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Biomedical subjects

A H Neufeld

Publications and source records attributed to A H Neufeld.

At least 19 recordsLinked 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↗
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↗
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↗
PubMed1979

Modification by timolol of catecholamine stimulation of chloride transport in isolated corneas.

In the isolated frog cornea, 10(-5)M timolol completely blocked the stimulation of chloride transport by 10(-6)M isoproterenol. In this preraration, timolol inconsistently modified the response to epinephrine. In some experiments, epinephrine added to the bathing medium after timolol caused a smaller than normal increase in chloride transport, but in other instances, epinephrine caused a decrease in chloride transport. In the isolated rabbit cornea, 10(-5)M timolol totally blocked the stimulation of chloride transport by 10(-6)M epinephrine. In vivo, topical treatment of rabbit eyes with 0.5% timolol resulted in corneas from these eyes having an inhibited response to epinephrine when incubated in vitro. This inability to stimulate chloride transport persisted for several days following termination of topical treatment with timolol.

Administration, Topical↗
PubMed1979

The rabbit cornea lacks cholinergic receptors.

Cholinergic receptors were studied in membranes prepared from rabbit cornea, iris-ciliary body, and retina, using 3H-quinuclidinyl benzilate (3H-QNB) to identify muscarinic receptors and 125I-alpha-bungarotoxin (125I-BGT) to identify nicotinic receptors. Muscarinic cholinergic receptors were not found in the cornea. As a positive control, muscarinic cholinergic receptors were characterized in preparations of the iris-ciliary body. Specific binding of 3H-QNB to iris-ciliary body membrane preparations was saturable, with a Kd of 1.3 nM QNB. Specificity of the assay for muscarinic receptors was confirmed by the relative abilities of the following compounds to displace 3H-QNB: atropine greater than pilocarpine greater than hexamethonium. Nicotinic cholinergic receptors were not found in the cornea. As a positive control, nicotinic cholinergic receptors were characterized in preparations of the retina. Specific binding of 1252-BGT to retinal membrane preparations was saturable with both high and low affinity receptors (Kd values of 1.0 nM and 93 nM BGT, respectively). Specificity of the assay for nicotinic receptors was confirmed by the relative abilities of the following compounds to prevent 125I-BGT binding: curare greater than or equal to nicotine greater than hexamethonium greater than atropine. The lack of cholinergic receptors in the cornea, which has high levels of acetylcholine and related enzymes, suggests either an extraordinary use or a lack of function for acetylcholine in this tissue.

Animals↗
PubMed1978

Topical epinephrine causes a decrease in density of beta-adrenergic receptors and cathecholamine-stimulated chloride transport in the rabbit cornea.

A single administration, or twice daily administration for 4.5 days, of topical 2% epinephrine to the rabbit eye in vivo causes a 30-40% decrease in the density of beta-adrenergic receptors on membranes prepared from the cornea. Such treatment also causes complete loss of the ability of excised corneas to respond to epinephrine in vitro with enhanced active chloride transport. These findings indicate that stimulation with a high concentration of catecholamine depresses the entire pathway from receptor to physiological response.

Amphotericin B↗
PubMed1978

Influences on the density of beta-adrenergic receptors in the cornea and iris--ciliary body of the rabbit.

By measurement of the specific binding of 3H-dihydroalprenolol, the densities of beta-adrenergic receptors on membranes prepared from homogenized corneas and iris--ciliary bodies of rabbits were studied. Sympathetic denervation, as a result of subconjunctival treatment with 6-hydroxydopamine, causes an increase in the density of beta-adrenergic receptors in membranes prepared from the ipsilateral iris--ciliary body but not the cornea. Topical treatment with epinephrine for 5 days causes a decrease in the density of beta-adrenergic receptors in membranes prepared from cornea and iris-ciliary body, whereas similar treatment with timolol causes an increase in the density of beta-adrenergic receptors. In the cornea, the decrease in receptor density that occurs following in vivo treatment with epinephrine is associated with a decreased ability to synthesize cyclic AMP, whereas the increase in receptor density that occurs following in vivo treatment with timolol is not associated with an altered ability to synthesize cyclic AMP. Our results indicate that the density of beta-adrenergic receptors in the anterior segment of the eye is inversely related to the level of adrenergic stimulation to the tissue but that the ability of a tissue to synthesize cyclic AMP does not necessarily parallel the change in receptor density.

Animals↗
PubMed1977

In vitro determination of the ability of drugs to bind to adrenergic receptors.

Alpha- and beta-adrenergic receptors were studied by measuring the binding of 3H-dihydroergocryptine and 3H-dihydroalprenolol, respectively, to membranes prepared from homogenized rabbit iris--ciliary bodies. The binding of 3H-dihydroergocryptine appears to be specific for alpha-adrenergic receptors, since adrenergic agents displace this radioligand with the following order of potency: phentolamine greater than epinephrine greater than or equal to norepinephrine greater than or equal to isoproterenol = propranolol. The binding of 3H-dihydroalprenolol appears to be specific for beta-adrenergic receptors, since adrenergic agents displace this radioligand with the following order of potency: propranolol greater than or equal to isoproterenol greater than or equal to epinephrine greater than norepinephrine greater than or equal to phentolamine. Clonidine and dopamine bind to the alpha-adrenergic receptor but have little activity at the beta-adrenergic receptor. Timolol, d-isoproterenol, and dipivalyl epinephrine bind to the beta-adrenergic receptor but have little activity at the alpha-adrenergic receptor. The results demonstrate that in vitro binding assays for alpha- and beta-adrenergic receptors are useful for studying the mechanism of drug action.

Alprenolol↗
PubMed1976

Differential reactivity of rabbit iris and ciliary process to topically applied prostaglandin E2 (dinoprostone).

The topical application of prostaglandin E2 (dinoprostone) is followed by massive swelling of the ciliary process, leading to substantial leakage of marker dye into the posterior chamber and directly into the region of the iris, where the primary ciliary processes insert. In contrast, blood vessels of the iris proper retain most of their normal barrier quality and do not leak Evans blue dye. The adrenergic innervation of the anterior segment remains normal in density and in quality after the topical application of PGE.

Administration, Topical↗