Identification of lymphatic endothelium in cranial arachnoid granulation-like dural gap.

The dynamics of cerebrospinal fluid (CSF) are essential for maintaining homeostasis in the central nervous system. Despite insufficiently detailed descriptions of their structural and molecular properties for a century, cranial arachnoid granulations (CAGs) on meninges have been thought to participate in draining CSF from the subarachnoid space into the dural sinuses. However, recent studies have demonstrated the existence of other types of CSF drainage systems, such as lymphatic vessels adjacent to dural sinus and paravascular space in the brain so-called glymphatic system. Therefore, the role of CAGs in CSF drainage has become dubious. To better understand CAG function, we analyzed the ultrastructure and molecular identity of CAG-like structure on meninges adjacent to the superior sagittal sinus of pigs. Transmission electron microscopy analysis revealed that this structure has a reticular conglomerate consisting of endothelial cells that resembles lymphatic linings. Furthermore, immunohistochemistry and immunoelectron microscopy showed that they express molecules specific to lymphatic endothelial cell. We coined a name 'CAG-like dural gap (CAG-LDG)' to this structure and discussed the physiological relevance in terms of CSF drainage.

Inhibitory Effect of ß-Elemene on Human Airway Granulation Tissue in vivo and in vitro.

BACKGROUND: Recurrent airway granulation hyperplasia and scar formation make airway stenosis a clinical challenge. Therefore, a new approach for the treatment of airway stenosis is necessary. OBJECTIVE: To explore the inhibitory effect of ß-elemene on the proliferation of fibroblasts and airway granulation. METHODS: In vivo: (1) study of the effect of local ß-elemene injection by bronchoscopy. (2) During bronchoscopy, granulation tissues both before and after treatment were obtained. HE staining was performed and the result compared. In vitro: (1) human airway primary fibroblasts were purified and characterized. (2) Fibroblasts were treated with ß-elemene and normal saline (NS) and then examined by optical and electron microscopy. (3) Fibroblasts treated with ß-elemene or NS were assessed for viability by tetrazolium salt assay. (4) Apoptotic rates were determined by flow cytometry. RESULTS: In vivo: (1) after local injection of ß- elemene, airway granulation tissue was reduced. (2) Granulation tissue was found to have less edema, and fibroblasts turned into mature fiber cells. In vitro: (1) human airway primary fibroblasts were successfully purified and cultured. (2) Compared with the control group, fibroblasts of the experimental group became clumped, the plasma granules were increased, and some fibroblasts lost their nucleus and organelles. (3) Compared with the control group, reduction of cell viability was detected with increased concentrations of ß-elemene. (4) With increased concentrations of ß-elemene, apoptotic rates of the fibroblasts were raised compared with the control group. CONCLUSIONS: ß-Elemene may induce apoptosis and necrosis of airway primary fibroblasts and inhibit the proliferation of fibroblasts and airway granulation. The results provide a new approach for the treatment of airway stenosis.

Effects of a local focus of granulation tissue formed in the bone marrow cavity on reparative osteogenesis.

The stimulatory effect of a local focus of granulation tissue, created in the bone marrow cavity, on reparative osteogenesis after tibial bone fracture was detected in experimental rats by microscopic examination of histological sections, scanning electron microscopy, and X-ray electron probe microanalysis.

Deep vascular imaging in wounds by two-photon fluorescence microscopy.

Deep imaging within tissue (over 300 µm) at micrometer resolution has become possible with the advent of two-photon fluorescence microscopy (2PFM). The advantages of 2PFM have been used to interrogate endogenous and exogenous fluorophores in the skin. Herein, we employed the integrin (cell-adhesion proteins expressed by invading angiogenic blood vessels) targeting characteristics of a two-photon absorbing fluorescent probe to image new vasculature and fibroblasts up to ≈ 1600 µm within wound (neodermis)/granulation tissue in lesions made on the skin of mice. Reconstruction revealed three dimensional (3D) architecture of the vascular plexus forming at the regenerating wound tissue and the presence of a fibroblast bed surrounding the capillaries. Biologically crucial events, such as angiogenesis for wound healing, may be illustrated and analyzed in 3D on the whole organ level, providing novel tools for biomedical applications.

Scab-inspired cytophilic membrane of anisotropic nanofibers for rapid wound healing.

This work investigates the influence of cytophilic and anisotropic nanomaterials on accelerated cell attachment and directional migration toward rapid wound healing. Inspired by the anisotropic protein nanofibers in scab, a polyurethane (PU) nanofibrous membrane with an aligned structure was fabricated. The membrane showed good affinity for wound-healing-related cells and could guide cell migration in the direction of PU nanofibers. Also, the morphology and distribution of F-actin and paxillin of attached cells were influenced by the underlying nanofibers. The randomly distributed PU nanofibers and planar PU membrane did not show a distinct impact on cell migration. This scab-inspired cytophilic membrane is promising in applications as functional interfacial biomaterials for rapid wound healing, bone repair, and construction of neural networks.

Ex vivo model of cerebrospinal fluid outflow across human arachnoid granulations.

PURPOSE: The brain's arachnoid membrane with granulations is an important biological barrier whose responsibilities include the transmission of cerebrospinal fluid (CSF) and the regulation of pressure. Membrane disturbance may cause changes that are difficult to replicate with animal models, suggesting the need for a model using human arachnoid membrane with granulations for the study of conditions such as Alzheimer disease, hydrocephalus, and pseudotumor cerebri. The authors detail the development and validation of an ex vivo model of CSF outflow across human arachnoid granulations (AGs) as an approximation of in vivo conditions. METHODS: Human AGs were perfused at normal physiological pressure in physiological and nonphysiological directions for permeability data. Fluorescent particle perfusion with electron microscopy identified outflow pathways through the AGs. RESULTS: This human ex vivo model demonstrated in vivo properties of unidirectionality, particle transport, and ultrastructure, similar to our 2005 in vitro model. The average baseline hydraulic conductivity in the physiological direction (n = 20) was 1.05 +/- 0.15 microL/min/mm Hg/cm(2) compared with 0.11 +/- 0.03 microL/min/mm Hg/cm(2) in the nonphysiological direction (n = 3) under statistically equivalent (P = 0.46) average normal physiological pressures (5.88 +/- 0.22 mm Hg and 6.14 +/- 0.23 mm Hg, respectively). CONCLUSIONS: The ex vivo model is feasible and herein demonstrated. These findings agree with in vivo CSF outflow. This model increases understanding of the clearance not only of CSF but also of metabolites through the arachnoid membrane. Additional evidence suggests, but does not yet prove, that CSF outflow may occur in a similar manner in the arachnoid membrane adjacent to the granulations, in addition to the flow through the AGs. This is a topic for further investigation.

Wound healing activity of Persea americana (avocado) fruit: a preclinical study on rats.

OBJECTIVE: Avocado (Persea americana) oil is rich in nutrient waxes, proteins and minerals, as well as vitamins A, D and E. It is an excellent source of enrichment for dry, damaged or chapped skin. This study aimed to evaluate the wound-healing activity of fruit extract of Persea americana in rats. METHOD: The effect of topical and oral administration of Persea americana fruit extract (300 mg/kg/day) on excision and dead space wound models was evaluated. The rats used in the excision wound model were divided into four groups of five each and received either topical or oral treatment. The rats used in the dead space wound model were divided into two groups of five each and were treated orally. Healing was assessed by the rate of wound contraction, period of epithelialisation, granulation tissue weight and hydoxyproline content. RESULTS: In the excision wound model, complete healing (full epithelialisation) was observed on average on day 14 in the rats who receive oral or topical treatment. In contrast, the controls took approximately 17 days to heal completely. The extract-treated wounds were found to epithelialise faster than the controls (p < 0.001). Wet and dry granulation tissue weight and the hydroxyproline content of the tissue obtained from extract-treated animals used in the dead space wound model were significantly higher (p < 0.05) compared with the controls. CONCLUSION: Rate of wound contraction, epithelialisation time together with the hydroxyproline content and histological observations support the use of Persea americana in the management of wound healing.

Granulación aracnoidea gigante / Giant arachnoid granulations

No disponible

Endothelial cell hypertrophy is associated with microvascular occlusion in horse wounds.

Wound repair in horse limbs is often complicated by excessive fibroplasia and scarring. Occlusion of the microvessels populating the granulation tissue appears to be involved in the excessive accumulation of extracellular matrix during the repair of limb wounds. This study aimed to determine whether endothelial cell hypertrophy or hyperplasia, or both, contribute to microvascular occlusion and whether the pericyte is involved in this anomaly. We created 5 wounds, each 2.5 x 2.5 cm, on both forelimbs and on the body of 6 horses. One limb was bandaged to stimulate excessive wound fibroplasia. Weekly biopsy specimens were evaluated by transmission electron microscopy to measure microvessel luminal diameters and the surface area of endothelial cells and to count endothelial cells and pericytes. Microvessels were occluded significantly more often in limb wounds than in body wounds. The surface area of endothelial cells lining occluded microvessels (mean +/- standard error, 28.4013 +/- 1.5154 microm2) was significantly greater (P = 0.05) than that of cells lining patent microvessels (26.2220 +/- 1.5268 microm2). Conversely, neither the number of endothelial cells nor the number of pericytes differed between patent and occluded microvessels or between limb and body wounds. Furthermore, the wound location and the status of the microvessels (patent or occluded) did not alter the ratio of endothelial cells to pericytes. These data suggest that endothelial cell hypertrophy might play a role in the microvascular occlusion present in granulation tissue of limb wounds in horses, but the contribution of the pericyte remains obscure.

Prefabricated engineered skin flap using an arteriovenous vascular bundle as a vascular carrier in rabbits.

BACKGROUND: The authors previously described induction of spontaneous tissue generation by implanting a collagen matrix and a ligated pedicle (arteriovenous bundle) into a hollow porous chamber in vivo in the rabbit. They hypothesized that increased tissue volume could be obtained by the application of basic fibroblast growth factor (bFGF) and/or by increasing the chamber size and porosity. METHODS: In rabbits, a saphenous arteriovenous pedicle and a collagen sponge were inserted into a porous chamber in the groin. Small-volume pore chambers (experiment 1, n = 7) and larger-volume, wider pore chambers (experiment 2, n = 13) were compared, and each was compared with and without bFGF. An additional three flaps of experiment 2 with bFGF were skin grafted, microsurgically transplanted to the ear, and evaluated at 6 months for stability. RESULTS: All patent chambers grew tissue; chambers with bFGF were almost filled, and those without were only half-filled. Histomorphometric analysis confirmed a significant difference. The larger-volume, larger-pore chambers produced more than twice the volume of tissue as the smaller chambers did, and this was significant. Tissue volume in both the control and bFGF groups of experiment 2 was significantly greater than that in the respective groups of experiment 1. Histology, angiography, and scanning electron microscopy confirmed greater vascularity in the bFGF groups and demonstrated vascular connections penetrating the chamber pores linking with angiogenic sprouts, probably from the vasa vasorum of the pedicle, to contribute to new growth. Transplanted flaps survived and appeared normal 6 months later. CONCLUSIONS: Patent pedicles, bFGF, large pore size, and larger-volume chambers all seemed to contribute to increased tissue growth in this model. The tissue is stable long term.

Influence of sea buckthorn (Hippophae rhamnoides L.) flavone on dermal wound healing in rats.

The present investigation was undertaken to determine the efficacy of topical administration of flavone of sea buckthorn (Hippophae rhamnoides L.) on cutaneous wound healing in rats. Four full-thickness excision wounds were created on the back of rat and 1.0% w/v flavone prepared in propylene glycol was applied topically. Control animals received the vehicle alone in an identical manner. The healing of the wound was assessed by the rate of wound contraction, period of epithelialization, hydroxyproline, hexosamine, antioxidants estimation and histopathology of the granulation tissue. The sea buckthorn flavone promoted the wound healing activity as indicated by improved rate of wound contraction, decreased time taken for epithelialization (16.3 days versus 24.8 days in controls) and significant increase in hydroxyproline (26.0%) and hexosamine (30.0%) content. These findings were also confirmed by histopathological examinations. In addition, it was observed that sea buckthorn flavone possesses potent antioxidant properties as evidenced by significant increase in reduced glutathione (55.0%), vitamin C (70.0%) and catalase (20.0%) activities in wound granulation tissue. The flavone treatment also resulted in significant decrease in lipid peroxide levels (39.0%). The results suggest that the sea buckthorn flavone promotes wound healing activity.

Modeling dermal granulation tissue with the linear fibroblast-populated collagen matrix: a comparison with the round matrix model.

BACKGROUND: Wound contraction typically is not symmetrical; for example, a square-shaped wound will not yield a square scar. Interestingly, the round fibroblast-populated collagen matrix has been used as a model of wound contraction, even though contraction in this model is mostly symmetrical. OBJECTIVE: We wanted to compare the round versus linear fibroblast-populated collagen matrix to see which would be a better model of dermal granulation tissue. METHODS: Gross and microscopic morphology, contraction kinetics, cytoskeletal architecture, and apoptotic and proliferative indices were compared between the round versus the linear fibroblast-populated collagen matrix. A rat excisional wound model was used as an in vivo standard of healing. RESULTS: The rate of contraction was similar between the two models, although the mode of contraction was grossly asymmetric in the linear while remaining symmetric in the round model. Cellular survival and proliferation were both dependent on matrix attachment in both models; this was analogous to the attachment-dependence of granulation tissue. In the attached (restrained) condition, the level of cellular organization was higher in the linear than in the round matrix; the tissue architecture of the linear matrix, moreover, mimicked that of the excisional wound model. CONCLUSION: The round versus linear fibroblast-populated collagen matrix displayed a similar proliferative and survival response to matrix attachment. The latter model, however, demonstrated tissue organization with attachment and asymmetrical contraction after detachment analogous to that of the in vivo wound model. The linear fibroblast-populated collagen matrix appears to be the better model of dermal granulation tissue.

Intracellular formation of collagen microfibrils in granulation tissue.

It is important to determine the biosynthesis process of collagen fibers to elucidate the mechanism by which granulation tissue is induced after injury. The purpose of this study is to investigate whether collagen microfibrils can be formed not only outside but also inside a cell. Fibroblast-like cells in granulation tissue resulting from incision and ligation were examined. The cells possessed vesicles containing collagen microfibrils. The vesicles were present in connection with Golgi apparatus or the rough endoplasmic reticulum. Furthermore, the vesicles were exhibited to be secretory granules with the secretory granule marker Rab3A. The fibroblast-like cells were also indicated to be myofibroblasts, using conventional transmission electron microscopy and immunoelectron microscopy for the myofibroblast marker alpha smooth muscle actin. In conclusion, it was demonstrated that collagen microfibrils could be formed in the cell in the case of collagen fiber overproduction.

[Lanthanides and microanalysis. Effects of oral administration of two lanthanides: ultrastructural and microanalytical study]. / Lanthanides et microanalyse. Effets de l'administration orale de deux lanthanides :etude ultrastructurale et microanalytique.

The subcellular localization of cerium and lanthanum in the intestinal mucosa was studied after oral administration of cerium chloride or lanthanum chloride or lanthanum chloride followed 30 minutes after of cerium chloride to young adults Wistar rats. Two methods of observation and microanalysis were used. The transmission electron microscopy revealed the presence of dense electron granulations in the lysosmes of the duodenum enterocyte, when these elements were administrated simultaneously. The ion mass microanalysis permits to detect the presence of La and Ce as bright points outlining the intestinal villi. These points correspond to the lysosomes containing the granulations previously described. These granulations are formed by the cerium and the lanthanum associated to the phosphor and forming probably insoluble salts of Ce/La phosphate.

Side-to-side sutureless vascular anastomosis with magnets.

OBJECTIVE: Abbe and Payr introduced vascular techniques and devices to facilitate vessel anastomosis over a century ago. Obora published the idea of a sutureless vascular anastomosis with use of magnetic rings in 1978. The purpose of this study was to assess the performance of a new magnetic device to perform a side-to-side arteriovenous anastomosis in a dog model. MATERIAL AND METHODS: Male fox hounds (25 kg) were treated preoperatively and daily postoperatively with clopidogrel bisulfate (Plavix) and aspirin. The femoral artery and vein were exposed unilaterally in 3 dogs and bilaterally in 4 dogs (n = 11 anastomoses). A 4-mm arteriotomy was performed, and 1 oval magnet 0.5 mm thick was inserted into the lumen of the artery and a second magnet was applied external to the artery, compressing and stabilizing the arterial wall to create a magnetic port. An identical venous magnetic port was created with another pair of oval magnets. When the 2 ports were allowed to approach each other, they self-aligned and magnetically coupled to complete the arteriovenous anastomosis. Patency was assessed for the first hour with direct observation, again after 9 weeks with duplex ultrasound scanning, and at 10 weeks under direct open observation. The anastomoses were explanted after 10 weeks. Hydrodynamic resistance was measured ex vivo on the final 8 anastomoses by measuring the pressure drop across an anastomosis with a known flow rate. RESULTS: After implantation, very high flow created visible turbulence and palpable vibration. All 11 anastomoses were patent under direct observation and palpation. Ten of 11 anastomoses were clearly patent on duplex scans, and patency of 1 anastomosis was questionable. Hydrodynamic resistance averaged 0.73 +/- 0.33 mm Hg min/mL (mean +/- SEM). CONCLUSIONS: Vascular anastomoses performed with magnets demonstrated feasibility; exhibited 100% patency after 10 weeks in a dog arteriovenous shunt model; lacked apparent aneurysm or other potentially catastrophic failure; demonstrated remodeling of the vessel wall after several weeks to incorporate the magnets, making the magnetic force unnecessary; and warrants further study in vessels with different sizes, flow rates, and locations. CLINICAL RELEVANCE: We present a magnet-based device used to perform side-to-side peripheral vascular anastomoses. Its advantages include the ability to anastomose vessels without requiring circumferential surgical exposure. Vascular anastomosis performed with these magnets demonstrated 100% patency in the dog, lacked apparent aneurysm or other potentially catastrophic failure, and demonstrated remodeling of the vessel wall after several weeks, to incorporate the magnets, making indefinite retention of field strength unnecessary. This technique could enable minimally invasive procedures, such as complex reconstructive and revascularizing surgery, and warrants further study in vessels with different sizes, flow rates, and locations.

A clinical model of dermal wound angiogenesis.

Full-thickness dermal biopsies were performed in healthy volunteers to establish the range of angiogenic responses in wound healing in a normal population. Four-millimeter punch biopsies were made in the forearms of 15 healthy volunteers. Each wound was evaluated microscopically 4-5 times per week for 2 weeks. A semiquantitative wound scoring system to evaluate the neovasculature at the wound periphery was investigated. A vascular score was calculated for each wound at each observation. Two independent observers analyzed the microscopic wound images using the scoring system. At the end of the 14-day period, repeat biopsies were performed on some of the volunteers, and the granulation tissue was stained with anti-CD31. The Kaplan-Meier method was used to estimate the distribution of the time to reach predetermined target average vascular scores. A mixed-effects regression model indicated that time, age, and observer were predictors for the average vascular score outcome. The pattern and time course for wound neovascularization was highly reproducible in this group of healthy volunteers, and the assay was feasible and well tolerated. This wound angiogenesis model may be useful for monitoring the effects of antiangiogenic agents on normal wound neovascularization.

Experimental study of reparative regeneration processes in the wound treated with bioactive dressings.

Quantitative and structural functional analysis of granulation tissue cells during treatment with protein-polysaccharide dressing Collahit F was carried out. The preparation effectively cleansed the wound from detritus, prevented secondary infection due to stimulation of the functional activity of macrophages and due to the effect of its antiseptic component (furagin), and stimulated proliferative activity of fibroblasts and granulation tissue microvessels on day 5 of treatment, thus promoting repair processes in the wound.

Association between hemosiderin deposition and blood vessel regression during involution of foreign-body granuloma. Histochemical and ultrastructural study.

We have characterized the sequence of events resulting in vessel occlusion and stasis of blood flow during involution of a foreign-body granuloma using histochemistry and electron microscopy. In the microvascular bed of the granulation tissue accompanying the progressive resorption of an implanted collagen sponge, endothelial cells protruded into the vascular lumen, resulting in the occlusion of the lumens of venules and capillaries. Examination of sections stained by the TUNEL method showed brown-yellow stained structures in the vascular lumen during regression of blood vessels. However, the ultrastructural profiles of endothelial cells effectively involved in the vessel occlusion showed none of the cardinal morphological features of apoptosis. These endothelial cells which displayed remarkable indentations of their nuclei in the form of nuclear pinches and/or deep pockets bulged conspicuously into the lumen. Such endothelial cells served as effective valves by protruding into the lumens of small blood vessels, and eventually the vessels were completely plugged by red blood cells. However, protruding endothelial cells subsequently shed into the vascular lumen by deviating themselves from the constitution of the vessel wall. The endothelial cells undergoing apoptosis were removed by intraluminal macrophages. Between the 130 and 140 day, the occurrence of small vessels tightly packed with erythrocytes reached a peak value in the granulation tissue and was accompanied by hemosiderin deposits. The plugged vessels were frequently associated with erythrocyte extravasation, leading to openings between degenerated endothelial cells due to the disappearance of endothelial cytoplasmic projections. Extravasated erythrocytes were rapidly eliminated by phagocytic cells such as mononuclear macrophages and multinucleated giant cells, and ended as hemosiderin deposits in granulation and/or scar tissue at the end of the experimental period (130-140 days). The morphological analysis of this regression sequence suggests that the protrusion of endothelial cells with the nuclear deformation is a mechanism contributing to the occlusion of blood vessels and consequently leads to erythrocyte extravasation.

Fimbria-mediated bacterial adhesion to human oral epithelium.

Oral mucosa biopsies and saliva samples from 12 individuals were processed for transmission (TEM) and scanning (SEM) electron microscopy with and without ruthenium red staining. Additionally performed microbiological estimations indicated in all bacteriological samples a facultative pathogenic flora. SEM and TEM investigation showed a diverse bacterial flora attached to the mucosal surface. Fimbriae comprising the glycocalyx and enabling bacterial attachment to the epithelial cells could be clearly visualised by ruthenium red. The only mode of bacterial attachment to the oral mucosa detected in the present investigation was fimbria-mediated adhesion and co-adhesion. The fimbria-mediated adhesion enables the bacterial persistence in the oral cavity and is the first step in the bacterial colonisation process.

The fibronexus in reactive and tumoral myofibroblasts: further characterisation by electron microscopy.

Forty two surgical specimens containing myofibroblasts were studied to clarify the criteria for identifying the fibronexus, an ultrastructural feature regarded as a marker for myofibroblastic differentiation. Granulation tissue, tumour stroma, fibro-proliferative lesions (nodular fasciitis, myofibromatosis, inflammatory myofibroblastic tumour) and malignancies (myofibrosarcoma and fibrosarcoma) were studied. Comparable results were found throughout these specimens, although fibronexus junctions were better developed in reactive compared with tumoral myofibroblasts. By electron microscopy, myofibroblasts were identified by abundant rough endoplasmic reticulum, peripheral smooth-muscle myofilaments with focal densities, and fibronexus junctions. The latter were recognised as the points of convergence on the myofibroblast surfaces of intracellular myofilaments and extracellular fibronectin fibrils. The fibronectin fibrils were often co-linear with myofilaments. Also, fibronectin fibrils were dark-staining, straight and rigid-looking, and had a longitudinal filamentous substructure. A striking feature was the tendency of fibronectin fibrils to project into the surrounding extracellular space, away from the myofibroblast surface: in these respects, they differed significantly from lamina ("basement membrane"). The presence of fibronectin fibrils correlated positively with fibronectin immunostaining by light and electron microscopy. Laminin and collagen IV showed variable and weak staining in the intercellular spaces in a minority of cases and never strongly stained myofibroblast surfaces. The data emphasise that the fibronexus has a number of distinctive features permitting identification, and constitute a reference-point for pathologists wishing to use electron microscopy to refine light microscopy diagnoses of putative myofibroblastic lesions. The role of the fibronexus in the definition of the myofibroblast is discussed.