Variation in salivary cortisol responses in yearling Thoroughbred racehorses during their first year of training.

Thoroughbred horses are bred for competitive racing and undergo intense training regimes. The maintenance of physical soundness and desirable behavioural characteristics are critical to the longevity of a racing career. Horses intended for Flat racing generally enter training as yearlings and undergo introductory training prior to exercise conditioning for racing. This period requires rapid adjustment to a novel environment. As a prey animal, a horse's 'fight-or-flight' response is highly adapted, in which a well-understood component of this response, the hypothalamic-pituitary-axis, is activated in response to a stress stimulus, releasing cortisol. In the Thoroughbred, a significant difference in salivary cortisol concentrations between pre- and post-first time ridden (i.e., first backing) by a jockey have previously been identified. Here, to test the hypothesis that salivary cortisol concentrations may be used to objectively detect individual variations in the acute physiological stress response we investigate individual variation in cortisol response to training milestones. Saliva samples were collected from a cohort of n = 96 yearling Flat racehorses, at the same training yard, across three timepoints at rest: before entering the training yard (n = 66), within three days of entry to the training yard (n = 67) and following 2-3 weeks in the training yard (n = 50). Salivary cortisol concentration was measured using an ELISA. There was no significant difference in cortisol concentration (ANOVA, P > 0.05) across the samples collected at timepoints at rest. Samples were also collected before and 30 minutes after exposure to three novel training events: first time long-reined (n = 6), first time backed by a jockey (n = 34), and first time ridden on the gallops (n = 10). Mean salivary cortisol concentration after all three novel training events was significantly higher than prior to the training event (Paired t-test, P <0.005). The ranges of post-event salivary cortisol concentration across all timepoints suggest individual variation in the measured stress response, reflecting individual differences in stress response to the early training environment. This measure may be used as an objective assessment of the stress response of Thoroughbred racehorses during training.

Integrative genomics analysis highlights functionally relevant genes for equine behaviour.

Behavioural plasticity enables horses entering an exercise training programme to adapt with reduced stress. We characterised SNPs associated with behaviour in yearling Thoroughbred horses using genomics analyses for two phenotypes: (1) handler-assessed coping with early training events [coping] (n = 96); and (2) variation in salivary cortisol concentration at the first backing event [cortisol] (n = 34). Using RNA-seq derived gene expression data for amygdala and hippocampus tissues from n = 2 Thoroughbred stallions, we refined the SNPs to those with functional relevance to behaviour by cross-referencing to the 500 most highly expressed genes in each tissue. The SNPs of high significance (q < 0.01) were in proximity to genes (coping - GABARAP, NDM, OAZ1, RPS15A, SPARCL1, VAMP2; cortisol - CEBPA, COA3, DUSP1, HNRNPH1, RACK1) with biological functions in social behaviour, autism spectrum disorder, suicide, stress-induced anxiety and depression, Alzheimer's disease, neurodevelopmental disorders, neuroinflammatory disease, fear-induced behaviours and alcohol and cocaine addiction. The strongest association (q = 0.0002) was with NDN, a gene previously associated with temperament in cattle. This approach highlights functionally relevant genes in the behavioural adaptation of Thoroughbred horses that will contribute to the development of genetic markers to improve racehorse welfare.

Robotic Resection of Gallbladder Cancer: A Single-Center Retrospective Comparative Study to Open Resection.

BACKGROUND: Minimally invasive surgery is gaining support for resection of gallbladder cancer (GBC). This study aims to compare operative and early outcomes of robotic resection (RR) to open resection (OR) from a single institution performing a high volume of robotic HPB surgery. METHODS: Twenty patients with GBC underwent RR from January 2013 to August 2019. Outcomes were compared to a historical control of 23 patients with OR. Radical cholecystectomy for suspected GBC and completion operations for incidental GBC after routine cholecystectomy were both included. RESULTS: Robotic resection had lower blood loss compared to OR (150 vs 350 mL, P = .002) and shorter postoperative length of stay (2.5 vs 6 days, P < .001), while median operative time was similar (193 vs 208 min, P = .604). There were no statistical differences in 30-day major complications or readmissions. No 30-day mortalities occurred. There was no statistical difference in survival trend (P = .438) or median lymph node harvest (5 vs 3, P = .189) for RR compared to OR. CONCLUSION: Robotic resection of GBC is safe and efficient, with lower length of hospital stay and blood loss compared to OR. Technical benefits of robotic-assisted surgery may prove advantageous though larger studies are still needed.

Treatment of spontaneously ruptured hepatocellular carcinoma: use of laparoscopic microwave ablation and washout.

BACKGROUND: Ruptured, or bleeding, hepatocellular carcinoma (rHCC) is a relatively rare disease presentation associated with high acute mortality rates. This study sought to evaluate outcomes following laparoscopic microwave ablation (MWA) and washout in rHCC. METHODS: A retrospective single-center review was performed to identify patients with rHCC (2008-2018). The treatment algorithm consisted of transarterial embolization (TAE) or trans-arterial chemoembolization (TACE) followed by laparoscopic MWA and washout. RESULTS: Fifteen patients with rHCC were identified (n = 5 single lesion, n = 5 multifocal disease, n = 5 extrahepatic metastatic disease). Median tumor size was 83 mm (range 5-228 mm), and 10 of 15 underwent TAE or TACE followed by laparoscopic MWA/washout. One patient required additional treatment for bleeding after MWA with repeat TAE. Thirty-day mortality was 6/15. For those patients discharged (n = 9), additional treatments included chemotherapy (n = 5), TACE (n = 3), and/or partial lobectomy (n = 2). Median follow-up was 18.2 months and median survival was 431 days (range 103-832) (one-year survival n = 7; two-year survival n = 4; three-year survival n = 3). Six patients had post-operative imaging from which one patient demonstrated recurrence. CONCLUSION: Using laparoscopic MWA with washout may offer advantage in the treatment of ruptured HCC. It not only achieves hemostasis but also could have oncologic benefit by targeting local tumor and decreasing peritoneal carcinomatosis risk.

Pathologic response translates to improved patient survival after locoregional treatment for hepatocellular carcinoma: the importance of minimally invasive microwave ablation.

BACKGROUND: Hepatectomy or transplantation can serve as curative treatment for early-stage hepatocellular carcinoma (HCC). Unfortunately, as progression remains a reality, locoregional therapies (LRT) for curative or bridging intent have become common. Efficacy on viability, outcomes, and accuracy of imaging should be defined to guide treatment. METHODS: Patients with HCC who underwent minimally invasive (MIS) microwave ablation (MWA), transarterial chemoembolization (TACE), or both (MIS-MWA-TACE) prior to hepatectomy or transplantation were identified. Tumor response and preoperative computed tomography (CT) accuracy were assessed and compared to pathology. Clinical and oncologic outcomes were compared between MIS-MWA, TACE, and MIS-MWA-TACE. RESULTS: Ninety-one patients, with tumors from all stages of the Barcelona Clinic Liver Cancer (BCLC) staging, were identified who underwent LRT prior to resection or transplant. Fourteen patients underwent MIS-MWA, 46 underwent TACE, and 31 underwent both neoadjuvantly. TACE population was older; otherwise, there were no differences in demographics. Fifty-seven percent of MIS-MWA patients had no viable tumor on pathology whereas only 13% of TACE patients and 29% of MIS-MWA-TACE patients had complete destruction (p = 0.004). The amount of remaining viable tumor in the explant was also significantly different between groups (MIS-MWA: 17.2%, TACE: 48.7%, MIS-MWA-TACE: 18.6%; p ≤ 0.0001). Compared with TACE, the MIS-MWA and MIS-MWA-TACE groups had significantly improved overall survival (MIS-MWA: 99.94 months, TACE: 75.35 months, MIS-MWA-TACE: 140 months; p = 0.017). This survival remained significant with stratification by tumor size. CT accuracy was found to be 50% sensitive and 86% specific for MIS-MWA. For TACE, CT had an 82% sensitivity and 33% specificity and for MIS-MWA-TACE, there was a 42% sensitivity and 78% specificity. CONCLUSION: The impact of locoregional treatments on tumor viability is distinct and superior with MIS-MWA alone and MIS-MWA-TACE offering significant advantage over TACE alone. The extent of this effect may be implicated in the improved overall survival.

A pre-operative platelet transfusion algorithm for patients with cirrhosis and hepatocellular carcinoma undergoing laparoscopic microwave ablation.

BACKGROUND: Thrombocytopenia is a common finding in patients with chronic liver disease. It is associated with poor clinical outcomes due to increased risk of bleeding after even minor procedures. We sought to determine an algorithm for pre-operative platelet transfusion in patients with cirrhosis and hepatocellular carcinoma (HCC) undergoing laparoscopic microwave ablation (MIS-MWA). METHODS: A retrospective review identified all patients with cirrhosis and HCC who underwent MIS-MWA at a single tertiary institution between 2007 and 2019. Demographics, pre-operative and post-operative laboratory values, transfusion requirements, and bleeding events were collected. The analyzed outcome of bleeding risk included any transfusion received intra-operatively or a transfusion or surgical intervention post-operatively. Logistic regression models were created to predict bleeding risk and identify patients who would benefit from pre-operative transfusion. RESULTS: There were 433 patients with cirrhosis and HCC who underwent MIS-MWA identified; of these, 353 patients had complete laboratory values and were included. Bleeding risk was evaluated through bivariate analysis of statistically and clinically significant variables. The accuracy of both models was substantiated through bootstrap validation for 500 iterations (model 1: ROC 0.8684, Brier score 0.0238; model 2: ROC 0.8363, Brier score 0.0252). The first model captured patients with both thrombocytopenia and anemia: platelet count < 60 × 109 / L (OR 7.75, p 0.012, CI 1.58-38.06) and hemoglobin < 10 gm/dL (OR 5.76, p 0.032, CI 1.16-28.63). The second model captured patients with thrombocytopenia without anemia: platelet count < 30 × 109/L (OR 8.41, p 0.05, CI 0.96-73.50) and hemoglobin > 10 gm/dL (OR 0.16, p 0.026, CI 0.031-0.80). CONCLUSION: The prediction of patients with cirrhosis and HCC requiring pre-operative platelet transfusions may help to avoid bleeding complications after invasive procedures. This study needs to be prospectively validated and ultimately may be beneficial in assessment of novel therapies for platelet-based clinical treatment in liver disease.

Oncologic Outcomes of Robotic Left Pancreatectomy for Pancreatic Adenocarcinoma: A Single-Center Comparison to Laparoscopic Resection.

BACKGROUND: Feasibility and safety of robotic surgery for pancreatic disease has been well demonstrated; however, there is scarce literature on long-term oncologic outcomes. We compared perioperative and oncologic outcomes between robotic left pancreatectomy (RLP) and laparoscopic left pancreatectomy (LLP) for pancreatic adenocarcinoma. METHODS: A retrospective review evaluated left pancreatectomies performed for pancreatic adenocarcinoma from 2009 to 2019 in a tertiary institution. Baseline characteristics, operative and oncologic outcomes were compared between RLP and LLP. RESULTS: There were 75 minimally invasive left pancreatectomy cases for pancreatic adenocarcinoma identified of which 33 cases were done robotically and 42 laparoscopically. Baseline characteristics demonstrated no difference in gender, age, BMI, T stage, N stage, neoadjuvant, or adjuvant chemotherapy. An analysis of operative variables demonstrated no difference in blood loss, increased duration, and higher lymph node yield with RLP (20 vs 12; P = .0029). Postoperatively, both cohorts had 30% pancreatic fistulas and no difference in complications. There were no differences in length of stay (LOS), 30- or 90-day readmission rates, or 90-day mortality. The analysis of oncologic outcomes demonstrated similar R0 resections (RLP: 72% vs OLP: 67%), recurrence rates (RLP: 36% vs OLP: 41%), and time to recurrence (RLP: 324 vs OLP 218 days). There was increased survival in the RLP cohort that was not significant (32 vs 19 months). CONCLUSION: This analysis demonstrates RLP is at least equivalent to LLP in perioperative and oncologic outcomes. The significantly higher lymph node yield and trend toward an improved survival suggests oncologic advantage. Randomized controlled studies are needed to clarify benefit.

Use of Artificial Intelligence Deep Learning to Determine the Malignant Potential of Pancreatic Cystic Neoplasms With Preoperative Computed Tomography Imaging.

BACKGROUND: Society consensus guidelines are commonly used to guide management of pancreatic cystic neoplasms (PCNs). However, downsides of these guidelines include unnecessary surgery and missed malignancy. The aim of this study was to use computed tomography (CT)-guided deep learning techniques to predict malignancy of PCNs. MATERIALS AND METHODS: Patients with PCNs who underwent resection were retrospectively reviewed. Axial images of the mucinous cystic neoplasms were collected and based on final pathology were assigned a binary outcome of advanced neoplasia or benign. Advanced neoplasia was defined as adenocarcinoma or intraductal papillary mucinous neoplasm with high-grade dysplasia. A convolutional neural network (CNN) deep learning model was trained on 66% of images, and this trained model was used to test 33% of images. Predictions from the deep learning model were compared to Fukuoka guidelines. RESULTS: Twenty-seven patients met the inclusion criteria, with 18 used for training and 9 for model testing. The trained deep learning model correctly predicted 3 of 3 malignant lesions and 5 of 6 benign lesions. Fukuoka guidelines correctly classified 2 of 3 malignant lesions as high risk and 4 of 6 benign lesions as worrisome. Following deep learning model predictions would have avoided 1 missed malignancy and 1 unnecessary operation. DISCUSSION: In this pilot study, a deep learning model correctly classified 8 of 9 PCNs and performed better than consensus guidelines. Deep learning can be used to predict malignancy of PCNs; however, further model improvements are necessary before clinical use.

Pure and Hybrid Deep Learning Models can Predict Pathologic Tumor Response to Neoadjuvant Therapy in Pancreatic Adenocarcinoma: A Pilot Study.

BACKGROUND: Neoadjuvant therapy may improve survival of patients with pancreatic adenocarcinoma; however, determining response to therapy is difficult. Artificial intelligence allows for novel analysis of images. We hypothesized that a deep learning model can predict tumor response to NAC. METHODS: Patients with pancreatic cancer receiving neoadjuvant therapy prior to pancreatoduodenectomy were identified between November 2009 and January 2018. The College of American Pathologists Tumor Regression Grades 0-2 were defined as pathologic response (PR) and grade 3 as no response (NR). Axial images from preoperative computed tomography scans were used to create a 5-layer convolutional neural network and LeNet deep learning model to predict PRs. The hybrid model incorporated decrease in carbohydrate antigen 19-9 (CA19-9) of 10%. Accuracy was determined by area under the curve. RESULTS: A total of 81 patients were included in the study. Patients were divided between PR (333 images) and NR (443 images). The pure model had an area under the curve (AUC) of .738 (P < .001), whereas the hybrid model had an AUC of .785 (P < .001). CA19-9 decrease alone was a poor predictor of response with an AUC of .564 (P = .096). CONCLUSIONS: A deep learning model can predict pathologic tumor response to neoadjuvant therapy for patients with pancreatic adenocarcinoma and the model is improved with the incorporation of decreases in serum CA19-9. Further model development is needed before clinical application.

Faster Return to Intended Oncologic Treatment (RIOT) After Trisectionectomy Does Not Translate to Better Outcomes.

BACKGROUND: Resection with trisectionectomy may necessitate liver molding for adequate future liver remnant (FLR), and subsequent complications can impact return to intended oncologic therapy (RIOT). This study evaluated whether a difference in RIOT exists with the use of molding and between liver molding techniques (associating liver partition and portal vein ligation for staged hepatectomy [ALPPS] and portal vein embolization [PVE]) with trisectionectomy. METHODS: A retrospective review evaluated trisectionectomies for malignancy. Outcomes were compared with and without molding, and RIOT was determined. RESULTS: Fifty-one patients underwent trisectionectomy: 11 ALPPS, 14 PVE, 26 without molding. 73% of ALPPS, 64% of PVE, and 58% without molding achieved RIOT (P = .971). There were no differences found in baseline characteristics, R0 rate, length of stay, readmission, complications, or mortality. Time to RIOT was significantly different (ALPPS: 3.3 months; PVE: 5.2 months; none: 2.4 months, P = .0203). There were no differences in recurrence or survival. CONCLUSIONS: Liver molding should not cause apprehension as there are no differences in achieving RIOT. Although technique alters time to RIOT, this does not translate into improved outcomes, implicating disease biology, and regeneration stimulus.

Vertical Compliance: A novel method of reporting patient specific ERAS compliance for real-time risk assessment.

BACKGROUND: ERAS protocol compliance is traditionally measured and reported as 'items compliance' which is retrospective longitudinal adherence of all patients to an index element. Reporting in this manner is restrictive and cannot impact care in real-time. In order to modify behavior effectively and instantaneously, we seek to introduce the novel concept of "vertical compliance". METHODS: Vertical compliance is defined as assessment of an individual's compliance along his/her own surgical pathway as ERAS index items are encountered. Prospectively entered data from the ERAS Interactive Audit System (EIAS) database was queried for all patients who underwent pancreatic or hepatic resections. Individual linear regression models were generated using compliance data from 46 ERAS index items against LOS. Significant items with p value < 0.05 were compiled into multivariable linear regression models; each with a unique coefficient that could be used to predict effect on LOS as well as control for the effect of the other items in the model. RESULTS: Compliance data from 483 patients who underwent pancreatic resections and 292 patients who underwent hepatic resections was compiled. Seven ERAS items for pancreatic resections and six ERAS items for hepatic resections were found to significantly impact LOS. Regression models were created for each of the items in an additive fashion. Calculations to determine predicted LOS as a patient progressed through a pathway was able to be determined. CONCLUSION: Vertical compliance is a novel metric, described in this study, that can provide significant and accurate patient-specific risk prediction to impact care in real-time. This can allow for creation of a variable echelon such that pathway items can be ranked by importance on clinical outcome effect and patient progress can be monitored and altered.

Polyhydroxyphenylvalerate/polycaprolactone nanofibers improve the life-span and mechanoresponse of human IPSC-derived cortical neuronal cells.

The physico-chemical characteristics of the extracellular matrix (ECM) cause mechanical cues that could elicit responses in the survival rate of cortical neuronal cells. Efficient neurite outgrowth in vitro, is critical for successful cultivation of cortical neuronal cells and the potential for attempts at regeneration of the central nervous system (CNS) in vivo. Relatively soft and hydrophilic, microbially synthesized aromatic polyester, polyhydroxyphenylvalerate (PHPV) was blended 50:50 with the stiff and hydrophobic polycaprolactone (PCL) and electrospun in microfibers for use in a 3D (CellCrown™) configuration and in a 2D coverslip coated configuration. This blend allows a 2.3-fold increase in the life-span of human induced pluripotent stem derived cortical neuronal cells (hiPS) compared to pure PCL fibers. HiPS-derived cortical neuronal cells grown on PHPV/PCL fibers show a 3.8-fold higher cumulative neurite elaboration compared to neurites grown on PCL fibers only. 96% of cortical neuronal cells die after 8 days of growth when plated on PCL fibers alone while >83% and 55% are alive on PHPV/PCL fibers on day 8 and day 17, respectively. An increased migration rate of cortical neuronal cells is also promoted by the blend compared to the PCL fibers alone. The critical survival rate improvement of hiPS derived cortical neuronal cells on PHPV/PCL blend holds promise in using these biocompatible nanofibers as implantable materials for regenerative purposes of an active cortical neuronal population after full maturation in vitro.

Robotic pancreaticoduodenectomy may offer improved oncologic outcomes over open surgery: a propensity-matched single-institution study.

BACKGROUND: The robotic platform in pancreatic disease has gained popularity in the hepatobiliary community due to significant advantages it technically offers over conventional open and laparoscopic techniques. Despite promising initial studies, there remains scant literature on operative and oncologic outcomes of robotic pancreaticoduodenectomy (RPD) for pancreatic adenocarcinoma. METHODS: A retrospective review evaluated all RPD performed for pancreatic adenocarcinoma from 2008 to 2019 in a single tertiary institution. RPD cases were matched to open cases (OPD) by demographic and oncologic characteristics and outcomes compared using Mann-Whitney U test, log rank tests, and Kaplan-Meier methods. RESULTS: Thirty-eight RPD cases were matched to 38 OPD. RPD had significantly higher lymph node (LN) yield (21.5 vs 13.5; p = 0.0036) and no difference in operative time or estimated blood loss (EBL). RPD had significantly lower rate of delayed gastric emptying (DGE) (3% vs 32%; p = 0.0009) but no difference in leaks, infections, hemorrhage, urinary retention ,or ileus. RPD had significantly shorter length of stay (LOS) (7.5 vs. 9; p = 0.0209). There were no differences in 30- or 90-day readmissions or 90-day mortality. There was an equivalent R0 resection rate and LN positivity ratio. There was a trend towards improved median overall survival in RPD (30.4 vs. 23.0 months; p = 0.1105) and longer time to recurrence (402 vs. 284 days; p = 0.7471). OPD had two times the local recurrent rate (16% vs. 8%) but no difference in distant recurrence. CONCLUSIONS: While the feasibility and safety of RPD has been demonstrated, the impact on oncologic outcomes had yet to be investigated. We demonstrate that RPD not only offers similar if not superior immediate post-operative benefit by decreasing DGE but more importantly may offer improved oncologic outcomes. The significantly higher LN yield and decreased inflammatory response demonstrated in robotic surgery may improve overall survival.

Novel use of REDCap to develop an advanced platform to display predictive analytics and track compliance with Enhanced Recovery After Surgery for pancreaticoduodenectomy.

BACKGROUND: Prediction models are increasingly being used with clinical practice guidelines to inform decision making. Enhanced Recovery After Surgery (ERAS®) protocols are standardized care pathways that incorporate evidence-based practices to improve patient outcomes. Predictive analytics incorporated within a data management system, such as Research Electronic Data Capture (REDCap), may help clinicians estimate risk probabilities and track compliance with standardized care practices. METHODS: Predictive models were developed from retrospective data on 400 patients who underwent pancreaticoduodenectomy from 2008 through 2014. The REDCap was programmed to display predictive analytics and create a data tracking system that met ERAS guidelines. Based on predictive scores for serious complication, 30-day readmission, and 30-day mortality, we developed targeted interventions to decrease readmissions and postoperative laboratory tests. RESULTS: Predictive models demonstrated a receiver-operating characteristic area (ROC) ranges of 641-856. After implementing the REDCap platform, the readmission rate for high-risk patients decreased 15.8% during the initial three months following ERAS implementation. Based on predictive outputs, patients with a low-risk score received a limited set of postoperative laboratory tests. Targeted interventions to decrease hospital readmission for high-risk patients included home care orders and post-discharge instructions. CONCLUSIONS: The REDCap platform offers hospitals a practical option to display predictive analytics and create a data tracking program that meets ERAS guidelines. Prediction models programmed into REDCap offer clinicians a support tool to assess the probability of patient outcomes. Risk calculations based on predictive scores enabled clinicians to titrate postoperative laboratory tests and develop post-discharge home care orders.

A novel 3-dimensional electromagnetic guidance system increases intraoperative microwave antenna placement accuracy.

BACKGROUND: Failure to locate lesions and accurately place microwave antennas can lead to incomplete tumor ablation. The Emprint™ SX Ablation Platform employs real-time 3D-electromagnetic spatial antenna tracking to generate intraoperative laparoscopic antenna guidance. We sought to determine whether Emprint™ SX affected time/accuracy of antenna-placement in a laparoscopic training model. METHODS: Targets (7-10 mm) were set in agar within a laparoscopic training device. Novices (no surgical experience), intermediates (surgical residents), and experts (HPB-surgeons) were asked to locate and hit targets using a MWA antenna (10-ultrasound only, 10-Emprint™ SX). Time to locate target, number of attempts to hit the target, first-time hit rate, and time from initiating antenna advance to hitting the target were measured. RESULTS: Participants located 100% of targets using ultrasound, with experts taking significantly less time than novices and intermediates. Using ultrasound only, successful hit-rates were 70% for novices and 90% for intermediates and experts. Using Emprint™ SX, successful hit rates for all 3-groups were 100%, with significantly increased first-time hit-rates and reduced time required to hit targets compared to ultrasound only. DISCUSSION: Emprint™ SX significantly improved accuracy and speed of antenna-placement independent of experience, and was particularly beneficial for novice users.

REST is a hypoxia-responsive transcriptional repressor.

Cellular exposure to hypoxia results in altered gene expression in a range of physiologic and pathophysiologic states. Discrete cohorts of genes can be either up- or down-regulated in response to hypoxia. While the Hypoxia-Inducible Factor (HIF) is the primary driver of hypoxia-induced adaptive gene expression, less is known about the signalling mechanisms regulating hypoxia-dependent gene repression. Using RNA-seq, we demonstrate that equivalent numbers of genes are induced and repressed in human embryonic kidney (HEK293) cells. We demonstrate that nuclear localization of the Repressor Element 1-Silencing Transcription factor (REST) is induced in hypoxia and that REST is responsible for regulating approximately 20% of the hypoxia-repressed genes. Using chromatin immunoprecipitation assays we demonstrate that REST-dependent gene repression is at least in part mediated by direct binding to the promoters of target genes. Based on these data, we propose that REST is a key mediator of gene repression in hypoxia.

Normal nerve striations are altered in the trembler-J mouse, a model of Charcot-Marie-Tooth disease.

INTRODUCTION: This study was initiated because it was noted that the peripheral nerves of Trembler-J mice (a model of human Charcot-Marie-Tooth disease) appear to lack normal striations. METHODS: We performed confocal microscopy of whole sciatic nerves and tested the effect of axial stress on impulse conduction. RESULTS: We found that the axons of mutant mice were longer than those of the wild-type (1.55 mm of axon/mm length of nerve vs. 1.28 mm/mm respectively). This axonal elongation altered the helical nerve striations (bands of Fontana). As nerves were stretched axially, the conduction distance became correspondingly shorter. The effect on latency was significantly greater in the more coiled nerves of Trembler-J mice (P = 0.038). CONCLUSIONS: The finding that mice with a mutated peripheral myelin protein 22 (PMP22) possess excessively long axons may be related to the excess Schwann cell numbers found in this disorder.

CX3CL1 is up-regulated in the rat hippocampus during memory-associated synaptic plasticity.

Several cytokines and chemokines are now known to play normal physiological roles in the brain where they act as key regulators of communication between neurons, glia, and microglia. In particular, cytokines and chemokines can affect cardinal cellular and molecular processes of hippocampal-dependent long-term memory consolidation including synaptic plasticity, synaptic scaling and neurogenesis. The chemokine, CX3CL1 (fractalkine), has been shown to modulate synaptic transmission and long-term potentiation (LTP) in the CA1 pyramidal cell layer of the hippocampus. Here, we confirm widespread expression of CX3CL1 on mature neurons in the adult rat hippocampus. We report an up-regulation in CX3CL1 protein expression in the CA1, CA3 and dentate gyrus (DG) of the rat hippocampus 2 h after spatial learning in the water maze task. Moreover, the same temporal increase in CX3CL1 was evident following LTP-inducing theta-burst stimulation in the DG. At physiologically relevant concentrations, CX3CL1 inhibited LTP maintenance in the DG. This attenuation in dentate LTP was lost in the presence of GABAA receptor/chloride channel antagonism. CX3CL1 also had opposing actions on glutamate-mediated rise in intracellular calcium in hippocampal organotypic slice cultures in the presence and absence of GABAA receptor/chloride channel blockade. Using primary dissociated hippocampal cultures, we established that CX3CL1 reduces glutamate-mediated intracellular calcium rises in both neurons and glia in a dose dependent manner. In conclusion, CX3CL1 is up-regulated in the hippocampus during a brief temporal window following spatial learning the purpose of which may be to regulate glutamate-mediated neurotransmission tone. Our data supports a possible role for this chemokine in the protective plasticity process of synaptic scaling.

Theta-burst stimulation of hippocampal slices induces network-level calcium oscillations and activates analogous gene transcription to spatial learning.

Over four decades ago, it was discovered that high-frequency stimulation of the dentate gyrus induces long-term potentiation (LTP) of synaptic transmission. LTP is believed to underlie how we process and code external stimuli before converting it to salient information that we store as 'memories'. It has been shown that rats performing spatial learning tasks display theta-frequency (3-12 Hz) hippocampal neural activity. Moreover, administering theta-burst stimulation (TBS) to hippocampal slices can induce LTP. TBS triggers a sustained rise in intracellular calcium [Ca2+]i in neurons leading to new protein synthesis important for LTP maintenance. In this study, we measured TBS-induced [Ca2+]i oscillations in thousands of cells at increasing distances from the source of stimulation. Following TBS, a calcium wave propagates radially with an average speed of 5.2 µm/s and triggers multiple and regular [Ca2+]i oscillations in the hippocampus. Interestingly, the number and frequency of [Ca2+]i fluctuations post-TBS increased with respect to distance from the electrode. During the post-tetanic phase, 18% of cells exhibited 3 peaks in [Ca2+]i with a frequency of 17 mHz, whereas 2.3% of cells distributed further from the electrode displayed 8 [Ca2+]i oscillations at 33 mHz. We suggest that these observed [Ca2+]i oscillations could lead to activation of transcription factors involved in synaptic plasticity. In particular, the transcription factor, NF-κB, has been implicated in memory formation and is up-regulated after LTP induction. We measured increased activation of NF-κB 30 min post-TBS in CA1 pyramidal cells and also observed similar temporal up-regulation of NF-κB levels in CA1 neurons following water maze training in rats. Therefore, TBS of hippocampal slice cultures in vitro can mimic the cell type-specific up-regulations in activated NF-κB following spatial learning in vivo. This indicates that TBS may induce similar transcriptional changes to spatial learning and that TBS-triggered [Ca2+]i oscillations could activate memory-associated gene expression.

Automated analysis of intracellular calcium fluorescence in rat organotypic hippocampal cultures: comparison to a manual, observer based method.

The technical advances made in microscopy have been matched by an increase in the application of fluorescent microscopy to answer scientific questions. While analysis of fluorescent microscopy images represents a powerful tool, one must be aware of the potential pitfalls. Frequently, the analysis methods applied involve at least some manual steps which are dependent on an observers input. Typically these steps are laborious and time consuming, but more importantly they are also influenced by an individual observer's bias, drift or imprecision. This raises concerns about the repeatability and definitiveness of the reported observations. Using calcium fluorescence in organotypic hippocampal slices as an experimental platform, we demonstrate the influence that manual interventions can exert on an analysis. We show that there is a high degree of variability between observers, and that this can be sufficient to affect the outcome of an experiment. To counter this, and to eliminate the disagreement between observers, we describe an alternative fully automated method which was created using EBImage package for R. This method has the added advantage of being fully open source and customisable, allowing for this approach to be applied to other analyses.