Biomedical subjects

A H Neufeld

Publications and source records attributed to A H Neufeld.

At least 37 records · Page 2Linked to original sources

PubMed1991

Localization of collagen types I and IV mRNAs in human optic nerve head by in situ hybridization.

Using in situ hybridization, individual cells expressing mRNAs for collagen types I and IV were localized in fixed-tissue sections of adult and fetal human optic nerve heads. Astroglial cells lining the cribriform plates and cells inside the cribriform plates of the lamina cribrosa had mRNA for collagen type IV. Cells in the glial columns, pial septa, and vascular wall also contained mRNA collagen type IV. Collagen type I mRNA was expressed by cells of the cribriform plates of the lamina cribrosa of adults. Few cells in the glial columns, pial septa, and blood vessels had mRNA for collagen type I. Scleral fibroblasts contained mRNA for collagen type I. These results indicated that the expression of mRNA for both collagen types I and IV paralleled the localization of these extracellular matrix proteins in the optic nerve head and suggested that both collagen types were synthesized in this tissue throughout life.

Adolescent↗

PubMed1990

Retinal pigment epithelium wound closure in vitro. Pharmacologic inhibition.

Early-passage bovine retinal pigment epithelium (RPE) cells were grown to confluence in 24-well plates, and a central defect was created mechanically in the monolayer within quadruplicate wells, sequentially over 9 days. Closure of the wounded area occurred by single-cell migration of elongated RPE cells from the edge of the wound and subsequent cell proliferation. Ten days after wounding, the cultures were fixed, stained, and photographed, and the residual wound area was quantified by computerized planimetry. Cell counts of unfixed cultures were determined with a Coulter counter. Wound closure was complete after 10 days. Using this technique, we assessed the response of RPE to various concentrations of 5-fluorouracil (5-Fu), colchicine (COL), and cytochalasin-B (CYT-B). 5-Fu (10 micrograms/ml) and COL (0.1 and 1 microgram/ml) inhibited migration and proliferation of RPE cells. CYT-B (5 micrograms/ml) inhibited migration. This model allows in vitro study of the response of RPE cells after loss of contact inhibition. The technique provides a quantitative model for assessing the dynamic capabilities of RPE cells in response to a localized mechanical defect and for assessing the pharmacologic modulation of these responses.

Animals↗

PubMed1990

Changes in the extracellular matrix of the human optic nerve head in primary open-angle glaucoma.

Using immunofluorescent staining, we were able to characterize the changes in composition and distribution of the macromolecules making up the extracellular matrix of the lamina cribrosa of the glaucomatous human optic nerve head. In tissue adjacent to the glaucomatous cups, there was marked disorganization and loss of fibers of elastin within the cores of the cribriform plates. Collagen type VI, normally sparse, increased in quantity considerably throughout the lamina cribrosa in glaucomatous eyes with all degrees of damage. Collagen type IV and other basement membrane macromolecules appeared to extend into nerve bundles, presumably filling in spaces previously occupied by nerves. There was no appreciable change in the postlaminar region, which indicates the specificity of the extracellular matrix changes in the lamina cribrosa. Our results indicate that changes in the extracellular matrix play an important role in the progression of the glaucomatous process and may be a causative agent of the disease.

Aged↗

PubMed1990

In vitro pharmacologic separation of corneal endothelial migration and spreading responses.

Repair of corneal endothelial wounds involves two forms of cell translocation: (1) "migration," in which individual cells at the wound edge break contacts with neighboring cells and move as individuals into the wound defect, and (2) "spreading," in which cells within the confluent monolayer adjacent to the wound move as a group into the wound area. The authors combined morphometric analysis of Giemsa-stained cultures, phase-contrast video microscopy, and Rh-phalloidin staining of actin filaments to study the effects of epidermal growth factor (EGF) and indomethacin on the migratory and spreading responses to wounding using an in vitro wound-closure model which mimics the amitotic state and general behavior of human corneal endothelium. They found that EGF stimulated the migration of individual cells from the wound edge, induced cellular elongation, and promoted a diffuse distribution of actin filaments. Indomethacin promoted spreading of the confluent monolayer into the wound defect, induced enlargement and flattening of cells, and promoted the formation of long, thick actin stress fibers. These results provide evidence that the migration and spreading responses of corneal endothelial cells to wounding can be pharmacologically separated. The findings suggest that migration of individual cells during wound repair may result from an endogenous form of EGF-like stimulation and that the elongated shape associated with this form of translocation results, at least in part, from an EGF-like alteration in actin-filament organization. Spreading of the confluent monolayer to cover the wound defect may result from a decrease in cyclic adenosine monophosphate induced by a transient reduction in prostaglandin E2 synthesis. This form of translocation may result, in part, from enlargement and flattening of corneal endothelial cells secondary to an enhancement of actin stress-fiber formation.

Actins↗

PubMed1989

Age-related changes in the extracellular matrix of the human optic nerve head.

By using immunofluorescent staining we were able to characterize the age-related changes in the macromolecules making up the extracellular matrix of the lamina cribrosa of the human optic nerve head. As the cores of the cribriform plates enlarged with age, there were age-related increases in the apparent density of collagen types I and III and elastin that constituted the connective tissue support of the nerve bundles. Collagen type IV coated the cribriform plates as basement membranes and was also present within the cores as a fine filamentous network, which increased in density with age as the cribriform plates expanded. As this tissue ages, individual differences leading to more or less of a particular macromolecule of the extracellular matrix may alter the support function of the lamina cribrosa and influence the degeneration of the optic nerve associated with glaucoma.

Aging↗

PubMed1989

Corneal endothelial wound closure in vitro. Effects of EGF and/or indomethacin.

In response to stress, the corneal endothelium must maintain or region its barrier function. To study cellular responses of the corneal endothelium, our laboratory has developed an in vitro model of rabbit corneal endothelial wound closure. When cells are free to divide, a 3 mm diameter wound closes within 4 days. 5-fluorouracil added to these cultures does not affect the cellular morphology or ultrastructure, but does inhibit cell division. In the presence of 5-fluorouracil, wounds close in approximately 7 days. These conditions mimic the amitotic state and general behavior of adult human corneal endothelium in vivo. Using this model, we studied the effects of epidermal growth factor (EGF) and/or indomethacin treatment on corneal endothelial wound closure in mitotically competent and inhibited cultures. EGF appeared to stimulate migration, whereas indomethacin appeared to enhance cell spreading in response to wounding, particularly in mitotically inhibited cultures. Treatment with the above agents at the time of wounding had little effect on wound closure rates, but did affect closure patterns. In contrast, pretreatment of cultures, particularly with indomethacin, significantly accelerated closure in mitotically inhibited cultures. In the presence of indomethacin, wounds closed in 3-4 days compared to 7-8 days for controls. These results indicate that the response of corneal endothelial cells to wounding can be pharmacologically manipulated, and perhaps accelerated, and suggest that the treatment of the endothelium with nonsteroidal anti-inflammatory drugs or EGF-like growth factors may be clinically useful.

Animals↗

PubMed1989

Effects of EGF and indomethacin on rabbit corneal endothelial wound closure in excised corneas.

Corneal endothelial cells of rabbit corneas stored in M-K medium at 37 degrees C were wounded by touching them lightly with a micropipet under video specular microscope observation. Three groups were studied: control, with EGF, and with EGF + indomethacin. The wound closure process (initial wound area about 8500 microns 2) was observed and recorded with time-lapse videography for 6 hr. The recorded video images were digitized and computer assisted morphometric analysis was performed. (1) Addition of either EGF (10 ng/ml) + indomethacin (1 microM), or EGF (10 ng/ml) alone to the M-K medium statistically significantly shortened the wound closure time as compared with the control group. (2) Both EGF + indomethacin and EGF alone resulted in a greater average percent relative change of the shape factor, more than three times greater with EGF + indomethacin and more than two times greater with EGF alone, than in the control group 150 min after wounding. (3) The maximum cell shape change occurred at about 150 min after wounding in the groups EGF + indomethacin and EGF alone, and at about 200 min in the control group. After this time in all three groups the cells began to approach a normal shape. (4) The cells near the wound boundary moved faster in the EGF + indomethacin and the EGF groups as compared with the control group. These results suggest that EGF and indomethacin may be of therapeutic value in promoting closure of traumatized human corneal endothelium.

Animals↗

PubMed1988

Pharmacological regulation of morphology and mitosis in cultured rabbit corneal endothelium.

Cultured rabbit corneal endothelial cells elongate when grown in the presence of epidermal growth factor (EGF) and indomethacin (INDO); whereas maintenance of the differentiated polygonal cell shape is apparently dependent upon endogenous synthesis of prostaglandin E2 (PGE2). In the current study, the authors demonstrate morphological changes in phenotypically altered cells and identify two intracellular pathways which interdependently regulate endothelial cells. Morphometric and mitotic analyses of cultures treated with a variety of pharmacological agents indicate that both protein kinases A- and C-dependent pathways regulate cell shape and cell division in corneal endothelial cells. Marked intracellular reorganization is associated with the morphological changes in the endothelial cells. When stained with rhodamine conjugated phallicidin, polygonal endothelial cells have circumferential bands of f-actin at their borders. EGF and/or INDO induce elongation and redistribution of f-actin into a diffuse cytoplasmic reticulum. Transmission electron microscopy demonstrates loss of several characteristic morphological markers for endothelial cells in response to pharmacologically induced elongation. The elongated cells lose intracellular junctions, apical/basal polarity and rough endoplasmic reticulum. These ultrastructural markers and circumferential f-actin bands are restored in cultures supplemented with exogenous PGE2. Modulation of these pathways in vivo may regulate cellular migration and mitosis during wound closure, stress, trauma and with age.

1-Methyl-3-isobutylxanthine↗

PubMed1988

Cell culture of the human lamina cribrosa.

The extracellular matrix of the lamina cribrosa may be important in the changes in the optic nerve head associated with glaucoma. To investigate the cell biology of this tissue, human lamina cribrosa was explanted in tissue culture and two cell types grown from this tissue were characterized. The most common cell type obtained was a large, flat, polygonal cell which was negative for glial fibrillar acidic protein (GFAP) and could be serially subcultured. This cell type synthesized collagens type III and type IV, fibronectin and elastin. Much less commonly grown was a cell type with conspicuous long processes and which was positive for GFAP. This presumed astrocyte synthesized collagen type IV and fibronectin. Fibroblastic cells were not obtained from this tissue but were easily grown from sclera. The cells that we have cultured from the human lamina cribrosa may produce the extracellular matrix present in the cribriform plates of this tissue and be important in the glaucomatous process.

Adult↗

PubMed1987

Extracellular matrix of the human lamina cribrosa.

We used immunoperoxidase staining and double immunofluorescent staining to demonstrate the macromolecular components of the extracellular matrix of the lamina cribrosa from young human donors. The cribriform plates were made up of a core of elastin fibers with a sparse, patchy distribution of collagen type III. The plates were coated with collagen type IV and laminin; these basement membrane components were presumably made by the astrocytes that were distributed on the surfaces of the plates. The insertion of the lamina cribrosa in the sclera was made up of concentric, circumferential elastin fibers that surrounded the lamina cribrosa and were continuous with the elastin in the cribriform plates. Astrocytic processes extended into the bundles of elastin fibers, whereas the basement membrane components extended into the sclera. The mechanical properties of the macromolecules of the extracellular matrix of the lamina cribrosa may make this tissue compliant and sensitive to intraocular pressure. Perhaps individual differences in the macromolecular components of this tissue contribute to the glaucomatous changes in the optic nerve head.

Adolescent↗

PubMed1987

Corneal epithelial wound closure in tissue culture: an in vitro model of ocular irritancy.

The influence of 13 test agents on the ability of cultured rabbit corneal epithelial cells to migrate and re-cover a wound has been utilized to evaluate an in vitro model of ocular irritancy. Cells were grown under standard conditions and monitored for adequate cell density. Seven days after subculture, replicate wounds were produced and cultures were exposed to varying concentrations of a test agent in culture medium for 24 hr. Following exposure, cultures were fixed and stained to reveal remaining wound areas which were quantitated by computerized planimetry and compared to evaluate the deleterious effects of the test agents. The test ranks irritants in an order similar to that described in the literature for both in vivo and in vitro tests. This tissue culture model is conceptually simple, quantitative, and an alternative to the corneal component of whole animal testing for ocular irritancy.

Animals↗

PubMed1987

The effects of forskolin on cyclic AMP, intraocular pressure and aqueous humor formation in rabbits.

Forskolin was used to study cyclic AMP-mediated regulation of aqueous humor dynamics in rabbits. Crystalline forskolin was solubilized in oil and its pharmacological effects were studied both in vitro and following topical ocular administration. In vitro, using cultured corneal epithelial cells, forskolin rapidly stimulated cyclic AMP production and in vivo increased cyclic AMP concentration in the aqueous humor 10-fold following topical administration. The effect of topical forskolin on intraocular pressure and aqueous humor formation was determined in vivo using pneumatonometry and fluorophotometry, respectively. Forskolin caused a prolonged reduction of intraocular pressure and decreased aqueous humor formation. The ability of forskolin to potentiate the ocular hypotensive effect of epinephrine was investigated. Forskolin in combination with epinephrine caused a decrease in intraocular pressure of longer duration than either 0.1% epinephrine or 1% forskolin administered separately. Forskolin caused a small but significant increase in the permeability of the blood-aqueous barrier at the time of maximal intraocular pressure reduction. This effect on the blood-aqueous barrier may explain the inhibitory effect of forskolin on aqueous humor formation.

Animals↗

PubMed1987

Effects of stimulation of the ocular sympathetic nerves on IOP and aqueous humor flow.

Ocular sympathetic nerves were stimulated chronically in awake rabbits using electrodes unilaterally implanted on the cervical sympathetic trunk. IOP was measured by pneumatonometry and aqueous inflow was measured by fluorophotometry. In each animal, continuous trains of 1 msec pulses were delivered by means of a portable electrical stimulator. Experiments were spaced by 1 week recovery periods. Stimulation was varied over a range of amplitudes (5-15 V) and frequencies (3-12 Hz). Continuous sympathetic stimulation produced an immediate sharp decrease in IOP followed by a gradual rise to pre-stimulation values which were attained 60-90 min after onset. A rebound increase in IOP occurred when stimulation was terminated. The magnitude of the initial IOP drop, the delay in the return to pre-stimulation IOP, and the rebound rise in IOP subsequent to termination of electrical stimulation were proportional to the stimulation frequency. Maximal effects were observed at 12 Hz, and stimulation with 8-10 Hz for 180 min caused a sustained reduction in anterior chamber aqueous humor flow. Topical 2% phentolamine 1 hr before stimulation markedly reduced IOP and abolished the acute IOP changes observed in untreated stimulated animals. Topical 1% timolol did not affect either the initial IOP drop or the rebound; however, the IOP recovered during stimulation to values greater than pre-stimulation IOP. We conclude that in rabbits the beta-adrenergic effect of prolonged sympathetic nerve stimulation is to decrease aqueous flow. Chronic electrical stimulation in awake animals provides an experimental model for studying the role of the ocular sympathetic nerves.

Animals↗

PubMed1987

Adrenergic stimulation of ciliary process epithelium causes surface membrane internalization.

An ultrastructural change induced in the nonpigmented epithelium (NPE) of the ciliary processes by adrenergic stimulation in the albino rabbit was studied. Thirty min after topical treatment with 2% isoproterenol, an extensive intracellular membranous network, previously reported to be smooth endoplasmic reticulum, was revealed by electron microscopy. It was postulated that this network originated from the plasma membrane. Using cationized ferritin (CF) as an ultrastructural tracer, freshly isolated anterior segments were incubated in buffer containing 10(-5) M isoproterenol and 0.2% CF. As early as 10 min, and for at least 30 min, the isoproterenol-treated NPE cells contained a membranous network that was morphologically similar to that which occurs in vivo. CF particles were present within the network, indicating that the membranous network had originated at the cell surface. This labeling was prevented by pretreatment with the beta-adrenergic antagonist timolol maleate. In both treated and control ciliary processes, CF was present in the ciliary canals between the NPE and the underlying pigmented epithelium after 10 min incubation. This suggests that the NPE is able to transport CF from its basilar to apical surface. These experiments imply that the NPE is able to internalize rapidly large amounts of plasma membrane in response to adrenergic stimulation. This response may be part of the mechanism of adrenergic receptor desensitization, alteration of aqueous humor production, or another adrenergic response.

Administration, Topical↗

PubMed1986

Extracellular matrix of the human optic nerve head.

Double-antibody immunofluorescent studies of sectioned human optic nerve head indicated the marked presence of collagen type IV and laminin in the extracellular matrix of the lamina cribrosa. These macromolecules were layered transversely across the nerve fascicles and appeared to constitute the cribriform plates. Relatively little collagen types III and I were present in the extracellular matrix of this tissue and fibronectin was not detected in appreciable amounts. These results indicated that the lamina cribrosa contains a specialized extracellular matrix of the central nervous system made up of plates of material resembling basement membrane. The major macromolecular components of the lamina cribrosa do not resemble those of sclera.

Adult↗

PubMed1986

Aqueous to cornea fluorescein distribution ratio in normal and swollen cornea.

Intravitreal sodium fluorescein was used to simulate equilibrium fluorescein kinetics, thereby allowing simple measurement of the aqueous to cornea fluorescein distribution ratio. Two groups of rabbit corneas were studied: normal corneas and corneas wounded by freezing. The aqueous to cornea fluorescein distribution ratio was approximately 0.4, was not significantly different in groups of normal or wounded eyes and little variability was noted. In addition, a comparison of in vivo and in vitro measurements of corneal fluorescein concentration in wounded eyes suggests that in vivo protein-bound fluorescein in the cornea fluoresces less efficiently than free fluorescein.

Animals↗

PubMed1986

A tissue culture assay of corneal epithelial wound closure.

Experimental assays have been developed using cultured tissue derived from rabbit corneal epithelium to study migration of epithelial sheets during wound closure and cell-substrate adhesion. To study wound closure, epithelial defects, 6 mm in diameter, were produced in vitro in 24 well multiplates by a local freezing technique, and the size of the remaining defect was quantitated over time by staining. To study adhesion, cultured cells were labeled with 3H-leucine, suspended, and added to fresh culture plates. At various times, adherent cells were lysed and the radioactivity of the lysate was determined. Serum enhances the closure of experimental defects, but laminin and fibronectin have no effect. Agents which alter mitotic rate, such as epidermal growth factor and 5-fluorouracil, do not influence the rate of wound closure in this assay. Compounds which elevate intracellular levels of cyclic AMP inhibit wound closure but promote cell-substrate adhesion. Thus, cultured corneal epithelial cells may be used to assay for influences on the migratory events governing closure of superficial epithelial wounds.

Animals↗

PubMed1986

Rabbit corneal endothelial cells in vitro: effects of EGF.

Rabbit corneal endothelial cells were grown in tissue culture. Epidermal growth factor (EGF) increased the mitotic rate during the growth phase by 70% over control without affecting the plating efficiency. Within 48 hr of exposure to EGF, the endothelial cells became spindle-shaped. This morphological change was quantitated by morphometry; cells treated with EGF had a major axis 1.5 X larger than that of non-EGF treated cells. The spindle-shaped morphological change did not occur in response to other growth factors, was not related to cell density, and was reversible within 24 hr after removal of EGF from the media or subculture in the absence of EGF. The addition of 5-fluorouracil blocked cell division but did not affect the EGF-induced morphological change. The appearance of the endothelial cells following EGF stimulation is similar to migrating cells closing a wound in vivo.

Animals↗