Sciatic nerve fascicle differentiation on high-resolution ultrasound with histological verification: An ex vivo study.

INTRODUCTION/AIMS: The development of high-resolution ultrasound (HRUS) has enabled the depiction of peripheral nerve microanatomy in vivo. This study compared HRUS fascicle differentiation to the structural depiction in histological cross-sections (HCS). METHODS: A human cadaveric sciatic nerve was marked with 10 surgical sutures, and HRUS image acquisition was performed with a 22-MHz probe. The nerve was excised and cut into five segments for HCS preparation. Selected HCS were cross-referenced to HRUS, with sutures to improve orientation. Sciatic nerve and fascicle contouring were performed to assess nerve and fascicular cross-sectional area (CSA), fascicle count, and interfascicular distances. Three groups were defined based on HRUS fascicle differentiation in comparison to HCS, namely single fascicle (SF), fascicular cluster (FC), and no depiction (ND) group. RESULTS: On cross-referenced HRUS to HCS images, 58% of fascicles were differentiated. On HRUS, significantly larger fascicle CSA and smaller fascicle count were observed compared with HCS. Group analysis showed that 41% of fascicles were defined as SF, 47% as FC, and 12% as ND. The mean fascicle CSA in the ND group was 0.05 mm2. Compared with the SF, the FC had significantly larger fascicle CSA (1.2 ± 0.7 vs. 0.6 ± 0.4 mm2; p < .001) and shorter interfascicular distances (0.1 ± 0.04 vs. 0.5 ± 0.3 µm; p < .001). DISCUSSION: While HRUS can depict fascicular anatomy, only half of the fascicles visualized on HRUS directly correspond to single fascicles observed on HCS. The amount of interfascicular epineurium appears to influence the ability of HRUS to differentiate individual fascicles.

Skeletal Muscle Microvascular Dysfunction in Obesity-Related Insulin Resistance: Pathophysiological Mechanisms and Therapeutic Perspectives.

Obesity is a worrisomely escalating public health problem globally and one of the leading causes of morbidity and mortality from noncommunicable disease. The epidemiological link between obesity and a broad spectrum of cardiometabolic disorders has been well documented; however, the underlying pathophysiological mechanisms are only partially understood, and effective treatment options remain scarce. Given its critical role in glucose metabolism, skeletal muscle has increasingly become a focus of attention in understanding the mechanisms of impaired insulin function in obesity and the associated metabolic sequelae. We examined the current evidence on the relationship between microvascular dysfunction and insulin resistance in obesity. A growing body of evidence suggest an intimate and reciprocal relationship between skeletal muscle microvascular and glucometabolic physiology. The obesity phenotype is characterized by structural and functional changes in the skeletal muscle microcirculation which contribute to insulin dysfunction and disturbed glucose homeostasis. Several interconnected etiologic molecular mechanisms have been suggested, including endothelial dysfunction by several factors, extracellular matrix remodelling, and induction of oxidative stress and the immunoinflammatory phenotype. We further correlated currently available pharmacological agents that have deductive therapeutic relevance to the explored pathophysiological mechanisms, highlighting a potential clinical perspective in obesity treatment.

Pathophysiological and Therapeutic Roles of Fascial Hyaluronan in Obesity-Related Myofascial Disease.

Myofascial disease is an important complication associated with obesity and one of the leading causes of physical disability globally. In the face of limited treatment options, the burden of myofascial disorders is predicted to increase along with the escalating prevalence of obesity. Several pathological processes in obesity contribute to modifications in fascial extracellular matrix mechanical and biological properties and functions. Changes in adipose tissue metabolism, chronic inflammatory phenotype, oxidative stress, and other mechanisms in obesity may alter the physiochemical and biomechanical properties of fascial hyaluronan. Understanding the pathophysiological importance of hyaluronan and other components of the fascial connective tissue matrix in obesity may shed light on the etiology of associated myofascial disorders and inform treatment strategies. Given its unique and favorable pharmacological properties, hyaluronan has found a broad range of clinical applications, notably in orthopedic conditions such as osteoarthritis and tendinopathies, which share important pathophysiological mechanisms implicated in myofascial diseases. However, while existing clinical studies uniformly affirm the therapeutic value of hyaluronan in myofascial disorders, more extensive studies in broader pharmacological and clinical contexts are needed to firmly validate its therapeutic adaptation.

Application of FTIR Spectroscopy to Detect Changes in Skeletal Muscle Composition Due to Obesity with Insulin Resistance and STZ-Induced Diabetes.

Age, obesity, and diabetes mellitus are pathophysiologically interconnected factors that significantly contribute to the global burden of non-communicable diseases. These metabolic conditions are associated with impaired insulin function, which disrupts the metabolism of carbohydrates, lipids, and proteins and can lead to structural and functional changes in skeletal muscle. Therefore, the alterations in the macromolecular composition of skeletal muscle may provide an indication of the underlying mechanisms of insulin-related disorders. The aim of this study was to investigate the potential of Fourier transform infrared (FTIR) spectroscopy to reveal the changes in macromolecular composition in weight-bearing and non-weight-bearing muscles of old, obese, insulin-resistant, and young streptozotocin (STZ)-induced diabetic mice. The efficiency of FTIR spectroscopy was evaluated by comparison with the results of gold-standard histochemical techniques. The differences in biomolecular phenotypes and the alterations in muscle composition in relation to their functional properties observed from FTIR spectra suggest that FTIR spectroscopy can detect most of the changes observed in muscle tissue by histochemical analyses and more. Therefore, it could be used as an effective alternative because it allows for the complete characterization of macromolecular composition in a single, relatively simple experiment, avoiding some obvious drawbacks of histochemical methods.

Horizontal deformation of skeletal muscle thick sections visualised by confocal microscopy.

Certain morphological parameters of the skeletal muscle tissue can be better understood via 3D considerations. Fluorescent confocal microscopy of thick tissue sections is a well-established method for visualising and measuring skeletal muscle fibres and surrounding capillaries in 3D. However, thick tissue sections are prone to deformations which may significantly influence some stereological and morphometric results like muscle fibre diameter and capillary length, but not dimensionless parameters like object number and Euler-Poincaré characteristics. To better understand this phenomenon, we studied the horizontal deformation of thick (100 µm) transverse skeletal muscle sections, by comparing the muscle fibre diameters measured on thick sections to muscle fibre diameters measured on thin (10 µm) sections of the same sample. Diameter changes were further correlated with shrinkage in the Z direction (axial shrinkage) and deviation of the muscle fibre preferential axis from the Z-axis. We showed that the thick sections dilated in horizontal and shrunk in Z direction, and that the magnitude of horizontal dilation was associated with the magnitude of shrinkage in the Z direction. The latter was more pronounced in transversely than obliquely cut tissue sections. The results emphasise that even when shrinkage in the Z direction can be corrected using calibration, it is important to optimise histological protocols to minimise the Z-axis collapse that could cause horizontal dilation. LAY DESCRIPTION: In skeletal muscle research, 3D analysis is especially important for studying the microvasculature. Laser scanning confocal microscopy of skeletal muscle thick tissue sections is a well-established method for visualising and measuring skeletal muscle fibres and surrounding capillaries in 3D. However, such sections are prone to deformations which may significantly influence the study results. To better understand this phenomenon, we studied the horizontal deformation of thick transverse skeletal muscle sections. We compared the average muscle fibre diameters measured on thick skeletal muscle sections, thin fixed skeletal muscle sections and immunohistochemically stained thin skeletal muscle sections with the muscle fibre diameters measured on thin native skeletal muscle sections of the same sample, with the latter condition serving as the standard diameters (ie the control condition). We further studied the association among muscle fibre diameter changes, shrinkage of the thick skeletal muscle sections in the Z direction and their sectioning angle. We showed that the thick skeletal muscle sections dilated in the horizontal direction and shrunk in the Z direction, and that the magnitude of horizontal dilation was associated with the magnitude of shrinkage in Z direction. The shrinkage in the Z direction was more pronounced in transversely than obliquely cut tissue sections. These results emphasise that even when shrinkage in the Z direction can be corrected using Z-axis calibration, it is very important to optimise histological protocols to minimise the Z-axis collapse that could cause horizontal dilation in order to enhance the integrity of study results.

Determination of bupivacaine tissue concentration in human biopsy samples using high-performance liquid chromatography with mass spectrometry.

In the present study, we developed a simple and rapid analytical method for the quantification of bupivacaine hydrochloride in human biopsy samples of adipose, muscle, neural, connective and cartilage tissue using liquid chromatography-mass spectrometry. Anesthetics were extracted from the tissue samples using 0.1% formic acid in acetonitrile for protein denaturation and hexane for removal of lipophilic impurities. Analytes were separated adequately on Phenomenex Luna Omega polar C18 column using a gradient mobile phase 0.1% formic acid in water and 0.1% formic acid in acetonitrile. The lower limits of quantification were ≤ 97 ng g-1 tissue for all studied tissues. Intra-day recoveries were between 48.2% and 82.1% with relative standard deviations (RSDs) between 1.47% and 14.28%, whereas inter-day recoveries were between 52.2% and 77.6% with RSDs between 2.98% and 14.79%. The calibration curve showed a linear fit with R2 higher than 0.99 in the concentration range from 0.16 to 100 µg g-1 . Lidocaine hydrochloride was tested as internal standard because its recoveries and matrix effects were comparable to bupivacaine hydrochloride. Post-analytical corrections of measured bupivacaine tissue concentrations can accordingly be made based on recovery of lidocaine as internal standard, with recoveries between 51.2% and 86.9% and RSDs between 1.99% and 16.88%. The developed method could be used to study time-dependent spread of bupivacaine locally or to more distant locations across tissue barriers.

Clinical and histologic tissue responses of skin incisions closed with glycomer 631 and lactomer 9-1 using a subcuticular technique in pigs.

BACKGROUND: Glycomer 631 and lactomer 9-1 are absorbable suture materials indicated for soft tissue approximation in non-infected wounds. Pigs are often used as surgical models in translational research; however, reports of tissue reactions to both suture materials in pigs are limited. The aim of this study was to assess clinical and histologic responses of skin incisions closed with a subcuticular technique using glycomer 631 and lactomer 9-1 in pigs. RESULTS: Skin incisions on 17 pigs were closed with glycomer 631 and lactomer 9-1, and a clinical reactive score (CRS) including erythema, swelling, discharge and dehiscence was calculated on postoperative days 7 and 14. Subcuticular tissue reaction was assessed histologically on postoperative day 14 (the presence of extravascular neutrophils, macrophages, multinucleated giant cells, lymphocytes, fibroblasts, bacterial colonies and the overall severity of the inflammatory response to the suture material), and the cumulative score of the variables was calculated as an aggregate tissue irritation score (ATIS). Tissue samples were examined for suture extrusion and evaluated microbiologically. The clinical reactive score did not differ between the suture materials. Only one ATIS variable, namely the overall severity of the inflammatory response, was lower (p = 0.029) when glycomer 631 was used. Suture extrusion was found in 10/17 of the incisions closed by glycomer 631 and in 7/13 of the incisions closed by lactomer 9-1. Trueperella pyogenes was isolated from the skin and from the area of tissue reaction in six pigs. CONCLUSIONS: No difference in CRS between the suture materials was observed, and thus both materials may be used for the subcuticular technique in pigs. Glycomer 631 induced less tissue reaction only in terms of the overall severity of the inflammatory response. Suture extrusion was observed in more than 50% of incisions regardless of the suture material, possibly due to a large amount of suture material in the wound. Trueperella pyogenes was the only pathogen isolated from the tissue surrounding the suture material.

Neurotoxicity of bupivacaine and liposome bupivacaine after sciatic nerve block in healthy and streptozotocin-induced diabetic mice.

BACKGROUND: Long-acting local anaesthetics (e.g. bupivacaine hydrochloride) or sustained-release formulations of bupivacaine (e.g. liposomal bupivacaine) may be neurotoxic when applied in the setting of diabetic neuropathy. The aim of the study was to assess neurotoxicity of bupivacaine and liposome bupivacaine in streptozotocin (STZ) - induced diabetic mice after sciatic nerve block. We used the reduction in fibre density and decreased myelination assessed by G-ratio (defined as axon diameter divided by large fibre diameter) as indicators of local anaesthetic neurotoxicity. RESULTS: Diabetic mice had higher plasma levels of glucose (P < 0.001) and significant differences in the tail flick and plantar test thermal latencies compared to healthy controls (P < 0.001). In both diabetic and nondiabetic mice, sciatic nerve block with 0.25% bupivacaine HCl resulted in a significantly greater G-ratio and an axon diameter compared to nerves treated with 1.3% liposome bupivacaine or saline (0.9% sodium chloride) (P < 0.01). Moreover, sciatic nerve block with 0.25% bupivacaine HCl resulted in lower fibre density and higher large fibre and axon diameters compared to the control (untreated) sciatic nerves in both STZ-induced diabetic (P < 0.05) and nondiabetic mice (P < 0.01). No evidence of acute or chronic inflammation was observed in any of the treatment groups. CONCLUSIONS: In our exploratory study the sciatic nerve block with bupivacaine HCl (7 mg/kg), but not liposome bupivacaine (35 mg/kg) or saline, resulted in histomorphometric indices of neurotoxicity. Histologic findings were similar in diabetic and healthy control mice.

3D analysis of capillary network in skeletal muscle of obese insulin-resistant mice.

In obesity, the skeletal muscle capillary network regresses and the insulin-mediated capillary recruitment is impaired. However, it has been shown that in the early stage of advanced obesity, an increased functional vascular response can partially compensate for other mechanisms of insulin resistance. The present study aimed to investigate the changes in the capillary network around individual muscle fibres during the early stage of obesity and insulin resistance in mice using 3D analysis. Capillaries and muscle fibres of the gluteus maximus muscles of seven high-fat-diet-induced obese and insulin-resistant mice and seven age-matched lean healthy mice were immunofluorescently labelled in thick transverse muscle sections. Stacks of images were acquired using confocal microscope. Capillary network characteristics were estimated by methods of quantitative image analysis. Muscle fibre typing was performed by histochemical analysis of myosin heavy chain isoforms on thin serial sections of skeletal muscle. Capillary length per muscle fibre length and capillary length per muscle fibre surface were increased by 27% and 23%, respectively, around small muscle fibres in obese mice, while there were no significant comparative differences around large fibres of obese and lean mice. Furthermore, the capillarization was larger around small compared to large fibres and there was a shift toward fast type myosin heavy chain isoforms, with no significant changes in muscle fibre diameters, tortuosity and anisotropy in obese mice. Overall, the results show that obese insulin-resistant mice have selective increase in capillarization around small predominantly intermediate muscle fibres, which is most likely related to the impaired glucose metabolism characteristic of type 2 diabetes.

Neurotoxicity of intraneural injection of bupivacaine liposome injectable suspension versus bupivacaine hydrochloride in a porcine model.

OBJECTIVE: To test whether neurotoxic effects of a bupivacaine liposome injectable suspension differ from those of a standard formulation of bupivacaine hydrochloride (HCl) after intraneural injection into the sciatic nerves in pigs. STUDY DESIGN: Prospective, randomized study. ANIMALS: Fifteen pigs, hybrids of Landrace and Large White. METHODS: After the National Ethics Committee approval, 15 pigs were randomly allocated to three groups (n = 5/group) to receive intraneural injections of 4 mL of 1.33% bupivacaine liposome injectable suspension, 0.5% bupivacaine HCl or normal saline. Serial neurologic examinations were conducted to detect sensory and motor response to noxious stimuli using a modified Thalhammer's scale at 2 hour intervals for the first 12 hours after injection and daily thereafter for 2 weeks. Fiber characteristics (density) of the harvested sciatic nerves were measured during histomorphometric analysis. Inflammatory response was studied using immunohistochemical analysis. Data were tested using analyses of variance; p values for paired comparisons were Bonferroni adjusted. RESULTS: Compared with bupivacaine HCl, bupivacaine liposome injectable suspension provided longer sensory (11.2 ± 1.8 hours versus 3.2 ± 1.1 hours, respectively, p < 0.0001) and motor (10.0 ± 2.0 hours versus 4.0 ± 1.4 hours respectively, p < 0.0001) blockade. Histomorphometric parameters were similar among the groups. No changes in axonal density or myelin structure indicative of injury to the sciatic nerves were observed in any of the groups. Number of immunopositive cells did not differ between the bupivacaine liposome injectable suspension (23 ± 6 cells per mm2) and the bupivacaine HCl groups (21 ± 4 cells per mm2), p > 0.90. CONCLUSIONS AND CLINICAL RELEVANCE: Intraneural injections of bupivacaine liposome injectable suspension or bupivacaine HCl in our porcine model did not result in evidence of neurotoxicity.

Unusual course and termination of common facial vein: a case report.

Given the common and increasing exploitation of superficial head and neck veins in a widening spectrum of clinical and surgical procedures, it is important to equally broaden understanding of the heterogeneity of the vascular anatomy of the region. We report an unusual course of the right common facial vein parallel to the course of external jugular vein, emptying into the ipsilateral subclavian vein in the lateral neck triangle behind the posterior border of the sternocleidomastoid muscle in a 78-year-old male cadaver. Such course may be hazardous for surgical procedures in the region given the high risk of profuse haemorrhage from any injury of the vessel. The variant anatomy may also cause problems in invasive techniques involving the vein, especially in emergency and intensive care settings. This index report, therefore, provides a new insight into the superficial head and neck vein anatomical variations in order to guide relevant clinical procedures in the region.

3D reconstruction of peripheral nerves from optical projection tomography images: A method for studying fascicular interconnections and intraneural plexuses.

The general microscopic characteristics of nerves are described in several textbooks of histology, but the specific microanatomies of most nerves that can be blocked by anesthesiologists are usually less well known. Our objective was to evaluate the 3D reconstruction of nerve fascicles from optical projection tomography images (OPT) and the ability to undertake an internal navigation exploring the morphology in detail, more specifically the fascicular interconnections. Median and lingual nerve samples were obtained from five euthanized piglets. OPT images of the samples were acquired and 3D reconstruction was performed. The OPT technique revealed the inner structure of the nerves at high resolution, including large and small fascicles, perineurium, interfascicular tissue, and epineurium. The fascicles were loosely packed inside the median nerve and more densely so in the lingual nerve. Analysis of the 3D models demonstrated that the nerve fascicles can show six general spatial patterns. Fascicular interconnections were clearly identified. The 3D reconstruction of nerve fascicles from OPT images opens a new path for research into the microstructure of the inner contents of fascicular nerve groups and their spatial disposition within the nerve including their interconnections. These techniques enable 3D images of partial areas of nerves to be produced and could became an excellent tool for obtaining data concerning the 3D microanatomy of nerves, essential for better interpretation of ultrasound images in clinical practice and thus avoiding possible neurological complications. Clin. Anat. 31:424-431, 2018. © 2017 Wiley Periodicals, Inc.

Complete occipitalization of the atlas with bilateral external auditory canal atresia.

Fusion of the atlas with the occipital bone is a rare congenital dysplasia known as occipitalization of the atlas, occipitocervical synostosis, assimilation of the atlas, or atlanto-occipital fusion. It is a component of the paraxial mesodermal maldevelopment and commonly associated with other dysplasias of the craniovertebral junction. External auditory canal atresia or external aural atresia is a rare congenital absence of the external auditory canal. It occurs as the consequence of the maldevelopment of the first pharyngeal cleft due to defects of cranial neural crest cells migration and/or differentiation. It is commonly associated with the dysplasias of the structures derived from the first and second pharyngeal arches including microtia. We present the coexistence of the occipitalization of the atlas and congenital aural atresia, an uncommon combination of the paraxial mesodermal maldevelopment, and defects of cranial neural crest cells. The association is most probably syndromic as minimal diagnostic criteria for the oculoariculovertebral spectrum are fulfilled. From the clinical point of view, it is important to be aware that patients with microtia must obtain also appropriate diagnostic imaging studies of the craniovetebral junction due to eventual concomitant occipitalization of the atlas and frequently associated C1-C2 instability.

Unilateral fenestration of the internal jugular vein: a case report.

The internal jugular vein (IJV) is a significant landmark that is encountered during dissection of the cervical lymph nodes in oncological surgery, central venous catheter insertion and interventional radiological procedures. The variations in the patterns of its course, and knowledge of these variations, are important. During the neck dissection of an approximately 75-year-old male cadaver, unilateral fenestration of the IJV, the medial branch of which received the submental and linguofacial veins, was found--a case that hitherto has not been reported. An embryological evaluation and the clinical implications of the anomaly are described. Clinicians and surgeons performing neck vascular or reconstructive surgery should be made aware of this variation of the IJV in the hope of preventing inadvertent injury.

A case of left-sided absence and right-sided fenestration of the external jugular vein and a review of the literature.

The external jugular vein is increasingly being used as the recipient vein in head and neck tissue transfers, and for cannulation to conduct diagnostic procedures or intravenous therapies. The variations in the patterns of its course, and knowledge of these variations, are therefore important. We report on a bilateral external jugular vein anomaly found during the neck dissection of an approximately 75-year-old female cadaver, a case which has hitherto not been reported. The vein was absent on the left and fenestrated on the right. An embryological evaluation and the clinical implications of the anomaly are described. Clinicians and surgeons performing vascular or reconstructive surgery should be made aware of this variation of the external jugular vein to prevent inadvertent injury.

Intraneural and perineural inflammatory changes in piglets after injection of ultrasound gel, endotoxin, 0.9% NaCl, or needle insertion without injection.

BACKGROUND: Ultrasound gel nerve inflammation has been reported. We evaluated the extent and nature of inflammation after gel injection with endotoxin (positive), saline, or dry needle puncture (negative) controls after peripheral blocks in piglets. METHODS: Selected nerves of 12 piglets were localized by landmarks and nerve stimulator. Forty-eight hours after injection, specimens were examined for immunohistochemical cell differentiation/quantification and cytokine expression by using quantitative polymerase chain reaction. RESULTS: Both gel and endotoxin injections resulted in a significantly higher density of inflammatory cells (lymphocytes/granulocytes) as compared with needle insertions and/or saline injections (both P < 0.001). Cytokines were not detected in any of the specimens. CONCLUSIONS: Perineural gel injections cause significant inflammation. The lack of cytokines suggests injectate-related changes rather than mechanical trauma.

Bilateral anomalous high position of the mandibular foramen: a case report.

The inferior alveolar nerve block is one of the most common techniques for delivering dental anesthesia. Its success depends on placing the needle tip in close proximity to the mandibular foramen (MF). In certain cases, however, this nerve block fails, even when performed by the most experienced clinician. Anatomical variability may be one source of local anesthetic failure and includes bone and nerve variations. A case is presented of a bilateral anomalous high position of the MF, identified from the panoramic radiograph. An adjusted anesthetic technique (the Vazirani-Akinosi technique) was used to achieve local anesthesia before extraction of a lower second molar following an unsuccessful conventional indirect technique with a higher entry point. A description of the embryological basis for the anomalous high positioned MF and its impact from a practical and clinical perspective, with particular reference to local anatomy, is included.

The Early Iron Age elite grave from the Kapiteljska njiva barrow cemetery (Novo mesto, Slovenia): an approach to dental profiling through non-invasive and minimally-invasive techniques.

Kapiteljska njiva is a prehistoric cemetery located in the town of Novo mesto in southern Slovenia. There is a long history of archaeological research at this site, as the first investigations date back to the end of the 19th century. In 2004, an Early Iron Age barrow XVI was investigated. The oldest surviving grave in the barrow is the central grave, numbered XVI/34, which according to its position and the richness of the grave goods, belongs to a woman of higher status. Most likely, she was the first member or initiator of a family that continued to bury its dead in the barrow for the next 300 years. There were no preserved skeletal elements; however, in the head part of the grave, remains of human teeth, mostly the tooth crowns and the shells of enamel from the tooth crowns, were found among the scattered amber beads of a necklace. Moreover, these tooth remains are one of the few human biological materials (mostly fragments of skull and long limb bones) unearthed in this cemetery, reflecting the influence of acidic soil from the burial site. This study aimed to create a dental profile of the deceased in a similar way as during a forensic investigation. The remains were examined macroscopically and stereo-microscopically. The morphological traits were scored following the ASUDAS protocol. Ancestry was estimated by entering these scores into a beta version of the web-based application rASUDAS. Brothwell's system was used for age at death estimation from occlusal attrition. Identification of sex was based on the analysis of sex-specific amelogenin isoforms in dental enamel by nanoflow liquid chromatography tandem mass spectrometry (nanoLC-MS/MS). The present study reveals that the dental remains from Kapiteljska njiva belong to the permanent dentition of a single individual and that only the right upper central incisor and third molar are completely absent. The remains of teeth exhibit a simple external morphology, characterised by the absence of morphological dental traits, with a notable exception of the two-rooted left lower canine. The probability of assigning this individual to the Western Eurasian ancestry group is 98%. According to the degree of dentine exposure on the occlusal surfaces of molars, the estimated age range is 17-25 years. Another line of evidence comes from the observation that the first signs of approximal attrition are present in lower third molars but absent in the only preserved upper counterpart, indicating that the age at which the lower third molars entered into occlusion represents a proxy of the individual's age at death. Data on the chronology of the lower third molar development and eruption in present-day European populations from forensic literature (Brkic et al. 2011; Olze et al. 2008; Selmanagic et al. 2013) and The London Atlas confirm the above age-range estimate. Caution is, however, needed in the interpretation of results, because reference data are not based on the population of the individual's origin. Proteomic analysis classified the individual as a female, which is in line with the archaeological evidence. No pathological lesions or indicators of systemic stress were identified; however, the absence of approximal attrition facets in upper anterior teeth indicates interdental spacing. The results of dental profiling are discussed in the context of historical background, today's clinical knowledge, and epidemiological data.

Myosin heavy chain-2b transcripts and isoform are expressed in human laryngeal muscles.

Three fast myosin heavy chain (MyHC) isoforms, i.e. MyHC-2a, -2x and -2b, are expressed in skeletal muscles of smaller mammals. In contrast, only MyHC-2a and -2x have been revealed in humans so far. The expression of MyHC isoforms is known to be wider in the functionally more specialized laryngeal muscles. Though mRNA transcripts of the MyHC-2b gene were found to be expressed in certain human skeletal and laryngeal muscles, the corresponding isoform has not been demonstrated in these muscles. To our knowledge, we are the first to demonstrate not only the expression of MyHC-2b transcripts using an in situ hybridization technique but also the corresponding protein, i.e. the MyHC-2b isoform, in some human laryngeal muscles by immunohistochemistry but not by polyacrylamide gel electrophoresis. Using a set of antibodies specific to MyHC isoforms, we demonstrated that MyHC-2b was always co-expressed with the major MyHC isoforms, not only with the fast ones (MyHC-2a and -2x) but with the slow isoform (MyHC-1) as well.