The mgΔlpn mouse model for Marfan syndrome recapitulates the ocular phenotypes of the disease.

PURPOSE: Fibrillin-1 and -2 are major components of tissue microfibrils that compose the ciliary zonule and cornea. While mutations in human fibrillin-1 lead to ectopia lentis, a major manifestation of Marfan syndrome (MFS), in mice fibrillin-2 can compensate for reduced/lack of fibrillin-1 and maintain the integrity of ocular structures. Here we examine the consequences of a heterozygous dominant-negative mutation in the Fbn1 gene in the ocular system of the mgΔlpn mouse model for MFS. METHODS: Eyes from mgΔlpn and wild-type mice at 3 and 6 months of age were analyzed by histology. The ciliary zonule was analyzed by scanning electron microscopy (SEM) and immunofluorescence. RESULTS: Mutant mice presented a significantly larger distance of the ciliary body to the lens at 3 and 6 months of age when compared to wild-type, and ectopia lentis. Immunofluorescence and SEM corroborated those findings in MFS mice, revealing a disorganized mesh of microfibrils on the floor of the ciliary body. Moreover, mutant mice also had a larger volume of the anterior chamber, possibly due to excess aqueous humor. Finally, losartan treatment had limited efficacy in improving ocular phenotypes. CONCLUSIONS: In contrast with null or hypomorphic mutations, expression of a dominant-negative form of fibrillin-1 leads to disruption of microfibrils in the zonule of mice. This in turn causes lens dislocation and enlargement of the anterior chamber. Therefore, heterozygous mgΔlpn mice recapitulate the major ocular phenotypes of MFS and can be instrumental in understanding the development of the disease.

Collagen morphology in human meniscal attachments: a SEM study.

Qualitative analysis of meniscal attachments from five human knees was completed using scanning electron microscopy (SEM). In addition, quantitative analysis to determine the collagen crimping angle and length in each attachment was done. Morphological differences were revealed between the distinct zones of the attachments from the meniscus transition to the bony insertion. Collagen fibers near to the meniscus appeared inhomogeneous in a radial cross-section view. The sheath surrounding the fibers seemed loose compared with the membrane wrapping around the fibers in the menisci. The midsubstance of human meniscal attachments was composed of collagen fibers running parallel to the longitudinal axis, with a few fibers running obliquely, and others transversely. The bony insertion showed that the crimping pattern vanishes as the collagen fibers approach the fibrocartilagenous enthesis. There were no differences between attachments for crimping angle or length. Collagen crimping angles for all attachments were similar with values of approximately 22°. Crimp length values tended to be smaller for the medial attachments (MA: 4.76 ± 1.95 µm; MP: 3.72 ± 2.31 µm) and higher for the lateral (LA: 6.49 ± 2.34 µm, LP: 6.91 ± 2.29 µm). SEM was demonstrated to be an effective method for revealing the morphology of fibrous connective tissue. The data of collagen fiber length and angle found in this study will allow for better development of microstructural models of meniscal attachments. This study will help to better understand the relation between the morphology and the architecture of collagen and the mechanical behavior of meniscal attachments.

Ultrastructural and immunocytochemical evaluation of the effects of extracorporeal shock wave treatment in the hind limbs of horses with experimentally induced suspensory ligament desmitis.

OBJECTIVE: To evaluate the effects of extracorporeal shock wave therapy (ESWT) on affected ligaments in the hind limbs of horses with experimentally induced suspensory ligament desmitis by use of ultrasonographic, ultrastructural, and immunocytochemical techniques. ANIMALS: 10 horses. PROCEDURE: Suspensory ligament desmitis was induced in both hind limbs of each horse by use of 2 collagenase injections (administered 2 weeks apart) in each suspensory ligament. Two weeks after the second injection, the right hind limb of each horse was treated with ESWT (3 treatments at 3-week intervals); the left hind limb was not treated (control limb). Periodically during the study, the healing process was monitored ultrasonographically and the proportions of ligaments affected with lesions were assessed. Four weeks after the last ESWT treatment, biopsy specimens were collected from all ligaments for ultrastructural evaluation and immunocytochemical analysis of transforming growth factor beta-1. RESULTS: The difference in the proportion of the lesion-affected ligament between ESWT-treated and control limbs was significant (P < 0.05) from 3 weeks after the second ESWT treatment to the end of the study. Compared with control ligaments, ESWT-treated ligaments had more small, newly formed collagen fibrils and greater expression of transforming growth factor beta-1 4 weeks after the last ESWT treatment was administered. CONCLUSIONS AND CLINICAL RELEVANCE: Results have indicated that ESWT appears to facilitate the healing process in horses with experimentally induced hind limb suspensory ligament desmitis.

Guinea-pig interpubic joint (symphysis pubica) relaxation at parturition: underlying cellular processes that resemble an inflammatory response.

BACKGROUND: At term, cervical ripening in coordination with uterine contractions becomes a prerequisite for a normal vaginal delivery. Currently, cervical ripening is considered to occur independently from uterine contractions. Many evidences suggest that cervical ripening resembles an inflammatory process. Comparatively little attention has been paid to the increased flexibility of the pelvic symphysis that occurs in many species to enable safe delivery. The aim of this study was to investigate whether the guinea-pig interpubic joint relaxation process observed during late pregnancy and parturition resembles an inflammatory process. METHODS: Samples of pubic symphysis were taken from pregnant guinea-pigs sacrificed along gestation, parturition and postpartum. Serial sections of paraffin-embedded tissues were used to measure the interpubic distance on digitalized images, stained with Giemsa to quantify leukocyte infiltration and to describe the vascular area changes, or studied by the picrosirius-polarization method to evaluate collagen remodeling. P4 and E2 serum levels were measured by a sequential immunometric assay. RESULTS: Data showed that the pubic relaxation is associated with an increase in collagen remodeling. In addition, a positive correlation between E2 serum levels and the increase in the interpubic distance was found. On the other hand, a leukocyte infiltration in the interpubic tissue around parturition was described, with the presence of almost all inflammatory cells types. At the same time, histological images show an increase in vascular area (angiogenesis). Eosinophils reached their highest level immediately before parturition; whereas for the neutrophilic and mononuclear infiltration higher values were recorded one day after parturition. Correlation analysis showed that eosinophils and mononuclear cells were positively correlated with E2 levels, but only eosinophilic infiltration was associated with collagen remodeling. Additionally, we observed typical histological images of dissolution of the connective tissue matrix around eosinophils. CONCLUSION: The present study shows that a timely regulated influx of infiltrating leukocytes is associated with an extensive collagen remodeling process that allows the pubic separation for a normal delivery in guinea-pig. Thus, the findings in this study support the hypothesis that the guinea-pig pubic symphyseal relaxation at parturition resembles an inflammatory process.

Aging of the elastic and collagen fibers in the human cervical interspinous ligaments.

BACKGROUND CONTEXT: The ligaments consist of collagen bands intermingled with elastic fibers that support hundreds of pounds of stress per square inch. In the spine the basic functional unit comprises vertebrae, intervertebral disc and ligament tissues. The interspinous ligaments with the function of limiting the spine flexion are exposed to a traumatic and degenerative process that promotes pain or instability. It has been shown that aging induces structural changes to capsular, fascial and ligamentous structures, mainly to the elastic and collagen fibers. However, the relative changes with age in elastic and collagen fibers have not been quantified. PURPOSE: Examine the changes in the arrangement and amount of the elastic and collagen fibers of the human cervical interspinous ligament and attempt to correlate them with age. STUDY DESIGN/SETTING: Histomorphometric analysis of ligament samples harvested during surgery. PATIENT SAMPLE: We studied the dorsal portion of this ligament from 17 patients aged 16 to 69 years. OUTCOME MEASURES: Fraction of collagen and elastic fibers with linear regression analysis correlating fraction versus age. METHODS: The elastic and collagen fibers were identified by selective staining methods, and a blinded investigator using an image analysis system performed the histomorphometry. RESULTS: There is an age-related progressive increase in collagen and mature and elaunin elastic fibers responsible to elasticity. However, these elastic fibers showed structural degenerative changes with aging. Furthermore, there is an age-related decrease of oxytalan elastic fibers responsible to resistance. CONCLUSIONS: The aged interspinous ligament showed loss of elasticity that could alter the flexion limiting of the vertebral column.

The osteochondral ligament: a fibrous attachment between bone and articular cartilage in Rana catesbeiana.

The anuran epiphyseal cartilage shows a lateral expansion that covers the external surface of the bone, besides other features that distinguish it from the corresponding avian and mammalian structures. The fibrous structure that attaches the lateral cartilage to the bone was characterized in this work. It was designated osteochondral ligament (OCL) and presented two main areas. There was an inner area that was closer to the periosteal bone and contained a layer of osteoblasts and elongated cells aligned to and interspersed with thin collagen fibers. The thin processes of the cells in this area showed strong alkaline phosphatase activity. The outer area, which was closer to the cartilage, was rich in blood vessels and contained a few cells amongst thick collagen fibers. TRITC-phaloidin staining showed the cells of the inner area to be rich in F-actin, and were observed to form a net around the cell nucleus and to fill the cell processes which extended between the collagen fibers. Cells of the outer area were poor in actin cytoskeleton, while those associated with the blood vessels showed intense staining. Tubulin-staining was weak, regardless of the OCL region. The main fibers of the extracellular matrix in the OCL extended obliquely upwards from the cartilage to the bone. The collagen fibers inserted into the bone matrix as Sharpey's fibers and became progressively thicker as they made their way through the outer area to the cartilage. Immunocytochemistry showed the presence of type I and type III collagen. Microfibrils were found around the cells and amongst the collagen fibrils. These microfibrils were composed of either type VI collagen or fibrilin, as shown by immunocytochemistry. The results presented in this paper show that the osteochondral ligament of Rana catesbeiana is a complex and specialized fibrous attachment which guarantees a strong and flexible anchorage of the lateral articular cartilage to the periosteal bone shaft, besides playing a role in bone growth.

Light and electron microscopy of the human hepato-duodenal ligament: a morpho-functional study.

The morpho-functional relationships between the hepato-duodenal ligament and the superior part of the duodenum are analysed. Twenty-four specimens were removed during necropsies of adults and prepared according to various mesoscopic, microscopic, and ultramicroscopical methods, i.e., whole-mounts, membrane preparations, thick and thin histological sections, and polarized light, scanning, and transmission electron microscopy were employed. The hepato-duodenal ligament is formed by longitudinally elongated, type I collagen fiber bundles which are interlinked by more delicate bundles of type III collagen fibers. Longitudinally disposed elastic fibers are the principal component of the elastic fiber system of the ligament. These are in continuity with the elaunin and oxytalan fibers which are intermingled with the muscle cells of the vessels and duodenal wall, and fat cells of the ligament. Part of the muscle bundles of the external, longitudinal, muscle layer of the duodenum is anchored in the adventitial and subserosal collagen and elastic fiber framework of the organ while part inserts directly into the fibrous framework of the ligament. The fibrous system of the ligament is continuous with that of the subserosa and adventitia of the duodenum.