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Biomedical subjects

Timothy W Olsen

Publications and source records attributed to Timothy W Olsen.

15 recordsLinked to original sources
PubMed2006

Cannulation of the suprachoroidal space: a novel drug delivery methodology to the posterior segment.

PURPOSE: To describe, test, and evaluate the pharmacokinetics of a novel posterior drug delivery system (PDS) by means of microcannulation of the suprachoroidal space in both the primate and pig animal model. DESIGN: Animal study. METHODS: A rhesus macaque (Macaca mulatta) (n = 1) and pig model (n = 93) were used to evaluate the PDS, a microcannula that combines a drug delivery channel with a fiber-optic illumination and optimal transition properties. The surgical technique, safety profile, histopathology, retinal and choroidal blood flow, injection of tracer dyes, and triamcinolone pharmacokinetics were studied. Pre- and postsurgical high-speed video confocal scanning laser ophthalmoscopy (cSLO) that used fluorescein and indocyanine green (ICG) imaging and wide-field fundus imaging studies were performed. Globes were enucleated for either histopathology or pharmacokinetics. RESULTS: Cannulation was performed in 93 of 94 animals. Complications included: endophthalmitis (1/94), choroidal tear (1/94), choroidal blood flow irregularities (4/94), postoperative inflammation (6/94), scleral ectasia (4/94), wound abscess (1/94), and others. Histopathology demonstrated normal anatomy in uncomplicated cases. Triamcinolone remains in the local ocular tissue for at least 120 days, and measurable at very low levels in the systemic circulation. CONCLUSIONS: Accessing the suprachoroidal space by the microcannulation system can be performed in a safe and reproducible manner by using careful surgical technique. Forceful PDS tip impact into connective tissues in the macular and optic nerve regions should be avoided. Triamcinolone pharmacokinetics are unique and suggest long-term local tissue levels with low systemic levels. PDS access to the suprachoroidal space represents a novel drug delivery method, applicable to a wide variety of pharmacotherapies to the macula, optic nerve, and posterior pole.

Animals↗
PubMed2006

Retinal proteins modified by 4-hydroxynonenal: identification of molecular targets.

The reactive aldehyde, 4-hydroxynonenal (HNE), is a product of lipid peroxidation that can covalently modify and inactivate proteins. Previously, we reported increased HNE modification of select retinal proteins resolved by one-dimensional gel electrophoresis in aged Fisher 344 x Brown Norway rats (Louie, J.L., Kapphahn, R.J., Ferrington, D.A., 2002. Proteasome function and protein oxidation in the aged retina. Exp. Eye Res. 75, 271-284). In the current study, quantitative assessment of HNE molar content using slot blot immunoassays showed HNE content is increased 30% in aged rat retina. In contrast, there was no age-related difference in HNE content in individual spots resolved by 2D gel electrophoresis suggesting the increased modification is likely on membrane proteins that are missing on 2D gels. The HNE-immunoreactive proteins resolved by 2D gel electrophoresis were identified by MALDI-TOF mass spectrometry. These proteins are involved in metabolism, chaperone function, and fatty acid transport. Proteins that were frequently modified and had the highest molar content of HNE included triosephosphate isomerase, alpha enolase, heat shock cognate 70 and betaB2 crystallin. Immunochemical detection of HNE adducts on retinal sections showed greater immune reaction in ganglion cells, photoreceptor inner segment, and the inner plexiform layer. Identification of HNE modified proteins in two alternative model systems, human retinal pigment epithelial cells in culture (ARPE19) and human donor eyes, indicated that triosephosphate isomerase and alpha enolase are generally modified. These results identify a common subset of proteins that contain HNE adducts and suggest that select retinal proteins are molecular targets for HNE modification.

Aging↗
PubMed2006

Proteomics of the retinal pigment epithelium reveals altered protein expression at progressive stages of age-related macular degeneration.

PURPOSE: Age-related macular degeneration (AMD) is characterized clinically by changes in the retinal pigment epithelium (RPE), formation of drusen between the RPE and the underlying vasculature, geographic atrophy, and choroidal neovascularization. Later clinical stages are accompanied by impaired central vision. A limited understanding of the molecular events responsible for AMD has constrained the development of effective treatments. A proteomics approach was used to investigate the underlying mechanisms of AMD and to identify proteins exhibiting significant changes in expression with disease onset and progression. METHODS: Human donor eyes were categorized into one of four progressive stages of AMD. Proteins from the RPE were resolved and quantified by two-dimensional (2-D) gel electrophoresis. Proteins exhibiting significant expression changes at different disease stages were identified by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry. 2-D and semiquantitative one dimensional (1-D) Western blot analyses were used to determine whether changes identified by the proteomic analysis were specific for a protein subpopulation or representative of the entire protein population. RESULTS: Proteins were identified from several critical pathways that changed at early and late disease stages, indicating potential causal mechanisms and secondary consequences of AMD, respectively. Proteins involved in protecting from stress-induced protein unfolding and aggregation, mitochondrial trafficking and refolding, and regulating apoptosis changed early in the disease process. Late-stage changes occurred in proteins that regulate retinoic acid and regeneration of the rhodopsin chromophore. CONCLUSIONS: These results provide the first direct evidence of AMD stage-specific changes in human RPE protein expression and provide a basis for functional investigation of AMD that may ultimately suggest new therapeutic strategies.

Adult↗
PubMed2006

The proteome of central and peripheral retina with progression of age-related macular degeneration.

PURPOSE: A growing understanding of the molecular events in age-related macular degeneration (AMD) has lead to targeted therapies for a select group of patients with advanced AMD. Development of therapies for the earlier stages requires further elucidation of disease mechanisms. In this study, a proteomics approach was used to identify proteins that had altered content in human donor eyes with progression of AMD. METHODS: The early molecular events associated with AMD were identified by comparing the proteome of the macular and peripheral neurosensory retina during four progressive stages of AMD. Proteins were resolved and quantified by two-dimensional gel electrophoresis. Twenty-six proteins exhibited changes in content and were identified by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry. Two-dimensional (2-D) and semiquantitative one-dimensional (1-D) Western blot analyses were used to determine whether changes identified by proteomic analysis were specific for a protein subpopulation or representative of the entire protein population. RESULTS: Twenty-six proteins were identified that exhibited changes at disease onset or with progression (indicating potential causal mechanisms) and at end-stage disease (indicating potential secondary consequences). These proteins are involved in key functional pathways, such as microtubule regulation and protection from stress-induced protein unfolding. Approximately 60% of the proteins exhibited changes specific to either the macula or periphery, with the remaining 40% changing in both regions. These results imply that both the macula and periphery are affected by AMD. CONCLUSIONS: This study provides the first direct evidence of AMD stage- and region-specific changes in retinal protein levels and highlights potential novel, disease-related proteins and biochemical pathways for future studies of AMD.

Adult↗
PubMed2005

Declines in arrestin and rhodopsin in the macula with progression of age-related macular degeneration.

PURPOSE: Biochemical analysis of age-related macular degeneration (AMD) at distinct stages of the disease will help further understanding of the molecular events associated with disease progression. This study was conducted to determine the ability of a new grading system for eye bank eyes, the Minnesota Grading System (MGS), to discern distinct stages of AMD so that retinal region-specific changes in rod photoreceptor protein expression from donors could be determined. METHODS: Donor eyes were assigned to a specific level of AMD by using the MGS. Expression of the rod photoreceptor proteins rhodopsin and arrestin was evaluated by Western immunoblot analysis in the macular and peripheral regions of the neurosensory retina from donors at different stages of AMD. RESULTS: A significant linear decline in both arrestin and rhodopsin content correlated with progressive MGS levels in the macula. In contrast, the peripheral region showed no significant correlation between MGS level and the content of either protein. CONCLUSIONS: The statistically significant relationship between decreasing macular rod photoreceptor proteins and progressive MGS levels of AMD demonstrates the utility of the clinically based MGS to correspond with specific protein changes found at known, progressive stages of degeneration. Future biochemical analysis of clinically characterized donor eyes will further understanding of the pathobiochemistry of AMD.

Adult↗
PubMed2005

Extraocular muscle insertions relative to the fovea and optic nerve: humans and rhesus macaque.

PURPOSE: To identify extraocular anatomic relationships of muscle insertions relative to the fovea and the optic nerve. METHODS: Thirty-eight human eye bank eyes and 10 rhesus macaque (Macaca mulatta) eyes were measured. Ten human volunteers were used to determine the horizontal rectus muscle-to-globe apposition in primary, left, and right gaze. RESULTS: External globe measurements (human/rhesus; mm +/- SD) from the temporal border of the optic nerve (ON) to the center of the fovea (F) were 3.7 +/- 0.6 and 2.6 +/- 0.2; F to the posterior border of the inferior oblique (IO) insertion, 2.5 +/- 0.8 and 0.5 +/- 0.4; ON to the posterior border of the IO, 5.6 +/- 0.9 and 2.8 +/- 0.3; horizontal axial plane (H) of the eye, defined by the long posterior ciliary artery, to the IO, 2.0 +/- 0.8 and 0.5 +/- 0.4; and H to F, 1.0 +/- 0.6 and 0.4 +/- 0.3, respectively. The IO insertion formed an arc, inferior to H, with an anterior-to-posterior cord insertion width of 9.2 +/- 0.7 and 7.7 +/- 0.3. The IO angle of insertion (theta) was 30 degrees in 84% (32/38) and 0 degrees in 16% (6/38) of human eyes and 25 to 30 degrees in all rhesus. In 20 human volunteers, from the ON to the apex of lateral rectus globe apposition was 13.9 +/- 1.1 in primary, 17.2 +/- 1.9 in lateral, and 9.3 +/- 1.7 in medial gaze. CONCLUSIONS: The fovea is located mostly superior and slightly posterior to the posterior border of the IO insertion. Topographic relationships of the extraocular muscles relative to the fovea are essential for the design of extraocular drug delivery systems.

Aged↗
PubMed2004

Identification and localization of major soluble vitreous proteins in human ocular tissue.

PURPOSE: To identify the major soluble proteins from human vitreous, and to establish a baseline for comparison of vitreous samples from eyes with various diseases. DESIGN: Laboratory investigation. METHODS: Normal vitreous was obtained from eight human donor eyes and from eight eyes of patients undergoing diabetic vitrectomy. Vitreous specimens were subjected to SDS-PAGE and MALDI-TOF-MS analysis. Six specific antibodies were used to identify proteins using Western blot. Four proteins were localized within ocular tissue in a normal donor eyebank eye. RESULTS: We found eight distinct bands on SDS-PAGE in normal vitreous and two additional bands (hemoglobin) in eyes with diabetic retinopathy. Proteins were identified using MALDI-TOF-MS and confirmed by Western blot. We established a quantitative analysis of relative protein concentrations of undiluted vitreous. Immunohistochemistry localized selected proteins within the posterior segment layers. CONCLUSIONS: We present a normal human vitreous protein profile using current technologies and provide a baseline for comparison to ocular disease states. Tissue distribution of vitreous proteins may help to elucidate more specific protein function.

Amino Acid Sequence↗
PubMed2004

Fluorescein angiographic lesion type frequency in neovascular age-related macular degeneration.

OBJECTIVE: To assess the frequency of lesion types using fluorescein angiography (FA) in neovascular age-related macular degeneration (nAMD). DESIGN: Cross-sectional study. PARTICIPANTS: Two hundred cases of nAMD. METHODS: Fluorescein angiograms from 908 patients (university-based, tertiary retinal referral practice [UP] = 478; comprehensive, community-based eye clinic [CC] = 430) were reviewed to identify 200 cases of nAMD (100 from each center). Two graders evaluated the frequency of angiographic subtypes. MAIN OUTCOME MEASURES: Identifying (1) the frequency of subfoveal nAMD lesions that meet the definition of "predominantly classic," "minimally classic," "occult with no classic"; (2) lesion location, size, and subtype; and (3) the intergrader agreement (kappa). RESULTS: There was little difference in the frequency of lesion type between the UP and the CC. Most nAMD lesions were subfoveal (78.5%, 157 of 200), and of these, 20% (32 of 157) were predominantly classic; whereas 73% (114 of 157) were occult with no classic, and 7% (11 of 157) were minimally classic. Of the 200 angiograms, 33 (16.5%) were juxtafoveal, and 10 (5%) were extrafoveal. Twenty of the 43 juxtafoveal and extrafoveal lesions (47%) were predominantly classic. Classic with no occult subfoveal lesions were smaller than minimally classic or occult with no classic (1.7 vs. 3.7 and 2.8 mm; P = 0.001 and 0.01, respectively). Of 114 subfoveal occult with no classic lesions, 54 (47%) had both smaller lesion size <==4 disc areas (DA) and lower visual acuity <20/50, whereas 107 (94%) had a smaller lesion or lower visual acuity. CONCLUSIONS: Most angiographic lesions of patients who undergo FA for nAMD are subfoveal and occult. We estimate that 20% of subfoveal lesions are predominantly classic. Approximately half of the juxtafoveal and extrafoveal lesions are predominantly classic. Nearly 30% of all nAMD lesions have both small occult lesions (size <==4 DA) and a visual acuity less than 20/50. We found minimal difference in lesion type between a UP and a CC.

Aged↗
PubMed2004

The Minnesota Grading System of eye bank eyes for age-related macular degeneration.

PURPOSE: The Minnesota Grading System (MGS) is a method to evaluate human eye bank eyes and determine the level of age-related macular degeneration (AMD), by using criteria and definitions from the Age-Related Eye Disease Study (AREDS). METHODS: Donor eyes (108 pairs) from the Minnesota Lions Eye Bank were cut circumferentially at the pars plana to remove the anterior segment. A 1000 +/- 2.5-microm ruby sphere was placed on the optic nerve as a size reference. A digital, high-resolution, color macular photograph was taken through a dissecting microscope. The neurosensory retina was removed from one globe of the pair. The underlying retinal pigment epithelium was rephotographed, localizing the fovea with a proportional triangle. A grid was superimposed in the macular photographs and images were graded according to AREDS criteria. Twenty pairs were dissected bilaterally and graded for symmetry. RESULTS: Eighty-eight globes were graded into one of four MGS categories. Nineteen (95%) of 20 globes had symmetric grades. CONCLUSIONS: The MGS provides a methodology to grade donor tissue from eye bank eyes to correspond to the AREDS classification system. Donor tissue may be used for subsequent molecular analysis, including genomics and proteomics.

Aged↗
PubMed2003

Stability of RNA from the retina and retinal pigment epithelium in a porcine model simulating human eye bank conditions.

PURPOSE: To assess RNA stability after death in a porcine model to simulate current human eye bank techniques. METHODS: Eye bank time interval data were collected from 191 donor specimens: death to refrigeration, enucleation, and tissue processing. A control porcine eye was enucleated, retina and RPE isolated, and specimens frozen (-80 degrees C). Fourteen porcine eyes remained at room temperature for 2 hours and then cooled to 4 degrees C. Retina and RPE were isolated and frozen (-80 degrees C) at 5, 12, 24, 29, 36, 48, and 72 hours. Four globes remained in a moist chamber, five whole and five sectioned globes were immersed in RNAlater (Ambion, Austin, TX) at 5, 12, 24, or 48 hours. RNA was isolated. The 28S and 18S rRNA peaks were analyzed by electrophoresis. RT-PCR was performed on each sample. Messenger RNA for GAPDH, beta-actin, mouse rhodopsin from retina (mRHO), and RPE-65 (from RPE) were analyzed with gel electrophoresis. RESULTS: The average time from death to refrigeration was 4.2 hours, to enucleation 6.4 hours, and to tissue processing 10.7 hours. RT-PCR gel electrophoresis patterns from retinal tissue had bands of similar intensity at each interval from beta-actin, GAPDH, and RHO. Band patterns from RPE demonstrated decay of the RT-PCR gene products after 5 hours. This decay was delayed by at least 24 hours with the use of RNAlater. The 28S rRNA decay was similar for retina and RPE. CONCLUSIONS: Retinal tissue RNA can be analyzed within the time constraints of current eye bank tissue processing, whereas analysis of RPE necessitates either rapid processing or use of RNAlater. These results should aid in future studies in which eye bank tissue is used for RNA analysis.

Actins↗
PubMed2002

Porcine sclera: thickness and surface area.

PURPOSE: To assess the thickness and surface area of porcine sclera. METHODS: One hundred twenty-eight porcine globes were sectioned from the center of the cornea to the region of the optic nerve. Photographs of the sectioned sclera including a millimeter scale were taken. Photographic slides were projected onto blank paper, and the scleral silhouette was traced. Perpendicular thickness measurements were taken at 1-mm intervals from the limbus to the optic nerve. The sclera of 18 porcine eyes were cut into small pieces, and the surface area was calculated with computerized digital tracing software. RESULTS: The scleral thickness near the corneal scleral limbus was 0.83 +/- 0.2, 0.91 +/- 0.17, and 1.12 +/- 0.23 mm in the small-, medium-, and large-sized pigs, respectively. Thickness decreased to minimum of 0.31 +/- 0.07, 0.35 +/- 0.1, and 0.43 +/- 0.16 mm at a distance of 5 mm from the limbus in the small- and medium-sized pigs and 6 mm in the large-sized pigs, respectively. The mean scleral surface area was 7.78 +/- 0.66, 9.66 +/- 0.75, and 11.92 +/- 1.57 cm(2) in the small-, medium-, and large-sized pigs, whereas the corneal surface area was 1.09 +/- 0.07, 1.15 +/- 0.09, and 1.40 +/- 0.19 cm(2), respectively. CONCLUSIONS: Porcine scleral thickness is very similar to human scleral thickness. The porcine model is an excellent model for studying transscleral drug delivery.

Animals↗
PubMedDate not supplied

Vitrectomy for dense vitreous hemorrhage in infancy.

PURPOSE: To report clinical data, including etiology and visual outcome, in newborns requiring vitrectomy for dense vitreous hemorrhage. METHODS: In this retrospective case series, we surveyed subscribers to the American Association for Pediatric Ophthalmology and Strabismus ListServe regarding patients under their care. RESULTS: A total of 28 eyes of 21 patients were included. Most common etiologies were thrombocytopenia, shaken baby syndrome, and birth trauma. In 9 cases (12 eyes), the vitreous hemorrhage was idiopathic. Mean time between diagnosis and surgery was 1.4 months. Complications included strabismus, cataract, glaucoma, high myopia, and retinal detachment. Recognition visual acuities were available for 8 eyes: 20/25 (2 eyes), 20/30, 20/40 (2 eyes), 20/60 (2 eyes), and 20/100. One eye had no light perception. CONCLUSIONS: The etiologies encountered in our patients were similar to those reported previously. Visual outcomes were much worse in cases with retinal complications. Other patients had better visual outcomes. Despite potential surgical and postoperative complications, this series demonstrates favorable visual outcomes can be achieved following early vitrectomy in this setting.

Birth Injuries↗