Biomedical subjects

Cindy K Bahler

Publications and source records attributed to Cindy K Bahler.

6 recordsLinked to original sources

PubMed2006

Perfusion of his-tagged eukaryotic myocilin increases outflow resistance in human anterior segments in the presence of aqueous humor.

PURPOSE: A previous study by the authors has shown that recombinant myocilin purified from a prokaryotic expression system increases outflow resistance in cultured human anterior segments. The present study was performed to determine whether full-length myocilin purified from a human trabecular meshwork cell expression system alters outflow resistance after infusion into human anterior segments. METHODS: A feline immunodeficiency virus vector encoding both full-length myocilin (amino acids 1-503 fused to C-terminal V5 and six-histidine epitopes) and puromycin resistance was used to transduce a transformed trabecular meshwork cell line (TM5). Stably expressing cells were selected with puromycin. Recombinant myocilin was purified from the media using nickel ion affinity chromatography. Control purifications were performed on media from parental TM5 cells. Anterior segments of human eyes were placed in organ culture and perfused with either Dulbecco's modified Eagle's medium (DMEM) or DMEM supplemented with 50% porcine aqueous humor. One eye received an anterior chamber exchange with recombinant myocilin (2 microg/mL), whereas the fellow eye received an equal volume of control. Immunohistochemistry was performed with anti-myocilin and anti-V5 antibodies. Native polyacrylamide gel electrophoresis was used to analyze myocilin complex formation in porcine aqueous humor. RESULTS: Recombinant myocilin in porcine aqueous humor increased outflow resistance in cultured human anterior segments (91% +/- 68% [mean +/- SD] versus 18% +/- 31% in fellow control eye; n = 9, P = 0.004). Maximum outflow resistance was obtained 5 to 17 hours after infusion and remained above baseline for >3 days. Recombinant myocilin also increased outflow resistance in eyes incubated in DMEM, but only if myocilin was preincubated with porcine aqueous humor (78% +/- 77% when preincubated in DMEM containing porcine aqueous humor versus 13% +/- 15% when preincubated with DMEM alone, n = 6, P = 0.03). Recombinant myocilin appears to form a complex in porcine aqueous humor with a heat-labile protein(s). Immunohistochemistry revealed the presence of myocilin in the juxtacanalicular region of the trabecular meshwork. CONCLUSIONS: Myocilin purified from human trabecular meshwork cells increased outflow resistance in cultured human anterior segments, but only after incubation with porcine aqueous humor. Recombinant myocilin appears to form a complex in porcine aqueous humor that enables it to bind specifically within the trabecular meshwork.

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PubMed2005

Cationic ferritin and segmental flow through the trabecular meshwork.

PURPOSE: To determine whether segmental labeling by the tracer molecule cationic ferritin (CF) is indicative of preferential patterns of fluid flow in the trabecular meshwork or of differences in cell and extracellular matrix properties. Nonlabeled regions could indicate no fluid entering that area, insufficient perfusion time, or that the cells and extracellular matrix differ in that region and cannot bind CF. METHODS: Six whole eyes (three normal and three with pseudoexfoliation [PEX]) syndrome were perfused with CF for 30 minutes to 4 hours. Wedges of trabecular meshwork were dissected and some wedges immediately fixed. Adjacent wedges were placed in a CF bath before fixation. Transmission electron microscopy was used to analyze CF labeling. RESULTS: CF increased in the trabecular meshwork with increasing perfusion time. At 30 minutes, CF labeled mainly the uveal and corneoscleral regions. By 4 hours, CF was found diffusely through the meshwork, although a few isolated nonlabeled areas were still present. Wedges immersed in the CF bath showed fewer nonlabeled regions at all time points. Clumps of PEX material labeled more heavily in the periphery than the center, suggesting the clumps were less permeable than surrounding regions. PEX eyes otherwise had similar labeling patterns. CONCLUSIONS: Segmental labeling with CF implies regions of preferential flow exist in the meshwork. With increasing perfusion time, there were fewer nonlabeled regions. CF labeling of most regions of bath-immersed tissue suggests that nonlabeled regions do not differ in the characteristics of the cells, but rather that CF does not reach these regions.

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PubMed2004

Trabecular bypass stents decrease intraocular pressure in cultured human anterior segments.

PURPOSE: To determine the effect on intraocular pressure (IOP) of bypassing the trabecular meshwork in cultured human anterior segments. DESIGN: Prospective laboratory investigation using normal human eyes obtained at autopsy. METHODS: Anterior segments from 21 eyes were placed in perfusion culture, and trabecular bypass stents were inserted through the trabecular meshwork, with the lumen of the tube opening into Schlemm's canal. Eyes received from one to four stents, placed equidistant apart. In eyes receiving one or two stents, additional stents were later added to a maximum of four per eye. RESULTS: Intraocular pressure was lowered after placement of a single stent, from 21.4 +/- 3.8 mm Hg to 12.4 +/- 4.2 (P < .001). This corresponded to an 84% increase in facility of outflow. Eyes receiving more than one stent had final IOP of 11.9 +/- 3.7 mm Hg. Nine eyes had sequential addition of stents, and seven of these had a further decrease of IOP (13.6 +/- 4.1 to 10.0 +/- 4.3; P = .02). Excision of the entire meshwork, between stents, dropped IOP to 6.3 +/- 3.2 mm Hg, indicating some residual meshwork or canal resistance remained even after placement of three stents. CONCLUSIONS: Bypass of the trabecular meshwork lowers IOP in cultured human anterior segments. One stent produced the greatest change in pressure. The sequential addition of more stents further lowered pressure in seven of nine eyes. This technique holds promise as a new clinical surgery for glaucoma.

Anterior Eye Segment↗

PubMed2004

Pharmacologic disruption of Schlemm's canal cells and outflow facility in anterior segments of human eyes.

PURPOSE: To determine the effect of disruption of Schlemm's canal cells on outflow facility. Pharmacologic agents that weaken the cytoskeleton or interfere with integrin binding may allow targeted disruption of the cells lining Schlemm's canal because of the transmural pressure gradient the cells face as aqueous passes into the canal. METHODS: Anterior segments of human eyes were placed in perfusion organ culture, and either single or sequential doses of H-7 or RGD peptide were added. Fellow eyes received vehicle or RGE peptide. Eyes were fixed and examined by light and electron microscopy. RESULTS: Both agents caused a partial loss of the endothelial lining of Schlemm's canal cells without disruption of trabecular cells in other regions. H-7 significantly increased outflow facility after single or sequential doses, with moderate cell loss of both the inner and outer wall canal cells: 20.0% +/- 10.5% of the width of the canal versus 5.2% +/- 3.7% in control meshworks (P = 0.05). No significant correlation between the amount of canal cell loss and outflow facility was found. RGD was associated with a variable loss of canal cells but did not change outflow facility. CONCLUSIONS: Pharmacologic disruption of Schlemm's canal cells appears possible. H-7 increased outflow facility, causing a partial loss of the endothelial lining of Schlemm's canal. A simple relationship between canal cells and outflow facility was not found; canal cells probably interact with the extracellular matrix in influencing outflow facility.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed2004

Factors influencing intraocular pressure in cultured human anterior segments.

PURPOSE: To determine why variations in intraocular pressure (IOP) occur in cultured human anterior segments despite a constant rate of infusion of culture medium. Two types of variations occur: an initial elevation of IOP and small changes in baseline IOP. METHODS: Anterior segments from human eyes were placed in perfusion organ culture. In cultures with initially high IOP, eyes were fixed at the high IOP level and histologic examination performed. In other cultures with high initial IOP, effluent medium was collected and subsequently reinfused after IOP had decreased to baseline. In cultures with stable baseline IOP, cell fragments from monolayer-cultured cells, or human genomic DNA, were infused at concentrations equivalent to 30,000 to 300,000 cells. RESULTS: Electron microscopy of initially high-pressure cultures revealed scattered cell debris throughout the meshwork in greater amounts than found in eyes without initially high IOP. Reinfusion of effluent media from cultures with high initial pressures caused elevation of IOP. Centrifugation of effluent media lessened this elevation of IOP. In cultures with stable baseline IOP, infusion of cell fragments or genomic DNA raised IOP in a dose-dependent manner, with elevation of IOP for a minimum of 24 hours. CONCLUSIONS: Cell debris can elevate IOP during the initial culture period, and after baseline pressures are established. Cell fragments and DNA increase IOP in a dose-dependent manner. The variations in baseline IOP seen during culture are probably caused by cell fragments and debris from dying cells in the meshwork, ciliary body, and other anterior segment tissues.

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PubMed2004

Long-term, targeted genetic modification of the aqueous humor outflow tract coupled with noninvasive imaging of gene expression in vivo.

PURPOSE: To address a problem impeding research into glaucoma-associated genetic mutations and glaucoma gene therapy and achieve permanent, targeted transgene expression in the trabecular meshwork (TM). Lentiviral vectors are known to transduce human donor eye TM ex vivo, but efficacy in vivo has not been shown. More generally in the field of gene therapy, the authors hypothesized that distinctive properties of the intraocular aqueous circulation could facilitate solving problems of accessibility, targeting, and scale that have hindered realization of gene therapy in other settings. METHODS: A domestic cat model was developed in which long-term in vivo studies were performed. After dose-response studies in primary human TM cells, 19 cats received anterior chamber (AC) injections of stepped doses (10(6)-10(8) transduction units) of lentiviral vectors encoding different marker transgenes (beta-galactosidase, Aequorea victoria green fluorescent protein [GFP], or Renilla reniformis GFP). Animals were monitored serially for transgene expression and IOP. RESULTS: High-grade, stable transgene expression in the TM was achieved and monitored noninvasively over time in living animals. Extensive expression resulted after a single transcorneal injection, persisted for at least 10 months (time of death in the present studies), and was targeted to the TM. The initial IOP did not differ significantly from the IOP at the end of the study (P = 0.4). Aequorea GFP was superior to Renilla GFP. Vectors were effective enough to cause GFP-specific overexpression cytotoxicity at the highest dose, which was solved by dose reduction. CONCLUSIONS: High-grade transgene expression in this large-animal model persisted stably for at least 10 months after a single transcorneal lentiviral vector injection, was highly targeted, and could be monitored serially and noninvasively in living animals. These studies provide a basis for developing realistic disease models and administering glaucoma gene therapy.

Animals↗