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H D Cavanagh

Publications and source records attributed to H D Cavanagh.

At least 73 records · Page 4Linked to original sources
PubMed1996

Quantitative three-dimensional confocal imaging of the cornea in situ and in vivo: system design and calibration.

A new depth encoding system (DES) is presented, which makes it possible to calculate, display, and record the z-axis position continuously during in vivo imaging using tandem scanning confocal microscopy (TSCM). In order to verify the accuracy of the DES for calculating the position of the focal plane in the cornea both in vitro and in vivo, we compared TSCM measurements of corneal thickness to measurements made using an ultrasonic pachymeter (UP, a standard clinical instrument) in both enucleated rabbit, cat, and human eyes (n = 15), and in both human patients (n = 7). Very close agreement was found between the UP and TSCM measurements in enucleated eyes; the mean percent difference was 0.50 +/- 2.58% (mean +/- SD, not significant). A significant correlation (R = 0.995, n = 15, p < 0.01) was found between UP and TSCM measurements. These results verify that the theoretical equation for calculating focal depth provided by the TSCM manufacturer is accurate for corneal imaging. Similarly, close agreement was found between the in vivo UP and TSCM measurements; the mean percent differences was 1.67 +/- 1.38% (not significant), confirming that z-axis drift can be minimized with proper applanation of the objective. These results confirm the accuracy of the DES for imaging of the cornea both ex vivo and in vivo. This system should be of great utility for applications where quantitation of the three-dimensional location of cellular structures is needed.

Adult↗
PubMed1996

Measurement of surgically induced corneal deformations using three-dimensional confocal microscopy.

The goal of this study was to develop and apply a new set of experimental techniques for measuring the local deformations induced by partial-thickness corneal incisions in situ. Eight adult cat eyes were enucleated and cannulated, with corneal viability maintained as close to in vivo conditions as possible and intraocular pressure (IOP) carefully controlled. Experimental measurements were made pre/post radial keratotomy (RK) surgery in situ at IOPs of 15, 30, and 45 mm Hg. Incision depth and cross-sectional profiles were measured at the midpoint of selected incisions using three-dimensional (3-D) tandem scanning confocal microscopy (TSCM); central corneal curvature was estimated using a commercial corneal topographical analysis system, and corneal thickness was assessed by both 3-D TSCM and ultrasonic pachymetry. Corneas were then processed for light microscopy and incision depth was measured histologically. Finite element models were developed for comparison with the experimental measurements. There was no significant change in central corneal thickness (-5.3 +/- 3.9%, n = 8) over the course of the experiments, demonstrating that normal endothelial cell function and normal stromal hydration was maintained. The in situ TSCM incision depth measurements were significantly correlated with the histological measurements (slope = 0.95, R = 0.854, p < 0.01, n = 13 incisions). Measured incision gape at the top (anterior) of the stroma was 64.9 +/- 13.4, 87.3 +/- 12.6, and 108.7 +/- 14 microns at IOPs of 15, 30, and 45 mm Hg, respectively. The 3-D incision profiles were nonlinear in shape; comparison with the finite element models suggests that the shape of the wound profile may provide unique information regarding the shear stiffness of the cornea. Overall, the data suggest that TSCM measurements of the cross-sectional profile of the incisions immediately after RK under controlled in situ conditions provide important data regarding the mechanical behavior of the cornea after refractive surgery. These data should provide the foundation for future studies into the relationships between local tissue mechanics and corneal wound healing.

Animals↗
PubMed1996

Chronic localized conjunctival chemosis.

Conjunctival chemosis is a commonly encountered clinical finding stemming from the presence of excess fluid in the conjunctiva. It is typically self-limiting or reversible if the underlying condition is treated. The authors present a series of seven cases of chronic localized conjunctival chemosis. Each patient had a localized area of dependent conjunctival edema for >or= 6 months. Evaluation of each patient included clinical examination, laboratory studies, and neuroimaging to attempt to elucidate the pathogenesis of the chemosis. Conjunctival biopsy was performed in six of the seven patients. In all of the patients studied, clinical examination uncovered no definitive signs of local inflammation. Laboratory evaluation was normal, and neuroimaging failed to confirm obstruction of venous or lymphatic drainage. Conjunctival biopsies showed chronic tissue inflammation or lymphangiectasia. The diagnosis of chronic localized conjunctival chemosis (CLCC) can be made if localized conjunctival edema persists for 6 months. Evaluation of biopsy specimens supports the theory that CLCC may result from scarring and structural alteration of conjunctival lymphatics, which appears to irreversibly affect the distribution of fluid in the conjunctiva.

Adult↗
PubMed1996

Induction of alpha-smooth muscle actin expression and myofibroblast transformation in cultured corneal keratocytes.

The effects of serum, transforming growth factor (TGF) beta 1, bFGF, and heparin on in vitro myofibroblast transformation was studied. Primary rabbit corneal keratocytes were grown under serum-free conditions or in media supplemented with serum (10% fetal calf serum), TGF beta 1 (0.1-10 ng/ml), basic fibroblast growth factor (bFGF) (0.1-10 ng/ml), or heparin (10 U/ml). Cells were analyzed for expression of alpha-smooth muscle actin (alpha-SM actin), alpha 5 beta 1 integrin (the high-affinity fibronectin receptor) and fibronectin by immunoprecipitation, Western blotting, and immunofluorescence. Corneal keratocytes grown in the presence of serum showed a typical fibroblast morphology with induction of alpha-SM actin expression in 1 to 10% of cells. Addition of bFGF blocked serum-induced alpha-SM actin expression, whereas addition of TGF beta 1 enhanced alpha-SM actin expression (100%), which in combination with heparin (10 U/ml), led to a pulling apart of the fibroblastic sheet, simulating contraction. Under serum-free conditions, with or without bFGF and heparin, primary corneal fibroblasts appeared morphologically similar to in situ corneal keratocytes, demonstrating a broad, stellate morphology with interconnected processes and no alpha-SM actin expression. Addition of TGF beta 1 to serum-free cultures resulted in a dramatic transformation of corneal keratocytes to spindle-shaped, fibroblast-like cells that expressed alpha-SM actin in 100% of cells and exhibited a 20-fold increase in fibronectin synthesis and a 13-fold increase in alpha 5 beta 1-integrin synthesis. These effects were blocked by the addition of neutralizing antibodies (16 micrograms/ml). Overall these data suggest that TGF beta 1 is a potent modulator of myofibroblast transformation under serum-free conditions. In addition, the growth of keratocytes in serum appears to mimic, in part, in vivo activation and myofibroblast transformation. We conclude that detailed study of TGF beta 1-induced myofibroblast transformation under defined serum-free conditions will provide important insights into the myofibroblast transformation process.

Actins↗
PubMed1996

Effects of basic FGF and TGF beta 1 on F-actin and ZO-1 organization during cat endothelial wound healing.

Previous studies suggest the existence of two separate and distinct mechanisms of endothelial wound healing (i.e., cell migration and cell spreading), which may be controlled by unique, injury-dependent, wound-related factors. The purpose of our study was to evaluate potential biologic mediators regulating healing of the growth arrested cat endothelium by using an ex vivo, organ-culture model. Three buttons were punched from each cornea of 11 cats with a 6-mm trephine. A 1- to 2-mm diameter endothelial scrape injury (SI) was made, and buttons were cultured in (a) serum-free media (SFM), (b) serum plus media (20% fetal calf serum), (c) SFM plus basic fibroblast growth factor (bFGF), (d) SFM plus bFGF and heparin, (e) SFM plus transforming growth factor-beta 1 (TGF beta 1), or (f) SFM plus TGF beta 1 and anti-TGF beta 1. At various times from 8-48 h after injury, buttons were stained with phalloidin and anti-ZO-1, and imaged by using laser scanning confocal microscopy. Evaluation of SI in cat corneal buttons under serum-free conditions showed maintenance of normal endothelial differentiation, indicating that the organ-culture SI model mimics in vivo SI. Addition of TGF beta 1 produced a dramatic reorganization of apical F-actin and development of stress fibers, as well as the loss of normal cell border-associated ZO-1 distribution. The effects of TGF beta 1 were blocked by the neutralizing antibodies to TGF beta 1. Addition of serum or bFGF produced much less pronounced changes in F-actin and ZO-1 distribution. These results suggest that TGF beta 1 may play a critical role in modulating the wound-healing response of the corneal endothelium.

Actins↗
PubMed1995

Basic science and applications of in vivo microscopy.

Confocal microscopy creates a scanned image from a point light source and point detection or a scanning slit to remove scattered light and improve optical resolution. This also results in optical sectioning of tissues. These capabilities can be employed to image structures in the human cornea, in vivo, both for research and for the diagnosis and treatment of human disease. Optical sections, when recombined, can lead to three-dimensional reconstructions from which very useful information is obtained. Investigators have found keratocyte density decreases from anterior to posterior in the stroma of the rabbit cornea. Surface epithelial desquamation can also be studied and the effects of contact lens use can be demonstrated. The instrument is also useful for diagnosing and guiding therapy for some human diseases such as Acanthamoeba keratitis. Colonies of bacteria may also be observed and treatment evaluated in patients with infectious crystalline keratopathy. Confocal microscopy can also image the retina.

Contact Lenses↗
PubMed1995

Assessment of f-actin organization and apical-basal polarity during in vivo cat endothelial wound healing.

PURPOSE: To assess the relationships between cytoskeletal changes and apical-basal polarity during healing of mechanical scrape injuries in the cat corneal endothelium. METHODS: Ten cats (20 eyes) were used in this study. One mechanical scrape injury was created in the corneal endothelium of each eye using a blunt olive tip cannula. Tandem scanning confocal microscopy (TSCM) was performed at sequential time points after injury for in vivo assessment of cell morphology and wound healing rates. In two eyes, scanning electron microscopy was performed to allow verification of TSCM observations. Ten eyes were collected between 6 and 48 hours after wounding for in situ labeling of f-actin, ZO-1, or both. RESULTS: Cat endothelial cell morphology observed using in vivo microscopy was identical to that shown using scanning electron microscopy. During healing, endothelial cells always remained attached to the endothelial sheet, although some showed extensions of lamellipodia into the open wound area. The in situ localization of f-actin also correlated with the TSCM in vivo wound morphology. Quantitative analysis showed that there was a decrease in the intensity of phalloidin-fluorescein isothiocyanate staining at the leading edge of the wound, suggesting a decrease in f-actin; a significant correlation was found between the relative intensity of f-actin staining and the distance from the wound margin (R = 0.98, P < 0.01). At 24 and 48 hours after injury, both ZO-1 and f-actin maintained an apical localization within cells immediately adjacent to the leading edge, despite the considerable distance of movement and dramatic decrease in the intensity of f-actin staining. CONCLUSIONS: Overall, these data demonstrate that after scrape injury in the cat, endothelial cells exhibit a pattern of healing in which total intracellular f-actin is reduced, but normal cell connectivity and apical-basal polarity are maintained throughout.

Actins↗
PubMed1995

Identification and subcellular distribution of muscarinic acetylcholine receptor-related proteins in rabbit corneal and Chinese hamster ovary cells.

PURPOSE: The authors examined the muscarinic acetylcholine receptor (mAChR) subtypes in rabbit corneal epithelial and endothelial cells and in subcellular fractions of these cell types. A Chinese hamster ovary (CHO) cell line (nontransfected CHO K1), expected to be a negative control, also was investigated. METHODS: Whole cell homogenate and subcellular fractions were labeled with the covalent-binding, mAChR-specific ligand [3H]propylbenzilylcholine mustard ([3H]PrBChM) and were analyzed by a combination of sodium dodecyl sulfate-polyacrylamide gel electrophoresis, or SDS-PAGE, and autoradiography. RESULTS: A pattern of multiple PrBChM-binding proteins was detected in homogenates of corneal epithelial and endothelial cells and, surprisingly, in the CHO cells. Ligand binding to all of these proteins is inhibited by the mAChR antagonists atropine sulfate and quinculidinyl benzilate. The sizes of four of the labeled protein bands are the same as the molecular masses deduced from mAChR sequence data for subtypes m3, m4, m5, and either m1 or m2. One band of 47 kd, smaller than any reported sequence, was also observed. Two of the [3H]PrBChM-binding proteins, one at 59 to 62 kd (corresponding to m5 in size) and another at 47 kd, clearly were present when highly purified nuclei were analyzed. CONCLUSIONS: The presence of multiple mAChR-like proteins at low concentrations in these disparate cell types suggests the possibility of a more general regulatory role for this type of receptor than was considered previously. Combined with other reports, the identification of proteins with the characteristics of mAChRs in purified nuclei adds support to data indicating the likelihood of G-protein-coupled signaling across the nuclear envelope.

Animals↗
PubMed1995

Temporal, 3-dimensional, cellular anatomy of corneal wound tissue.

We have evaluated temporally the 3-dimensional cellular anatomy of corneal wound tissue in the rabbit eye using in vivo tandem scanning confocal microscopy. In vivo microscopic studies showed that corneal fibroblast migrated into the wound as an interconnected cellular meshwork with long, thin, randomly oriented cell processes. Interconnection of fibroblasts was further confirmed by localisation of monoclonal antibodies to connexin 43 which demonstrated prominent staining of putative gap junctions between fibroblasts. Temporal observations indicated that the interconnected cells and cellular processes undergo sequential positional changes leading to orientation of cells and interconnected cell processes parallel to the wound margin. Laser scanning confocal microscopy of en bloc, phalloidin-stained corneal wounds showed prominent intracellular f-actin bundles (i.e. stress fibres) within cell processes which formed an extensive interwoven pattern within the wound.

Actins↗
PubMed1995

Expression of alpha-smooth muscle (alpha-SM) actin during corneal stromal wound healing.

PURPOSE: The purpose of this study was to correlate the temporal expression of alpha-smooth muscle specific actin (alpha-SM actin), a molecular marker for myofibroblast transformation, with corneal wound contraction. METHODS: After full-thickness, central corneal injury in rabbit eyes, the anterior width of the wound (wound gape) was measured in the same animals using in vivo confocal microscopy. In addition, animals were sacrificed at various times after injury for the determination of alpha-SM actin expression by immunofluorescent microscopy using a mouse monoclonal antibody specific for human alpha-actin. Antibody specificity was confirmed by Western blot analysis of normal and wound fibroblasts. Expression of alpha-SM actin also was related spatially to f-actin and the wound margin by co-localization with phalloidin and DTAF (5([4,6-dichlorotriazin-2yl]amino)fluorescein), a fluorescent marker bound to the wound margin. RESULTS: Wound contraction was most evident from days 7 to 42, when wound gape progressively decreased from 574 +/- 120 microns to 250 +/- 61 microns. Thereafter, the wound remained stable to day 84 (304 +/- 58 microns). Expression of alpha-SM actin directly correlated with wound contraction--appearing across the wound at day 7, the full thickness of the wound at day 14, and the posterior wound at day 28. alpha-SM actin was localized exclusively to phalloidin-stained, f-actin microfilament bundles or stress fibers within wound healing fibroblasts, and the disappearance of alpha-SM actin correlated with the concomitant disappearance of stress fibers at days 28 to 42. Staining of the wound margin with DTAF confirmed that the expression of alpha-SM actin was limited to fibroblasts within the wound. CONCLUSIONS: The expression of alpha-SM actin was directly correlated to corneal wound contraction, appearing at the initiation of and disappearing at the completion of the contraction process. Furthermore, the exclusive expression of alpha-SM actin by fibroblasts present only within the wound suggests that local environmental factors unique to the wound may play an important role in myofibroblast transformation.

Actins↗
PubMed1995

Quantitative assessment of anteroposterior keratocyte density in the normal rabbit cornea.

The anteroposterior keratocyte density distribution in the rabbit cornea was measured. Unsectioned tissue blocks from the central cornea of five rabbits were stained with propidium iodide and imaged using a Leica laser scanning confocal microscope. A z-series of images was acquired confocal microscope. A z-series of images was acquired in each sample, from anterior to posterior stroma in either 3- or 8-microns steps. Software was developed to allow interactive marking of the keratocyte nuclei within each section of the z-series and for calculating cell density. For convenience, cell density was expressed as the number of cells per corneal volume element (CVE), where CVE is a newly defined volume unit with x, y, and z dimensions of 250, 250, and 10 microns, respectively. The calculated keratocyte density was 20.2 +/- 1.0 cells/CVE (n = 5), which is equivalent to 32,360 +/- 1,660 cells/mm3. The greatest density was underneath the epithelium (26.3 +/- 2.5 cells/CVE), the density then decreased linearly with depth to 15.2 +/- 1.4 cells/CVE; there was a slight increase in density pre-Descemets membrane to 18.5 +/- 3.5 cells/CVE. A 30% decrease in cell density over the entire anteroposterior stromal thickness was observed. To facilitate statistical analysis, the cell density was averaged over 5% thickness intervals from anterior to posterior cornea. A significant difference in mean cell density of these intervals was found (ANOVA, n = 20, p < 0.01). To further assess the density distribution, linear regression analysis was performed. A significant correlation was found between keratocyte density and stromal depth (R = -0.94, n = 20, p < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

The spatial organization of corneal endothelial cytoskeletal proteins and their relationship to the apical junctional complex.

PURPOSE: To determine the spatial organization of the major cytoskeletal proteins and their relationship to the apical junctional complex (AJC) in the normal rabbit corneal endothelium. METHODS: Normal endothelial cytoskeletal structure in three dimensions was studied in rabbit eyes by laser scanning confocal microscopy after en bloc immunocytochemical staining of whole corneal tissue with various antibodies and fluorescent probes; specificity of antibodies to rabbit corneal endothelial cell proteins was established by Western blot analysis. RESULTS: Normal actin microfilament network organization was seen predominantly as a complex apical array forming a circumferential bundle. The tight junction-associated protein ZO-1 was positive at the apical junctions, forming a hexagonal pattern that was localized between and just proximal to the circumferential actin microfilament bundles. The distribution of ZO-1 was discontinuous around the cell, with the largest gaps (1 micron in diameter) occurring at the Y-junction between adjacent endothelial cells; transmission electron microscopy of the apical face of the endothelium confirmed the existence of 1-micron diameter gaps in the adherens junctions located at the Y-junction. Antivimentin antibodies showed a ring of intermediate filaments located just below the circumferential actin microfilament band. This ring appeared to be continuous with a basal mat of filaments, which together formed a basketlike structure within endothelial cells. An intricate cytoplasmic, perinuclear network of microtubules was observed by antitubulin antibodies that appeared unrelated either to the apical circumferential actin microfilament bundle or to intermediate vimentin filament ring. Staining of endothelial cells with NBD-ceramide identified a prominent, perinuclear Golgi complex suggesting an association between microtubules and Golgi. CONCLUSIONS: The organization of cytoskeletal elements and the tight junction-associated protein ZO-1 is similar to the classical AJC of transporting epithelia, comprised of a zonulae occludens (ZO) located apical to a zonulae adherens (ZA) and desmosomes. The organizational pattern seen in corneal endothelial cells, however, is distinct from transporting epithelia in that the ZO and ZA are discontinuous, with large gaps in the ZO-1 distribution at the Y-junction between adjacent endothelial cells. The authors propose that the structural differences in the AJC underlie the functional differences between classical transporting epithelia, which actively pump fluid from the lumen to the mucosa, and the corneal endothelium, which has a "pump-leak" fluid transport mechanism.

Animals↗
PubMed1995

Transplantation of corneal tissue from donors with diseases of the central nervous system.

A great deal of controversy and concern exists over potential transmission of central nervous system diseases by corneal transplant. The purpose of this study was to evaluate the available data relative to this question, pertaining especially to transmission of infectious dementia. From these data, determination of conveyance risks are possible, and rational policies for donor inclusion criteria can be constructed. Retrospective analysis of available published data regarding transmission of infectious dementias was performed. Risk of disease transmission was calculated from population data. Of the various forms of dementia, only rabies, hepatitis B, and Creutzfeldt-Jakob disease (CJD) have been transmitted by corneal transplantation. Transmission of the first two viruses is preventable by serologic testing. Prevention of CJD transmission relies on clinical history. Despite the possibility of transmission and the lack of available testing, slow virus disease (CJD) has been transmitted only once. That this case represents an extremely rare event is supported by a lack of successful transmission via corneal transplant in monkeys; lower levels of infectious agent in cornea than in brain; lack of successful transmission of similar human dementias, including Alzheimer's disease to primates; the apparent requirement for homozygosity at codon 129 of chromosome 20 for transmission; lack of transmission in 5-10% of CJD cases even after brain inoculation; and low numerical risk of transmission based on population data. Only 0.5-4 CJD infected donors per year would be expected. Current Eye Bank Association of America criteria for donor exclusion based on suspicious history are adequate to protect against accidental conveyance of transmissible dementia.

Animals↗
PubMed1995

The impact on tissue utilization of screening donor corneas by specular microscopy at the University of Texas Southwestern Medical Center.

To determine how frequently specular microscopy results affect the outcome of eye-bank judgments on the transplantability of donor corneas, 1,011 consecutive donor records from a 3-year period at the Transplant Services Center of the University of Texas Southwestern Medical Center at Dallas were analyzed. Specular microscopy cell counts from each decade of donor age were determined, and it was found that there were no cell counts < 2,000 mm2 for any donor age < 40. Above age 40, the percentage of cell counts < 2,000 per mm2 rose from 3.9% for donors in their forties of 6% for donors in their seventies. For donors between 40 and 69 years, specular microscopy was used to rule out unacceptable tissue in an additional 3.3% of a prescreened pool of corneas evaluated by current Eye Bank Association of America standards. While corneas from donors over age 69 were initially presumed to be unacceptable for transplant at this eye bank, routine specular microscopic examination helped to clear for transplant of 31 corneas from donors of this age group.

Academic Medical Centers↗