New anatomical findings in the mandibular body region: Two parts that make up bucco-mandibular space.

Soft tissue spaces not only enable gliding by contraction of the facial muscles, but they also cause drooping of the superficial fat due to gravity in the upright position. This study was performed to clarify the structures around the bucco-mandibular space (BMS) and to apply this anatomical knowledge to clinical practice. Four sides of the face were dissected using a conventional gross anatomical dissection technique, and 10 sides (5 horizontal and 5 frontal sections) of the removed semi-facial soft tissue were dissected using the stretched tissue dissection (STD) method. Histological examination of the mandible was performed on two sides to confirm the findings of conventional gross anatomical dissection and STD. In all cases, both gross dissection and STD revealed that the BMS was composed of two parts. The superficial part was filled with adipose tissue containing nerves and vessels, including the marginal mandibular branch of the facial nerve, facial artery, facial vein, and mental nerve. We named this part the adipo-neuromandibular part. By contrast, the deep part was separated from the adipo-neurovascular part by facial deep fascia and composed of loose connective tissue. We named this deep part the loose connective tissue part. The STD method enabled us to obtain detailed anatomical findings of the mandibular region and elucidate two parts of the BMS in which the neurovasculature is distributed. We believe that these findings provide new insights into facial anatomy by resolving existing anatomical uncertainties and will contribute to safer surgical treatment in the facial region.

A Methodology for the Enzymatic Isolation of Embryonic Hypothalamus Tissue and Its Acute or Post-Culture Analysis by Multiplex Hybridisation Chain Reaction.

The hypothalamus is an evolutionarily ancient part of the vertebrate ventral forebrain that integrates the dialogue between environment, peripheral body, and brain to centrally govern an array of physiologies and behaviours. Characterizing the mechanisms that control hypothalamic development illuminates both hypothalamic organization and function. Critical to the ability to unravel such mechanisms is the skill to isolate hypothalamic tissue, enabling both its acute analysis and its analysis after explant and culture. Tissue explants, in which cells develop in a manner analogous to their in vivo counterparts, are a highly effective tool to investigate the extrinsic signals and tissue-intrinsic self-organising features that drive hypothalamic development. The hypothalamus, however, is induced and patterned at neural tube stages of development, when the tissue is difficult to isolate, and its resident cells complex to define. No single molecular marker distinguishes early hypothalamic progenitor subsets from other cell types in the neural tube, and so their accurate dissection requires the simultaneous analysis of multiple proteins or mRNAs, techniques that were previously limited by antibody availability or were arduous to perform. Here, we overcome these challenges. We describe methodologies to precisely isolate early hypothalamic tissue from the embryonic chick at three distinct patterning stages and to culture hypothalamic explants in three-dimensional gels. We then describe optimised protocols for the analysis of embryos, isolated embryonic tissue, or cultured hypothalamic explants by multiplex hybridisation chain reaction. These methods can be applied to other vertebrates, including mouse, and to other tissue types. Key features • Detailed protocols for enzymatic isolation of embryonic chick hypothalamus at three patterning stages; methods can be extended to other vertebrates and tissues. • Brief methodologies for three-dimensional culture of hypothalamic tissue explants. • Optimised protocols for multiplex hybridisation chain reaction for analysis of embryos, isolated embryonic tissues, or explants.

The assesment of intraoperative use of mesna in canal wall-up cholesteatoma surgery - preliminary report.

<b>Introduction:</b> Surgery is still the method of choice in chronic otitis media with cholesteatoma. Except for some specific clinical situations, classic canal wall up technique (CWU), remains a gold standard as a primary treatment in most departments. Unfortunately, the risk of recurrence in such an approach is estimated at 9 to even 70%. This fact prompts researchers to look for ways to reduce those unfavourable statistics. One of the recognized methods supporting the removal of cholesteatoma is the intraoperative use of mesna (sodium 2-mercaptoethanesulfonate). This synthetic sulphur compound disrupts disulfide bridges in polypeptide chains, thanks to which it facilitates matrix preparation.</br></br> <b>Aim:</b> To evaluate the effect of intraoperative use of mesna on the treatment outcomes in patients with chronic otitis media with cholesteatoma operated on by means of the canal wall up technique (CWU).</br></br> <b>Material and methods:</b> 459 surgical reports of patients with middle ear cholesteatoma were analyzed. In total, 52 adult patients with no history of previous ear surgery operated on by means of the CWU technique by the same experienced otosurgeon with all follow-up data available were included in the study. Twenty-six were operated on with the use of mesna (mesna group) and 26 by means of the classic CWU technique (control / no-mesna group). There were 28 women and 24 men with a mean age of 41 years.</br></br> <b>Main Outcome Measure(s):</b> Postoperative hearing results and cholesteatoma recidivism rate.</br></br> <b>Results:</b> Overall recidivism rate was 21.15 %. It was higher in the no-mesna (26.9%) than in the mesna group (15.4%) - although the outcomes were better in the mesna group, the difference was not statistically significant (P = 0.49715). Hearing gain was better in the mesna than in the no-mesna group (10 dB vs 7 dB), but the difference was not statistically significant (P = 0.20089).</br></br> <b>Conclusions:</b> Our preliminary results show that mesna reduces recidivism rates in patients with cholesteatoma. Further study with the analysis of a larger group of patients is needed to prove it statistically.

Performance and learning rate prediction models development in FLS and RAS surgical tasks using electroencephalogram and eye gaze data and machine learning.

OBJECTIVE: This study explored the use of electroencephalogram (EEG) and eye gaze features, experience-related features, and machine learning to evaluate performance and learning rates in fundamentals of laparoscopic surgery (FLS) and robotic-assisted surgery (RAS). METHODS: EEG and eye-tracking data were collected from 25 participants performing three FLS and 22 participants performing two RAS tasks. Generalized linear mixed models, using L1-penalized estimation, were developed to objectify performance evaluation using EEG and eye gaze features, and linear models were developed to objectify learning rate evaluation using these features and performance scores at the first attempt. Experience metrics were added to evaluate their role in learning robotic surgery. The differences in performance across experience levels were tested using analysis of variance. RESULTS: EEG and eye gaze features and experience-related features were important for evaluating performance in FLS and RAS tasks with reasonable results. Residents outperformed faculty in FLS peg transfer (p value = 0.04), while faculty and residents both excelled over pre-medical students in the FLS pattern cut (p value = 0.01 and p value < 0.001, respectively). Fellows outperformed pre-medical students in FLS suturing (p value = 0.01). In RAS tasks, both faculty and fellows surpassed pre-medical students (p values for the RAS pattern cut were 0.001 for faculty and 0.003 for fellows, while for RAS tissue dissection, the p value was less than 0.001 for both groups), with residents also showing superior skills in tissue dissection (p value = 0.03). CONCLUSION: Findings could be used to develop training interventions for improving surgical skills and have implications for understanding motor learning and designing interventions to enhance learning outcomes.

A Comparison of Two Different FFPE Tissue Dissection Methods for Routine Diagnostics in Molecular Pathology: Manual Macrodissection versus Automated Microdissection Using the Roche "AVENIO Millisect" System.

Currently, in routine diagnostics, most molecular testing is performed on formalin-fixed, paraffin-embedded tissue after a histomorphological assessment. In order to find the best possible and targeted individual therapy, knowing the mutational status of the tumour is crucial. The "AVENIO Millisect" system Roche introduced an automation solution for the dissection of tissue on slides. This technology allows the precise and fully automated dissection of the tumour area without wasting limited and valuable patient material. In this study, the digitally guided microdissection was directly compared to the manual macrodissection regarding the precision and duration of the procedure, their DNA concentrations as well as DNA qualities, and the overall costs in 24 FFPE samples. In 21 of 24 cases (87.5%), the DNA yields of the manually dissected samples were higher in comparison to the automatically dissected samples. Shorter execution times and lower costs were also benefits of the manual scraping process. Nevertheless, the DNA quality achieved with both methods was comparable, which is essential for further molecular testing. Therefore, it could be used as an additional tool for precise tumour enrichment.

Expression Microdissection for the Analysis of miRNA in a Single-Cell Type.

Cell-specific microRNA (miRNA) expression estimates are important in characterizing the localization of miRNA signaling within tissues. Much of these data are obtained from cultured cells, a process known to significantly alter miRNA expression levels. Thus, our knowledge of in vivo cell miRNA expression estimates is poor. We previously demonstrated expression microdissection-miRNA-sequencing (xMD-miRNA-seq) to acquire in vivo estimates, directly from formalin-fixed tissues, albeit with a limited yield. In this study, we optimized each step of the xMD process, including tissue retrieval, tissue transfer, film preparation, and RNA isolation, to increase RNA yields and ultimately show strong enrichment for in vivo miRNA expression by qPCR array. These method improvements, such as the development of a noncrosslinked ethylene vinyl acetate membrane, resulted in a 23- to 45-fold increase in miRNA yield, depending on the cell type. By qPCR, miR-200a increased by 14-fold in xMD-derived small intestine epithelial cells, with a concurrent 336-fold reduction in miR-143 relative to the matched nondissected duodenal tissue. xMD is now an optimized method to obtain robust in vivo miRNA expression estimates from cells. xMD will allow formalin-fixed tissues from surgical pathology archives to make theragnostic biomarker discoveries.

Spatially resolved transcriptomics: a comprehensive review of their technological advances, applications, and challenges.

The ability to explore life kingdoms is largely driven by innovations and breakthroughs in technology, from the invention of the microscope 350 years ago to the recent emergence of single-cell sequencing, by which the scientific community has been able to visualize life at an unprecedented resolution. Most recently, the Spatially Resolved Transcriptomics (SRT) technologies have filled the gap in probing the spatial or even three-dimensional organization of the molecular foundation behind the molecular mysteries of life, including the origin of different cellular populations developed from totipotent cells and human diseases. In this review, we introduce recent progresses and challenges on SRT from the perspectives of technologies and bioinformatic tools, as well as the representative SRT applications. With the currently fast-moving progress of the SRT technologies and promising results from early adopted research projects, we can foresee the bright future of such new tools in understanding life at the most profound analytical level.

Three sublayers in the SMAS: Observations of facial soft tissue using the stretched tissue dissection method focusing on the SMAS.

The characteristics of the superficial musculoaponeurotic system (SMAS), including the morphology of each part and the connection between tissues, remain controversial. The purpose of this study is to clarify the anatomy of the SMAS using our new dissection method. In this study, six hemi-sides of heads from formalin-preserved cadavers were used. Three were used for creating a horizontal section and three were used for creating the section along the axial line perpendicular to the surface of the skin, resulting in a gradual change from the coronal section at the lateral to the sagittal section at the median. The resected head was cut into slices with widths of 7 mm. The stretched tissue dissection method was performed by fixing a tissue slice to a board and pulling the skin outward to stretch the soft tissue. Blunt dissection was then performed under a microscope. The SMAS comprises three layers: superficial, intermediate, and deep. The superficial layer is a thin membrane directly connecting to the septa in the subcutaneous fat. The deep layer is the connective tissue in contact with the sub-SMAS structure. The layer surrounded by the superficial and deep layer of the SMAS is the intermediate layer, containing connective tissue, adipose tissue, and facial muscles. The detailed findings of the SMAS obtained using this method resolve theoretical discrepancies and provide important insight for the field of facial surgery.

Gross anatomical study of the subcutaneous structures that create the three-dimensional shape of the buttocks.

The purpose of this study was to clarify the subcutaneous structures involved in the morphology of the gluteal region for clinical application. Thirty-seven formalin-fixed cadavers and one soft-fixed cadaver were used in this study. Gluteal tissue was removed from five formalin-fixed cadavers. A horizontal section and sections parallel to the long axis of the body were made from the excised tissue, and the subcutaneous fat was removed to observe the fibrous structure within the subcutaneous fat. Two formalin-fixed cadavers and one soft-fixed cadaver were used to perform conventional gross anatomical dissection and histological examination. On 30 formalin-fixed cadavers, the thickness of the subcutaneous fat was measured in various areas of the buttocks. The thickness of subcutaneous fat was thicker in the center of the buttocks and thinner on the lateral buttocks. Superficial fascia (SF) was found only in the upper buttock, being indistinct in the lower buttock. In the sacral and coccygeal areas, the dermis was tightly adhered to the bone as a single mass. Fibers arose from around the iliac crest to the SF. On the medial side of the gluteal fold, a strong fiber arose from the sciatic tubercle and inserted into the gluteus maximus and dermis. By identifying the characteristic subcutaneous structures of the gluteal region, we were able to identify the anatomical structures that shape the three-dimensional morphology of the buttocks. These findings may be useful in surgical treatments such as improving the buttock shape.

A Comparison of Tissue Dissection Techniques for Diagnostic, Prognostic, and Theragnostic Analysis of Human Disease.

Histopathology has historically been the critical technique for the diagnosis and treatment of human disease. Today, genomics, transcriptomics, and proteomics from specific cells, rather than bulk tissue, have become key to understanding underlying disease mechanisms and rendering useful diagnostic information. Extraction of desired analytes, i.e., nucleic acids or proteins, from easily accessible formalin-fixed paraffin-embedded tissues allows for clinically relevant activities, such as sequencing biomarker mutations or typing amyloidogenic proteins. Genetic profiling has become routine for cancers as varied as non-small cell lung cancer and prostatic carcinoma. The five main tissue dissection techniques that have been developed thus far include: bulk scraping, manual macrodissection, manual microdissection, laser-capture microdissection, and expression microdissection. In this review, we discuss the importance of tissue dissection in clinical practice and research, the basic methods, applications, as well as some advantages and disadvantages for each modality.

Clear Cornea Femto DALK: a novel technique for performing deep anterior lamellar keratoplasty.

PURPOSE: A new femtosecond laser assisted deep anterior lamellar keratoplasty technique (Clear Cornea Femto DALK or CCF DALK) can be performed with less technical challenges compared to conventional procedures. This paper reports on a preliminary case series to evaluate the technique. METHOD: First, through a clear cornea approach, Descemet's membrane (DM) is completely separated from posterior stroma by injection of balanced salt solution/viscoelastic substance through a special cannula inserted into the deep stroma and positioned right above the DM without perforating. The injection creates a liquid chamber that detaches the DM while preserving a reasonably transparent corneal stroma. Afterwards, a complete posterior/anterior trephination of the stroma, from the liquid chamber to the epithelium, is done using a femtosecond laser system under optical coherence tomography control. RESULTS: This technique was successfully performed in a preliminary series of 10 eyes/10 patients. All patients had the DM completely bared and kept their own endothelial cell population with minimal cell loss (< 15%) after 6 months. Postoperative interface reaction was minimal, and no immune reactions were observed thus far. CONCLUSION: Clear Cornea Femto DALK is a promising alternative to previous Femto DALK procedures with good acceptance of the tissue seen to date.

Tissue dissection with saline injection: application in thyroid surgery.

Postoperative hypoparathyroidism is a thyroidectomy complication. The effect of this complication cannot be accurately quantified. The incidence of hypoparathyroidism after total thyroidectomy has high variability in the literature, between 7 and 37%. Data from 78 patients who underwent total thyroidectomy with Tissue Dissection with Solution Injection (TDSI group) from December 2018 to August 2019 were retrospectively reviewed. These patients were compared with 78 patients to whom the technique was not applied (non-TDSI group), and they were treated from January 2018 to September 2018. All thyroidectomies were performed by the same surgeon. The mean duration of a thyroidectomy was 1 hour. The reduction of the incidence of postoperative hypoparathyroidism in the group of patients was applied in respect of the technique of tissue dissection with saline injection. TDSI technique paves the way for further application to other tissues and surgeries.

Development and validation of an objective scoring tool to evaluate surgical dissection: Dissection Assessment for Robotic Technique (DART).

Purpose: Evaluation of surgical competency has important implications for training new surgeons, accreditation, and improving patient outcomes. A method to specifically evaluate dissection performance does not yet exist. This project aimed to design a tool to assess surgical dissection quality. Methods: Delphi method was used to validate structure and content of the dissection evaluation. A multi-institutional and multi-disciplinary panel of 14 expert surgeons systematically evaluated each element of the dissection tool. Ten blinded reviewers evaluated 46 de-identified videos of pelvic lymph node and seminal vesicle dissections during the robot-assisted radical prostatectomy. Inter-rater variability was calculated using prevalence-adjusted and bias-adjusted kappa. The area under the curve from receiver operating characteristic curve was used to assess discrimination power for overall DART scores as well as domains in discriminating trainees (≤100 robotic cases) from experts (>100). Results: Four rounds of Delphi method achieved language and content validity in 27/28 elements. Use of 3- or 5-point scale remained contested; thus, both scales were evaluated during validation. The 3-point scale showed improved kappa for each domain. Experts demonstrated significantly greater total scores on both scales (3-point, p< 0.001; 5-point, p< 0.001). The ability to distinguish experience was equivalent for total score on both scales (3-point AUC= 0.92, CI 0.82-1.00, 5-point AUC= 0.92, CI 0.83-1.00). Conclusions: We present the development and validation of Dissection Assessment for Robotic Technique (DART), an objective and reproducible 3-point surgical assessment to evaluate tissue dissection. DART can effectively differentiate levels of surgeon experience and can be used in multiple surgical steps.

Safety and feasibility of a novel non-thermal device for tissue dissection: A preliminary study of the DD1 differential dissector.

Energy devices can cause significant thermal damage to surrounding tissues causing unanticipated organ trauma. To evaluate the safety and feasibility of a novel electric device (DD1) for soft tissue dissection. Three series of measurements were performed in a pig model. First, macro- and microscopic tissue damage was compared between the DD1 and an electric scalpel (ES). Second, the time course of tissue temperature was measured for the DD1 and three other energy devices (ES, Harmonic and LigaSure). Third, the time required for mobilization of a peripheral artery of the intestine was compared between the DD1 and manual, non-energized forceps. First, the tissue damage area caused by ES was significantly larger compared to that in the DD1 at all time points (p < 0.0001). The number of damaged cells due to thermal damage was significantly larger for ES than for DD1, even when the DD1 was applied to a single point at maximum power for 60 sec (p < 0.0001). Second, the maximum temperature of Harmonic was 160°C 3 sec after use and dropped to 68°C after 10 sec. At the same time points after use, we observed: ES (84°C, 45°C), LigaSure (61°C, 49°C), and DD1 (30.5°C, 29°C). Third, the median dissection time for the artery using DD1 was significantly shorter than that for dissecting forceps (DD1: 100 sec, range 70-205 sec vs. forceps: 130 sec, range 90-210 sec, p = 0.0325). DD1 was a safe non-thermal device which causes less tissue damage while also providing shorter dissection times than manual dissection.

Isolating Spermathecae and Determining Mating Status of Drosophila suzukii: A Protocol for Tissue Dissection and Its Applications.

The spotted wing drosophila, Drosophila suzukii (Diptera: Drosophilidae), is an emerging invasive pest, which attacks a wide variety of fruits and berries. Although previous studies have focused on different aspects of D. suzukii reproductive biology, there are no protocols available for determining the mating status of D. suzukii females and drosophilids in general. In this study, a step-by-step protocol for tissue dissection, isolating spermathecae, and determining the mating status of females was developed specifically for D. suzukii. This protocol is an effective and relatively quick method for determining female mating status. It has important applications from exploring reproductive output of D. suzukii females to understanding the biology of D. suzukii winter morph, which presumably plays the main role in the overwintering of this invasive species. We demonstrated applicability of this protocol for both field collected flies and flies reared in the lab, including fly specimens stored on a long-term basis.

Notochord isolation using laser capture microdissection.

BACKGROUND: Chordoma are malignant tumors of the axial skeleton, which arise from remnants of the notochord. The Notochord (chorda dorsalis) is an essential embryonic structure involved in the development of the nervous system and axial skeleton. Therefore, the notochord seems to be the most biologically relevant control tissue to study chordoma in molecular biology research. Nevertheless, up to now mainly different tissues but not the notochord have been used as control for chordoma, due to difficulty of isolating notochordal tissue. Here, we describe a fast and precise method of isolating notochordal cells. METHODS: Examination of human fetuses, with a gestation of 9, 11 and 13 weeks, using (immuno)histochemical methods was performed. To isolate pure notochord cells for further molecular biology investigation five flash frozen fetuses between 9 and 10 weeks of gestation were dissected by microtome slicing. Thereafter pure notochord cells for further molecular biology investigation where harvested by using laser capture microdissection (LCM). RNA was extracted from these samples and used in quantitative PCR. RESULTS: This study illustrates notochord of embryonic spines in three different stages of gestation (9-11-13 weeks). Immunohistochemical staining with brachyury showed strong staining of the notochord, but also weak staining of the intervertebral disc and vertebral body. LCM of notochord slices and subsequent total RNA extraction resulted in a good yield of total RNA. qPCR analysis of two housekeeping genes confirmed the quality of the RNA. CONCLUSION: LCM is a fast and precise method to isolate notochord and the quality and yield RNA extracted from this tissue is sufficient for qPCR analysis. Therefore early embryo notochord isolated by LCM is suggested to be the gold standard for future research in chordoma development, classification and diagnosis.

Postmarketing Analysis of a Novel, Cordless Ultrasonic Dissector.

Introduction Tissue dissection and vessel sealing is performed using a variety of energy sources and surgical devices. We describe the postmarketing analysis of a cordless ultrasonic dissector and vessel sealer in a series of general and gynecological procedures. Methods Patients were prospectively screened and consented for participation. Data collected included demographics, device activations/seals and failures, and patient complications. Surgeons were surveyed following each case. Data was analyzed using standard statistical methods. Results A total of 110 patients were consented and participated in the study. The most frequently performed procedures were bilateral salpingo-oophorectomy (n = 48) and total laparoscopic hysterectomy (n = 36). Mean age was 54.2 years and 79.2% were female. The most frequent number of device activations per case was between 26 and 50 (36.6%). Five failed seals occurred out of 4858 total estimated seals (0.11%). Failed seals were felt to be due to thickened, scarred tissue not amenable to device compression. There were no patient intraoperative complications related to the device itself. Overall, surgeons felt the device was extremely easy to use (97.6%) and no visual obstruction due to steam from the device was encountered (95%). Ninety-five percent of surgeons felt the device was beneficial for soft tissue dissection and vessel sealing. Conclusion Sonicision is safe and effective for use in dissection of soft tissues and vessel sealing in a variety of laparoscopic and open procedures. In this study, there were no complications related to the device itself. The remarkable cordless design of this device enhances its ease of use with overall excellent effectiveness.

Using Drosophila for Studies of Intermediate Filaments.

Drosophila melanogaster is a useful organism for determining protein function and modeling human disease. Drosophila offers a rapid generation time and an abundance of genomic resources and genetic tools. Conservation in protein structure, signaling pathways, and developmental processes make studies performed in Drosophila relevant to other species, including humans. Drosophila models have been generated for neurodegenerative diseases, muscular dystrophy, cancer, and many other disorders. Recently, intermediate filament protein diseases have been modeled in Drosophila. These models have revealed novel mechanisms of pathology, illuminated potential new routes of therapy, and make whole organism compound screens feasible. The goal of this chapter is to outline steps to study intermediate filament function and model intermediate filament-associated diseases in Drosophila. The steps are general and can be applied to study the function of almost any protein. The protocols outlined here are for both the novice and experienced Drosophila researcher, allowing the rich developmental and cell biology that Drosophila offers to be applied to studies of intermediate filaments.

Early results of dual approach hernioplasty (DAH): an innovative laparoscopic inguinal hernioplasty technique.

BACKGROUND: In this study, the proposed technique is combining the invented method of extraperitoneal dissection utilizing gas insufflation through Veress needle introduced from the groin with the transabdominal approach. Such a combination minimizes operative demands, achieves major goals, and reduce operative time in an attempt to improve laparoscopic hernioplasty techniques. METHODS: The study recruited 211 patients having primary reducible inguinal hernia upon first diagnosis. Abdominal ultrasound examination achieved to exclude any additional pathology and confirming clinical diagnosis. The new technique is applied on all patients without any modification through the whole series. RESULTS: Patients' epidemiology, operative characteristics, and follow-up are all tabulated. Results showed no complications nor conversion to open procedure. CONCLUSIONS: The proposed procedure showed preliminary encouraging results regarding technique, clinical outcome, time-saving, and patients' safety. Combination of extraperitoneal gas-derived dissection, transabdominal field review, and preperitoneal mesh application offers an innovative and promising laparoscopic hernioplasty technique. The study is introducing the technique and as well invites further trials on wider scale to verify the technique.