Auditory epithelial migration on the human tympanic membrane: II. The existence of two discrete migratory pathways and their embryologic correlates

The pathways of movement of dye-markings on the tympanic membranes of 30 ears of 15 volunteers were investigated by photography with a Hopkin's rod. Two discrete pathways were identified. Movement on the pars tensa was centrifugal in all directions from the edge of the handle of malleus surface. This supported our previous suggestion that pars tensa epithelial migration is based on growth of the meatal plate. Movement on the more central part of the handle surface itself, however, was always upwards to the pars flaccida epithelium and then, with all dye on the latter, in a posterosuperior direction only. The covering epithelium of the pars flaccida/handle of malleus arises from the fundus of the early embryonic external canal. To explore the possibility that its migration may also commence early, the fundus was examined histologically in 12 embryo ears. It was shaped like an inverted pear to conform to the primordial head of the malleus above and its handle below. There was distinct flattening of the whole fundal epithelium in contrast to the side wall of the external canal, lending support to the concept of a primordial flux of epithelium in the main fundus and its inferior extension. The meatal plate grew from the rim of the fundus producing the pars tensa and deep external canal epithelia. This process, however, was not associated with significant cytologic alterations or increased expression of the human proliferation antigen, Ki-67, at that rim. This suggests that the pars tensa epithelium grows and moves without the active involvement of the epithelium of the pars flaccida/handle of malleus.     

Auditory development in the mouse: Structural maturation of the middle ear

The development of middle-ear structures in the mouse was examined in nine groups of pups between 1 and 45 days of age. The area of the tympanic membrane (pars tensa and pars flaccida), the length of the lever arms of the malleus and incus, the surface area of the oval window, and the volume of the bulla all showed systematic changes during neonatal life. The area of the oval window reached maturity first and the lever arms achieved 90% of their adult size on day 11. The tympanic membrane achieved the same criterion on day 18. These data help us further to understand the processes that contribute to the functional ontogeny of the middle ear.     

Postnatal development of the sound-transporting apparatus in the middle ear of rabbits

The middle ear apparatus realized a multiplicatory function during the transfer of the vibrational energy, depending on the area ratio of pars tensa of tympanic membrane and of stapes footplate as well as the lever ratio of long arms of manubrium and of incus. These structures exhibit a size increase during the postnatal development, but the sequel on the multiplicatory function in rabbits is unknown. The middle ear structures of 46 rabbits, 1 to 30 d old, were prepared and measured. The area of pars tensa and the levers of malleus and incus increase with age. After the 10th d of life, no statistical significant growth were measurable. But the calculated multiplicatory factor of single animals indicate the end of the development at the 15th postnatal d. In contrast, the cochlear function attains the adult values not till the 26th d of life. It is 10 d longer than the middle ear growth.     

Microvascular networks in tympanic membrane, malleus periosteum, and annulus perichondrium of neonatal mongrel dog: a vasculoanatomic model for surgical considerations

India-ink-imaged blood-vessel networks in cleared tympanic membranes and adnexa from ten neonatal dogs were examined microscopically and photographed. The major significance of the study lies in documentation of a dual source of arterial supply, a bilaminar relationship of arterial and venous plexuses intrinsic to the tympanic membrane, and a consistent major venous pathway relative to a definite locus (pars flaccida of the membrane). Illustration of all three types of blood pathways (arteries, veins, and capillaries) provides new vasculoanatomic data that are essential to ear surgery, specifically--to myringotomies and myringoplasties. A comparison was made between dog and human tympanic membrane structures and their arterial supplies. Close similarities suggested that dog tympanic membrane might serve as a suitable model for development of innovative surgical procedures and as a model for rehearsal of difficult techniques. The results of this investigation provide a valuable caveat to otologists.     

The ear in subterranean insectivora and rodentia in comparison with ground-dwelling representatives. I. Sound conducting system of the middle ear.

Compared to acoustically unspecialized mammals (soricids and murids), the middle ear of subterranean insectivores and rodents (twelve species of six families examined) was clearly distinguished and characterized by many common features: rather round and relatively larger eardrum without a pars flaccida; reduced gonial; loose or no connection between the malleus and the tympanic bone; reduced and straightened transversal part of the malleus; enlarged incus; increased and rather flat incudo-mallear joint; rather parallel position of the mallear manubrium and incudal crus longum in some species (and their fusion in bathyergids); reduced or even missing middle ear muscles. Convergent occurrence of these structural features in taxa of different origin and their generally derived character suggest that they cannot be categorized as degenerative. The form of the stapes can be considered as a non-adaptive trait; it was taxon specific yet remarkably polymorphous in some species and exhibited no convergent features among subterranean mammals. Structural retrogression resulting in a columella-like stapes was observed in some species lacking the stapedial artery. The stapedial base was relatively larger than in unspecialized mammals. The subterranean mammals did not exhibit conspicuously enlarged eardrums as would be required for sensitive tuning to low frequencies. It is, however, argued that while selective pressures in the subterranean ecotope promoted hearing of low frequencies, hearing sensitivity did not have to be enhanced.     

Development of the stratified squamous epithelium of the human tympanic membrane and external canal: the origin of auditory epithelial migration

The development of the stratified squamous epithelium of the tympanic membrane and external canal was studied in 167 embryonic, fetal, and postnatal human ears. It originates as a tube derived from the epithelium of the fundus of the primary external canal (zone 1). The tube is composed of a thin, flat epithelium on the medial side (zone 2), continuous with a thicker one (zone 3) on the lateral side; zone 3 thereafter merges with the external epithelium of the primary external canal (zone 4). Proliferative activity, as indicated by a thickened epithelium, with rete ridges in later fetal life, is present mainly in zones 1 and 3. Cornification at 18 weeks gestation is followed by clearing of keratinous debris to the exterior. Subsequently the canal widens, zone 1 now covering the pars flaccida region, a tongue-shaped area passing inferiorly from it and a part of the postero-superior deep canal adjacent to it; zone 2 covers the pars tensa and zone 3 most of the deep external canal. On the basis of the original embryonic growth, migratory epithelial movement throughout life is postulated to be generated in zone 1 by mitotic interposition and then to pass to zone 2. It then moves en masse through to zone 3, where unilateral progression by mitotic means takes the epithelium up to the cartilaginous canal. Such a pathway is approximately that seen in the marked, living eardrum and canal.     

Immunohistochemical investigation of lymphatic vessel formation control in mouse tooth development: lymphatic vessel-forming factors and receptors in tooth development in mice

The presence of lymphatic vessels in dental pulp has recently been controversial, and no conclusion has been reached. In this study, we investigated the control of lymphangiogenesis with dental pulp development in the mouse mandibular molar using VEGF-C, VEGF-D, and VEGFR-3 as indices of lymphatic vessel-controlling factors. In addition, to distinguish blood and lymphatic vascular epithelial cells, we performed immunohistochemical analysis using von Willebrand factor (vWF) and statistical analysis. In dental papilla in the bell-stage non-calcified period, mesenchymal cells positive for VEGF-C, VEGF-D, and VEGFR-3 increased and lumen-forming endothelial cells were noted, but vWF was negative, suggesting that these were actively forming lymphatic vessels. Positive undifferentiated mesenchymal cells, an increase in endothelial cells in dental pulp, and lumen expansion were noted early after birth. Positivity was also detected in the odontoblast layer and sheath of Hertwig after birth, suggesting that these factors also play important roles in odontoblast differentiation and maturation and periodontal ligament and tooth root formation. We embryologically clarified lymphatic vessel formation in dental pulp and a process of lymphatic vessel formation from blood vessels, suggesting involvement of the surrounding tissue, odontoblasts, and sheath of Hertwig in vessel formation.     

Intralobular lymphatic vessels and their relationship to blood vessels in the mouse thymus

Summary The spatial distribution and fine structure of the lymphatic vessels within the thymic lobules of normal and hydrocortisone-injected mice were studied by light- and electron microscopy. The lymphatic vessels of the cortex and medulla of normal thymus are irregularly shaped spaces closely associated with branches of the intralobular artery and vein. The overall distribution of these vessels in the greatly involuted thymus of hydrocortisone-treated mice is essentially the same as in the normal thymus. The wall of the lymphatic vessels consists of only a layer of endothelial cells supported by underlying reticular cells. The luminal surface of the endothelial cell is smooth, but trabecular processes are often seen. There are three morphological types of intercellular contacts between contiguous cells, namely, end-to-end, overlapping and interdigitating. The lymphatic vessel has anchoring filaments and collagen fibrils, but a basal lamina is either absent, or if present, is discontinuous. This is in contrast to the continuous basal lamina of the venule. The perivascular space surrounding the postcapillary venule opens into a terminal lymphatic vessel at the cortico-medullary junction and in the medulla. Lymphocytes are seen penetrating the lymphatic endothelium, particularly in acutely involuted thymuses. These findings suggest that the intralobular lymphatic vessels may originate from the vacuities that surround the postcapillary venules, and the lymphatic system may function as a pathway for the migration of lymphocytes into or out of the lymphatic circulation.     

Basement membrane protein distribution in LYVE-1-immunoreactive lymphatic vessels of normal tissues and ovarian carcinomas

The endothelial cells of blood vessels assemble basement membranes that play a role in vessel formation, maintenance and function, and in the migration of inflammatory cells. However, little is known about the distribution of basement membrane constituents in lymphatic vessels. We studied the distribution of basement membrane proteins in lymphatic vessels of normal human skin, digestive tract, ovary and, as an example of tumours with abundant lymphatics, ovarian carcinomas. Basement membrane proteins were localized by immunohistochemistry with monoclonal antibodies, whereas lymphatic capillaries were detected with antibodies to the lymphatic vessel endothelial hyaluronan receptor-1, LYVE-1. In skin and ovary, fibrillar immunoreactivity for the laminin α4, β1, β2 and γ1 chains, type IV and XVIII collagens and nidogen-1 was found in the basement membrane region of the lymphatic endothelium, whereas also heterogeneous reactivity for the laminin α5 chain was detected in the digestive tract. Among ovarian carcinomas, intratumoural lymphatic vessels were found especially in endometrioid carcinomas. In addition to the laminin α4, β1, β2 and γ1 chains, type IV and XVIII collagens and nidogen-1, carcinoma lymphatics showed immunoreactivity for the laminin α5 chain and Lutheran glycoprotein, a receptor for the laminin α5 chain. In normal lymphatic capillaries, the presence of primarily α4 chain laminins may therefore compromise the formation of endothelial basement membrane, as these truncated laminins lack one of the three arms required for efficient network assembly. The localization of basement membrane proteins adjacent to lymphatic endothelia suggests a role for these proteins in lymphatic vessels. The distribution of the laminin α5 chain and Lutheran glycoprotein proposes a difference between normal and carcinoma lymphatic capillaries.     

Static versus dynamic gerbil tympanic membrane elasticity: derivation of the complex modulus

An accurate estimation of tympanic membrane stiffness is important for realistic modelling of middle ear mechanics. Tympanic membrane stiffness has been investigated extensively under either quasi-static or dynamic loading conditions. It is known that biological tissues are sensitive to strain rate. Therefore, in this work, the mechanical behaviour of the tympanic membrane was studied under both quasi-static and dynamic loading conditions. Experiments were performed on the pars tensa of four gerbil tympanic membranes. A custom-built indentation apparatus was used to perform in situ tissue indentations and testing was done applying both quasi-static and dynamic sinusoidal indentations up to 8.2?Hz. The unloaded shape of the tympanic membrane was measured and used to create specimen-specific finite element models to simulate the experiments. The frequency dependent Young's modulus of each specimen was then estimated by an inverse analysis in which the error between experimental and simulated indentation data was optimised for each indentation frequency separately. Using an 8?μm central region thickness, we found Young's moduli between 71 and 106?MPa (n = 4) at 0.2?Hz indentation frequency. A standard linear viscoelastic model and a viscoelastic model with a continuous relaxation spectrum were used to derive a complex modulus in the frequency domain. Due to experimental limitations, the indentation frequency upper limit was 8.2?Hz. The average relative modulus increase in this domain was 14% and the increase was the strongest below 6?Hz.     

领春木茎次生木质部中导管穿孔板的变异

领春木Euptelea pleiosperma Hook. f. &; Thoms.隶属领春木科Eupteleaceae。该科为东亚特有的单型科,其系统位置一直颇有争议。本文对中国产领春木茎次生木质部中导管穿孔板的变异进行了观察,以期为它的系统位置提供进一步的解剖学证据。结果表明,领春木茎次生木质部中包括无明显穿孔板的管胞状导管和典型的导管两种类型。在无明显穿孔板的导管中,穿孔中的纹孔膜全部或部分消失,但穿孔无规则排列或聚集,不形成具典型的形态特征的穿孔板;在典型的导管中,穿孔板形态变异较大,包括几个类型:网状穿孔板(含麻黄式穿孔板)、网状和梯状混合型穿孔板、梯状穿孔板、梯状穿孔板向单穿孔板的过渡。在上述导管穿孔板类型中,只有梯状穿孔板的穿孔中可以观察到纹孔膜的残余。在领春木次生木质部中也观察到了端壁多穿孔板及侧壁穿孔板。根据观察结果,我们认为领春木次生木质部导管穿孔板的许多特征说明该科可能处于毛茛目中比较原始的系统位置。     

Three-Dimensional Imaging of Prox1-EGFP Transgenic Mouse Gonads Reveals Divergent Modes of Lymphangiogenesis in the Testis and Ovary

The lymphatic vasculature forms a specialized part of the circulatory system, being essential for maintaining tissue fluid homeostasis and for transport of hormones, macromolecules, and immune cells. Although lymphatic vessels are assumed to play an important role in most tissues, their morphogenesis and function in the gonads remains poorly understood. Here we have exploited a lymphatic-specific Prox1-EGFP reporter mouse model and optical projection tomography technology to characterize both the temporal and spatial development of the lymphatic vessel network in mouse testes and ovaries. We find that lymphangiogenesis in the testis is initiated during late gestation, but in contrast to other organs, lymphatic vessels remain confined to the testis cap and, unlike blood vessels, do not infiltrate the entire organ. Conversely, lymphatic vessels invade the ovarian tissue, beginning postnatally, and sprouting from preexisting lymphatic vessels at the extraovarian rete. The ovary develops a rich network of lymphatic vessels, extending from the medulla into the surrounding cortex adjacent to developing follicles. This study reveals distinct patterns of lymphangiogenesis in the testes and ovaries and will serve as the basis for the identification of the divergent molecular pathways that control morphogenesis and the function of the lymphatic vasculature in these two organs.     

Kinetics of fluid flux in the rat diaphragmatic submesothelial lymphatic lacunae

The specific role of loops and/or linear segments in pleural diaphragmatic submesothelial lymphatics was investigated in seven anesthetized, paralyzed, and mechanically ventilated rats. Lymphatic loops lay peripherally above the diaphragmatic muscular plane, whereas linear vessels run over both the muscular and central tendineous regions. Lymph vessel diameter, measured by automatic software analysis, was significantly greater (P < 0.01) in linear vessels [103.4 +/- 8.5 microm (mean +/- SE), n = 18] than in loops (54.6 +/- 3.3 microm, n = 21). Conversely, the geometric mean of intraluminal flow velocity, obtained from the speed of distribution of a bolus of fluorescent dextrans injected into the vessel, was lower (P < 0.01) in linear vessels (26.3 +/- 1.4 microm/s) compared with loops (51.3 +/- 3.2 microm/s). Lymph flow, calculated as the product of flow velocity by vessel cross-sectional area, was similar in linear vessels and in individual vessels of a loop, averaging 8.6 +/- 1.6 nl/min. Flow was always directed from the diaphragm periphery toward the medial tendineous region in linear vessels, whereas it was more complex and evidently controlled by intraluminal unidirectional valves in loops. The results suggest that loops might be the preferential site of lymph formation, whereas linear vessels would be mainly involved in the progression of newly formed lymph toward deeper collecting diaphragmatic ducts. Within the same hierarchic order of diaphragmatic lymphatic vessels, the spatial organization and geometrical arrangement of the submesothelial lacunae seem to be finalized at exploiting the alternate contraction/relaxation phases of diaphragmatic muscle fibers to optimize fluid removal from serosal cavities.     

Lymphatic vessels in the developing diaphragm of the rat.

Diaphragms of fetal, neonatal and young albino rats have been observed both under light and electron microscopes to examine the presence and distribution of lymphatic vessels and their morphological features. In fetal diaphragms of between 18 and 22 days of gestation, no normal lymphatic vessels can be seen; only after birth, specifically in neonatal and 2-day-old rats, small lymphatic vessels appear; they are in close proximity to the blood vessels in the inner areas of the muscle. As the rats get older, lymphatic vessels are also observed in the subserosa where an abundant connective tissue is present. The fine structure of diaphragmatic lymphatic vessels is different at different ages. In neonatal rats of up to 2 days, the endothelial wall is very thin and often holed. The relationships between contiguous endothelial cells are characterized by simple end-to-end or overlapping structures. The basement membrane is virtually absent. Within the first week of life, the endothelial wall becomes more complex; along the wall, complex interdigitations between two contiguous endothelial cells often touch. A discontinuous basement membrane and collagen and elastic fibers surround the vessels. In the older rats (from 14 to 25 to 140 days), next to the complex interdigitations which characterize the junction between two contiguous endothelial cells, cellular flaps interdigitate forming a channel which opens out either to the exterior or the interior of the vessel. Dense bundles of elastic and collagen fibers are closely apposed to the endothelial wall.     

Rotation of middle ear ossicles during cetacean development

Cetacean middle ears are unique among mammals in that they have an elongated tympanic membrane, a greatly reduced manubrium mallei, and an incudal crus longum that is shorter than the crus breve. Elongation of the tympanic membrane and reduction of the manubrium is thought to be related to an evolutionary rotation of the incus and malleus out of the plane of the tympanic membrane. We examined if rotation also occurs during ontogeny by comparing the middle ears of two species of dolphins (Delphinus delphis, Stenella attenuata) at different stages of development. We observed that: the incus has the body and crural proportions as in terrestrial mammals early in development; the incudomallear complex rotates approximately 90 degrees following ossification; the tympanic membrane is not elongated until relatively late in development. Therefore, some of the unique characteristics of the cetacean middle ear develop as modifications of an initially terrestrial-like morphology.     

Origin and nature of vessels in monocotyledons. 5. Araceae subfamily Colocasioideae

Tracheary elements from macerations of roots and stems of one species each of five genera of Araceae subfamily Colocasioideae were studied by means of SEM (scanning electron microscopy). All of the genera have vessel elements not merely in roots, as previously reported for the family as a whole, but also in stems. The vessel elements of stems in all genera other than Syngonium are less specialized than those of roots; stem vessel elements are tracheid-like and have porose pit membrane remnants in perforations. The perforations with pit membrane remnants demonstrate probable early stages in evolution of vessels from tracheids in primary xylem of monocotyledons. The vessel elements with such incipient perforation plates lack differentiation in secondary wall thickenings between perforation plate and lateral wall, and such vessel elements cannot be identified with any reliability by means of light microscopy. The discrepancy in specialization between root and stem vessel elements in genera other than Syngonium is ascribed to probable high conductive rates in roots where soil moisture fluctuates markedly, in contrast with the storage nature of stems, in which selective value for rapid conduction is less. Syngonium stem vessels are considered adapted for rapid conduction because the stems in that genus are scandent. Correlation between vessel element morphology and ecology and habit are supported. Although large porosities in vessel elements facilitate conduction, smaller porosities may merely represent rudimentary pit membrane lysis.     

Bapx1 regulates patterning in the middle ear: altered regulatory role in the transition from the proximal jaw during vertebrate evolution

The middle ear apparatus is composed of three endochondrial ossicles (the stapes, incus and malleus) and two membranous bones, the tympanic ring and the gonium, which act as structural components to anchor the ossicles to the skull. Except for the stapes, these skeletal elements are unique to mammals and are derived from the first and second branchial arches. We show that, in combination with goosecoid (Gsc), the Bapx1 gene defines the structural components of the murine middle ear. During embryogenesis, Bapx1 is expressed in a discrete domain within the mandibular component of the first branchial arch and later in the primordia of middle ear-associated bones, the gonium and tympanic ring. Consistent with the expression pattern of Bapx1, mouse embryos deficient for Bapx1 lack a gonium and display hypoplasia of the anterior end of the tympanic ring. At E10.5, expression of Bapx1 partially overlaps that of Gsc and although Gsc is required for development of the entire tympanic ring, the role of Bapx1 is restricted to the specification of the gonium and the anterior tympanic ring. Thus, simple overlapping expression of these two genes appears to account for the patterning of the elements that compose the structural components of the middle ear and suggests that they act in concert. In addition, Bapx1 is expressed both within and surrounding the incus and the malleus. Examination of the malleus shows that the width, but not the length, of this ossicle is decreased in the mutant mice. In non-mammalian jawed vertebrates, the bones homologous to the mammalian middle ear ossicles compose the proximal jaw bones that form the jaw articulation (primary jaw joint). In fish, Bapx1 is responsible for the formation of the joint between the quadrate and articular (homologues of the malleus and incus, respectively) enabling an evolutionary comparison of the role of a regulatory gene in the transition of the proximal jawbones to middle ear ossicles. Contrary to expectations, murine Bapx1 does not affect the articulation of the malleus and incus. We show that this change in role of Bapx1 following the transition to the mammalian ossicle configuration is not due to a change in expression pattern but results from an inability to regulate Gdf5 and Gdf6, two genes predicted to be essential in joint formation.     

The intraorganic lymphatic bed of the mucous membrane of the tongue

The intraorganic lymphatic bed of the canine tongue mucous membrane is presented by the superficial and deep capillary networks and by the plexuses of the intraorganic lymphatic vessels. The capillaries of the superficial network in the thickness of the mucous membrane of the dorsal surface of the lingual tip, body and root form the lymphatic bed of the mechanical and gustatory papillas. The diameter of the capillaries in the both networks and in the intraorganic lymphatic vessels is greater in the mucous membrane of the ventral surface of the tip and of the lateral surfaces of the lingual body, and density of their arrangement is higher in the mucous membrane on the dorsal surface of the lingual tip, body and root. The capillaries of the superficial network in all areas of the lingual mucous membrane are thinner than those of the deep network, and the loops formed by the capillaries of the superficial network are less than the loops of the deep network.     

Assembling a functional tympanic membrane: signals from the external acoustic meatus coordinate development of the malleal manubrium

In terrestrial mammals, hearing starts with the perception of acoustic pressure by the tympanic membrane. Vibrations in this membrane are then transduced into the inner ear by the ossicle chain of the middle ear, composed of the malleus, incus and stapes. The proper connection of the ossicle chain with the tympanic membrane, provided by the insertion of the manubrium of the malleus into the eardrum, is essential for the functionality of the hearing apparatus. We describe here the mechanisms regulating the development of the manubrium and its integration into the tympanic membrane. We show that the external acoustic meatus (EAM), which eventually forms the outer epithelium of the tympanic membrane, plays an essential role in this developmental process. Histological and expression analyses indicate that the manubrium develops close to the EAM with a similar temporal sequence. In addition, when the middle ear ossicles are allowed to develop in vitro under conditions that do not support further EAM development, the manubrium develops only up to the stage of its induction at the time of explantation. Moreover, genetically or teratogenically derived alterations in the EAM also have an effect on manubrial development. Finally, we show that the EAM is the source of two quite opposite activities, one that induces chondrogenesis and another that represses it. The combination of these two activities results in the proper positioning of the manubrium.     

国产五味子科植物导管分子的比较解剖

对五味子科 2属 1 9种植物导管分子的结构进行了比较观察。结果表明 :2属均以具缘纹孔导管为主 ,少有梯纹和螺纹导管 ,南五味子属多数种还有梯孔纹导管 ;2属导管长度和宽度差异很小 ,长 /宽比值有差异 ;根据穿孔板的结构可分为 2种形式 :( 1 )单穿孔板 ,这类穿孔板较普遍存在 ;( 2 )梯形穿孔板 ,发现 2属中的红花五味子 (Schisandrarubriflora)、五味子 (S .chinensis)、翼梗五味子 (S .henryi)、铁箍散 (S .propinquavar.sinensis)和黑老虎 (Kadsuracoccinea) 5种植物的导管分子具有此类穿孔板。其中五味子属中的五味子导管分子只具有梯形穿孔板 ,无单穿孔板 ,但横条较少 ,多为 2～ 3条。