safe procedure for liver ablation.

Perductal electrolytic ablation of the porcine pancreas: a minimally invasive option-studies of morbidity and mortality.

BACKGROUND: Pancreatic cancer has a dismal prognosis. Few patients are suitable for surgical resection, leaving the majority requiring symptom palliation. Current palliative techniques such as surgical bypass and endoscopic retrograde cholangiopancreatography (ERCP) are imperfect. A novel palliative therapy combining the symptom control of surgical bypass with the minimally invasive nature of ERCP is required. METHODS: Perductal electrolytic ablation of pancreatic tissue, in a porcine model, was performed. There were two survival groups of 2 weeks (n = 4) and 8 weeks (n = 4). Postoperatively, serum biochemistry, amylase and C-reactive protein (CRP) were assessed. Histological examination of the pancreas, lungs, and kidneys was performed to determine the presence of acute pancreatitis or systemic inflammatory response. RESULTS: An immediate transient increase in both amylase and CRP was seen. Although pancreatic histology demonstrated localised necrosis at the electrolytic site at 2 weeks, there was no evidence of generalized pancreatitis or a systemic inflammatory response at either 2 or 8 weeks. CONCLUSIONS: This study suggests that, although there is localized pancreatic necrosis and transient hyperamylasemia, perductal pancreatic electrolytic ablation is safe, with neither generalized pancreatitis nor a systemic inflammatory response, in the medium and long term. Although performed in normal porcine pancreas, because of the absence of a large-animal model of pancreatic cancer, this study suggests that electrolytic pancreatic ablation is safe. This technique may have a role in the palliation of pancreatic cancer, especially if delivered via a minimally, invasive approach, and warrants further investigation.

Segmental nature of the porcine liver and its potential as a model for experimental partial hepatectomy.

BACKGROUND: In-depth knowledge of pig liver anatomy allows potential research into segmental liver resections and hepatic regeneration, as well as liver transplantation techniques. The segmental anatomy, however, remains largely unknown. This study aimed to delineate the segmental anatomy of the porcine liver in comparison with that of the human. METHODS: The segmental anatomy of the porcine liver was determined using acrylic injection casting of ex vivo pig livers, allowing the arterial, venous and biliary supply to be visualized directly. This was correlated using multi-slice computed tomography (CT) and three-dimensional reconstructions. RESULTS: Although the external morphology of the porcine liver differs from that of the human, the segmental anatomy is remarkably similar in term of its vascularity and biliary tree. CONCLUSION: Acrylic casting of the porcine liver accurately delineates the vascular and biliary anatomy, and is a useful tool for performing experimental liver surgery. The similarities between porcine and human segmental anatomy allow domestic swine to be used as a comparable model. Three-dimensional CT reconstructions can also accurately visualize the anatomy and may be used to perform virtual surgery, or to assess segmental volumes.

Palliation of pancreatic cancer using electrolytic ablation.

BACKGROUND: Inoperable pancreatic cancer has a dismal prognosis. Palliation involves either stenting or surgical bypass. Stenting does not relieve gastric outlet obstruction, and surgical bypass is a major procedure. A minimally invasive procedure is needed that relieves both gastric outlet and biliary obstruction, with the potential for relieving pain. METHODS: In an experimental model, pancreatic electrolysis was investigated. The pancreatic duct was cannulated via a transduodenal approach with an electrode catheter. In 6 animals an electrolytic "lesion" was created using a direct current generator. Six animals were controls. The local and systemic effects of electrolysis were assessed using histological and biochemical parameters. RESULTS: The pancreatic duct was cannulated in all animals and treatment was uneventful. Electrolytic lesions comprised a central area of necrosis with a sharp demarcation between necrotic and viable pancreas. All animals developed transient hyperamylasemia after electrolysis. There was no significant difference between treatment and controls. Importantly, no animal had clinical, biochemical, or histological evidence of pancreatitis. CONCLUSIONS: This experimental study suggested that electrolytic palliation of inoperable pancreatic cancer via the gastrointestinal tract is potentially safe. In patients, this treatment could be performed during endoscopic retrograde cholangiopancreatography and may have therapeutic advantages when compared to stenting or biliary bypass.

The mystery of liver regeneration.

BACKGROUND: Partial hepatectomy is the strongest stimulator of hepatic regeneration. The process of initiation and the control of the final size of the regenerated liver have been the subject of research for many years. A better understanding of this process and the effect of disease may allow better selection of patients for partial hepatectomy. It may also allow an insight into the possible application of clinical stimulation of regeneration. METHODS: Data were reviewed from the published literature using the Medline database. RESULTS: Most knowledge comes from in vitro studies and the study of resection in the rat model. A variety of cytokines, hormones and growth factors are involved in regeneration but very few have been found capable of stimulating regeneration in vitro. The exact interactions are not known, but there is probably a cascade involving different factors at differing stages of regeneration. CONCLUSION: Further in vivo research should allow greater understanding of liver regeneration, thereby providing a potential therapeutic tool in patients for whom regeneration has failed, or is likely to fail. Such research is also important in respect of liver support devices, which may inhibit liver regeneration by filtration of many of the factors involved.

Wallerian degeneration of injured axons and synapses is delayed by a Ube4b/Nmnat chimeric gene.

Axons and their synapses distal to an injury undergo rapid Wallerian degeneration, but axons in the C57BL/WldS mouse are protected. The degenerative and protective mechanisms are unknown. We identified the protective gene, which encodes an N-terminal fragment of ubiquitination factor E4B (Ube4b) fused to nicotinamide mononucleotide adenylyltransferase (Nmnat), and showed that it confers a dose-dependent block of Wallerian degeneration. Transected distal axons survived for two weeks, and neuromuscular junctions were also protected. Surprisingly, the Wld protein was located predominantly in the nucleus, indicating an indirect protective mechanism. Nmnat enzyme activity, but not NAD+ content, was increased fourfold in WldS tissues. Thus, axon protection is likely to be mediated by altered ubiquitination or pyridine nucleotide metabolism.

Nerve regeneration in Wld(s) mice is normalized by actinomycin D.

Injured nerves of Wld(s) mice neither degenerate nor regenerate for several weeks. We have conjectured that Wld(s) axons have the ability to regenerate but its expression is impaired by the Schwann cells of the undegenerated distal stump. To test this conjecture, transcription was locally arrested with actinomycin D (ActD), nerves were crushed, and regrowth was evaluated. In normal CD1 nerves injected with ActD 3 days before the crush, the rate of elongation was not affected but the delay of regrowth was shortened. In sharp contrast, ActD normalized the elongation of Wld(s) axons. When Wld(s) nerves were crushed past the treated segment, axons did not regenerate. After 7, but not 4, days of treatment, intact CD1 and Wld(s) axons presented a local sprouting response. We conclude that Wld(s) axons can regenerate in a normal way but do not do so because the undegenerated Schwann cells of the distal stump repress the regrowth program. We present a model axon that includes a destruction program and a post-transcriptional trophic regulation of its phenotype.

Drug accumulation and elimination in Calliphora vicina larvae.

Calliphora vicina larvae were fed on drug-laden muscle from three suicides involving amitriptyline, temazepam and a combination of trazodone and trimipramine; triplicate daily harvestings were analysed. The limit of detection for all four drugs was 0.01 micrograms drug/g larvae. Mean drug concentrations (microgram/g) in the initial muscle were:amitriptyline, 2.68; temazepam, 4.04; trazodone, 21.56; and trimipramine, 19.58. Larval rearings for days 4-8 (15 larval samples per drug) had mean and ranges of drug concentrations (microgram/g) of 0.10 (r, 0.02-0.24) for amitriptyline; 0.52 (r, 0.26-0.78) for temazepam; 0.13 (r, 0.05-0.32) for trazodone; and 0.28 (r, 0.10-0.59) for trimipramine. After day 8 there was a precipitous fall in larval drug concentrations associated with pupariation. At day 11 ranges of drug concentrations (microgram/g) were: amitriptyline, < 0.01-0.01; temazepam, 0.01-0.08; trazodone, < 0.01-0.01; and trimipramine, 0.04-0.04. Day 16 pupae had corresponding ranges (microgram/g) of < 0.01, 0.01-0.01, < 0.01 and < 0.01-0.02. Transfer to drug-free food at day 5 led to similar falls in drug concentrations (microgram/g) from day 5 to day 6: 0.08-0.03 for amitriptyline, 0.61-0.09 for temazepam, 0.13-0.01 for trazodone, and 0.30-0.02 for trimipramine. The results show considerable variation in larval drug concentrations, both at the same developmental stage and at different stages of the life cycle, under conditions which closely reflect case situations. In practice, the precipitous decrease in drug concentrations in non-feeding larvae and at pupariation make it desirable to sample only larvae actively feeding on a corpse.