Gastric Mucosal Devitalization (GMD): Using the Porcine Model to Develop a Novel Endoscopic Bariatric Approach.

BACKGROUND AND AIMS: As the pig model has similar gastrointestinal anatomy and physiology to humans, we used pigs to create a gastric mucosal devitalization (GMD) model in preparation for clinical translation of this technique as an endoscopic bariatric therapy (EBT). The aims of this study were to determine the ablation parameters and technique for a successful, safe, and feasible large surface area GMD that produces weight loss. METHODS: We performed GMD using argon plasma coagulation (APC) in 3 phases. Phase 1 assessed the ablation energy required to accomplish selective mucosal ablation using ex vivo pig stomachs (n = 2). Phase 2 assessed the optimal percentage of mucosal surface area to be treated and was performed on 10 pigs. Phase 3 assessed feasibility, efficacy, and safety with 8 pigs randomized into GMD (n = 4) or sham (SH, n = 4) and survived for 1 month. Body weights (GMD, n = 4, SH, n = 4) were measured daily in phase 3 for 1 month, and relative body weights were calculated and analyzed using one-tailed Student's t-test. Percent body fat was compared between GMD and SH at baseline and 1 month post-GMD. RESULTS: Phase 1 identified the optimal ablation parameters (120 W) that were then used in phase 2. Phase 2 revealed a trend that was suggestive that the optimal percent surface area to ablate was similar to that which is removed at laparoscopic sleeve gastrectomy. In phase 3, GMD was performed over 70% surface area of the greater curvature of the stomach in four pigs. GMD pigs had significantly lower relative body weight increase compared to SH at 1 month (1.375 ± 0.085 vs 1.575 ± 0.047, p = 0.0435). MRI showed a significantly lower body fat mass at 1 month in GMD pigs (5.9 ± 0.4% vs 12.7 ± 2.3%, p = 0.026) compared to SH. CONCLUSIONS: GMD resulted in decreased weight gain in the GMD group as evidenced by a lower relative body weight at 1 month. GMD in an animal model appears to show promise as a potential weight loss therapy.

Gastric mucosal devitalization (GMD): translation to a novel endoscopic metabolic therapy.

Background and study aims The metabolic effects of bariatric surgery may partially result from removal of the gastric mucosa, an often underappreciated endocrine organ. Using argon plasma coagulation (APC), we may be able to selectively devitalize (ablate) the mucosa. The aim of this study was to identify the optimal tissue color that would correspond to selective gastric mucosal devitalization (GMD) using ex-vivo human stomach specimens. Patients and methods Stomach specimens were obtained at sleeve gastrectomy. Prior to APC application, a submucosal fluid cushion was created. APC was then applied over a 2 × 2-cm area to the fundus and body, aiming for the three indicator colors (white, golden, brown). Pathological analysis was then performed independently and in a blinded fashion by two pathologists to determine the depth of mucosal and submucosal percent thermal injury and mucosal percent cell death. Results Six patients were enrolled. There was a significant correlation between tissue color and mucosal percent thermal injury. The highest percent mucosal thermal injury was seen with brown (99.6 %, 95 % CI: 98.7, 100), followed by golden (92.5 %, 95 % CI: 85.5, 99.5), and then white (75.2 %, 95 % CI: 58.3, 92.1, P  < 0.01). Submucosal thermal injury was seen in 88.9 % of the slides. Greater than minimal submucosal injury (> 10 % depth) was found significantly more with brown tissue color (91.6 %) than golden (75 %) or white (33.3 %, P  < 0.05). However, 91.7 % of the entire sample set < 50 % injury. Conclusion GMD is achievable using APC without thermal injury to muscularis propria. A golden color results in sufficient mucosal injury with only superficial injury to the submucosa.

Gentamicin concentration gradients in scala tympani perilymph following systemic applications.

It has been shown in prior studies that round window membrane (RWM) application of gentamicin produced a robust basal-apical concentration gradient in the perilymph of scala tympani (ST) with peak concentrations in the basal turn of ST. These gradients potentially contribute to the clinical efficacy and safety of intratympanic gentamicin applications for the treatment of Ménière's disease. The present study aimed to establish the distribution of gentamicin along ST perilymph after systemic applications. Gentamicin sulfate was applied intravenously in the amounts of 100, 300 and 600 mg/kg body weight (BW) over a period of 3 h or as a 300 mg/kg BW subcutaneous bolus injection. At 3 and 5 h after the start of the application perilymph of ST was aspirated from the cochlea apex of the right and left cochlea, respectively, and 10 sequential 1-µl perilymph samples from the apex of each cochlea were quantitatively analyzed using a fluorescence polarization immunoassay. In contrast to local RWM delivery, systemic application of gentamicin resulted in the highest perilymph levels in the apex of the cochlea with decreasing concentrations towards the basal regions of ST. The absolute gentamicin concentrations increased with the amount of drug applied and time before sampling. While it is likely that the basal-apical gradient measured after local drug applications to the round window niche is the result of the direct uptake of drugs into the perilymph of the ST, distribution by diffusion and a very low perilymph flow towards the cochlear apex, computer simulations suggested that the apical-basal gradient observed with these systemic applications can be explained by higher entry rates of gentamicin in the apex compared to the basal turns of the cochlea. It is also possible that gentamicin enters perilymph indirectly from the blood via the endolymph. In this case the faster kinetics in apical turns could be due to the smaller cross-sectional area of ST relative to endolymph in the apical turns.

Dexamethasone levels and base-to-apex concentration gradients in the scala tympani perilymph after intracochlear delivery in the guinea pig.

HYPOTHESIS: To determine whether intracochlearly applied dexamethasone will lead to better control of drug levels, higher peak concentrations, and lower base-to-apex concentration gradients in the scala tympani (ST) of the guinea pig than after intratympanic (round window [RW]) application. BACKGROUND: Local application of drugs to the RW results in substantial variation of intracochlear drug levels and significant base-to-apex concentration gradients in ST. METHODS: Two microliters of dexamethasone-phosphate (10 mg/ml) were injected into ST either through the RW membrane, which was covered with 1% sodium hyaluronate gel or through a cochleostomy with a fluid tight seal of the micropipette. Perilymph was sequentially sampled from the apex at a single time point for each animal, at 20, 80, or 200 min after the injection ended. Results were mathematically interpreted by means of an established computer model and compared with previous experiments performed by our group with the same experimental techniques but using intratympanic applications. RESULTS: Single intracochlear injections of 20 minutes resulted in approximately 10 times higher peak concentrations (on average) than 2 to 3 hours of intratympanic application to the RW niche. Intracochlear drug levels were less variable and could be measured for over 220 minutes. Concentration gradients along the scala tympani were less pronounced. The remaining variability in intracochlear drug levels was attributable to perilymph and drug leak from the injection site. CONCLUSION: With significantly higher, less variable drug levels and smaller base-to-apex concentration gradients, intracochlear applications have advantages to intratympanic injections. For further development of this technique, it is of importance to control leaks of perilymph and drug from the injection site and to evaluate its clinical feasibility and associated risks.

A novel buoyancy technique optimizes simulated microgravity conditions for whole sensory organ culture in rotating bioreactors.

Whole-organ culture of a sensory organ in a rotating wall vessel bioreactor provides a powerful in vitro model for physiological and pathophysiological investigation as previously demonstrated for the postnatal inner ear. The model is of specific relevance as a tool for regeneration research. In the immature inner ear explant, the density was only 1.29 g/cm(3). The high density of 1.68 g/cm(3) of the functionally mature organ resulted in enhanced settling velocity and deviation from its ideal circular orbital path causing enhanced shear stress. The morphometric and physical properties, as well as the dynamic motion patterns of explants, were analyzed and numerically evaluated by an orbital path index. Application of a novel buoyancy bead technique resulted in a 6.5- to 14.8-fold reduction of the settling velocity. The deviation of the explant from its ideal circular orbital path was adjusted as indicated by an optimum value for the orbital path index (-1.0). Shear stress exerted on the inner ear explant was consequently reduced 6.4- to 15.0-fold. The culture conditions for postnatal stages were optimized, and the preconditions for transferring this in vitro model toward mature high-density stages established. This buoyancy technique may also be useful in tissue engineering of other high-density structures.

Whole organ culture of the postnatal sensory inner ear in simulated microgravity.

Among the three major biological in vitro models, cell culture, tissue culture, and organ culture, the latter provides the closest approximation to the in vivo situation, but also requires the most demanding culture conditions. Due to its small size and complex tissue architecture, the mammalian inner ear provides a particular challenge to the development of whole organ culture. Using a rotating bioreactor system with simulated microgravity conditions, the entire mouse inner ear organ can be maintained in culture for up to seven days with preservation of sensory organ morphology and robust marker protein expression in sensory hair cells. Controlled sensory cell lesions can be induced by the ototoxic agent, neomycin sulphate, as a toxicologic model of hair cell degeneration and hair cell loss. The results demonstrate that simulated microgravity organ culture of the inner ear affords an in vitro model for the investigation of developmental, regulatory, and differentiation processes, as well as toxicological, biotechnological, and pharmaceutical screening applications within the normal and pathologic sensory hearing organ.

Cochlear microdialysis for quantification of dexamethasone and fluorescein entry into scala tympani during round window administration.

Before new drugs for the treatment of inner ear disorders can be studied in controlled clinical trials, it is important that their pharmacokinetics be established in inner ear fluids. Microdialysis allows drug levels to be measured in perilymph without the volume disturbances and potential cerebrospinal fluid contamination associated with fluid sampling. The aims of this study were to show: (i) that despite low recovery rates from miniature dialysis probes, significant amounts of drug are removed from small fluid compartments, (ii) that dialysis sampling artifacts can be accounted for using computer simulations and (iii) that microdialysis allows quantification of the entry rates through the round window membrane (RWM) into scala tympani (ST). Initial experiments used microdialysis probes in small compartments in vitro containing sodium fluorescein. Stable concentrations were observed in large compartments (1000 microl) but significant concentration declines were observed in smaller compartments (100, 10 and 5.6 microl) comparable to the size of the inner ear. Computer simulations of these experiments closely approximated the experimental data. In in vivo experiments, sodium fluorescein 10 mg/ml and dexamethasone-dihydrogen-phosphate disodium salt 8 mg/ml were simultaneously applied to the RWM of guinea pigs. Perilymph concentration in the basal turn of ST was monitored using microdialysis. The fluorescein concentration reached after 200 min application (585+/-527 microg/ml) was approximately twice that of dexamethasone phosphate (291+/-369 microg/ml). Substantial variation in concentrations was found between animals by approximately a factor of 34 for fluorescein and at least 41 for dexamethasone phosphate. This is, to a large extent, thought to be the result of the RWM permeability varying in different animals. It was not caused by substance analysis variations, because two different analytic methods were used and the concentration ratio between the two substances remained nearly constant across the experiments and because differences were apparent for the repeated samples obtained in each animal. Interpretation of the results using computer simulations allowed RWM permeability to be quantified. It also demonstrated, however, that cochlear clearance values could not be reliably obtained with microdialysis because of the significant contribution of dialysis to clearance. The observed interanimal variation, e.g., in RWM permeability, is likely to be clinically relevant to the local application of drugs in patients.

In vitro expansion of human nasoseptal chondrocytes reveals distinct expression profiles of G1 cell cycle inhibitors for replicative, quiescent, and senescent culture stages.

In vitro expansion of chondrocytes for tissue-engineering applications is limited by forms of growth arrest known as quiescence and replicative senescence. At the molecular level cyclin-dependent kinase inhibitors (CDKIs) are involved in mediating growth arrest in the G1 phase of the cell cycle. Using ribonuclease protection assays and immunocytochemical staining methods, we quantitatively analyzed expression profiles of G1 cell cycle inhibitors at the mRNA and protein levels. These inhibitors included the CDKIs of the CIP/KIP family (p21CIP1 p27KIP1, and p57KIP2) and the INK4 family (p15INK4b, p16INK4a, p18INK4c, and p19INK4d) as well as the retinoblastoma protein-family (pRb, p107, and p130) and the tumor suppressor p53. Analysis was carried out in proliferating, quiescent, and senescent states of primary cultures of adult human nasoseptal chondrocytes. The most pronounced effect (p < 0.0001) between cultures in proliferation and cultures in growth arrest was an increased expression of the CDKIs p57KIP2 and p15INK4b for quiescent growth arrest, and of p16INK4a, p15INK4b, and p57KIP2 for senescent growth arrest. Thus, these cell cycle inhibitors represent potential candidates for selective intervention to promote cellular multiplication of chondrocytes undergoing in vitro expansion for tissue-engineering applications. Possible methods of modulation include the targeted elimination of specifically identified cell cycle inhibitors by antisense technologies.

[1D-and 3D- computer simulation for experimental planning and interpretation of pharmacokinetic studies in the inner ear after local drug delivery]. / 1D- und 3D-Computersimulationen zur Versuchsplanung und -interpretation bei pharmakokinetischen Studien am Innenohr nach lokaler Medikamentenapplikation.

The local delivery of drugs to the cochlea is a promising alternative to systemic treatment of inner ear disorders. Whilst new drugs are being developed for this purpose, it is important to determine the time course and total dose required for the various target regions within the inner ear. Due to the small fluid spaces of the inner ear and the resulting experimental and analytical difficulties, many animal studies have only obtained one sample per animal. This results in limited information about drug time courses at specific locations in the inner ear. We show here how computer models considering general pharmacokinetic principles and inner ear geometry are used for application of the 3R-principle in animal research while avoiding experimental sampling artefacts. This can be achieved by: (1) careful planning and interpretation of experiments to study pharmacokinetics in the inner ear, (2) optimising volume sampling techniques, (3) facilitating the use of advantageous, continuous sampling methods like microdialysis and (4) developing a 3D-model that will permit consideration of the complex geometry of the inner ear when transferring results from one species to another.