Search PubMed⌕ Search

Biomedical subjects

H D Cavanagh

Publications and source records attributed to H D Cavanagh.

At least 91 records · Page 5Linked to original sources
PubMed1994

The relation between contact lens oxygen transmissibility and binding of Pseudomonas aeruginosa to the cornea after overnight wear.

PURPOSE: To assess adverse effects of contact lens-induced hypoxia on the rabbit cornea in vivo and determine the relation between binding of Pseudomonas aeruginosa and oxygen transmissibility for rigid and hydrogel lenses. METHODS: Six rigid lenses with Dk/Ltotal values between 0 and 97 x 10(-9) (cm/second) (ml O2/ml mmHg) and four hydrogel lenses (Dk/Ltotal 9, 20, 39, 51) were tested. All lenses had 14.0-mm diameters and a thickness (parallel) of 0.12 or 0.15 mm. Tear lactate dehydrogenase activity and tandem scanning confocal microscopy determinations were performed after the lens was worn for 24 hours. Binding of P. aeruginosa then was separately determined by the colony-forming unit method. Scanning electron microscopy was used to confirm in vivo tandem scanning confocal microscopy findings. RESULTS: Lens oxygen transmissibility determines binding of P. aeruginosa to the cornea after the lens is worn for 24 hours; epithelial damage produced by lenses of lower Dk/Ltotal appears to be the dominant biologic factor for P. aeruginosa binding and not lens rigidity. CONCLUSIONS: These results suggest that the risk of P. aeruginosa keratitis developing with overnight wear will be enhanced significantly for contact lenses with Dk/Ltotal values less than 50 x 10(-9) (cm/second) (ml O2/ml mmHg) (human equivalent oxygen percentage < or = 15%), and this risk will increase with further decreases in oxygen transmissibility. Because no hydrogel lenses approved by the Food and Drug Administration are available with oxygen transmission at this level, patients should be made aware of the increased risk of infectious keratitis associated with the overnight wear of current extended wear hydrogel lenses. Results of this study also demonstrate that quantitative clinical tandem scanning confocal microscopy imaging and tear lactate dehydrogenase activity measurements can provide prospective, noninvasive methods for assessing the ongoing interaction between contact lens and cornea in vivo.

Animals↗
PubMed1994

In vivo osmotic pertubation of intercellular fluid channels in the rabbit corneal endothelium.

An in vivo rabbit corneal model was used to evaluate morphological changes in the corneal endothelium associated with osmotically increasing fluid movement from the anterior chamber into the stroma. When the corneal stroma is rendered more hypertonic than normal by immersing the scraped epithelial side of the cornea in a hypertonic sucrose solution, intercellular channels and apical pores at the Y-junctions between endothelial cells become greatly enlarged. The foregoing changes are reversible and do not appear to result in damage to the corneal endothelium. These observations suggest that specific intercellular channels in the corneal endothelium may provide pathways for the movement of fluid from the aqueous humor into the stroma.

Animals↗
PubMed1994

Effects of rigid lens extended wear on lactate dehydrogenase activity and isozymes in rabbit tears.

Effects of and recovery from continuous wear of four rigid gas permeable (RGP) contact lenses was assessed by noninvasive measurement of lactate dehydrogenase (LDH) activity and isozyme pattern in rabbit tears. Oxygen transmissibility (Dk/L) of lenses used was 27, 44, 84, and 97 x 10(-9) (cm/s)(ml O2/ml mm Hg); lens thickness (0.15 mm) and diameter (14.0 mm) were standardized. Lenses were worn continuously for 90 days; recovery was assessed 30 days after cessation of lens wear. LDH activity was measured by UV rate assay; isozyme subtypes were determined by agarose gel electrophoresis. Light and scanning electron microscopy (LM, SEM) were used with the determination of total protein as additional measures of lens effects. LDH levels were inversely correlated with lens Dk/L values; low Dk/L values increased the anaerobic (LDH4,5)/aerobic (LDH1,2,3) subtypen ratio indicating in vivo metabolic shift. SEM observations were consistent with these results. There was no significant difference in the total cell content of tears or total tear protein levels between control and RGP test-wear groups. Measurement of tear LDH activity and isozyme ratios appears to provide a sensitive, noninvasive assessment of the effects of RGP lens-induced hypoxia over time on the corneal surface. A level of Dk/L of > or = 84 appears best for maintaining corneal physiology during extended wear. Recovery from chronic lens-induced hypoxia is characterized by a return to normal tear LDH levels and isozyme subtypes.

Animals↗
PubMed1994

The effects of daily wear of rigid gas permeable contact lenses treated with contact lens care solutions containing preservatives on the rabbit cornea.

We evaluated the effects on the rabbit cornea of daily wear of rigid gas permeable (RGP) contact lenses treated with preserved care solutions by measuring concomitant tear lactate dehydrogenase (LDH) activity followed by in vivo tandem scanning confocal microscopy (TSCM). In vivo morphologic changes were confirmed by in vitro scanning electron microscopy (SEM). Two standard commercial RGP lens wetting and soaking solutions from the same manufacturer were tested: solution A with 0.004% benzalkonium chloride (BAK) and solution B with 0.003% chlorhexidine digluconate (CHX) and 0.002% thimerosal. Two experimental PBS-based wetting and soaking solutions were also tested: solution C with 0.005% BAK and 2% hydroxypropylmethylcellulose (HPMC) and solution D with 0.005% BAK without HPMC. Instillation of solution A without contact lens wear caused significant (P < 0.01) increases in desquamation of the superficial corneal epithelium and tear LDH activity compared with control eyes. After 3 weeks of RGP contact lens daily wear (8 hours/day), modified Draize scores of ocular surface lesions on the eyes wearing RGP lenses treated with solution A increased according to the duration of lens wear. Solution B did not produce significant change. With daily wear for 4 days (8 hours/day), RGP lenses treated with solution C and solution D produced increased corneal epithelium desquamation and an increase of LDH activity in tears. These effects were greater with HPMC (solution C) than without HPMC (solution D).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Rapid assay of lactoferrin in keratoconjunctivitis sicca.

We compared measurement of lactoferrin concentration by the LactoCard solid phase ELISA assay with the LactoPlate radial immunodiffusion assay in tears of normal patients and those with keratoconjunctivitis sicca. The LactoCard, a new lactoferrin assay, allows rapid determination of tear lactoferrin concentration in 10-15 min, a notable improvement over the 3 days required of the LactoPlate. There was no statistically significant difference between the accuracy of the two assays in normal patients nor in those patients with a diagnosis of keratoconjunctivitis sicca. Both assays showed a significant decrease in tear lactoferrin concentration in patients with severe keratoconjunctivitis sicca when compared to normal patients. The LactoCard is a rapid and reliable means of measuring tear lactoferrin concentration in a clinical setting.

Adult↗
PubMed1994

Corneal keratocytes: in situ and in vitro organization of cytoskeletal contractile proteins.

PURPOSE: Recent studies of corneal wound healing suggest that activated corneal keratocytes develop myofibroblast-like characteristics including a putative contractile apparatus comprised, in part, of intracellular microfilament bundles (i.e., stress fibers) containing f-actin, myosin, and alpha-actinin; extracellular fibronectin fibrils; and fibronectin surface membrane receptors (alpha 5 beta 1 integrin). The purpose of this study was to determine the expression and organization of specific components of the contractile apparatus in normal, quiescent (in situ) corneal keratocytes, and to compare the in situ organization with that of activated, tissue culture (in vitro) corneal keratocytes that potentially mimic wound healing fibroblasts. METHODS: Cat corneal tissue was obtained immediately after sacrifice and was either fixed for in situ studies or cultured with MEM supplemented with 10% fetal calf serum for in vitro studies. Keratocytes (in situ and in vitro) were stained with the following probes: phalloidin, a mushroom toxin that specifically binds to f-actin; rabbit anti-bovine aortic myosin; monoclonal anti-human alpha-actinin; monoclonal anti-human vimentin; rabbit anti-human alpha 5 beta 1 integrin; monoclonal anti-human alpha 5 integrin; monoclonal anti-human connexin 43; and goat anti-human fibronectin. The cytoskeletal organization and co-localization were evaluated using epifluorescent and confocal microscopy. RESULTS: Normal, quiescent corneal keratocytes were distributed within the cornea as a lattice network, interconnected by broad, cellular processes extending from a flattened cell body. The f-actin distribution of in situ keratocytes was predominantly cortical and appeared to be closely associated with the plasma membrane. In addition, punctate areas that appeared to correlate with the localization of adhesion sites were identified. These punctate regions appeared to stain with antibodies to alpha 5 beta 1 but to not alpha 5. These data suggest that the fibronectin receptor, alpha 5 beta 1 integrin, is not present on normal corneal keratocytes. Based on co-localization studies, rabbit anti-bovine aortic myosin and monoclonal anti-alpha-actinin staining had similar distributions to FITC-phalloidin. Interconnections between keratocytes also showed staining for connexin 43, indicating the presence of gap junctions. By contrast, activated, cultured (in vitro) keratocytes showed an FITC-phalloidin staining pattern localized predominantly along intracellular stress fibers not detected in normal, quiescent keratocytes. Myosin and alpha-actinin staining had a similar stress fiber distribution, arranged in alternating bands and suggesting a sarcomeric distribution. Associated with stress fibers there was both anti-alpha 5 beta 1 and anti-alpha 5 staining, indicating the presence of focal adhesions. CONCLUSIONS: This study demonstrates that there are major structural differences in the organization of contractile cytoskeletal proteins between normal, quiescent (in situ), and activated (in vitro) keratocytes. In situ, contractile proteins appear to be associated with the cortical f-actin network, probably related to maintenance of cell shape and interconnectivity. Alternatively, activated keratocytes were characterized by the presence of a putative contractile apparatus comprised of f-actin, myosin, and alpha-actinin organized into sarcomeric, muscle-like bundles (stress fibers) associated with focal contacts containing alpha 5 beta 1 integrin. These data suggest that activation of keratocytes, i.e. myofibroblast transformation, must involve the reorganization of cytoplasmic contractile proteins as well as the expression of alpha 5 beta 1 integrin and the formation of focal contacts.

Animals↗
PubMed1993

The application of confocal microscopy to the study of living systems.

A unique tandem confocal microscope (TSCM) has been developed that permits noninvasive imaging in vivo of the eye and many other organ systems in real time in situ. The application to the study of microphysiological processes in vivo is described and illustrated for the cornea, kidney, liver, epididymis, muscle, and adipose tissue. Novel applications are shown for studying the healing of wounds in four dimensions (x, y, z, t) in single animals over time at the cellular level. Application to clinical diagnostic use in humans is also demonstrated. When combined with Laser Scanning Confocal fluorescence microscopy, the TSCM offers a unique new imaging paradigm for experimental biology and medicine with great potential for use in neuroscience and many other disciplines.

Animals↗
PubMed1993

Clinical and diagnostic use of in vivo confocal microscopy in patients with corneal disease.

BACKGROUND: The purpose of this article is to introduce the practicing ophthalmologist to the optical principles and images produced by a tandem scanning confocal microscope (recently approved by the Food and Drug Administration for general clinical use). The tandem scanning confocal microscope allows real-time viewing of structures in the living cornea at the cellular level in four dimensions (x, y, z, and time). METHODS: Nine patients (2 males, 7 females), ranging in age from 7 to 52 years, were examined. Images were recorded on super VHS videotape, digitized and processed on a computer workstation, and photographed for presentation. RESULTS: Two-dimensional (x, y) 400 x 400-microns images (9-microns z-axis thickness) are presented for normal corneal structures and for the clinical conditions of herpetic keratitis, wound healing after myopic excimer ablation, Acanthamoeba infection, corneal dystrophies (granular, Reis-Buckler), contact lens abrasion, and the irido-corneal endothelial syndrome. CONCLUSION: Clinical confocal microscopy has the unique potential of providing noninvasive assessment of corneal injury and disease at the cellular level that is not available currently from other technologies.

Acanthamoeba Keratitis↗
PubMed1993

In vivo confocal microscopic studies of endothelial wound healing in rabbit cornea.

Corneal endothelial wound healing in living rabbit eyes after mechanical scrape (MS) and transcorneal freeze (TCF) injury was studied using tandem scanning confocal microscopy (TSCM). MS injury was created on the central corneal endothelium with an olive tip cannula; TCF injury was created using a 3-mm-diameter stainless steel probe cooled with liquid nitrogen. In vivo observation of wound healing using TSCM was correlated with scanning electron microscopy (SEM) for fixed tissues. At 6 h after MS, migrating endothelial cells at the leading edge showed lamellipodial processes on in vivo TSCM and SEM. After 24 h, the denuded area was almost fully resurfaced by migrating endothelial cells showing wide spaces between nuclei by TSCM. After 28 days, resurfaced endothelial cells showed normal hexagonal mosaic appearance with enlarged cells by TSCM and SEM. TCF injury produced fibroblastic changes in the endothelial cells with elongation and spreading by 24 h after injury. After 3 days, the wounded area was resurfaced with two cell types: (a) migrating endothelial cells at the peripheral area, which appeared polygonal in shape with wide intracellular spaces and (b) fibroblast-like cells at the center of the wound, which formed a retrocorneal fibrous membrane (RCFM). The RCFM was posteriorly covered with normal endothelium after 28-60 days. TSCM of the stroma demonstrated spindle-shaped, activated keratocytes migrating into the wounded stroma at 3-14 days. In conclusion, TSCM allows viewing of dynamic four-dimensional morphologic changes (x, y, z, and time) during in vivo cellular repair of corneal wound healing after either MS or TCF injury.

Animals↗
PubMed1993

Three-dimensional imaging of corneal cells using in vivo confocal microscopy.

Confocal microscopy is a unique and powerful imaging paradigm which allows optical sectioning through intact tissue. Real-time tandem scanning confocal microscopy has previously been used to generate high-magnification two-dimensional (2-D) images of cells in living organ systems. Inherent problems with movement, however, have prevented the in vivo acquisition of complete 3-D datasets. The development of a new objective lens, used in combination with specialized real-time image acquisition procedures, has allowed sequential serial sections to be obtained in vivo from the rabbit cornea for the first time. These sections can be digitally registered and stacked on the computer to provide a 3-D reconstruction of the corneal cells. This technique should serve as a useful method for studying 3-D structures and analysing 4-D phenomena at the cellular level in living animals. Three-dimensional images of a stromal nerve in normal rabbit cornea and of fibroblasts within a rabbit corneal wound are presented as examples of current capabilities.

Animals↗
PubMed1993

Quantitative analysis of stress fiber orientation during corneal wound contraction.

Previous studies of actin and actin-binding proteins in corneal myofibroblasts suggest the development of a contractile apparatus composed, in part, of F-actin micro-filament bundles, i.e. stress fibers. To better understand the mechanics of wound contraction and the relationship between microfilament bundles and wound closure, we have analyzed the spatial and temporal organization of stress fibers during the process of corneal wound healing. Rabbit corneas (26 eyes) received 6 mm full-thickness, central incisions and were studied at various times for F-actin organization using en bloc (whole cornea) staining with FITC-phalloidin, as well as conventional histological techniques. 3-D datasets (z-series of 40 en face optical sections, 1 micron steps) were collected using the Biorad MRC-600 laser scanning confocal microscope at various regions within the wound. At 7 days, 3-D analysis showed randomly oriented, interconnected F-actin filament bundles (stress fibers). Between 7 and 28 days, stress fibers appeared to organize gradually into planes parallel to the wound surface, with a large population achieving a final orientation nearly parallel to the long axis of the wound. Using Fourier Transform analysis techniques, an orientation index (OI) was calculated to quantitate global fiber orientation at each time point. Analysis of variance demonstrated a significant change (P < 0.001) in overall stress fiber orientation from a random distribution at day 7 to an alignment more parallel to the lateral wound borders at day 28. Overall, these data suggest that stress fibers undergo temporal changes in spatial organization that correlate with wound closure, and that wound closure does not involve the development of previously described contractile or tractional forces aligned directly across the wound.

Actin Cytoskeleton↗
PubMed1993

Actin filament organization during endothelial wound healing in the rabbit cornea: comparison between transcorneal freeze and mechanical scrape injuries.

PURPOSE: To compare and contrast the in vivo mechanism of wound healing after mechanical scrape and transcorneal freeze (TCF) injury in a rabbit eye model by examining changes in the cytoskeletal organization of contractile, filamentous actin (f-actin) microfilaments as relates to differences in cell migration or translocation during endothelial repair. METHODS: Endothelial wound healing after mechanical scrape and transcorneal freeze injury was studied in rabbit eyes using laser scanning confocal microscopy (LSCM). Central corneal mechanical scrape injury was made using an olive tip cannula, and TCF injury was made using a 3-mm diameter stainless steel probe cooled with liquid nitrogen. Cytoskeletal changes in f-actin stained with phalloidin-FITC were observed during wound healing using LSCM. RESULTS: At 6 hours after mechanical scrape, the leading edge of the migrating sheet showed a decrease in the intensity of phalloidin-FITC staining, suggesting a decrease in cortical f-actin. Migrating endothelial cells in vivo did not appear to develop stress fibers after mechanical scrape, which is consistent with an in vitro cell spreading mechanism of endothelial wound healing. By 24 hours, the denuded area was almost fully resurfaced by migrating endothelial cells. On the other hand, TCF injury produced fibroblastic changes in the endothelial cells with extension and elongation of spindle-shaped endothelial cells at the leading edge by 24 hours after injury. Fibroblastic endothelial cells developed prominent actin stress-fibers, which is consistent with an in vitro cell migration mechanism of endothelial wound healing. Three days after TCF, the wounded area was resurfaced with two cell types: rough, fibroblast-like cells forming a retrocorneal fibrous membrane having prominent f-actin bundles or stress fibers with few cell-cell junctions, and smooth, polygonal-shaped endothelial cells having tight cell junctions with a cortical distribution of f-actin. After 28 days the retrocorneal fibrous membrane was posteriorly covered with normal endothelium. CONCLUSIONS: These data support the hypothesis that endothelial wound healing involves two separate, injury-dependent, mechanisms--cell spreading and cell migration.

Actins↗
PubMed1993

Nuclear muscarinic acetylcholine receptors in corneal cells from rabbit.

PURPOSE: Previous studies have indicated that muscarinic acetylcholine receptors (mAChR) may be present in an unexpected, unique location and play a singular role in cellular growth regulation of rabbit corneal epithelium that may be of general physiologic significance if found in other cells. The purpose of this study was to examine rabbit corneas and corneal cells in culture to determine mAChR location and tissue distribution. METHODS: Using [3H]-propylbenzilylcholine mustard ([3H]PrBChM), which binds covalently to the active site of mAChR, rabbit corneal cross-sections, cultured corneal keratocytes, epithelial and endothelial cells, as well as nuclei isolated from these cultured corneal cells were labeled, stained, and autoradiographed. Nuclei labeled with [3H]PrBChM were further analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. RESULTS: Direct visual confirmation of the localization of mAChRs was obtained. MAChR were found in epithelial and endothelial layers of fresh-frozen corneal cross-sections, in cultured rabbit epithelial and endothelial cells, and on isolated rabbit epithelial and endothelial cell nuclei. mAChR were not detectable in keratocytes with these techniques. When [3H]PrBChM-labeled nuclei from cultured corneal cells were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis, epithelial and endothelial samples showed specific mAChR binding, whereas binding to keratocyte nuclei was not detectable. CONCLUSIONS: As a result of these findings, a revised hypothesis is suggested for the locations and possible functions of mAChR in regulation of growth in corneal and other cells.

Acetylcholine↗
PubMed1993

Effects of rigid gas permeable contact lens extended wear on rabbit cornea assessed by LDH activity, MDH activity, and albumin levels in tear fluid.

We used noninvasive biochemical techniques to study the effects on rabbit corneas of 7-day extended wear of rigid gas permeable (RGP) contact lenses of varying oxygen transmissibilities. Corneal effects were assessed through measurement of lactate dehydrogenase (LDH) and malate dehydrogenase (MDH) activities and albumin levels in tears. The RGP contact lenses used had Dk/Ltotal values ranging from 33 to 64 x 10(-9) (cm/sec) (mL O2/mL mmHg) and were of uniform 0.15 mm center thickness. Extended wear of high Dk (Dk/Ltotal = 34) and super high Dk (Dk/Ltotal = 56) lenses caused an increase in tear LDH activity from 1,190 U/L (before lens wear) to over 18,000 U/L during 7 days of continuous wear. These contact lenses also caused gradual increases in tear MDH activity from 431 U/L (before lens wear) to over 750 U/L after 7 days of continuous wear. Extended wear of the ultra high Dk lens (Dk/Ltotal = 64), however, caused no significant increase in LDH or MDH activity in tears. Tear albumin levels in all contact lens wearing eyes increased after 1 day of lens wear, then gradually recovered to normal values after 2 days of continuous wear. The changes in albumin levels did not correlate with Dk/Ltotal values of lenses worn.(ABSTRACT TRUNCATED AT 250 WORDS)

Adaptation, Ocular↗
PubMed1993

Morphological and biochemical evaluation for rigid gas permeable contact lens extended wear on rabbit corneal epithelium.

We studied the effects of 24-hour wear of rigid gas permeable (RGP) contact lenses of varying oxygen transmissibilities on the rabbit cornea by measuring concomitant lactate dehydrogenase (LDH) activity in tears and by in vivo tandem scanning confocal microscopy (TSCM). We used a PMMA lens and rigid gas permeable (RGP) lenses that had Dk/L values ranging from 7 to 64 x 10(-9) (cm/sec) (mL O2/mL mmHg) and a uniform 0.15 mm thickness. After 6- and 24-hour contact lens wear, rabbit tear LDH activity increased according to the decrease in the Dk of RGP lenses. Tear LDH activity after 24 hours of lens wear was higher than after 6 hours. The observed increase in tear LDH activity was correlated with in vivo corneal epithelial morphology by tandem scanning confocal microscopy. The observed severity of desquamation and swelling of corneal epithelial cells was dependent upon the Dk/Ltotal of contact lenses worn, which directly related to the contact lens induced corneal hypoxia. Based on the results of this study, we conclude that: 1) a nap or accidental overnight wear of contact lenses with less than 20 x 10(-9) Dk/Ltotal could cause severe corneal epithelial damage; 2) the ultra high Dk lens appeared to alter the ocular surface least; and 3) TSCM accompanied with tear LDH assay is an objective, non-invasive in vivo method to assess the effect of contact lens wear on the ocular surface over time at the cellular level.

Animals↗
PubMed1992

In vivo confocal microscopy in clinical dental research: an initial appraisal.

Until recently, the in vivo microscopic investigation of intraoral tissues at high resolution has been virtually impossible. Confocal microscopy enables high-resolution imaging to be achieved below semitransparent surfaces in intact living specimens, but this may still be impractical for intraoral applications because of the need to stabilize the sample. The development of a steadying objective (x 240 overall mag.) which is held against the sample surface and is focused by moving internal elements, avoids the need for fine adjustment of the living sample under the microscope to achieve a change of focus. It is therefore more comfortable and also reduces the problems of movement due to the pulse. The objective was used with a tandem scanning microscope, with images recorded via a SIT video camera. Using this system internal tooth structure (e.g. enamel prisms/adhesive restoration interfaces) and the lining cells of the gingival crevice through to the junctional epithelium may be examined. It is also possible to image the oral mucous membrane, focusing to the capillary loops in the basal layers, where streaming red blood cells can be seen. Access is limited to the anterior regions as far back as the premolar teeth. Applications could include caries research, soft and hard tissue responses to biomaterials (e.g. implants), wound healing and monitoring the effect of periodontal treatment regimens. This new technique offers numerous exciting opportunities for the microscopic investigation of many clinical operative procedures in vivo, allowing the response of the tissues to be non-destructively monitored, over time, at high resolution.

Composite Resins↗