Human enteric nervous system progenitor transplantation improves functional responses in Hirschsprung disease patient-derived tissue.

OBJECTIVE: Hirschsprung disease (HSCR) is a severe congenital disorder affecting 1:5000 live births. HSCR results from the failure of enteric nervous system (ENS) progenitors to fully colonise the gastrointestinal tract during embryonic development. This leads to aganglionosis in the distal bowel, resulting in disrupted motor activity and impaired peristalsis. Currently, the only viable treatment option is surgical resection of the aganglionic bowel. However, patients frequently suffer debilitating, lifelong symptoms, with multiple surgical procedures often necessary. Hence, alternative treatment options are crucial. An attractive strategy involves the transplantation of ENS progenitors generated from human pluripotent stem cells (hPSCs). DESIGN: ENS progenitors were generated from hPSCs using an accelerated protocol and characterised, in detail, through a combination of single-cell RNA sequencing, protein expression analysis and calcium imaging. We tested ENS progenitors' capacity to integrate and affect functional responses in HSCR colon, after ex vivo transplantation to organotypically cultured patient-derived colonic tissue, using organ bath contractility. RESULTS: We found that our protocol consistently gives rise to high yields of a cell population exhibiting transcriptional and functional hallmarks of early ENS progenitors. Following transplantation, hPSC-derived ENS progenitors integrate, migrate and form neurons/glia within explanted human HSCR colon samples. Importantly, the transplanted HSCR tissue displayed significantly increased basal contractile activity and increased responses to electrical stimulation compared with control tissue. CONCLUSION: Our findings demonstrate, for the first time, the potential of hPSC-derived ENS progenitors to repopulate and increase functional responses in human HSCR patient colonic tissue.

Enteric nervous system regeneration and functional cure of experimental digestive Chagas disease with trypanocidal chemotherapy.

Digestive Chagas disease (DCD) is an enteric neuropathy caused by Trypanosoma cruzi infection. There is a lack of evidence on the mechanism of pathogenesis and rationales for treatment. We used a female C3H/HeN mouse model that recapitulates key clinical manifestations to study how infection dynamics shape DCD pathology and the impact of treatment with the front-line, anti-parasitic drug benznidazole. Curative treatment 6 weeks post-infection resulted in sustained recovery of gastrointestinal transit function, whereas treatment failure led to infection relapse and gradual return of DCD symptoms. Neuro/immune gene expression patterns shifted from chronic inflammation to a tissue repair profile after cure, accompanied by increased cellular proliferation, glial cell marker expression and recovery of neuronal density in the myenteric plexus. Delaying treatment until 24 weeks post-infection led to partial reversal of DCD, suggesting the accumulation of permanent tissue damage over the course of chronic infection. Our study shows that murine DCD pathogenesis is sustained by chronic T. cruzi infection and is not an inevitable consequence of acute stage denervation. The risk of irreversible enteric neuromuscular tissue damage and dysfunction developing highlights the importance of prompt diagnosis and treatment. These findings support the concept of treating asymptomatic, T. cruzi-infected individuals with benznidazole to prevent DCD development.

New quality outcome indicators for bone metastases: expert consensus analysis of patients, their families and specialist healthcare professionals.

OBJECTIVES: As workload increases, surgical care for patients with bone metastases is increasingly decentralised, with a shift in management away from primary bone tumour units to local centres. We must ensure that patients have similar outcomes regardless of where they receive their treatment. The aim was to develop and validate a set of quality outcome indicators (QOIs) to evaluate treatment success for patients undergoing surgery for bone metastases. METHODS: Outcome recommendations were adapted from the literature and field tested in a retrospective patient cohort to determine feasibility. The provisional outcome indicators were assessed during a modified RAND/Delphi consensus process by a group of patients, relatives and healthcare professionals with validated targets added. RESULTS: 1534 articles were reviewed. 38 quality objectives were extracted and assessed for feasibility using clinical records for 117 patients. 28 provisional outcome indicators proceeded to expert consensus and were reviewed by a group of 22 panellists including 10 patients and 4 relatives/carers. After two rounds, 15 QOIs were generated, with validated targets based on expert consensus. These included specific statements such as 'surgery improves pain and reduces the need for morphine, target: at follow-up, pain is documented in 80% of individuals and 50% of these have reduced need for morphine'. CONCLUSIONS: The published evidence and guidelines were adapted into a set of outcome indicators validated by patients, their family/carers and healthcare professionals. These can be used to compare care between centres and identify units of excellence in maximising good outcome after surgery for bone metastases.

Pulmonary vein dose and risk of atrial fibrillation in patients with non-small cell lung cancer following definitive radiotherapy: An NI-HEART analysis.

BACKGROUND AND PURPOSE: Symptomatic arrhythmia is common following radiotherapy for non-small cell lung cancer (NSCLC), frequently resulting in morbidity and hospitalization. Modern treatment planning technology theoretically allows sparing of cardiac substructures. Atrial fibrillation (AF) comprises the majority of post-radiotherapy arrhythmias, but efforts to prevent this cardiotoxicity have been limited as the causative cardiac substructure is not known. In this study we investigated if incidental radiation dose to the pulmonary veins (PVs) is associated with AF. MATERIAL AND METHODS: A single-centre study of patients completing contemporary (chemo)radiation for NSCLC, with modern planning techniques. Oncology, cardiology and death records were examined, and AF events were verified by a cardiologist. Cardiac substructures were contoured on planning scans for retrospective dose analysis. RESULTS: In 420 eligible patients with NSCLC treated with intensity-modulated (70%) or 3D-conformal (30%) radiotherapy with a median OS of 21.8 months (IQR 10.8-35.1), there were 26 cases of new AF (6%). All cases were grade 3 except two cases of grade 4. Dose metrics for both the left (V55) and right (V10) PVs were associated with the incidence of new AF. Metrics remained statistically significant after accounting for the competing risk of death and cardiovascular covariables for both the left (HR 1.02, 95%CI 1.00-1.03, p = 0.005) and right (HR 1.01 (95%CI 1.00-1.02, p = 0.033) PVs. CONCLUSION: Radiation dose to the PVs during treatment of NSCLC was associated with the onset of AF. Actively sparing the PVs during treatment planning could reduce the incidence of AF during follow-up, and screening for AF may be warranted for select cases.

Moving towards in pouch diagnostics for ostomy patients: exploiting the versatility of laser induced graphene sensors.

The development of a 3D printed sensor for direct incorporation within stoma pouches is described. Laser induced graphene scribed on either side of polyimide film served as the basis of a 2 electrode configuration that could be integrated within a disposable pouch sensor for the periodic monitoring of ileostomy fluid pH. The graphene sensors were characterised using electron microscopy, Raman spectroscopy, DekTak profilometry with the electrochemical properties investigated using both cyclic and square wave voltammetry. Adsorbed riboflavin was employed as a biocompatible redox probe for the voltammetric measurement of pH. The variation in peak position with pH was found to be linear over pH 3-8 with a sub Nernstian response (43 mV/pH). The adsorbed probe was found to be reversible and exhibited minimal leaching through repeated scanning. The performance of the system was assessed in a heterogeneous bacterial fermentation mixture simulating ileostomy fluid with the pH recorded before and after 96 h incubation. The peak profile in the bacterial medium provided an unambiguous signal free from interference with the calculated pH before and after incubation (pH 5.3 to 3.66) in good agreement with that obtained with commercial pH probes. Supplementary Information: The online version contains supplementary material available at 10.1007/s10853-023-08881-x.

Association between statin therapy dose intensity and radiation cardiotoxicity in non-small cell lung cancer: Results from the NI-HEART study.

INTRODUCTION: Radiation cardiotoxicity is a dose-limiting toxicity and major survivorship issue for patients with non-small cell lung cancer (NSCLC) completing curative-intent radiotherapy, however patients' cardiovascular baseline is not routinely optimised prior to treatment. In this study we examined the impact of statin therapy on overall survival and post-radiotherapy cardiac events. METHODS: Patients treated between 2015-2020 at a regional center were identified. Clinical notes were interrogated for baseline patient, tumor and cardiac details, and both follow-up cancer control and cardiac events. Three cardiologists verified cardiac events. Radiotherapy planning scans were retrieved for application of validated deep learning-based autosegmentation. Pre-specified Cox regression analyses were generated with varying degrees of adjustment for overall survival. Fine and Gray regression for the risk of cardiac events, accounting for the competing risk of death and cardiac covariables was undertaken. RESULTS: Statin therapy was prescribed to 59% of the 478 included patients. The majority (88%) of patients not prescribed a statin had at least one indication for statin therapy according to cardiovascular guidelines. In total, 340 patients (71%) died and 79 patients (17%) experienced a cardiac event. High-intensity (HR 0.68, 95%CI 0.50-0.91, p = 0.012) and medium-intensity (HR 0.70, 95%CI 0.51-0.97, p = 0.033) statin therapy were associated with improved overall survival after adjustment for patient, cancer, treatment, response and cardiovascular clinical factors. There were no consistent differences in the rate or grade of cardiac events according to statin intensity. CONCLUSIONS: Statin therapy is associated with improved overall survival in patients receiving curative-intent radiotherapy for NSCLC, and there is evidence of a dose-response relationship. This study highlights the importance of a pre-treatment cardiovascular risk assessment in this cohort. Further studies are needed to examine if statin therapy is cardioprotective in patients undergoing treatment for NSCLC with considerable incidental cardiac radiation dose and a low baseline cardiac risk.

A pulmonary vein atlas for radiotherapy planning.

BACKGROUND AND PURPOSE: Cardiac arrhythmia is a recognised potential complication of thoracic radiotherapy, but the responsible cardiac substructures for arrhythmogenesis have not been identified. Arrhythmogenic tissue is commonly located in the pulmonary veins (PVs) of cardiology patients with arrhythmia, however these structures are not currently considered organs-at-risk during radiotherapy planning. A standardised approach to their delineation was developed and evaluated. MATERIALS AND METHODS: The gross and radiological anatomy relevant to atrial fibrillation was derived from cardiology and radiology literature by a multidisciplinary team. A region of interest and contouring instructions for radiotherapy computed tomography scans were iteratively developed and subsequently evaluated. Radiation oncologists (n = 5) and radiation technologists (n = 2) contoured the PVs on the four-dimensional planning datasets of five patients with locally advanced lung cancer treated with 1.8-2.75 Gy fractions. Contours were compared to reference contours agreed by the researchers using geometric and dosimetric parameters. RESULTS: The mean dose to the PVs was 35% prescription dose. Geometric and dosimetric similarity of the observer contours with reference contours was fair, with an overall mean Dice of 0.80 ± 0.02. The right superior PV (mean DSC 0.83 ± 0.02) had better overlap than the left (mean DSC 0.80 ± 0.03), but the inferior PVs were equivalent (mean DSC of 0.78). The mean difference in mean dose was 0.79 Gy ± 0.71 (1.46% ± 1.25). CONCLUSION: A PV atlas with multidisciplinary approval led to reproducible delineation for radiotherapy planning, supporting the utility of the atlas in future clinical radiotherapy cardiotoxicity research encompassing arrhythmia endpoints.

Intravenous Tranexamic Acid Given at Femoral Fragility Fracture Surgery Reduces Blood Transfusion Requirements Fourfold.

AIMS: This study aims to determine whether intraoperative intravenous (IV) tranexamic acid (TXA) affects blood loss following the surgical management of femoral fragility fractures (FFF). METHODS: This was a single centre (university teaching hospital) non-randomised case-control study. There were 361 consecutive patients with FFF admitted over a 4-month period were included (mean age 81.4yrs; mean BMI 23.5; 73.7% female). Patient demographics, comorbidities, preoperative anticoagulation use, surgical management, intravenous TXA use, perioperative haemoglobin (Hb) and haematocrit, and requirement for blood transfusion were recorded. The primary outcome was postoperative blood transfusion requirement. Secondary outcomes included postoperative day one calculated blood loss (CBL) (using the Nadler and Gross formulae) and fall in Hb (percentage) from preoperative levels; and the incidence of thrombotic events and mortality up to 30 days. RESULTS: Groups were well matched in terms of patient demographics, comorbidities, preoperative anticoagulation use, injury types and surgical management. Intravenous TXA 1 g given at the beginning of surgery at the discretion of the operating team: 178 (49%) received TXA and 183 (51%) did not. The requirement for postoperative blood transfusion was significantly less in the TXA group: 15/178 (8.4%) compared to 58/183 (31.7%) (p < 0.001; Chi square). TXA significantly reduced both the percentage fall in Hb (mean difference 4.3%, p < 0.001) and the CBL (mean difference -222 ml, p < 0.001). There was no difference in VTE (2 vs 1, p = 0.620) or other thrombotic events (2 vs 0, p = 0.244) between groups. CONCLUSION: 1 g of intraoperative intravenous TXA during the surgical management of FFF was associated with reduced rate of transfusion, CBL and the percentage drop in HB. The use of TXA in this study was not randomised, so there could be un-quantifiable bias in the patient selection.

Synchronisation of apical constriction and cell cycle progression is a conserved behaviour of pseudostratified neuroepithelia informed by their tissue geometry.

Neuroepithelial cells balance tissue growth requirement with the morphogenetic imperative of closing the neural tube. They apically constrict to generate mechanical forces which elevate the neural folds, but are thought to apically dilate during mitosis. However, we previously reported that mitotic neuroepithelial cells in the mouse posterior neuropore have smaller apical surfaces than non-mitotic cells. Here, we document progressive apical enrichment of non-muscle myosin-II in mitotic, but not non-mitotic, neuroepithelial cells with smaller apical areas. Live-imaging of the chick posterior neuropore confirms apical constriction synchronised with mitosis, reaching maximal constriction by anaphase, before division and re-dilation. Mitotic apical constriction amplitude is significantly greater than interphase constrictions. To investigate conservation in humans, we characterised early stages of iPSC differentiation through dual SMAD-inhibition to robustly produce pseudostratified neuroepithelia with apically enriched actomyosin. These cultured neuroepithelial cells achieve an equivalent apical area to those in mouse embryos. iPSC-derived neuroepithelial cells have large apical areas in G2 which constrict in M phase and retain this constriction in G1/S. Given that this differentiation method produces anterior neural identities, we studied the anterior neuroepithelium of the elevating mouse mid-brain neural tube. Instead of constricting, mid-brain mitotic neuroepithelial cells have larger apical areas than interphase cells. Tissue geometry differs between the apically convex early midbrain and flat posterior neuropore. Culturing human neuroepithelia on equivalently convex surfaces prevents mitotic apical constriction. Thus, neuroepithelial cells undergo high-amplitude apical constriction synchronised with cell cycle progression but the timing of their constriction if influenced by tissue geometry.

Intervention rates are low after direct discharge from the Edinburgh trauma triage clinic: Outcomes of 6,688 patients.

AIM: The Edinburgh Trauma Triage clinic (TTC) is an established form of Virtual Fracture clinic (VFC) that permits the direct discharge of simple, isolated fractures from the Emergency Department (ED). Small, short-term cohort studies of similar systems have been published, but to detect low rates of complications requires a large study sample and longer-term follow-up. This study details the outcomes of all patients with injuries suitable for a direct discharge protocol over a four-year period, reviewed at a minimum of three years after attendance. PATIENTS: All TTC records between February 2014 and December 2017 were collated from a prospective database. Fractures of the radial head, little finger metacarpal, fifth metatarsal, toe phalanges and mallet finger injuries were included. TTC outcome, including any deviations from a well-established direct discharge protocol, were noted. All records were re-assessed at a minimum of 36 months after TTC triage (mean 54 months) to ascertain which injuries attended the trauma clinic after initial discharge. Reasons for attendance, the source of referral and any subsequent surgical procedures were identified. RESULTS: There were 6688 patients with fractures of the radial head (1861), little finger metacarpal (1621), fifth metatarsal (1916), toe phalanges (920) and mallet finger injuries (370). 298 (6%) patients were re-referred after direct discharge and attended trauma clinic at a mean time after injury of 11.9 weeks, of whom 11 (0.2%) underwent a surgical intervention. Serious adverse events, defined as those in which a patient may not have come to harm if early clinical review had been undertaken, occurred in 1 patient (0.01%). CONCLUSION: Intervention after direct discharge of simple pre-defined injuries of the elbow, hand and foot is low. Within a TTC system, patients with these injuries can be safely discharged without routine follow-up.

Validation of an established deep learning auto-segmentation tool for cardiac substructures in 4D radiotherapy planning scans.

Background: Emerging data suggest that dose-sparing several key cardiac regions is prognostically beneficial in lung cancer radiotherapy. The cardiac substructures are challenging to contour due to their complex geometry, poor soft tissue definition on computed tomography (CT) and cardiorespiratory motion artefact. A neural network was previously trained to generate the cardiac substructures using three-dimensional radiotherapy planning CT scans (3D-CT). In this study, the performance of that tool on the average intensity projection from four-dimensional (4D) CT scans (4D-AVE), now commonly used in lung radiotherapy, was evaluated. Materials and Methods: The 4D-AVE of n=20 patients completing radiotherapy for lung cancer 2015-2020 underwent manual and automated cardiac substructure segmentation. Manual and automated substructures were compared geometrically and dosimetrically. Two senior clinicians also qualitatively assessed the auto-segmentation tool's output. Results: Geometric comparison of the automated and manual segmentations exhibited high levels of similarity across parameters, including volume difference (11.8% overall) and Dice similarity coefficient (0.85 overall), and were consistent with 3D-CT performance. Differences in mean (median 0.2 Gy, range -1.6-0.3 Gy) and maximum (median 0.4 Gy, range -2.2-0.9 Gy) doses to substructures were generally small. Nearly all structures (99.5 %) were deemed to be appropriate for clinical use without further editing. Conclusions: Cardiac substructure auto-segmentation using a deep learning-based tool trained on a 3D-CT dataset was feasible on the 4D-AVE scan, meaning this tool is suitable for use on 4D-CT radiotherapy planning scans. Application of this tool would increase the practicality of routine clinical cardiac substructure delineation, and enable further cardiac radiation effects research.

Multiple Roles of Ret Signalling During Enteric Neurogenesis.

The majority of the enteric nervous system is formed by vagal neural crest cells which enter the foregut and migrate rostrocaudally to colonise the entire length of the gastrointestinal tract. Absence of enteric ganglia from the distal colon are the hallmark of Hirschsprung disease, a congenital disorder characterised by severe intestinal dysmotility. Mutations in the receptor tyrosine kinase RET have been identified in approximately 50% of familial cases of Hirschsprung disease but the cellular processes misregulated in this condition remain unclear. By lineage tracing neural crest cells in mice homozygous for a knock-in allele of Ret (Ret51/51), we demonstrate that normal activity of this receptor is required in vivo for the migration of enteric nervous system progenitors throughout the gut. In mutant mice, progenitors of enteric neurons fail to colonise the distal colon, indicating that failure of colonisation of the distal intestine is a major contributing factor for the pathogenesis of Hirschsprung disease. Enteric nervous system progenitors in the ganglionic proximal guts of mutant mice are also characterised by reduced proliferation and differentiation. These findings suggest that the functional abnormalities in Hirschsprung disease result from a combination of colonic aganglionosis and deficits in neuronal circuitry of more proximal gut segments. The reduced neurogenesis in the gut of Ret51/51 mutants was reproduced in the multilineage enteric nervous system progenitors isolated from these animals. Correction of the molecular defects of such progenitors fully restored their neurogenic potential in culture. These observations enhance our understanding of the pathogenesis of Hirschsprung disease and highlight potential approaches for its treatment.

Corrigendum: TALPID3/KIAA0586 Regulates Multiple Aspects of Neuromuscular Patterning During Gastrointestinal Development in Animal Models and Human.

[This corrects the article DOI: 10.3389/fnmol.2021.757646.].

Age, personal and family history are independently associated with venous thromboembolism following acute Achilles tendon rupture.

PURPOSE: The primary aim was to determine independent patient, injury and management-related factors associated with symptomatic venous thromboembolism (VTE) following acute Achilles tendon rupture (ATR). The secondary aim was to suggest a clinical VTE risk assessment tool for patients with acute ATR. METHODS: From 2010-2018, 984 consecutive adults (median age 47yrs, 73% [n = 714/984] male) sustaining an acute ATR were retrospectively identified. Ninety-five percent (n = 939/984) were managed non-operatively in a below-knee cast (52%, n = 507/984) or walking boot (44%, n = 432/984), with 5% (n = 45/984) undergoing primary operative repair (<6wks post-injury). VTE was diagnosed using local medical records and national imaging archives, reviewed at a mean 5yrs (range 1-10) post-injury. Multivariate logistic regression was performed to determine independent factors associated with VTE. RESULTS: The incidence of VTE within 90 days of ATR was 3.6% (n = 35/984; deep vein thrombosis 2.1% [n = 21/984], pulmonary embolism 1.9% [n = 19/984]), and the median time to VTE was 24 days (interquartile range 15-44). Age ≥50yrs (adjusted OR [aOR] 2.3, p = 0.027), personal history of VTE/thrombophilia (aOR 6.1, p = 0.009) and family history of VTE (aOR 20.9, p<0.001) were independently associated with VTE following ATR. These non-modifiable risk factors were incorporated into a VTE risk assessment tool. Only 23% of patients developing VTE (n = 8/35) had a relevant personal or family history, but incorporating age ≥50yrs into the VTE risk assessment tool (alongside personal and family history) identified 69% of patients with VTE (n = 24/35). Non weight-bearing for ≥2wks after ATR was also independently associated with VTE (aOR 3.2, p = 0.026). CONCLUSIONS: Age ≥50 years, personal history of VTE/thrombophilia and a positive family history were independently associated with VTE following ATR. Incorporating age into our suggested VTE risk assessment tool enhanced its sensitivity in identifying at-risk patients. Early weight-bearing in an appropriate orthosis may be beneficial to all patients in VTE risk reduction.

Dynamic integration of enteric neural stem cells in ex vivo organotypic colon cultures.

Enteric neural stem cells (ENSC) have been identified as a possible treatment for enteric neuropathies. After in vivo transplantation, ENSC and their derivatives have been shown to engraft within colonic tissue, migrate and populate endogenous ganglia, and functionally integrate with the enteric nervous system. However, the mechanisms underlying the integration of donor ENSC, in recipient tissues, remain unclear. Therefore, we aimed to examine ENSC integration using an adapted ex vivo organotypic culture system. Donor ENSC were obtained from Wnt1cre/+;R26RYFP/YFP mice allowing specific labelling, selection and fate-mapping of cells. YFP+ neurospheres were transplanted to C57BL6/J (6-8-week-old) colonic tissue and maintained in organotypic culture for up to 21 days. We analysed and quantified donor cell integration within recipient tissues at 7, 14 and 21 days, along with assessing the structural and molecular consequences of ENSC integration. We found that organotypically cultured tissues were well preserved up to 21-days in ex vivo culture, which allowed for assessment of donor cell integration after transplantation. Donor ENSC-derived cells integrated across the colonic wall in a dynamic fashion, across a three-week period. Following transplantation, donor cells displayed two integrative patterns; longitudinal migration and medial invasion which allowed donor cells to populate colonic tissue. Moreover, significant remodelling of the intestinal ECM and musculature occurred upon transplantation, to facilitate donor cell integration within endogenous enteric ganglia. These results provide critical evidence on the timescale and mechanisms, which regulate donor ENSC integration, within recipient gut tissue, which are important considerations in the future clinical translation of stem cell therapies for enteric disease.

Local association of Trypanosoma cruzi chronic infection foci and enteric neuropathic lesions at the tissue micro-domain scale.

Digestive Chagas disease (DCD) is an enteric neuropathy caused by Trypanosoma cruzi infection. The mechanism of pathogenesis is poorly understood and the lack of a robust, predictive animal model has held back research. We screened a series of mouse models using gastrointestinal tracer assays and in vivo infection imaging systems to discover a subset exhibiting chronic digestive transit dysfunction and significant retention of faeces in both sated and fasted conditions. The colon was a specific site of both tissue parasite persistence, delayed transit and dramatic loss of myenteric neurons as revealed by whole-mount immunofluorescence analysis. DCD mice therefore recapitulated key clinical manifestations of human disease. We also exploited dual reporter transgenic parasites to home in on locations of rare chronic infection foci in the colon by ex vivo bioluminescence imaging and then used fluorescence imaging in tissue microdomains to reveal co-localisation of infection and enteric nervous system lesions. This indicates that long-term T. cruzi-host interactions in the colon drive DCD pathogenesis, suggesting that the efficacy of anti-parasitic chemotherapy against chronic disease progression warrants further pre-clinical investigation.

Automated computational analysis reveals structural changes in the enteric nervous system of nNOS deficient mice.

Neuronal nitric oxide synthase (nNOS) neurons play a fundamental role in inhibitory neurotransmission, within the enteric nervous system (ENS), and in the establishment of gut motility patterns. Clinically, loss or disruption of nNOS neurons has been shown in a range of enteric neuropathies. However, the effects of nNOS loss on the composition and structure of the ENS remain poorly understood. The aim of this study was to assess the structural and transcriptional consequences of loss of nNOS neurons within the murine ENS. Expression analysis demonstrated compensatory transcriptional upregulation of pan neuronal and inhibitory neuronal subtype targets within the Nos1-/- colon, compared to control C57BL/6J mice. Conventional confocal imaging; combined with novel machine learning approaches, and automated computational analysis, revealed increased interconnectivity within the Nos1-/- ENS, compared to age-matched control mice, with increases in network density, neural projections and neuronal branching. These findings provide the first direct evidence of structural and molecular remodelling of the ENS, upon loss of nNOS signalling. Further, we demonstrate the utility of machine learning approaches, and automated computational image analysis, in revealing previously undetected; yet potentially clinically relevant, changes in ENS structure which could provide improved understanding of pathological mechanisms across a host of enteric neuropathies.

Prostate cancer metastasis to the testis: an unexpected presentation of a solitary recurrence.

A 74-year-old man with a history of prostate cancer was referred to the urology team with a new left sided testicular lump. He had a background of prostate cancer 4 years previous which had been treated with external beam radiotherapy and androgen deprivation therapy, both of which had been completed. Concurrently, he also had evidence of biochemical recurrence of prostate cancer with a rising prostate-specific antigen (PSA). He underwent a left radical orchidectomy. Following histopathological analysis, this was found to be metastatic spread from his prostate cancer. Subsequent staging showed no evidence of metastatic spread elsewhere. The patient made a good recovery following surgery and his PSA levels returned to undetectable levels. He received no further treatment for metastatic prostate cancer.

Combined treatment with enteric neural stem cells and chondroitinase ABC reduces spinal cord lesion pathology.

BACKGROUND: Spinal cord injury (SCI) presents a significant challenge for the field of neurotherapeutics. Stem cells have shown promise in replenishing the cells lost to the injury process, but the release of axon growth-inhibitory molecules such as chondroitin sulfate proteoglycans (CSPGs) by activated cells within the injury site hinders the integration of transplanted cells. We hypothesised that simultaneous application of enteric neural stem cells (ENSCs) isolated from the gastrointestinal tract, with a lentivirus (LV) containing the enzyme chondroitinase ABC (ChABC), would enhance the regenerative potential of ENSCs after transplantation into the injured spinal cord. METHODS: ENSCs were harvested from the GI tract of p7 rats, expanded in vitro and characterised. Adult rats bearing a contusion injury were randomly assigned to one of four groups: no treatment, LV-ChABC injection only, ENSC transplantation only or ENSC transplantation+LV-ChABC injection. After 16 weeks, rats were sacrificed and the harvested spinal cords examined for evidence of repair. RESULTS: ENSC cultures contained a variety of neuronal subtypes suitable for replenishing cells lost through SCI. Following injury, transplanted ENSC-derived cells survived and ChABC successfully degraded CSPGs. We observed significant reductions in the injured tissue and cavity area, with the greatest improvements seen in the combined treatment group. ENSC-derived cells extended projections across the injury site into both the rostral and caudal host spinal cord, and ENSC transplantation significantly increased the number of cells extending axons across the injury site. Furthermore, the combined treatment resulted in a modest, but significant functional improvement by week 16, and we found no evidence of the spread of transplanted cells to ectopic locations or formation of tumours. CONCLUSIONS: Regenerative effects of a combined treatment with ENSCs and ChABC surpassed either treatment alone, highlighting the importance of further research into combinatorial therapies for SCI. Our work provides evidence that stem cells taken from the adult gastrointestinal tract, an easily accessible source for autologous transplantation, could be strongly considered for the repair of central nervous system disorders.