Vascularization and Innervation of Connective Tissue Grafts in the Treatment of Gingival Recessions: A Histologic and Immunohistochemical Study.

The goal of this study was to histologically evaluate the healing process of subepithelial connective tissue graft in humans. A sample of 16 patients with Miller Class I or II localized gingival recession defects were consecutively treated with a bilaminar technique. At baseline and after 2 months, tissue samples were taken from the donor and recipient sites. The revascularization and reinnervation of the graft were examined by means of hematoxylin-eosin and immunohistochemical techniques. After 2 months of healing, the integration of the grafted tissue was evident with the connective tissue expressing features belonging to both the recipient (cellular component) and the donor site (extracellular matrix) in the inner and superficial layers, respectively. This was accompanied by increased neovascularization and reduction in intraepithelial free nerve endings.

Richly innervated soft tissues covering the superficial aspect of the extensor origin in patients with chronic painful tennis elbow - Implication for treatment?

BACKGROUND: Tennis elbow is difficult to treat. The results of surgical treatments are not convincing. Treatment studies on Achilles and patellar tendinopathy targeting the richly innervated and vascularized soft tissues outside the tendon have shown promising outcomes. The innervation patterns in the fibrous/fatty tissues superficially to the elbow extensor origin have not been clarified. METHODS: Nine tissue specimens from the fibrous/fatty tissue covering the extensor origin was taken from seven patients (mean age: 45 years) undergoing surgical treatment for chronic painful tennis elbow. The specimens were stained for morphology (haematoxylin and eosin, H and E) and immunohistochemically for general nerve marker protein gene product 9.5 (PGP 9.5) and markers for sympathetic (tyrosine hydroxylase, TH) and sensory nerve fibres (calcitonin gene-related peptide, CGRP). RESULTS: All specimens contained multiple blood vessels and nerve structures indicated by morphology and immunoreactions. There was a frequent occurrence of TH reactions, especially peri-vascularly, but also in nerve fascicles. Immunoreactions for CGRP were seen in nerve fascicles and isolated nerve fibres. CONCLUSION: The results provide new information on the innervation patterns of the superficial tissues of the extensor origin and their potential as source of tennis elbow pain. LEVEL OF EVIDENCE: IV.

Subparaneurial ganglion cysts of the fibular and tibial nerves: A new variant of intraneural ganglion cysts.

Over the last decade, the mechanism of formation of intraneural ganglion cysts has been established through a meticulous review of clinical findings and correlation with patterns produced on magnetic resonance imaging (MRI). Pathognomonic imaging patterns distinguish these rare lesions from the more common extraneural variants in almost all cases. In this report, we present a new pattern of cyst occurrence in the subparaneurial compartment of the nerve and provide potential anatomic explanations for its pathogenesis. Using an anatomic framework of connective tissue compartments of the nerve, we reviewed 63 (56 fibular and seven tibial) intraneural ganglion cysts in the knee region evaluated at our institution and all reports with MRI in the world's literature for evidence of cyst occurrence in the subparaneurial compartment. We identified six cases (five in the common fibular nerve and one in the tibial nerve) at our institution that had MR evidence of cyst in the subparaneurial compartment with a new complex lobulated pattern. All cases had articular branch connections to the superior tibiofibular joint, which at operation were resected along with the joints. Follow-up revealed complete recovery in all instances and no clinical or radiological signs of recurrence. Three cases out of 80 in the literature exhibited the new complex lobulated MRI pattern. We present a new pattern of intraneural ganglion cyst occurrence in a potential space that surrounds peripheral nerves--the subparaneurial compartment. We believe that the unifying articular theory applies to the pathogenesis and management of these rare variants.

Does Laser Surgery Interfere with Optical Nerve Identification in Maxillofacial Hard and Soft Tissue?--An Experimental Ex Vivo Study.

The protection of sensitive structures (e.g., nerves) from iatrogenic damage is of major importance when performing laser surgical procedures. Especially in the head and neck area both function and esthetics can be affected to a great extent. Despite its many benefits, the surgical utilization of a laser is therefore still limited to superficial tissue ablation. A remote feedback system which guides the laser in a tissue-specific way would provide a remedy. In this context, it has been shown that nerval structures can be specifically recognized by their optical diffuse reflectance spectra both before and after laser ablation. However, for a translation of these findings to the actual laser ablation process, a nerve protection within the laser pulse is of utmost significance. Thus, it was the aim of the study to evaluate, if the process of Er:YAG laser surgery--which comes with spray water cooling, angulation of the probe (60°) and optical process emissions--interferes with optical tissue differentiation. For the first time, no stable conditions but the ongoing process of laser tissue ablation was examined. Therefore, six different tissue types (nerve, skin, muscle, fat, cortical and cancellous bone) were acquired from 15 pig heads. Measurements were performed during Er:YAG laser ablation. Diffuse reflectance spectra (4500, wavelength range: 350-650 nm) where acquired. Principal component analysis (PCA) and quadratic discriminant analysis (QDA) were calculated for classification purposes. The clinical highly relevant differentiation between nerve and bone was performed correctly with an AUC of 95.3% (cortial bone) respectively 92.4% (cancellous bone). The identification of nerve tissue against the biological very similar fat tissue yielded good results with an AUC value of 83.4% (sensitivity: 72.3%, specificity: of 82.3%). This clearly demonstrates that nerve identification by diffuse reflectance spectroscopy works reliably in the ongoing process of laser ablation in spite of the laser beam, spray water cooling and the tissue alterations entailed by tissue laser ablation. This is an essential step towards a clinical utilization.

Chievitz' juxtaparotid organ, free from cancer.

INTRODUCTION: Up to the half of twentieth century, Chievitz organ was considered an embryonal organ, disappearing with growth. But Zenker, in 1953, demonstrated the existence of this organ in adult life, too4. REVIEW: In this article we review the embryology, the macroscopic and microscopic anatomy, the ultrastructure, the functional significance and the pathology of the Chievitz'Juxtaparotid Organ (CJO). The CJO is not a macroscopic apparent organ, but it looks like a nerve. The CJO takes connections with buccinator muscle, at the level of the parotid duct, and the medial pterygoid muscle. The cell parenchyma is enveloped by the connective tissue, that is divided into three layers15, 16: the inner layer -"stratum fibrosum internum"-, composed of collagenous and elastic microfibrils; the middle layer - "stratum nervosum" - containing a lamellar inner core and Ruffini SNF5; the external layer - "stratum fibrosum externum", that is a collagen capsule. The parenchymal cells show a rich enzyme activity. The parenchymal cells may play the same role as glomus cells of the 1st type and Merkel cells20, 21. When a surgical resection is performed for an oral carcinoma, the CJO may be present in the specimen25. The CJO may be wrongly diagnosed as perineural invasion by carcinoma26, 27, 28. CONCLUSION: We report that Chievitz' organ is the only organ in which the cancer does not occur. KEY WORDS: Chievitz' organ, Juxtaoral organ, Parotid gland.

Morphometric analysis of connective tissue sheaths of sural nerve in diabetic and nondiabetic patients.

One of the most common complications of diabetes mellitus is diabetic neuropathy. It may be provoked by metabolic and/or vascular factors, and depending on duration of disease, various layers of nerve may be affected. Our aim was to investigate influence of diabetes on the epineurial, perineurial, and endoneurial connective tissue sheaths. The study included 15 samples of sural nerve divided into three groups: diabetic group, peripheral vascular disease group, and control group. After morphological analysis, morphometric parameters were determined for each case using ImageJ software. Compared to the control group, the diabetic cases had significantly higher perineurial index (P < 0.05) and endoneurial connective tissue percentage (P < 0.01). The diabetic group showed significantly higher epineurial area (P < 0.01), as well as percentage of endoneurial connective tissue (P < 0.01), in relation to the peripheral vascular disease group. It is obvious that hyperglycemia and ischemia present in diabetes lead to substantial changes in connective tissue sheaths of nerve, particularly in peri- and endoneurium. Perineurial thickening and significant endoneurial fibrosis may impair the balance of endoneurial homeostasis and regenerative ability of the nerve fibers. Future investigations should focus on studying the components of extracellular matrix of connective tissue sheaths in diabetic nerves.

The connective tissue changes of Crohn's disease.

Although the inflammatory pathology of Crohn's disease is manifestly its most important attribute, the connective tissue changes are important in the genesis of the more chronic features of the disease, and yet these have received little attention from clinicians, pathologists, and scientists. Fat-wrapping appears to be pathognomonic of Crohn's disease, and is an important marker of disease for surgeons. There is evidence of a complex interplay between the effector inflammatory cells of Crohn's disease and adipocytes, hyperplasia of which results in fat-wrapping. Pathologically, this is exhibited in the close relationship between the transmural inflammation that is so characteristic of Crohn's disease and fat-wrapping. Fibrosis and muscularization are also important components of the chronic changes of intestinal Crohn's disease. Neuronal and vascular changes make up the remaining connective tissue changes: these constitute a distinctive feature, and are even specific for Crohn's disease. For pathologists, the combination of these connective changes will allow a diagnosis of chronic 'burnt-out' Crohn's disease, even in the absence of its highly characteristic inflammatory features. The connective tissue changes of Crohn's disease form an important part of its long-term pathology. They deserve more attention from clinicians, diagnostic pathologists and researchers alike.

Experimental confirmation that vibrations at soft tissue conduction sites induce hearing by way of a new mode of auditory stimulation.

BACKGROUND: A new mode of auditory stimulation has been demonstrated which is through soft tissue conduction (STC). It involves evoking auditory sensations by applying the clinical bone vibrator to the skin over soft tissue (not over bone) sites on the head and neck. METHODS: This study was designed to show that stimulation by STC excites the cochlea in a way similar to that of air conduction (AC) and bone conduction (BC). RESULTS: It is shown here that auditory nerve brainstem evoked response (ABR) thresholds in mice and in the fat sand rat to AC, to BC and to STC stimulation are all elevated following administration of drugs (salicylic acid and furosemide) which depress the cochlear amplifier. In addition, the present study brings evidence that STC stimulation is not a variant of BC since the sound pressures recorded in the occluded external auditory canal (the occlusion effect) in response to STC are significantly smaller than that to BC stimulation, though both are of equal loudness. CONCLUSIONS: This new mode, STC, therefore appears to bypass the middle ear mechanisms and consequently may contribute to auditory diagnosis.

SEM and neurohistological observations of nerve endings in the middle region of the tongue of the collared peccary (Tayassu tajacu): a silver impregnation method.

The presence of lingual papillae and the nerve endings in the middle region of the tongue mucosa of collared peccary (Tayassu tajacu) were studied using scanning electron microscopy and light microscopy, based upon the silver impregnation method. The middle region of tongue mucosa revealed numerous filiform and fungiform papillae. The thick epithelial layer showed epithelial cells and a dense connective tissue layer containing nerve fibre bundles and capillaries. The sensory nerve endings, intensely stained by silver impregnation, were usually non-encapsulated and extended into the connective tissue of the filiform and fungiform papillae very close to the epithelial cells. In some regions, the sensory nerves fibres formed a dense and complex network of fine fibrils. The presence of these nerve fibrils may characterize the mechanisms of transmission of sensitive impulses to the tongue mucosa.

Nerve cell bodies and small ganglia in the connective tissue stroma of human submandibular glands.

The objective of the study was to investigate the presence and distribution of nerve cell bodies and small ganglia in the stroma of human submandibular gland. A retrospective immunohistochemical study in 13 human submandibular glands, fixed in neutral buffered formalin and embedded in paraffin wax, was undertaken. Six glands were excised in the course of radical neck dissection for oral squamous cell carcinoma and were disease-free, six showed sialadenitis, and one was involved by tuberculosis. Primary antibodies applied were neuron specific enolase, synaptophysin, and glial fibrilliary acidic protein. Neuron specific enolase and synaptophysin positive nerve cell bodies and small ganglia were found in 8/13 and 13/13 glands, respectively. They were found in the interlobular connective tissue stroma of human SMG, in close association to salivary parenchymal cells and blood vessels, and some of them were incorporated in GFAP positive peripheral nerves. To our knowledge, nerve cell bodies and small ganglia have been described only in the connective tissue stroma of autotransplanted human SMG and their functional importance is not clear.

The other mechanism of muscular referred pain: the "connective tissue" theory.

Muscular referred pain, that is, pain perceived in a somatic area other than the site of the noxious stimulation, takes place on a specific place to each muscle in constant and predictable pattern. The central hyperexcitability theory focused on spinal cord, the most proper theory at present, can explain well the segmental pattern of referred pain showing delayed onset. But it is hard to explain the non segmental pattern of referred pain areas of superficial-seated or limb girdle and limb muscles. Referred pain areas of limb girdle and limb muscles appear on the skin above a belt of synergistic muscles beyond the segmental areas. In the case of forearm and calf muscles, referred pain shows up on the palm and sole, the point of force application to the outer object. This finding reflects biomechanical relationship between muscle and its referred pain area. From the phylogenetic perspective, aquatic vertebrated animals (e.g. fish) use myoseptum surrounding myomere, connected to skin to keep tensile strength with it for effective swimming. Likewise, in terrestrial vertebrated animals, there are skin parts weakly interconnected with muscles, though the tensile property of nearly all the skin devolutes except the points of action with the outside. These points are dynamic maximal skin tension areas connected with muscles through superficial fascia, in other words, referred pain areas. Referred pain of deep-seated or truncal muscles appears on the trunk segmentally via spinal cord (the central hyperexcitability theory), but superficial-seated or limb girdle and limb muscles elicit referred pain on dynamic maximal skin tension area through connective tissue (the "connective tissue" theory).

[Histophysiology of epithelial and connective tissue components of the vaginal portion of the uterine cervix].

This review provides an analysis of current data on the tissue and cell components of the vaginal portion of the uterine cervix (ectocervix - EC) in human female and on their interrelations under normal conditions and in the development of some pathological processes. Detailed histological and immunocytochemical characteristic is given to two types of the epithelium--stratified squamous and columnar; the regularities and mechanisms of their junction displacement during various age periods are described. The data on the relation between the proliferation activity and apoptosis intensity in EC epithelium at different developmental stages are discussed. The data are presented on connective tissue component (stroma), its vascular and nervous structures. Cellular elements of the immune system contained in EC are examined. The significance of hormones and other biologically active molecules in the control of EC tissue functions and in coordination of their interaction, is emphasized.

[Juxtaoral organ of Chievitz].

This review presents the analysis of the systematized data on human juxtaoral organ (JOO) development, structure and function based on the results of classical and recent morphological studies. JOO morphogenesis is traced, including the appearance of its anlage at the bottom of the primitive mouth, epithelial invagination into the mesenchyme, JOO detachment from the oral epithelium, its innervation, connective tissue capsule formation, and final maturation. The analysis of the results of macroscopical, histological, electron microscopical, histochemical and immunohistochemical studies is presented, suggesting high metabolic and synthetic activity of its epithelium, which expresses several neural markers, and emphasizing a rich innervation of both its epithelial and stromal components. The findings supporting the concepts of JOO secretory and mechanosensory functions, are examined. The data on the differential diagnosis between JOO and tumoral processes are discussed, as well as the pathological changes of JOO itself and their significance for the diagnosis of the diseases.

Vanilloid receptor TRPV1, sensory C-fibers, and activation of adventitial mast cells. A novel mechanism involved in adventitial inflammation.

The immunological mechanisms on adventitial inflammation has received much attention, while the contribution of nerves to adventitial inflammation has largely been ignored. Although the mechanism of initial chemotaxis of the adventitial inflammatory cells remains unknown, vascular nerves were frequently found in the inflammatory lesions of coronary adventitia and adventitial mast cells connect with sensory nerve fibers in atherosclerotic coronary arteries. The sensory nerves in contact with adventitial mast cells contained the neuropeptides SP and CGRP. These neuropeptides play an important role in the amplification of tissue injury by the increase of both vascular permeability and neutrophil recruitment, and the term ''neurogenic inflammation'' has been coined. Activation of adventitial mast cells, with ensuing release of vasoactive compounds, may cause vasoconstriction in atherosclerotic coronary segments. Therefore, we hypothesize that adventitial vanilloid receptor TRPV1 and sensory C-fibers may play a pistol role for adventitial inflammation.

A neurovascular transmission model for acupuncture-induced nitric oxide.

Acupuncture is the practice of inserting needles into the body to reduce pain or induce anesthesia. More broadly, acupuncture is a family of procedures involving the stimulation of anatomical locations on or in the skin by a variety of techniques. Employing acupuncture to treat human disease or maintain bodily condition has been practiced for thousands of years. However, the mechanism(s) of action of acupuncture at the various meridians are poorly understood. Most studies have indicated that acupuncture is able to increase blood flow. The acupuncture points have high electrical conductance and a relationship of the acupuncture points and meridians with the connective tissue planes and the perivascular space has also been suggested. Several studies employing the human and animal models have shown that acupuncture enhances the generation of nitric oxide (NO) and increases local circulation. Specifically, electroacupuncture (EA) seems to prevent the reduction in NO production from endothelial NO synthetase (eNOS) and neuronal NO synthase (nNOS) that is associated with hypertension and this process involves a stomach-meridian organ but not a non-stomach-meridian organ such as the liver. How can we explain the phenomena of EA and meridian effect? Here, we proposed a neurovascular transmission model for acupuncture induced NO. In this proposed model, the acupuncture stimulus is able to influence connective tissue via mechanical force transfer to the extracellular matrix (ECM). Through the ECM, the mechanotransduction stimulus can be translated or travel from the acupuncture points, which involve local tissue and cells. Cells in the local tissue that have received mechanotransduction induce different types of NO production that can induce changes in blood flow and local circulation. The local mechanical stress produced is coupled to a cyclic strain of the blood vessels and this could then change the frequency of resonance. According to the resonance theory, an oscillatory pattern of NO formation might occur in that specific organ. Therefore, the artery tree would then change the blood distribution and microcirculation of various organs and as a result further affect the production of NO.

Identification of nerve plexi in connective tissues of the sea cucumber Holothuria glaberrima by using a novel nerve-specific antibody.

The echinoderm nervous system is one of the least studied among invertebrates, partly because the tools available to study the neurobiology of this phylum are limited. We have now produced a monoclonal antibody (RN1) that labels a nervous system component of the sea cucumber Holothuria glaberrima. Western blots show that our antibody recognizes a major band of 66 kDa and a minor band of 53 kDa. Immunohistological experiments show that, in H. glaberrima, the antibody distinctly labels most of the known nervous system structures and some components that were previously unknown or little studied. A surprising finding was the labeling of nervous plexi within the connective tissue compartments of all organs studied. Double labeling with holothurian neuropeptides and an echinoderm synaptotagmin showed that RN1 labeled most, if not all, of the fibers labeled by these neuronal markers, but also a larger component of cells and fibers. The presence of a distinct connective tissue plexus in holothurians is highly significant since these organisms possess mutable connective tissues that change viscosity under the control of the nervous system. Therefore, the cells and fibers recognized by our monoclonal antibodies may be involved in controlling tensility changes in echinoderm connective tissue.

Stichopin-containing nerves and secretory cells specific to connective tissues of the sea cucumber.

Stichopin, a 17-amino acid peptide isolated from a sea cucumber, affects the stiffness change of the body-wall catch connective tissues and the contraction of the body-wall muscles. The localization of stichopin in sea cucumbers was studied by indirect immunohistochemistry using antiserum against stichopin. Double staining was performed with both stichopin antiserum and 1E11, the monoclonal antibody specific to echinoderm nerves. A stichopin-like immunoreactivity (stichopin-LI) was exclusively found in the connective tissues of various organs. Many fibres and cells with processes were stained by both the anti-stichopin antibody and 1E11. They were found in the body-wall dermis and the connective tissue layer of the cloacae and were suggested to be connective tissue-specific nerves. Oval cells with stichopin-LI (OCS) without processes were found in the body-wall dermis, the connective tissue sheath of the longitudinal body-wall muscles, the connective tissue layer of the tube feet and tentacles, and the connective tissue in the radial nerves separating the ectoneural part from the hyponeural part. Electron microscopic observations of the OCSs in the radial nerves showed that they were secretory cells. The OCSs were located either near the well-defined neural structures or near the water-filled cavities, such as the epineural sinus and the canals of the tube feet. The location near the water-filled cavities might suggest that stichopin was secreted into these cavities to function as a hormone.

Adventitia contribution to vascular contraction: hints provided by tissue-engineered substitutes.

It is well accepted that the adventitia is much more than a simple elastic membrane which surrounds the media. However, the extent of its contribution to vascular physiology, as well as the mechanisms involved, remains to be clearly established and characterised. Investigation into these topics is hampered by a few technical challenges, like the paucity of available healthy human vascular samples and the variability such samples can display. Another challenge is the isolation and preparation of intact adventitia without contaminating cells from the media. For those reasons, although other models have proved useful to address these questions, data from tissue-engineered vascular substitutes can also provide quite valuable answers. Results from such substitutes indicate that a reconstructed adventitial layer can respond to classic vasoactive agents such as endothelin and sodium nitroprusside.

The effect of ovariectomy on rat vaginal tissue contractility and histomorphology.

INTRODUCTION: Ovarian hormones have an important role in age-related genital arousal disorders; however, our knowledge regarding possible vaginal wall morphology and contractility changes in low-hormonal states is limited. AIMS: To investigate morphological and functional alterations in the vaginal tissue in a rat ovariectomy model and to show the differences between proximal and distal vagina. METHODS: Six weeks following ovariectomy, vaginal tissues were examined under light and electron microscopy. Circularly cut distal and proximal tissues were studied in the organ bath under isometric tension and compared with age-matched controls. Contractile responses to electrical field stimulation (EFS), phenylephrine, carbachol, and the effects of alpha-1 and alpha-2 blockade on EFS-induced contractility were investigated. Relaxation responses to EFS and vardenafil were investigated in precontracted strips. MAIN OUTCOME MEASURES: Differences between control and ovariectomy groups in terms of vaginal tissue contractility and histomorphological properties. RESULTS: Distal vagina showed different epithelial characteristics and a better-developed muscularis compared with proximal vagina. Ovariectomy caused thinning of the epithelium, severe degeneration in epithelial architecture, and smooth muscle atrophy. Contraction and relaxation responses of distal strips were significantly lower in ovariectomized rats. Contractile responses to neuropharmacological stimulation were insignificant in proximal strips of both groups. EFS-induced contractions in distal strips diminished significantly after alpha-1 and alpha-2 adrenergic blockade. EFS caused frequency-dependent relaxation responses in precontracted distal strips, which were significantly decreased after nitric oxide synthase inhibition. CONCLUSIONS: Ovariectomy causes significant alteration in rat vaginal tissue morphology and contractility. Contraction and relaxation responses of distal vagina are significantly greater compared with morphologically distinct proximal vagina. Alpha-1 and alpha-2 receptors are the main mediators of contraction in distal rat vaginal tissue whereas nitric oxide pathway may have at least a partial role in relaxation. Main mediators of the rat vaginal tissue relaxation and the effect of ovariectomy on this regulation are yet to be defined.

Organization of the coelomic lining and a juxtaposed nerve plexus in the suckered tube feet of Parastichopus californicus (Echinodermata: Holothuroida).

The coelomic lining of the water-vascular canal in a suckered tube foot from the sea cucumber, Parastichopus californicus, is a pseudostratified myoepithelium consisting of flagellated adluminal cells and myofilament-bearing retractor cells. The bodies of adluminal cells flank the water-vascular canal and send basal processes between the underlying retractor cells to confront the podial connective tissue. Retractor cells have a contractile apparatus of unregistered thick and thin myofilaments. The contractile apparatus is confined to the medullary sarcoplasm and oriented parallel to the primary axis of a tube foot. The bodies and processes of retractor cells intermingle with the basal processes of adluminal cells at the basal lamina of the coelomic lining. A ganglionated nerve plexus in the podial connective tissue approximates the basal lamina. Neuronal connectives link the ganglia to one another and to the nerve plexus in deep sectors of the podial epidermis. External laminae enveloping the ganglia and connectives in the podial connective tissue are continuous with the basal lamina of the epidermis. The adventitial nerve plexus, since it merges with the epidermal nerve plexus, is a component of the ectoneural division of the echinoderm nervous system.