Outer Connective Tissue Layer Research Articles

Case Report: An Encapsulated Mass of the Gluteal Region Found During Cadaveric Dissection

We describe a large, encapsulated mass containing friable tissue – likely a partially healed hematoma or abscess – in the right gluteal region of a 93-year-old female cadaver, which potentially caused impaired function of the ipsilateral tensor fasciae latae. We first observed this mass between the right gluteus medius and gluteus minimus muscles during dissection lab for the Physical Therapy DPT students. This structure was roughly circular (~11 cm in maximum width) and completely encapsulated by dense connective tissue. Since the inferior aspect appeared to pass through the sciatic foramen, we hypothesized that this was caused by a sciatic hernia. However, this was rejected based on the later dissection of the abdominal and pelvic cavities. None of the structures that can potentially cause a sciatic hernia – colon, ovaries, uterine tube, ureter – were protruding through the sciatic foramen. Additionally, dissection of the gluteal region revealed that the dense connective tissue covering the mass was not passing through the sciatic foramen, rather it was adhered to the ilium near the greater sciatic notch. Dissection of the structure itself showed that its margins were limited to the borders of the gluteus medius and it was composed of two layers of dense connective tissue with clear fluid between them. The outer layer of connective tissue had the appearance of thickened intermuscular fascia. Within the second layer, completely encapsulated, was friable tissue. This suite of characteristics is consistent with a partially healed intermuscular hematoma or abscess, but the pathologic diagnoses of this mass cannot be confirmed without histologic evaluation. Intermuscular hematomas and abscesses in the gluteal region can be caused by traumatic damage to the superior gluteal artery, trigger point injections, and hip arthroplasty. A plausible side effect of this mass lesion is compromised function of the superior gluteal nerve and artery. The tensor fasciae latae on the right side was 9.6% shorter and 13.4% lower in mass than on the left side. There was symmetry and no evidence of degeneration to the gluteus medius and gluteus minimus muscles, so it is possible that there was only a disruption to the innervation or blood supply to the ipsilateral tensor fasciae latae.

Influence of Mesenchymal Stem Cells Seeding on Morphology of Tissue-Engineered Vascular Graft Based on Poly (L-lactide) Scaffold

Introduction - The problem of small-diameter vascular grafts still exists in modern vascular surgery. Autologous or synthetic vascular grafts are used routinely for arterial bypass in patients with cardiovascular diseases. However, some patients either lack suitable autologous tissue or cannot receive synthetic grafts. Such patients could benefit from a vascular graft produced by tissue engineering. The aim of the study is to evaluate the effects of adipose-derived mesenchymal stem cells (AD-MSC) on the formation of tissue-engineered vascular graft (TEVG). Methods - Tubular scaffolds 1.1 mm inner diameter were obtained by the electrospinning method on the basis of nano- and microfibers of poly(L-lactide) (PLLA). AD-MSC were seeded on the scaffolds by the developed filtration method. The subsequent culturing for 14 days was carried out in the constructed flow bioreactor. Cell distribution in the scaffold wall was assessed by the fluorescence microscopy. Obtained grafts were implanted into rat aorta: group 1 without AD-MSC (n=36) with follow-up till 16 months, group 2 with seeded AD-MSC (n=36) with follow-up till 16 months. The material was subjected to histological examination, electron microscopy, immunohistochemistry CD 31+, aSMA and morphometric analysis (cell counting, neointima and neoadventia thickness). To track AD-MSC in vivo PKH-26 labeling and subsequent fluorescence microscopy were performed in 2, 7 and 14 days. Results - Filtration seeding and subsequent cultivation in the flow bioreactor led to a uniform distribution of AD-MSC in the scaffold wall. Graft patency in the first group was 86%, in the second 96%. All grafts formed neointima, with no signs of hyperplasia in the area of anastomoses. The total resorption of PLLA fibers in group 1 was observed by histological examination, new vascular wall consisted of endothelial lining and connective tissue without smooth muscle cells (SMC); in all cases aneurysm formation was detected. Total cells number of implant wall was more in group 2, including SMC that formed neomedia. Also the outer connective tissue layer was much thicker. Histology identified the uniform formation of new tissues in group 2 with no signs of aneurysm. Conclusion - AD-MSC seeding leads to the formation of TEVG, morphologically similar to the natural structure of the vessels. The study was supported by Russian Science Foundation (project 14-33-00003)

327 PORCINE AMNION: A SOURCE OF EPITHELIAL STEM CELLS

The use of pig models for preclinical testing is well established, and the availability of stem cells from this species would open the way to preclinical studies for application of cell therapy. According to the developmental stage from which they are obtained, stem cells are classified as being embryonic, fetal, or adult. Embryonic stem cells have unlimited self-renewing capacity and multilineage differentiation potential, but their clinical application seems to be hindered by the high tumorigenic rate after transplantation. Mesenchymal stem cells (MSC) derived from adult tissues are considered to be more limited in their potential and the risk of the immunological rejection of the transplanted stem cells by the recipient is an important limiting factor. The MSC derived from extra-fetal tissues could overcome many of these restrictions. Indeed, in veterinary medicine, MSC isolated from equine term placenta were the ideal candidates for tendon disease treatment, specifically for their plasticity and their reduced immunogenicity compared to bone marrow-derived cells. Extra-fetal derived MSC in porcine have been isolated from the umbilical cord matrix and amniotic fluid. The aim of this work was to provide, for the first time, an isolation protocol and the characterisation of stem cells from porcine amniotic membrane, which could hold potential uses in regenerative medicine. The amnion is a thin, avascular membrane made of an epithelial layer and an outer layer of connective tissue. From 3 samples of allanto-amnion retrieved at delivery, each amniotic membrane was stripped from the overlying allantois and, for isolation of the epithelial cells, it was digested with trypsin. After removal of epithelial cells, the stromal layer was digested with collagenase to obtain amniotic mesenchymal cells. The cellular yield from term amnion resulted only in epithelial cells (AEC) at a concentration of 10 × 106 for 1 g of digested tissue while no MSC were obtained. Histology, indeed, revealed very few cells in the stromal layer. The AEC readily attached to plastic culture dishes. Culture was established in DMEM-HG medium, supplemented with 10% serum and 10 ng mL–1 of EGF where the cells proliferated robustly. The AEC displayed typical cuboidal morphology. These cells showed a mean of 31 ± 0.24 cell population doublings after 31 days. The mean frequency of colony-forming unit fibroblasts was 1 for each of the 75 plated cells. The AEC expressed MSC mRNA markers (CD29, CD166, CD90, CD73, CD117) and pluripotent markers (Nanog and Oct4), while were negative for CD34 and MHC-II. Osteogenic, adipogenic, and neurogenic differentiations were confirmed by von Kossa, Red Oil, and Nissle stains, respectively, and by expression of specific markers (osteocalcin and osteopontin for osteogenic differentiation, adiponectin and leptin for adipogenic differentiation, and glial fibrillary acid protein and nestin for neurogenic differentiation). We conclude that porcine amnion contain unique and primitive cells whose potential is as yet undefined. Ease of collection and propagation of AEC make this tissue an attractive candidate as a resource for stem cell biotechnology and biomedical research.

Functional ultrastructure of the parenchymatic capsules of the cestode Thysanotaenia congolensis (Cyclophyllidea, Anoplocephalidae, Inermicapsiferinae).

Proglottids from adult Thysanotaenia congolensis from naturally infected black rats Rattus rattus from Santiago Island, Cape Verde, were examined by transmission electron microscopy (TEM). The uterus in mature proglottids is composed of an ephemeral transverse tube or sac that breaks down, releasing eggs into the parenchyma where they are sequestered in groups and encapsulated by parenchymatous layers. In gravid proglottids, eggs accumulate in groups of 6-12 at the distal end of sac-like uterine ducts. As eggs accumulate, the end of the uterine ducts expands until it pinches off, releasing groups of eggs into the parenchyma surrounded by remnants of uterine epithelium. These epithelium-bound groups of eggs remain in the parenchyma until they are encapsulated with several parenchymatous layers, forming parenchymatic egg capsules, typical for mature and gravid proglottids of Inermicapsiferinae. The parenchymatic capsules originate from the medullary parenchyma of immature proglottids, which undergoes differentiation into the three layers of gravid proglottids: (1) an outer connective tissue layer composed of long delicate filaments of unknown chemical nature embedded in a granular extracellular matrix; (2) a middle layer appearing as an accumulation of large closely packed PAS-positive mucous goblets that are intensely metachromatic after toluidine blue staining and (3) an inner compact layer composed of lipid-containing cells, muscle cells with elongated muscle fibres and cells of various sizes and shapes forming or containing calcareous corpuscles. The mature hexacanths of T. congolensis are surrounded by reduced oncospheral envelopes consisting of remnants of a very thin membranous layer of degenerating embryophore with long, irregularly shaped cytoplasmic processes and by remnants of uterine epithelium extending as numerous apical microlamellae into the parenchymatic capsule lumen.

Engineered skeletal muscle units for repair of volumetric muscle loss in the tibialis anterior muscle of a rat.

Volumetric muscle loss (VML) is the traumatic, degenerative, or surgical loss of muscle tissue, which may result in function loss and physical deformity. To date, clinical treatments for VML--the reflected muscle flap or transferred muscle graft--are limited by tissue availability and donor site morbidity. To address the need for more innovative skeletal muscle repair options, our laboratory has developed scaffoldless tissue-engineered skeletal muscle units (SMUs), multiphasic tissue constructs composed of engineered skeletal muscle with engineered bone-tendon ends, myotendinous junctions, and entheses, which in vitro can produce force both spontaneously and in response to electrical stimulation. Though phenotypically immature in vitro, we have shown that following 1 week of implantation in an ectopic site, our muscle constructs develop vascularization and innervation, an epimysium-like outer layer of connective tissue, an increase in myosin protein content, formation of myofibers, and increased force production. These findings suggest that our engineered muscle tissue survives implantation and develops the interfaces necessary to advance the phenotype toward adult muscle. The purpose of this study was to evaluate the potential of our SMUs to restore muscle tissue to sites of acute VML. Our results indicate that our SMUs continue to mature in vivo with longer recovery times and have the potential to repair VML sites by providing additional muscle fibers to damaged muscles. We conclude from this study that our SMUs have the potential to restore lost tissue volume in cases of acute VML.

High‐Resolution Median Nerve Sonographic Measurements

To study relationships between median wrist and forearm sonographic measurements and median nerve conduction studies. The study population consisted of a prospective convenience sample of healthy adults. Interventions included high-resolution median nerve sonography and median motor and sensory nerve conduction studies. Main outcome measures included median motor nerve compound muscle action potential amplitude, distal latency, and conduction velocity; sensory nerve action potential amplitude and distal latency; and sonographic median nerve cross-sectional area. Median motor nerve and sensory nerve conduction studies of the index finger were performed using standard published techniques. A second examiner blinded to nerve conduction study results used a high-frequency linear array transducer to measure the cross-sectional area of the median nerve at the distal volar wrist crease (carpal tunnel inlet) and forearm (4 cm proximally), measured in the transverse plane on static sonograms. The outer margin of the median nerve was traced at the junction of the hypoechoic fascicles and adjacent outer connective tissue layer. Fifty median nerves were evaluated in 25 participants. The compound muscle action potential amplitude with wrist stimulation was positively related to the cross-sectional area, with the area increasing by 0.195 mm(2) for every millivolt increase in amplitude in the dominant hand (95% confidence interval, 0.020, 0.370 mm(2); P < .05) and 0.247 mm(2) in the nondominant hand (95% confidence interval, 0.035, 0.459 mm(2); P < .05). There was no significant linear association between the wrist median cross-sectional area and median motor and sensory distal latencies. Conduction velocity through the forearm was not significantly linearly associated with the forearm area or forearm-to-wrist area ratio (tapering ratio). The wrist area was inversely related to the sensory nerve action potential amplitude. Although associations were found between median nerve conduction study amplitudes and sonographic nerve measurements, they were not found for other parameters. Studying these relationships may increase our understanding of when to best use these procedures.

Effect of implantation on engineered skeletal muscle constructs

The development of engineered skeletal muscle would provide a viable tissue for replacement and repair of muscle damaged by disease or injury. Our current tissue-engineering methods result in three-dimensional (3D) muscle constructs that generate tension but do not advance phenotypically beyond neonatal characteristics. To develop to an adult phenotype, innervation and vascularization of the construct must occur. In this study, 3D muscle constructs were implanted into the hindlimb of a rat, along the sciatic nerve, with the sural nerve isolated, transected and sutured to the construct to encourage innervation. Aortic ring anchors were sutured to the tendons of the biceps femoris muscle so that the construct would move dynamically with the endogenous muscle. After 1 week in vivo, the constructs were explanted, evaluated for force production and stained for muscle, nerve and collagen markers. Implanted muscle constructs showed a developing capillary system, an epimysium-like outer layer of connective tissue and an increase in myofibre content. The beginning of α-bungarotoxin clustering suggests that neuromuscular junctions (NMJs) could form on the implanted muscle, given more time in vivo. Additionally, the constructs increased maximum isometric force from 192 ± 41 μN to 549 ± 103 μN (245% increase) compared to in vitro controls, which increased from 276 ± 23 μN to 329 ± 27μN (25% increase). These findings suggest that engineered muscle tissue survives 1 week of implantation and begins to develop the necessary interfaces needed to advance the phenotype toward adult muscle. However, in terms of force production, the muscle constructs need longer implantation times to fully develop an adult phenotype.

Patients, cells, and organelles: The intersection of science and serendipity

Patients, cells, and organelles: The intersection of science and serendipity

1555 THE RISK OF IATROGENIC POSITIVE MARGIN IN RADICAL PROSTATECTOMY: AN EXPENDED CRITERION TO EVALUATE THE QUALITY OF SURGICAL RESECTION

You have accessJournal of UrologyProstate Cancer: Localized VI1 Apr 20101555 THE RISK OF IATROGENIC POSITIVE MARGIN IN RADICAL PROSTATECTOMY: AN EXPENDED CRITERION TO EVALUATE THE QUALITY OF SURGICAL RESECTION Christian Barré, Geneviève Aillet, and Matthieu Thoulouzan Christian BarréChristian Barré Nantes, France More articles by this author , Geneviève AilletGeneviève Aillet Nantes, France More articles by this author , and Matthieu ThoulouzanMatthieu Thoulouzan Toulouse, France More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2010.02.1320AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Conventionally, the rate of positive surgical margins (PSM) for pT2 tumors, i.e. iatrogenic positive margins (IPM), is taken as the technical error rate. However, this is not a good indicator of technical error as it concerns dissection errors involving only malignant and not benign tissue. The technical error rate should also include the risk of IPM i.e. all incisions of benign tissue within the histological limits of the prostate defined by McNeal because the incised tissue might just as well have been malignant as benign. Even if the benign proved to be benign, this did not eliminate the risk that was run. The safety of a technique does not depend only on the known consequences of an error such as a PSM (″adverse events″) but also on the number of times errors with potentially deleterious consequences are made (″near-misses″). The aim of this prospective study was to establish expanded criteria for evaluating the quality of surgical resection in patients undergoing radical retropubic prostatectomy (RRP). METHODS Prospective study (Jan 2005-Dec 2007), 507 consecutive patients with localised prostate cancer underwent RRP. We determined the rate of IPM and PSM. Pathological study: whole-mount 3-mm serial sections using the Stanford technique. Where capsule was absent (apex and bladder neck) no malignant or benign gland must be in contact with the inked margin. In the capsular zone, we defined 1st degree capsule incision (1st DCI): loss of the outer connective tissue layer and 2nd degree capsule incision (2nd DCI): loss of both capsule layers with malignant or benign gland in contact with ink. RESULTS Patient characteristics were mean age 61.3 years; disease stage: pT2 73%, pT3 25.8%; bilateral nerve-sparing n=273, 88.1%, unilateral n=37, 11.9%. The risk of IPM was 5%: incision of benign glandular tissue was 2.8% (apex: 43%; bladder neck: 57%), 1st DCI was 2.2% and 2nd DCI was 0%. The overall PSM rate was 6.3%: 2.2 % (8/370) for pT2, 14.5 % (19/131) for pT3, and 83.3% for pT4. It was 4.8% (15/310) in patients undergoing nerve-sparing surgery. PSM with pT2 tumours occurred at apex (1.7%), postero-lateral 0%, bladder neck (0.5%). PSMs with pT3a/b tumours occurred at: apex (6.1%), postero-lateral (3.8%) and bladder neck (6.9%). PSM size distribution for pT2: PSM≤1 mm: n=4; 1<PSM≤4 mm: n=2; PSM >4 mm: n=2, and for pT3: PSM≤1 mm: n=6; 1<PSM≤4 mm: n=5; PSM>4 mm: n=8. Biochemical recurrence rate (PSA >0.10 ng/ml) at 1 year was 2.6%. CONCLUSIONS The safety of an RP procedure is given not only by the PSM rate for pT2 tumours but also by the percentage of times there is a risk of a PSM. © 2010 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 183Issue 4SApril 2010Page: e600 Advertisement Copyright & Permissions© 2010 by American Urological Association Education and Research, Inc.MetricsAuthor Information Christian Barré Nantes, France More articles by this author Geneviève Aillet Nantes, France More articles by this author Matthieu Thoulouzan Toulouse, France More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

386 HORSE AMNION: A SOURCE OF MESENCHYMAL (AMSC) AND EPITHELIAL STEM CELLS

According to the developmental stage from which they are obtained, stem cells are classified as being embryonic, fetal, or adult. Embryonic stem cells have unlimited self-renewing capacity and multilineage differentiation potential, but the separation of these cells requires destruction of the embryo. Moreover, their clinical application seems to be hindered by the high tumorigenic rate after transplantation. Stem cells derived from adult tissues are considered to be more limited in their potential; although, they are currently the more versatile cells in the clinical field. However, the risk of the immunological rejection of the transplanted stem cells by the recipient is an important limiting factor. In human medicine, stem cells isolated from term placenta are the ideal candidates for disease treatment, specifically because of their plasticity and reduced immunogenicity. The aim of this work was to provide, for the first time, an isolation protocol and the characteristics of the stem cells from horse amniotic membrane, which hold potential uses in equine clinical regenerative medicine. Minimal criteria for stemness definition are adherence to plastic culture dish, formation of fibroblast colony forming units (CFU-F), specific pattern of surface antigen expression, and differentiation potential toward one or more lineages. The amnion is a thin, avascular membrane composed of an epithelial layer and an outer layer of connective tissue. From 3 samples of allantoamnion retrieved at delivery, each amniotic membrane was stripped from the overlying allantois and, for isolation of the epithelial cells, digested with trypsin. After removal of epithelial cells, the AMSC population was obtained by digestion with collagenase and DNase. The cellular yield from term amnion was 10-fold more epithelial cells than AMSC. Isolated cells readily attached to plastic culture dishes. Culture was established in DMEM-HG medium, supplemented with 10% serum and EGF, where the cells proliferated robustly. Epithelial cells displayed typical cuboidal morphology, whereas AMSC were fibroblast-like. Normally, 5 to 6 passages were achieved before proliferation decreased, with a mean of 13.08 and 26.5 cell population doublings after 31 days, respectively, for epithelial cells and AMSC. The mean frequency of CFU-F was, respectively, 1 : 283 and 1:111 for epithelial cells and AMSC. The 2 cellular lines expressed MSC mRNA markers (CD29, CD105, CD44) and were negative for CD34, which was expressed at the fifth passage in both cellular types. Osteogenic differentiation of epithelial stem cells and AMSC was confirmed by von Kossa stain and by an increased expression of osteocalcin and osteopontin. Our preliminary data showed that equine amnion holds apparent potential as a source of presumptive stem cells, which might have widespread clinical applications, but aspects including immunohistochemical study, preclinical experimentation, and immunological properties must be studied.

Microscopical characterization of the salivary glands of the carnivorous cephalaspidean Philinopsis depicta (Mollusca, Opisthobranchia)

AbstractCephalaspideans are a group of opisthobranch gastropods comprising carnivorous and herbivorous species, allowing an investigation of the relationship between these diets and the morphofunctional features of the salivary glands.In this study, the salivary glands of the carnivorous cephalaspidean Philinopsis depicta were observed by light microscopy using semithin sections and by transmission electron microscopy. A central duct runs along the length of these thin ribbon-shaped glands dividing them in two halves, each formed by a single row of tubules perpendicularly attached to the central duct. The simple epithelium of the central duct and lateral tubes contains ciliated cells and two types of secretory cells, named granular cells and cells with apical vacuole (Fig. 1). A very thin outer layer of connective tissue covers the epithelium (Fig. 1). The ciliated cells are numerous but very thin, forming small clusters between secretory cells. The nucleus, several mitochondria and a few lysosomes are located in the apical region were the cells are wider. A very thin cytoplasmic stalk reaches the base of the epithelium and contains bundles of filaments in addition to some mitochondria.

Multiterminal proprioceptors in Squilla mantis (Stomatopoda, Crustacea).

A series of morphologically simple multiterminal proprioceptors is described in the thoracopods of the hoplocaridian crustacean, Squilla mantis, which is a primitive offshoot of the main malacostracan evolutionary line. The description given here is primarily for the largest and most complex receptor that occurs in the second thoracopod, the raptorial limb. These stretch receptors, which lie distally in the coxopodite, consist of single multiterminal sensory neurons with widely branching dendrites innervating broad connective tissue strands. The single primary dendrite divides dichotomously or, occasionally, trichotomously to produce a large number of terminal dendrites. The terminal dendrites, which exhibit a beaded structure, run parallel to the connective tissue strands' longitudinal axis. Dendrites possess an elaborate connective tissue sheath consisting of a single inner glial layer and a multiple outer connective tissue layer. The presence of the beaded structure has been confirmed by transmission electron microscopy (EM), and, because microtubule density does not vary with dendrite diameter, the beads are not fixation artifacts. Beads have high numbers of mitochondria and glycogen deposits, which suggests that they have a high metabolic activity. Dendritic tips are not associated with beads, arise from the terminals of smaller dendrites, and are embedded in the extracellular ground matrix of the connective tissue strand, where they lie parallel to and surround bundles of elastic fibrils. The different degree of development within the receptor series allows comparison between functionally different limbs, and comparison is made with other segmental receptor series. The evolution of multiterminal proprioceptors is discussed.

MR appearance of the normal and abnormal vagina after hysterectomy.

To define the MR appearance of the vagina after hysterectomy, we reviewed the MR examinations of 43 women who had undergone hysterectomy for a variety of benign and malignant indications. In eight of the patients, MR examinations showed a mass lesion involving the vagina. The masses included four recurrent primary gynecologic malignant neoplasms (one endometrial, one ovarian, and two cervical carcinomas) and four primary vaginal carcinomas. The remaining 35 patients had no evidence of vaginal disease. Of these 35 patients, the repaired vaginal apex, or vaginal cuff, was linear and smooth in appearance in 23 and partially obscured by surgical clip artifacts in seven. In the other five patients, the vaginal cuff had a nodular appearance that was indistinguishable from a vaginal mass on T1-weighted images. However, the vagina had a normal appearance in these patients on T2-weighted images, on which the low-signal-intensity layer of vaginal smooth muscle could be distinguished from the bright outer layer of connective tissue. In patients with recurrent vaginal tumors, the tumor was relatively high in signal intensity on T2-weighted images and obliterated the low-signal-intensity vaginal muscularis. Our experience shows that the normal posthysterectomy vagina may have a nodular appearance on T1-weighted images mimicking a vaginal mass. This appearance can be distinguished from that of a true vaginal mass on the basis of different signal intensity characteristics on T2-weighted images.

Cells in the intestinal system of holoturians (echinodermata) express cholecystokinin-like immunoreactivity

The presence of cholecystokinin (CCK), originally isolated from porcine small intestine, has been reported in a diversity of invertebrates ranging, from cnidarians to protochordates, but so far, not in echinoderms. We have used immunohistochemical techniques to demonstrate the presence of cells expressing CCK-like immunoreactivity in the intestine of three species of sea cucumber: Holothuria mexicana, H. glaberrima, and Stichopus badionotus. The immunoreactivity was observed within the cytoplasm of these cells, in what appeared to be granular or vesicle-like structures. The cell bodies were present in the outer connective tissue layer of the intestine and had a neuronal appearance, sending an axon-like structure into the circular muscle and internal connective tissue. A plexus of fibers expressing CCK-like immunoreactivity was found overlying the muscle layer. Contractility of H. mexicana intestinal strips was studied under partially isometric conditions. CCK and related peptides induced relaxation of the basal muscle tension, and of tension induced by ACh application, suggesting a role for this agent in the intestinal physiology of holothurians.

Hepatobiliary Cystadenoma with Mesenchymal Stroma

In a detailed review of cystic hepatobiliary neoplasms, we identified a subset of 50 cases in which tumors were characterized by the presence of a mesenchymal cell layer interposed between an inner epithelial lining and an outer connective tissue layer. We have recently seen three such patients, making a total of 53 patients reported in the English literature. All of the patients were female, 44 of whom, with an average age of 41 years, had benign tumors. The average age of the remaining nine patients was 57 years and these patients had malignant tumors. In seven patients, the malignancy arose from the epithelial layer, but in two patients sarcomatous changes were identified in the mesenchymal tissues. The older age of the patients with malignant tumors with adequate serial biopsies in two cases supported the thesis that malignant transformation may occur in the benign tumors. Moreover the location of the tumor in one of our patients in whom the resected tumor was associated with anomalous right hepatic ducts and portal veins supported the theory that these tumors develop embryologically from nests of primitive hepatobiliary endodermal and mesodermal cells. Although surgical treatment was performed in all patients, 25% of the patients with benign hepatobiliary cystadenoma with mesenchymal stroma (CMS), and 33% of the patients with malignant CMS had tumor recurrence after primary resection. Ninety per cent of these patients had an incomplete resection at the time of their initial operations. Forty-four per cent of the patients with malignant CMS died after a mean follow-up of 17 months. We conclude that CMS (Edmonson's tumor) occurs uniquely in young female patients, develops from nests of primitive embryonal cells, has the potential for malignant transformation, and should be completely resected at primary operation to avoid recurrence.

An anatomical basis for the resistance and capacitance in series with excitable membrane of the squid giant axon.

To correlate periaxonal tissue layer resistance with Schwann cell layer anatomy, cross and longitudinal sections of giant axons of Loligo pealei were examined by transmission electron microscopy. Measurements were made of the width and frequency of mesaxonal clefts entering the Schwann cell layer from the periaxonal space and leaving the cell layer adjacent to the basal lamina. The average mesaxonal cleft width is 10.5 nm. One cm2 of the giant axon surface is enclosed by a single cell layer containing about 690 000 Schwann cells. One cm2 of axon surface has a sheath mesaxonal area of 0.002 cm2 at the periaxonal surface and 0.016 cm2 at the basal lamina, the mesaxons branching frequently as they cross the sheath. The volume of the Schwann cell layer extracellular space was estimated to be roughly 1% of the Schwann cell layer volume. Several models were used to predict the resistance R, across the Schwann cell layer. Assuming the mesaxonal clefts contain seawater, and can be lumped into volume conductors having simple geometries, then (normalized for one cm2 of axon surface) R was estimated to be between 0.4 and 0.9 omega cm2. This compares favourably with electrophysiological estimates of the periaxonal tissue resistance (current clamp value = 0.9 omega cm2 and the voltage clamp value = 1.4 omega cm2) as these electrically measured values include the resistance across the outer connective tissue layer as well as the Schwann cell layer. The value of the Schwann cell membrane capacity was estimated to be approximately 0.7 muF/cm2.

Feeding and gut physiology in Acanthopleura spinigera (Mollusca)

The morphology and histology of the alimentary canal of the rock chiton Acanthopleura spinigera are described and the ability of regions of the gut to digest specific substrates investigated. The oesophagus is produced into a pair of thin‐walled lateral pouches, the salivary glands or “sugar glands” which empty into the stomach. Folds of the capacious stomach are almost obscured by the large digestive gland over which is coiled the intestine. Histologically the gut consists of an outer layer of connective tissue, an inner muscular layer and a ciliated epithelium which varies in thickness from one region to the next. Proteases are most active in the stomach, digestive gland and anterior intestine at pH 6·5 and in the posterior intestine at pH 7·5‐8·5. The digestion of lipoidal substance was greatest in the stomach and digestive gland and least in anterior intestine. There was little increase in the amount of digestion product obtained after 20 hours incubation. All regions of the alimentary canal and salivary gland were capable of digesting carbohydrates except that many low molecular weight carbohydrates were digested by salivary gland extracts only. The amylases were most active at pH 6–6·5. It is concluded that digestive enzymes are distributed throughout the intestinal tract but the amount of enzyme present varies from region to region, and is greatest just after feeding.

CHOLESTEATOMA OF THE EXTERNAL AUDITORY MEATUS

The so-called cholesteatoma of the middle ear is the result of an ingrowth of the stratified squamous epithelium of the external auditory meatus into the middle ear. There are several ways in which a cholesteatoma may develop, the cholesteatoma always being characterized by an accumulation of concentric layers of horny epithelial lamellas within a cystlike sac which communicates through a perforation in the tympanic membrane with the external canal. The wall of the sac, the matrix of the cholesteatoma, is formed by an inner layer of stratified, squamous epithelium with abundant superficial keratinization and an outer layer of connective tissue. In rare instances similar formations are found in the external canal. For a long time it was doubted that a cholesteatoma could occur in the external canal without the simultaneous presence of a cholesteatoma of the middle ear; but lately it has been histologically shown by Mayer 1 that isolated cholesteatoma-like

THE SACCULE: OBSERVATIONS ON A DIFFERENTIATED REENFORCED AREA OF THE SACCULAR WALL IN MAN

Ever since Scarpa 1 described the membranous vesicles in the vestibule of the labyrinth their walls have generally been considered as undifferentiated except for the areas of the maculas. Thus it is customary for modern anatomists, in describing the membranous labyrinth, to include the walls of the utricle, the saccule and the membranous semicircular canals in a short general statement. Such a description has been given by the following authors: Kolmer, 2 A. Eckert Mobius, 3 Shambaugh 4 and Maximow and Bloom. 5 A general agreement exists that these membranous walls are made up of two thin layers of cells separated by a basement membrane, an outer connective tissue or mesothelial layer and an inner layer of exceedingly thin (3 to 4 microns) polygonal squamous cells. Occasional branched melanophores are found in the outer layer of connective tissue, especially in lower mammalian forms. While examining a large number of sections of human temporal bones I