Terpenes in Cannabis sativa Inhibit Capsaicin Responses in Rat DRG Neurons via Na+/K+ ATPase Activation.

Terpenes in Cannabis sativa exert analgesic effects, but the mechanisms are uncertain. We examined the effects of 10 terpenes on capsaicin responses in an established model of neuronal hypersensitivity. Adult rat DRG neurons cultured with neurotrophic factors NGF and GDNF were loaded with Fura2AM for calcium imaging, and treated with individual terpenes or vehicle for 5 min, followed by 1 µMol capsaicin. In vehicle treated control experiments, capsaicin elicited immediate and sustained calcium influx. Most neurons treated with terpenes responded to capsaicin after 6-8 min. Few neurons showed immediate capsaicin responses that were transient or normal. The delayed responses were found to be due to calcium released from the endoplasmic reticulum, as they were maintained in calcium/magnesium free media, but not after thapsigargin pre-treatment. Terpene inhibition of calcium influx was reversed after washout of medium, in the absence of terpenes, and in the presence of the Na+/K+ ATPase inhibitor ouabain, but not CB1 or CB2 receptor antagonists. Thus, terpenes inhibit capsaicin evoked calcium influx by Na+/K+ ATPase activation. Immunofluorescence showed TRPV1 co-expression with α1ß1 Na+/K+ ATPase in most neurons while others were either TRPV1 or α1ß1 Na+/K+ ATPase positive.

The Endocannabinoid Analgesic Entourage Effect: Investigations in Cultured DRG Neurons.

Background: The endocannabinoid 2-Arachidonyl glycerol (2-AG) exerts dose-related anti-nociceptive effects, which are potentiated by the related but inactive 2-palmitoyl glycerol (2-PG) and 2-linoleoyl glycerol (2-LG). This potentiation of analgesia and other in vivo measures was described as the "entourage effect". We investigated this effect on TRPV1 signalling in cultured dorsal root ganglion (DRG) nociceptors. Methods: Adult rat DRG neurons were cultured in medium containing NGF and GDNF at 37°C. 48 h later cultures were loaded with 2 µM Fura2AM for calcium imaging, and treated with 2-AG, 2-PG and 2-LG, individually or combined, for 5 min, followed by 1 µMol capsaicin. The amplitude and latency of capsaicin responses were measured (N=3-7 rats, controls N=16), and analysed. Results: In controls, 1 µMol capsaicin elicited immediate calcium influx in a subset of neurons, with average latency of 1.27 ± 0.2 s and amplitude of 0.15 ± 0.01 Units. 2-AG (10-100 µMol) elicited calcium influx in some neurons. In the presence of 2-AG (0.001-100 µMol), capsaicin responses were markedly delayed in 64% neurons by up to 320 s (P<0.001). 2-PG increased capsaicin response latency at 0.1 nMol-100 µMol (P<0.001), in 60% neurons, as did 2-LG at 0.1-100 µMol (P<0.001), in 76% neurons. Increased capsaicin response latency due to 2-AG and 2-PG was sensitive to the CB2 but not to the CB1 receptor antagonist. Combined application of 1 µMol 2-AG, 5 µMol 2-PG and 10 µMol 2-LG, also resulted in significantly increased capsaicin response latency up to 281.5 ± 41.5 s (P<0.001), in 96% neurons, that was partially restored by the CB2, but not the CB1 antagonist. Conclusion: 2-AG, 2-LG and 2-PG significantly delayed TRPV1 signalling in the majority of capsaicin-sensitive DRG neurons, that was markedly increased following combined application. Further studies of these endocannabinoids are required to identify the underlying mechanisms.

Visual gaze patterns in trainee endoscopists - a novel assessment tool.

BACKGROUND: Colonoscopy proficiency is significantly influenced by skills achieved during training. Although assessment scores exist, they do not evaluate the impact of visual search strategies and their use is time and labour intensive. Eye-tracking has shown significant differences in visual gaze patterns (VGPs) between expert endoscopists with varying polyp detection rates, so may provide a means of automated assessment and guidance for trainees. This study aimed to assess the feasibility of eye-tracking as a novel assessment method for trainee endoscopists. METHODS: Eye-tracking glasses were used to record 26 colonoscopies from 12 endoscopy trainees who were assessed with directly observed procedural scores (DOPS), devised by the Joint Advisory Group (JAG) on GI endoscopy, and a visual analogue score of overall competence. A 'total weighted procedure score' (TWPS) was calculated from 1 to 20. Primary outcomes of fixation duration (FixD) and fixation frequency (FixF) were analysed according to areas of interest (AOIs) with the bowel surface and lumen represented by three concentric rings. Correlation was assessed using Pearson's coefficient. Significance was set at p<.050. RESULTS: Trainees displayed a significant positive correlation between TWPS and FixD (R = 0.943, p<.0001) and FixF (R = 0.936, p<.0001) in the anatomical bowel mucosa peripheries. Conversely, they had significant negative correlations between TWPS and the anatomical bowel lumen (FixD: R= -0.546, p=.004; FixF: R= -0.568, p=.002). CONCLUSIONS: Higher objective performance scores were associated with VGPs focussing on bowel mucosa. This is consistent with prior analysis showing peripheral VGPs correspond with higher polyp detection rates. Analysis of VGPs, therefore, has potential for training and assessment in colonoscopy.

Real World Evidence in Medical Cannabis Research.

BACKGROUND: Whilst access to cannabis-based medicinal products (CBMPs) has increased globally subject to relaxation of scheduling laws globally, one of the main barriers to appropriate patient access remains a paucity of high-quality evidence surrounding their clinical effects. DISCUSSION: Whilst randomised controlled trials (RCTs) remain the gold-standard for clinical evaluation, there are notable barriers to their implementation. Development of CBMPs requires novel approaches of evidence collection to address these challenges. Real world evidence (RWE) presents a solution to not only both provide immediate impact on clinical care, but also inform well-conducted RCTs. RWE is defined as evidence derived from health data sourced from non-interventional studies, registries, electronic health records and insurance data. Currently it is used mostly to monitor post-approval safety requirements allowing for long-term pharmacovigilance. However, RWE has the potential to be used in conjunction or as an extension to RCTs to both broaden and streamline the process of evidence generation. CONCLUSION: Novel approaches of data collection and analysis will be integral to improving clinical evidence on CBMPs. RWE can be used in conjunction or as an extension to RCTs to increase the speed of evidence generation, as well as reduce costs. Currently, there is an abundance of potential data however, whilst a number of platforms now exist to capture real world data it is important the right tools and analysis are utilised to unlock potential insights from these.

Cannabinoids in the landscape of cancer.

INTRODUCTION: Cannabinoids are a group of terpenophenolic compounds derived from the Cannabis sativa L. plant. There is a growing body of evidence from cell culture and animal studies in support of cannabinoids possessing anticancer properties. METHOD: A database search of peer reviewed articles published in English as full texts between January 1970 and April 2021 in Google Scholar, MEDLINE, PubMed and Web of Science was undertaken. References of relevant literature were searched to identify additional studies to construct a narrative literature review of oncological effects of cannabinoids in pre-clinical and clinical studies in various cancer types. RESULTS: Phyto-, endogenous and synthetic cannabinoids demonstrated antitumour effects both in vitro and in vivo. However, these effects are dependent on cancer type, the concentration and preparation of the cannabinoid and the abundance of receptor targets. The mechanism of action of synthetic cannabinoids, (-)-trans-Δ9-tetrahydrocannabinol (Δ9-THC) and cannabidiol (CBD) has mainly been described via the traditional cannabinoid receptors; CB1 and CB2, but reports have also indicated evidence of activity through GPR55, TRPM8 and other ion channels including TRPA1, TRPV1 and TRPV2. CONCLUSION: Cannabinoids have shown to be efficacious both as a single agent and in combination with antineoplastic drugs. These effects have occurred through various receptors and ligands and modulation of signalling pathways involved in hallmarks of cancer pathology. There is a need for further studies to characterise its mode of action at the molecular level and to delineate efficacious dosage and route of administration in addition to synergistic regimes.

Cannabis-based medicines and pain: a review of potential synergistic and entourage effects.

The recent legalization of medicinal cannabis in several jurisdictions has spurred the development of therapeutic formulations for chronic pain. Unlike pure delta-9-tetrahydrocannabinol (THC), full-spectrum products contain naturally occurring cannabinoids and have been reported to show improved efficacy or tolerability, attributed to synergy between cannabinoids and other components in the cannabis plant. Although 'synergy' indicates that two or more active compounds may produce an additive or combined effect greater than their individual analgesic effect, potentiation of the biological effect of a compound by related but inactive compounds, in combination, was termed the 'entourage effect'. Here, we review current evidence for potential synergistic and entourage effects of cannabinoids in pain relief. However, definitive clinical trials and in vitro functional studies are still required.

Lay abstract Cannabis-based medicines have been used for millennia, and recent studies have identified their main constituents for pain relief, delta-9-tetrahydrocannabinol and cannabidiol (CBD). However, cannabis contains hundreds of other potentially active compounds, and their combined effects may underlie the reported preference of some patients for cannabinoid extracts, rather than pure delta-9-tetrahydrocannabinol. Further, cannabis-based drugs may interact with endocannabinoids, which are produced within the body and are related to the compounds found in cannabis. We have reviewed the evidence for cannabinoids in combination, and with other drugs, for pain relief. Although there is some evidence for an advantage of combinations, basic research and clinical studies are still required.

CBD Effects on TRPV1 Signaling Pathways in Cultured DRG Neurons.

INTRODUCTION: Cannabidiol (CBD) is reported to produce pain relief, but the clinically relevant cellular and molecular mechanisms remain uncertain. The TRPV1 receptor integrates noxious stimuli and plays a key role in pain signaling. Hence, we conducted in vitro studies, to elucidate the efficacy and mechanisms of CBD for inhibiting neuronal hypersensitivity in cultured rat sensory neurons, following activation of TRPV1. METHODS: Adult rat dorsal root ganglion (DRG) neurons were cultured and supplemented with the neurotrophic factors NGF and GDNF, in an established model of neuronal hypersensitivity. Neurons were stimulated with CBD (Adven 150, EMMAC Life Sciences) at 1, 10, 100 nMol/L and 1, 10 and 50 µMol/L, 48 h after plating. In separate experiments, DRG neurons were also stimulated with capsaicin with or without CBD (1 nMol/L to10 µMol/L), in a functional calcium imaging assay. The effects of the adenylyl cyclase activator forskolin and the calcineurin inhibitor cyclosporin were determined. We also measured forskolin-stimulated cAMP levels, without and after treatment with CBD, using a homogenous time-resolved fluorescence (HTRF) assay. The results were analysed using Mann-Whitney test. RESULTS: DRG neurons treated with 10 and 50 µMol/L CBD showed calcium influx, but not at lower doses. Neurons treated with capsaicin demonstrated robust calcium influx, which was dose-dependently reduced in the presence of low dose CBD (IC50 = 100 nMol/L). The inhibition or desensitization by CBD was reversed in the presence of forskolin and cyclosporin. Forskolin-stimulated cAMP levels were significantly reduced in CBD treated neurons. CONCLUSION: CBD at low doses corresponding to plasma concentrations observed physiologically inhibits or desensitizes neuronal TRPV1 signalling by inhibiting the adenylyl cyclase - cAMP pathway, which is essential for maintaining TRPV1 phosphorylation and sensitization. CBD also facilitated calcineurin-mediated TRPV1 inhibition. These mechanisms may underlie nociceptor desensitization and the therapeutic effect of CBD in animal models and patients with acute and chronic pain.

Pain, well-being, body image and cosmesis: a comparison of single-port and four-port laparoscopic cholecystectomy.

BACKGROUND: This study aims to compare post-operative pain, well-being, body image and cosmesis in SILS cholecystectomy and four-port laparoscopic cholecystectomy (FPLC). MATERIAL AND METHODS: Forty-two consecutive patients (15 SILS, 27 FPLC) undergoing elective cholecystectomy were included in the study. Peri-operative pain, well-being, body image and cosmesis were evaluated using validated assessment tools. RESULTS: Significantly lower pain scores were reported one week post-operatively in the SILS group (5.6 vs 8.3; p = 0.035). No significant difference was found in analgesic requirements, physical or mental well-being at any time interval. Significantly higher (favourable) body image questionnaire scores were reported in the SILS group at one week (5.4 v 4.5; p < 0.01), two weeks (5.6 vs 4.8; p < 0.01) and one month (5.7 vs 5.0; p < 0.01) post-operatively. CONCLUSION: SILS patients have significantly reduced one-week pain scores and there was no significant difference in well-being between the two groups. Patients who underwent SILS had improved body image and cosmesis. If both techniques are found to be equivalent concerning safety, cost, learning curve and availability, SILS may play a key role in the new era of patient choice.

A randomized controlled trial evaluating endoscopic and laparoscopic training in skills transfer for novices performing a simulated NOTES task.

BACKGROUND: The NOSCAR white paper lists training as an important step to the safe clinical application of natural orifice translumenal endoscopic surgery (NOTES). The aim of this randomized controlled trial was to evaluate whether training novices in either a laparoscopic or endoscopic simulator curriculum would affect performance in a NOTES simulator task. METHODS: A total of 30 third-year medical undergraduates were recruited. They were randomized to 3 groups: no training (control; n = 10), endoscopy training on a validated colonoscopy simulator protocol (n = 10), and training on a validated laparoscopy simulator curriculum (n = 10). All participants subsequently completed a simulated NOTES task, consisting of 7 steps, on the ELITE (endoscopic-laparoscopic interdisciplinary training entity) model. Performance was assessed as time taken to complete individual steps, overall task time, and number of errors. RESULTS: The endoscopy group was significantly faster than the control group at accessing the peritoneal cavity through the gastric incision (median 27 vs 78 s; P = .015), applying diathermy to the base of the appendix (median 103.5 vs 173 s; P = .014), and navigating to the gallbladder (median 76 vs 169.5 s; P = .049). Endoscopy participants completed the full NOTES procedure in a shorter time than the laparoscopy group (median 863 vs 2074 s; P < .001). CONCLUSION: This study highlights the importance of endoscopic training for a simulated NOTES task that involves both navigation and resection with operative maneuvers. Although laparoscopic training confers some benefit for operative steps such as applying diathermy to the gallbladder fossa, this was not as beneficial as training in endoscopy.

The effects of natural orifice translumenal endoscopic surgery (NOTES) on cardiorespiratory physiology: a systematic review.

INTRODUCTION: Despite the increasing awareness of natural orifice translumenal endoscopic surgery (NOTES), there remain obstacles to its wider implementation. We aim to examine the current evidence for the effects of variable uncontrolled insufflation pressures using either air or carbon dioxide in NOTES, as well the effects of NOTES procedures themselves, on physiological parameters. METHOD: We undertook a systematic review of all publications relating to the effects of NOTES on physiology, in particular compared with laparoscopy. RESULTS: Eleven reports were identified comprising comparative trials involving a total of 237 pigs. With one exception, no difference was found between the effect of NOTES and laparoscopic surgery on cardiopulmonary parameters. No difference was found also for the effect on inflammatory mediators such as interleukin-6. Two studies also assessed the effects choice of insufflation gas, with no change in inflammatory markers. Two further studies assessing the effects of variable pneumoperitoneal pressures found negative cardiorespiratory effects with higher pressures. DISCUSSION: Existing evidence is limited to small-sample animal trials but appears to show noninferiority for NOTES compared with laparoscopy. The development of a pressure regulation device for NOTES must be considered.

Single-incision Laparoscopic Surgery (SILS) in general surgery: a review of current practice.

Single-incision laparoscopic surgery (SILS) aims to eliminate multiple port incisions. Although general operative principles of SILS are similar to conventional laparoscopic surgery, operative techniques are not standardized. This review aims to evaluate the current use of SILS published in the literature by examining the types of operations performed, techniques employed, and relevant complications and morbidity. This review considered a total of 94 studies reporting 1889 patients evaluating 17 different general surgical operations. There were 8 different access techniques reported using conventional laparoscopic instruments and specifically designed SILS ports. There is extensive heterogeneity associated with operating methods and in particular ways of overcoming problems with retraction and instrumentation. Published complications, morbidity, and hospital length of stay are comparable to conventional laparoscopy. Although SILS provides excellent cosmetic results and morbidity seems similar to conventional laparoscopy, larger randomized controlled trials are needed to assess the safety and efficacy of this novel technique.

A hidden Markov model-based analysis framework using eye-tracking data to characterise re-orientation strategies in minimally invasive surgery.

Natural Orifice Translumenal Endoscopic Surgery (NOTES) represents a new paradigm in minimally invasive surgery whereby an operator uses a flexible instrument to navigate within an anatomical space. The flexible instrument is introduced via a natural body orifice such as the mouth to perform an operative intervention controlled by the surgeon without making incisions in the anterior abdominal wall. It differs from laparoscopic (traditional keyhole) surgery in two main ways: (1) the instruments used in NOTES are not rigid as in laparoscopic surgery and not specifically designed for this application; and (2) there are no incisions in the anterior abdominal wall. Disorientation during NOTES is a significant problem because as in laparoscopic surgery the operator typically uses a 2D camera-monitor interface for visualisation by the camera at the site of the operation. Furthermore, the positional cue offered by the external component (outside the body) of the camera is absent, and camera navigation is more cumbersome. When the operator is disorientated, it is important to be able to re-orientate quickly to minimise potential surgical errors. It is hypothesised that when surgeons become disorientated, there exist discrete patterns in psychophysical behaviour which are associated with effective re-orientation, and that these patterns are recognisable. In this study, we examine visual re-orientation behaviour in 18 subjects using eye-tracker data in a model comprised of selective image manipulation of everyday objects in a box trainer. We characterise effective behaviour using a fixation sequence similarity-based hidden Markov model. We show that the output of this algorithm is reliable in differentiating visual behavioural sequences, and that there are specific behavioural patterns and strategies associated with successful re-orientation in this model. Good re-orientation strategy appears to rely on identification and focus on a central object within the scene and judging position of its surrounding peripheral objects, suggesting integration of both geometric and feature information in a systematic way. Using selective, inconsistent feature cues for re-orientation were associated with less effective performance.