2nd Annual international Conference: Vision Restoration: Regenerative Medicine in Ophthalmology
May 11, 2012, University Club, Pittsburgh, pennsylvania
Posters displayed at the conference
In-Vivo Visualization of the Corneal Epithelial Stem Cell Niche by Optical Coherence Tomography
Kira L. Lathrop, Larry Kagemann, Divya Gupta, Nirmala SundarRaj, Joel S Schuman
Purpose: To demonstrate in-vivo visualization of the Palisades of Vogt, which are the corneal epithelial stem cell niche, by Optical Coherence Tomography
Methods: In this pilot study a Cirrus HD OCT system (Carl Zeiss Meditec) and a modified Bioptigen spectral-domain optical coherence tomography system (Bioptigen Inc, USA; SuperLum LTD, Ireland) were used to acquire images of the corneal limbus. The Bioptigen system had 3.0- to 3.5-μm axial image resolution and an imaging speed of 25,000 axial scans per second, and the Cirrus OCT had 5-μm resolution and a speed of 27,000 axial scans per second. 3D analysis of the POV was conducted using two different techniques: contour modeling C-mode using a custom software of our own design and 3D segmentation using a custom FIJI plugin.
Results: Reconstruction of the corneal limbal region via 3D OCT image sets revealed the configuration of the Palisades of Vogt in-vivo.
Conclusions: OCT is the only non-contact imaging method currently capable of visualizing the Palisades of Vogt, which are the corneal epithelial stem cell niche. The field acquired by OCT is large enough to describe a broad region of the palisades, and may, for the first time allow physicians to identify and track changes in the configuration of the palisades over time and during different treatments and may aid in surgical guidance. Further exploration and refinement of this technique is warranted.
Bioengineering gelatin hydrogel for corneal endothelial cell regeneration
Guoguang Niu1, Jin San Choi1, Min Jeong Kim1, Matthew Giegengack2, Shay Soker1
1 Institute for Regenerative Medicine, Wake Forest University, 2Department of Ophthalmology, Wake Forest School of Medicine
Purpose/Hypothesis: Bioengineering of corneas is a promising method to address the shortage of cornea donation for vision restoration. Corneal endothelium can be cultured in vitro, and together with appropriate scaffolding material, transplanted inside the patient's eyes to repair the dysfunctional endothelium. Researchers have reported on the fabrication of collagen sheets as corneal implants, using thermal gelation, vitrification and chemical cross-linking. One of drawbacks of collagen sheet is that the scaffolds have a low transparency. Gelatin is a hydrolysed product of collagen, and has been extensively used in medical applications. Compared with collagen, gelatin is cheaper and can be more easily dissolved in aqueous solutions. The purpose of this study was to develop a bioengineering gelatin hydrogel for corneal endothelial cell repair.
Methods: Gelatin hydrogel was obtained by cross-linking with 1-Ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC) / N-hydroxysuccinimide (NHS). The transparency, mechanical property and the microstructure of gelatin hydrogel were characterized respectively by UV-spectrophotometer, Instron, and SEM. Human corneal endothelial cells (hCECs) isolated from discarded donor corneas were seeded on the gelatin hydrogel and cell attachment and proliferation on the gelatin hydrogel were examined by SEM, haematoxylin and eosin (H&E) and Alizarin red staining. ZO-1 and K+-Na+-ATPase markers of hCEC were checked also. New Zealand rabbit was used for the implantation of gelatin hydrogel covered with/without hCEC.
Results: A flexible hydrogel was obtained after cross-linking gelatin film with EDC/NHS at room temperature. The hydrogel has proper mechanical properties, with a modulus of 3.2 MPa, tensile strain of 75% and the tensile stress at break around 1.4Mpa. Gelatin hydrogel showed a high transparency, 95% within the visible light scope, which was higher than human cornea. Gelatin hydrogel had a porous structure with a pore size of 20-30 um. The water content of Gelatin hydrogel was about 70-80%, which was very close to that of natural cornea stroma. hCECs were seeded directly on gelatin hydrogel, and tight confluent cell layer was formed on hydrogel surface after several days culture. The growth rate of hCECs on gelatin hydrogel was similar to that of cells cultured in tissue culture plates. ZO-1 and K+/Na+-ATPase were expressed by hCEC seeded on gelatin hydrogel. Round shaped gelatin hydrogels with a 7 mm diameter were easily implanted inside rabbit eyes with only 2-3mm incision, and no obvious inflammation and rejection responses were noted.
Conclusions: Gelatin hydrogels have the appropriate transparency, mechanical property and biocompatibility to serve as scaffolds for cornea bioengineering. The Gelatin hydrogels, seeded with hCECs can be used to create corneas for transplantation and overcome the shortage of donated corneas.
Different Parameters Contributing to the Iris Contour and Anterior Chamber Angle during Pupil Dilation
Rouzbeh Amini1, Sara Jouzdani2, Victor H. Barocas3.
1Deptartment of Bioengineering, University of Pittsburgh, Pittsburgh, PA 2Dept of Mechanical Engineering, University of Minnesota, Minneapolis, MN
3Dept of Biomedical Engineering, University of Minnesota, Minneapolis, MN
The purpose of this study was to examine the distinctive effects of dynamic pupillary block, iris volume change, and anatomical shape of the dilator on the narrowing of the anterior chamber angle during dilation. A mathematical model of the anterior segment was developed to simulate contraction of the dilator muscle. The aqueous humor and iris were modeled as an incompressible Newtonian fluid and a neo-Hookean solid, respectively. To change the pupil diameter from 3 mm to 6 mm during 12 seconds, active dilator contraction was applied by imposing stress in the dilator region. Dilation was simulated with changing three parameters: (1) a compressible vs. an incompressible iris to identify the effects of iris volume change, (2) a thin dilator (4 μm, 1% of full thickness) vs. a thick dilator covering the full thickness iris to identify the effects of dilator anatomy, and (3) in the presence vs. absence of pupillary block to identify the effect of dynamic motion of aqueous humor from the posterior to anterior chamber. The changes in the apparent iris-lens contact and AOD500 were calculated for each case.
When the entire thickness of the iris was modeled as the contracting dilator muscle and in the presence of pupillary block, the iris curvature initially increased from -0.03 mm to +0.01 mm when iris lens gap was at its minimum and eventually decreased to -0.06 mm as the iris fully dilated for incompressible model. AOD500 was decreased by 5% and 6% as pupil diameter reached 4 mm for the thick dilator case in the presence and in the absence of pupillary block, respectively. The predicted changes were more pronounced when the dilator was modeled as a thin layer on the posterior surface of the iris. For example, AOD500 decreased by 6% and 12% (pupil diameter reaching 4 mm for both case) in the presence and in the absence of pupillary block, respectively. Dilation simulation with a compressible iris with a thin dilator in the presence of pupillary block predicted that as the pupil diameter increased from 3 mm to 4 mm, the iris-lens contact decreased from 0.4 mm to 0.29 mm.
Our simulations predicted the most dramatic change in AOD500 when the dilator muscle was modeled as a thin layer in the posterior surface of the iris, the iris was molded as an incompressible material, and the pupillary block was prevented. In addition, the iris incompressibility (i.e. lack or iris volume change) led to a narrower anterior chamber angle following dilation simulation, a result consistent with clinical observation (Quigley et al., J Glaucoma, 18:173-9, 2009). The change in AOD500 was more pronounced when the iris was incompressible, while the decrease in iris lens contact was more pronounced in the presence of pupillary block.
An injectable bevacizumab-laden thermal gel for treatment of age-related macular degeneration
Britta M. Rauck1a, Carlos Medina1b, Veeral Shah1bThomas R. Friberg1b, Yadong Wang1a
aDepartment of Bioengineering, bOphthalmology, 1University of Pittsburgh, Pittsburgh, PA
Abstract:
The wet form of age-related macular degeneration (AMD) is characterized by excessive blood vessel growth beneath the retina resulting in bleeding, vision loss and scarring. Current treatment requires bi-monthly intravitreal injections of anti-angiogenic drugs such as bevacizumab (Avastin) to prevent further vision loss. Controlled release of such therapeutics intraocularly is highly desirable, as it reduces injection frequency, minimizes deleterious side effects and maintains drug concentration in the therapeutic window for a longer period of time. We designed a biodegradable reverse thermal gel (ESHU) that transitions from liquid to gel at body temperature. Upon intravitreal injection ESHU quickly gels and sinks out of the visual field. At the same dosage, ESHU is capable of sustaining Avastin levels for at least 6 weeks in vivo compared to less than 4 weeks when Avastin is injected alone. This delivery system remains in the eye for at least 10 weeks and elicits a minimal inflammatory response. Taken together, these results suggest that ESHU provides a promising platform for intraocular drug delivery.
Covariations between microstructural characteristics
of the human lamina cribrosa
NJ Jan1, J.L. Grimm2, DJ Brown3, K Reid3, I.A. Sigal1,2
1 Department of Bioengineering, Swanson School of Engineering,
University of Pittsburgh, Pittsburgh, PA, USA.
2 University of Pittsburgh Medical Center, Eye and Ear Institute, Department of Ophthalmology,
University of Pittsburgh, Pittsburgh, PA, USA.
3 Gavin Herbert Eye Institute, University of California, Irvine, CA, USA.
Purpose: From second harmonic generated (SHG) microscopy images we measured lamina cribrosa (LC) microstructural parameters and analyzed their relationships.
Methods: SHG images (Zeiss 510 Meta LSM, lateral resolution 1.76 to 4.97 µm/pixel) were taken of the LC of 6 normal human eyes (54 to 82 yo) at normal intraocular pressures (10 or 15 mmHg). Maximum intensity projections the LCs were analyzed to determine beam thickness (BT) and pore size (PS) diameters, connective tissue density (TD) and the number of beam insertions into the canal wall (IC) normalized by canal wall arc length. Parameter covariations were tested by ANOVA, for all eyes (pooled) as well as per eye (individual).
Results: Pooled parameter averages ± SD were: PS 54.2±21.6 µm, BT 47.7±21.3 µm, IC 27.9±25.5 insertions/mm, and TD 51.4±5.5%. Pooled analyses revealed significant positive parameter associations while individual analyses were more variable. For example, in the pooled analysis PS and BT had a positive correlation, whereas individual associations were positive in 1 eye and negative in 2 eyes. Similar covariations were identified between PS and TD, BT and TD, BT and IC and IC and TD.
Conclusions: In the pooled analysis larger PS was associated with increased BT and higher IC, but no significant association was found with TD. One possible reason is that thicker beams and more beam insertions into the canal wall provide the structural and nutritional support needed by larger pores. The results suggest that generalizing or particularizing conclusions based on microstructural characteristics of the LC should be done cautiously.
Eye-specific high-resolution measurements of the effects of intraocular pressure on the beams and pores of the human lamina cribrosa
J.L. Grimm1, NJ Jan2, DJ Brown3, K Reid3, I.A. Sigal1,2
1 University of Pittsburgh Medical Center, Eye and Ear Institute, Department of Ophthalmology,
University of Pittsburgh, Pittsburgh, PA, USA.
2 Department of Bioengineering, Swanson School of Engineering,
University of Pittsburgh, Pittsburgh, PA, USA.
3 Gavin Herbert Eye Institute, University of California, Irvine, CA, USA.
Purpose: To measure the mechanical effects of intraocular pressure (IOP) within the human lamina cribrosa (LC).
Methods: The LCs of five human eyes from three donors aged 54 to 82 were scanned from the posterior side using second harmonic generated (SHG) imaging (Zeiss 510 Meta LSM). Images up to a depth of 300 µm were acquired at IOPs ranging from 10 to 50 mmHg. Resolution varied from1.76 to 4.97 µm/pixel. Digital image correlation techniques were used to calculate the deformation mapping between maximum intensity projection images at low and at elevated IOP.
Results: IOP-increases produced substantial displacements and biologically relevant deformations within all the eyes. There were significant variations between eyes (P’s<0.0001, nested ANOVA) and between regions of a given eye (P’s<0.0001). Larger displacements, stretch, compression and shear often did not colocalize. Median shear strain was strongly and significantly correlated with both median beam thickness (adjusted-R2= 0.96, P<0.003) and median pore size (0.95, P<0.003) separately, and simultaneously (0.999, P<0.007).
Conclusions: We measured eye-specific IOP-induced displacements and deformations of the pores and beams of the LC in uncut and unfixed human eyes with unprecedented detail. Median pore size and beam thickness were excellent predictors of median LC shear and modest predictors of median stretch and compression, suggesting that LCs with thick beams and/or large pores may be more sensitive to elevated IOP.
TelerehA Tele-Rehabiltation Pilot Study for Artificial Vision Devices
Author Block: Amy C. Nau, Jacqueline Fisher. Ophthalmology, UPMC Eye Center, Pittsburgh, PA.
Abstract:
Purpose: The BrainPort™ (Wicab, Madison WI) pairs a camera to an electrode display which rests on the tongue. The device improves orientation and mobility for the blind by providing information about the proximal environment. Like all blind skills, practice is required to attain proficiency, but a lack of trained therapists, geographic and financial barriers are issues that must be overcome. Without a rehabilitation infrastructure to support long term training, rates of abandonment are likely to be high for artificial vision devices. Creative methods to overcome this deficiency must be urgently explored. The Rehabilitation Engineering Research Center at the University of Pittsburgh has developed a secure telerehabilitation portal termed Visyter. The purpose of this pilot study was to: 1) develop a year long rehabilitation protocol for advanced BrainPort training using existing Visyter infrastructure 2) determine the barriers to tele-rehabilitation in a blind cohort and 3) deploy a smartphone application allowing a sighted person to assist with training at home.
Methods: 3 adult, male blind subjects were enrolled in this pilot study. Baseline psychophysical and mobility tests used in previous BrainPort studies were conducted on day 1 and again after approximately 15 hours of training at the UPMC Eye Center. Upon completion, subjects were sent home with a BrainPort, a Droid smartphone (Verizon) allowing a sighted person to directly view images displayed on the tongue, a camera and a speaker. The clinical interactions between the subject and therapist consisted of increasingly advanced skills and homework tasks covered during 30 minute sessions for 6 months. Subjects used their own computers with a high speed internet connection. Our primary outcome measure included development of a prototype tele-rehab conference capability.
Results: We successfully deployed the Visyter software program to all subjects. Barriers included the need for a sighted person to assist with hardware and software set up, and ideally be present for training sessions. Computers with high speed internet access are needed. All subjects and therapists felt the portal was useful and the smartphone application to be very helpful. All subjects and therapists felt the interaction was as productive as face to face interaction.
Conclusions: This study showed that telerehabilitation represents a feasible strategy to overcome the barriers to successful long term rehabilitation with artificial vision devices. Future studies will explore use of smartphone cameras to allow for remote mobility training in outdoor settings as well as to investigate use of tele-rehabilitation portals for low vision populations.
Strategies for Enhanced Peripheral Nerve Repair
Bliley JM,1 Lin YC,1 Minteer DM,2 Oh S,1 Kaplan DL,3
Van Dyke M,4 Smith T,4 Pixley S,5 Rubin JP,1,2 Marra KG1,2
1Division of Plastic Surgery, University of Pittsburgh, Pittsburgh, PA
2Department Bioengineering, University of Pittsburgh, Pittsburgh, PA
3Department Biomedical Engineering, Tufts University, Medford, MA
4Wake Forest Institute for Regenerative Medicine, Winston-Salem, NC
5Department of Cancer and Cell Biology, University of Cincinnati, Cincinnati, OH
Background: Nerve injuries can occur due to trauma, tumor removal, and accidental surgical resection. Standard care for a peripheral nerve injury involves excising a portion of a non-essential sensory nerve (i.e., autograft), which is usually the sural nerve. This donor nerve is then transplanted into the defect. Although these autografts are considered the clinical standard of peripheral nerve repair, they also pose significant disadvantages for patients. Nerve autografts contribute to loss of sensory function and the production of neuromas at the donor site. In addition, surgeons are also limited in the number and diameter of nerves that they can harvest. This can complicate trauma situations in which multiple peripheral nerve injuries are sustained. When an autograft is not possible, nerve guides are often used to reconstruct defects less than 3 cm. To date, there is no commercially available guide that is approved to repair long gap peripheral nerve defects (i.e. gaps greater than 3 cm). Our research has aimed to counteract this deficit in care by investigating nerve conduit therapies that are able to promote repair in long gaps.
Methods: Both synthetic (polycaprolactone) and biologic (silk) nerve conduits were investigated in combination with a number of different treatment modalities in a critical size rat sciatic nerve defect model. Trophic factors, including glial-derived neurotrophic factor (GDNF), were embedded in double-walled microspheres to enhance repair. Additionally, several internal scaffolds were investigated for these conduits, including an ERC-funded project involving keratin hydrogel filler and magnesium wires.
Conclusions: Peripheral nerve repair involves multiple biological mechanisms. Our laboratory has investigated a number of these combined techniques in a small animal model with the goal of finding a nerve guide that can promote nerve regeneration for long gaps (>3 cm). We believe that combined that a combined therapy may be the best way to promote long gap peripheral nerve repair. We are now examining the most promising novel nerve guides in a non-human primate median nerve defect model, with the goal to begin clinical studies within the next 3-5 years.
Aqueous Humor Outflow Structures in the Nasal and Temporal Quadrants
Authors: Larry Kagemann1,2, Gadi Wollstein1, Hiroshi Ishikawa1,2, Ian A. Sigal1,2, Zachary Nadler1, Jessica E. Nevins1, Jonathan Grimm1, Richard A. Bilonick1,3, Joel S. Schuman1,2.
Affiliations: 1UPMC Eye Center, Eye and Ear Institute, Ophthalmology and Visual Science Research Center, Department of Ophthalmology, University of Pittsburgh School of Medicine, Pittsburgh, PA; 2Department of Bioengineering, Swanson School of Engineering, University of Pittsburgh, Pittsburgh, PA; 3Department of Biostatistics, Graduate School of Public Health, University of Pittsburgh, Pittsburgh, PA
Purpose: We previously published the first virtual visualization of the functioning outflow system in cadaveric eyes, finding distinct aqueous venous plexuses (AVP) in the superior and inferior (SI) quadrants1 (Figure). The purpose of the present study was to further explore the presence of the AVP’s in the nasal and temporal (NT) regions.
Methods: Volumetric spectral domain optical coherence tomography (Bioptigen, Durham, NC) scans of the limbus were obtained in NT and SI quadrants in 1 randomly selected eye in each of 6 healthy volunteers. Image processing was described previously1 with the addition of localized limited adaptive histogram equalization. Two layers of AVP’s were subjectively identified using 2D visualization. The distance from the surface of the limbus to each AVP was measured in the 2D slices. The diameters of aqueous veins in each AVP were measured. Processing and measurements were made in FIJI (ImageJ 1.45R, NIH, http://imagej.nih.gov/ij). Mean diameters and AVP depths in the NT versus SI quadrants were compared by unpaired t-test.
Results: Enhanced imaging revealed an AVP of small-diameter aqueous veins obscured by noise in the NT quadrants (Figure). The superficial AVP was located statistically significantly more posterior to the limbus in the NT than the SI quadrants (124.24 ± 21.02μm (NT) vs. 105.00 ± 24.86μm (SI), p = 0.039, Figure yellow arrows). There was no difference in the location of the deeper layer between SI and NT AVP (277.10 ± 57.03μm (NT) vs. 317.10 ± 63.67μm (SI), p = 0.10, Figure orange arrows). Aqueous vein diameters were statistically significantly larger in NT quadrants than SI quadrants in the superficial AVP (35.58 ± 8.44μm vs. 27.52 ± 9.03μm, p=0.02), and statistically significantly larger in SI than NT quadrants in the deep AVP (58.5 ± 23.53μm vs. 43.99 ± 8.79μm, p = 0.04).
Conclusions: The NT quadrants of the limbus have deeper layers of smaller aqueous vein diameter compared to the SI quadrants. Their location and size may contribute to challenges in their visualization.
1Kagemann L, Wollstein G, Ishikawa H, et al. 3D visualization of aqueous humor outflow structures in-situ in humans. Exp Eye Res. 2011;93:308-15.
Latent Class Regression (LCR) Analysis for Detecting Glaucoma Progression
Authors: Gadi Wollstein1, Richard A. Bilonick1,2, Hiroshi Ishikawa1,3, Larry Kagemann1,3, Ian A. Sigal1,3, Jay S. Duker4, Joel S. Schuman1,3.
Affiliations: 1UPMC Eye Center, Eye and Ear Institute, Ophthalmology and Visual Science Research Center, Dept. of Ophthalmology, University of Pittsburgh School of Medicine, Pittsburgh, PA; 2Department of Biostatistics, Graduate School of Public Health, University of Pittsburgh, Pittsburgh, PA; 3Department of Bioengineering, Swanson School of Engineering, University of Pittsburgh, Pittsburgh, PA; 4Ophthalmology, New England Eye Center, Boston, MA.
Purpose: Detection of glaucoma progression poses a major clinical challenge. LCR is a statistical method that allows the identification of groups within population by extracting the shared latent information among various parameters. The purpose of this study was to use LCR with longitudinal structural and functional measurements to characterize glaucoma progression grouping.
Methods: 125 eyes from healthy, glaucoma suspects and glaucoma subjects were enrolled all with !5 reliable visual field (VF; Carl Zeiss Meditec (CZM)) tests and good quality optical coherence tomography (OCT; Stratus, CZM) scans all acquired with a maximum interval of 6 months. VF parameters used for the analysis included mean deviation (MD), pattern standard deviation (PSD) and visual field index (VFI). Mean retinal nerve fiber layer (RNFL) thickness was used from OCT. Akaike information criterion analysis determined the optimal number of groups in the LCR analysis model. LCR analysis was employed with the 4 responses handled as a function of subject age.
Results: Five distinctive groups were detected and labeled as Stable low (low rate of change; 43 eyes) and high (38), and Progressing low (20), moderate (11) and high (13) (Figure). The groups showed a gradual steepening rate of change and intercept for both VF and RNFL for stable and progressing eyes and among their subgroups (with the exception of the Progression high group for some parameters). Most VF parameters slopes were significantly different (p<0.05) among the subgroups while RNFL was significantly different in between the Stable groups and between Stable hi and Progression moderate groups only.
Conclusions: LCR analysis allows grouping of eyes sharing latent structural and functional information. This could be of importance in situations such as assessment of glaucoma progression - and potentially the prediction of glaucoma progression - especially since widely accepted criteria for progression are lacking.
Signal Normalization Reduced Systematic Differences in Retinal Nerve Fiber Layer (RNFL) Thickness Measurements Between Spectral Domain Optical Coherence Tomography (SD-OCT) Devices
Authors: Chieh-Li Chen1,2, Hiroshi Ishikawa1,2, Gadi Wollstein1, Richard A. Bilonick1,3, Juan Xu1, James G. Fujimoto4, Joel S. Schuman1,2.
Affiliations: 1UPMC Eye Center, Eye and Ear Institute, Ophthalmology and Visual Science Research Center, Department of Ophthalmology, University of Pittsburgh School of Medicine, Pittsburgh, PA; 2Department of Bioengineering, Swanson School of Engineering, University of Pittsburgh, Pittsburgh, PA; 3Department of Biostatistics, Graduate School of Public Health, University of Pittsburgh, Pittsburgh, PA; 4Department of Electrical Engineering and Computer Science and Research Laboratory of Electronics, Massachusetts Institute of Technology, Cambridge, MA.
Purpose: Differences in RNFL thickness measurements between SD-OCT devices pose significant difficulty in both clinical and research settings where multiple devices are in use. The purpose of this study was to develop and test a novel signal normalization method that reduced systematic differences.
Methods: Ninety-six eyes (32 glaucomatous, 33 glaucoma suspect, and 31 healthy eyes) from 51 subjects were scanned with two SD-OCT devices on the same day (Cirrus HD-OCT; Carl Zeiss Meditec, Inc., Dublin, CA; Optic Disc Cube 200x200 scan pattern, and RTVue; Optovue, Fremont, CA; RNFL 3.45 Circle scan pattern). RTVue data had 768 pixels for 2.3 mm scan length on each A-scan, which was oversampled and padded with minimum signal to match with the Cirrus specification (1024 pixels for 2.0 mm). Then both the original Cirrus signal and RTVue derived Cirrus signal were normalized in amplitude so that meaningful signal range was stretched to the full gray scale range of 0 to 255. To compensate signal strength difference, custom high dynamic range (HDR) processing was also applied to poor signal strength images (lowest 20 percentile of the entire set of images). Conventional global mean circumpapillary RNFL thicknesses were measured automatically using software of our own design and then compared to the original device outputs. Measurement error models were used to analyze the absolute RNFL thickness difference between original device outputs and our software outputs after signal normalization. P<0.05 was considered as statistically significant.
Results: The mean absolute difference in RNFL thicknesses between Cirrus and RTVue were significantly different before normalization (10.5 ± 4.1 μm), but not after normalization (6.1 ± 4.7 μm). The absolute difference of RNFL thickness between Cirrus and RTVue was statistically significantly reduced by signal normalization. The median of the absolute difference of the RNFL thickness after normalization was
4.7 μm, which is within the inherent device measurement variability.
Conclusions: The reported novel signal normalization method successfully reduced the systematic difference in RNFL thickness measurement between Cirrus and RTVue to the level of the inherent device measurement variability. Enabling direct comparison of RNFL thickness obtained from multiple devices would broaden the use of OCT technology in both clinical and research applications.
Circuit Architecture to Eliminate Residual Electrode Polarization During Neural Stimulation
Ashwati Krishnan1, Shawn K. Kelly1,2
1Department of Electrical and Computer Engineering, Carnegie Mellon University
2Center for Innovative Visual Rehabilitation, Department of Veterans Affairs
Electrical stimulation of neural tissue is used in a variety of implantable electronic devices, including neural stimulators for Parkinson’s disease, pain management, and depression, as well as cochlear implants for the deaf and, more recently, retinal implants for the blind. The circuits that provide this stimulus have traditionally been designed to deliver biphasic, charge-balanced currents. However, when the electrode-tissue interface is modeled by a capacitive element, representing charge storage at the interface, in parallel with a resistive element, representing Faradaic reduction and oxidation reactions at the interface, it quickly becomes clear that delivering a charge-balanced stimulus results in a residual voltage on the electrode. This residual polarization can degrade the electrodes over time, reducing the lifespan of the implanted device and potentially damaging the surrounding nerve tissue.
We took voltage measurements of a 400μm diameter electrode coated with sputtered iridium oxide film (SIROF) in a saline bath. We extracted an electrical model of the electrode-saline interface, and then modeled the result of driving it with a charge-balanced biphasic current. We then built a prototype stimulator on a lab bench, and verified the simulations with voltage measurements from the pulsed electrode.
Using the measured residual voltage, we created a feedback loop to adjust the timing of the current pulses to leave zero residual voltage. This proof-of-concept system reduces the residual electrode polarization from 14 mV to less than 1 mV. We plan to implement this feedback system in an integrated circuit suitable for future use in implantable neural stimulation devices.
Schlemm's Canal (SC) Narrowing in Response to Intraocular Pressure (IOP) elevation observed by Spectral Domain Optical Coherence Tomography (SD-OCT)
Authors: L. Kagemann1,2, G. Wollstein1, H. Ishikawa1,2, R.A. Bilonick1, I.A. Sigal1,2, J.E. Nevins1, Z. Nadler1, J. Grimm1, N.-J. Jan1, J.S. Schuman1,2.
Affiliations: 1UPMC Eye Center, Eye and Ear Institute, Ophthalmology and Visual Science Research Center, Department of Ophthalmology, University of Pittsburgh School of Medicine, Pittsburgh, PA; 2Department of Bioengineering, University of Pittsburgh, Swanson School of Engineering, Pittsburgh, PA.
Purpose: The inner wall of SC bows outward toward the inner wall resulting in narrowing of SC as IOP is elevated in bovine eyes.1 The purpose of this study was to determine if SC narrowing is observable by SD-OCT in human eyes with IOP elevation.
Methods: Two cadaveric eyes in a constant IOP flow model2 were imaged by SD-OCT (Bioptigen Inc., Durham, NC) at perfusion IOPs of 10 and 25mmHg. Individual B-scans were examined to identify identical location pairs at low and high pressures (Figure). Visualization was subjectively optimized and all measurements performed in Fiji (ImageJ 1.45q, NIH, http://imagej.nih.gov/ij). SC length, representative width, and area were measured at low and high IOP and compared by paired t-test.
Results: Volumetric scans allowed the identification of fifteen paired locations at the 2, 4, 8, 9, and 10 o’clock positions. Bowing of the inner wall was observed subjectively (Figure). Width (39±16 to 25±11μm, mean±SD, p = 0.0007) and area(15,742±5,930 to 12,352±5,122μm2, p < 0.0001) were significantly reduced by IOP elevation, with no change in length (347±66 to 345 ± 87μm). Similar reductions were observed subjectively at all examined clock hours.
Conclusions: SD-OCT demonstrated narrowing of the SC with elevated IOP in human cadaveric eyes. These morphometric changes can be observed by SD-OCT. These findings agree with those in the animal model, though the relationship between outflow structure morphology and outflow facility is yet to be determined.
1Battista SA, Lu Z, Hofmann S, et al. Reduction of the available area for aqueous humor outflow and increase in meshwork herniations into collector channels following acute IOP elevation in bovine eyes. Invest Ophthalmol Vis Sci 2008;49(12):5346- 52.
2Kagemann L, Wollstein G, Ishikawa H, et al. 3D visualization of aqueous humor outflow structures in-situ in humans. Exp Eye Res 2011;93(3):308-15.
Ordinal Measurement Error Model for Assessing Agreement Among Raters for Glaucoma Progression
Authors: Yun Ling1,2, Richard A. Bilonick1,2, Hiroshi Ishikawa1,3, Gadi Wollstein1, Joel S. Schuman1,3.
Affiliations: 1UPMC Eye Center, Eye & Ear Institute, Ophthalmology and Visual Science Research Center, Dept. Ophthalmology, U. Pittsburgh School of Medicine, Pittsburgh, PA; 2Dept. Biostatistics, U. Pittsburgh Graduate School of Public Health, Pittsburgh, PA; 3Dept. Bioengineering, Swanson School of Engineering, U. Pittsburgh, Pittsburgh, PA.
Purpose: An ordinal measurement error model (OMEM) that describes agreement in terms of a slope (polychoric correlation) and intercept (threshold) was used to describe the agreement among 5 raters for glaucoma progression. This is an improvement over using kappa because, in general, agreement cannot be described as a single parameter.
Methods: The agreement among 5 raters (A, B, C, D, E) judging progression in eyes of 60 subjects was modeled using a common factor measurement error model for ordinal data. It assumes that subjects had an unobserved (latent) progression factor on a continuous scale. Each rater had a latent continuous scale for judging but only ordinal outcomes (no progression, progression) were observed. The model for agreement includes a scale (slope, -1 < β < 1) that also measures the polychoric correlation between the common factor and each observed rating and simultaneously describes the precision. Thresholds (intercepts, α) convert the unobserved rater's continuous judgment to ordinal outcomes. Since there are two rating categories, one threshold is needed per rater. The probability of making a judgment of progression increases with the amount of true progression μ:
P(rating = progression) = P(z > α - β*μ)
where the probabilities come from a unit standard normal distribution. R software with OpenMx was used to describe the structural equation model (SEM) for the OMEM (Figure 1) and estimate the parameters.
Results: Full information maximum likelihood (FIML) estimates of the most important and relevant functions of the model parameters are shown in Table 1. Rater E has the steepest slope, indicating E is the fastest in transition from non-progressor to progressor. Also E happens to have the lowest intercept, indicating E tends to rate more subjects as progressors. The estimated correlation between both eyes was ρ = 0.29, 95% CI (-0.06, 0.64). Subjects A, B, and C were very similar in their ratings and different from D and E.
Conclusions: SEM accounts for the correlations between both eyes of each subject, and, when FIML is used, missing ratings. Correctly describing agreement requires two parameters: a scale that describes the rate of transition and thresholds that control when the transitions occur.
Identifying the Best Structural and Functional Parameters to Detect Glaucoma Progression
Authors: Zach Nadler1, Richard A. Bilonick2, Michele Iester1, Gadi Wollstein1, Hiroshi Ishikawa1,3, Larry Kagemann1,3, Jessica Nevins1, Ian A. Sigal1,3, Jay S. Duker4, Joel S. Schuman1,3.
Affiliations: 1UPMC Eye Center, Eye and Ear Institute, Ophthalmology and Visual Science Research Center, Dept. of Ophthalmology, University of Pittsburgh School of Medicine, Pittsburgh, PA; 2Department of Biostatistics, Graduate School of Public Health, University of Pittsburgh, Pittsburgh, PA; 3Department of Bioengineering, Swanson School of Engineering, University of Pittsburgh, Pittsburgh, PA; 4Ophthalmology, New England Eye Center, Boston, MA.
Purpose: To identify the best parameters to detect glaucoma progression among visual field (VF) and optic nerve head (ONH) parameters.
Methods: 110 eyes of 60 subjects (24 healthy, 48 glaucoma suspects and 38 glaucomatous eyes) had comprehensive ocular examination and ≥4 visits with reliable VF testing (Carl Zeiss Meditec, Dublin, CA) and good quality spectral-domain optical coherence tomography (SD-OCT; RTVue-100, Optovue, Freemont, CA) and scanning laser ophthalmoscopy (SLO; HRT III, Heidelberg Engineering, Heidelberg, Germany). Structural equation models (SEM) were constructed to compute the common latent factor measurement error for each device using standardized global parameters as provided by the devices along with clinical diagnosis and baseline age.
Results: The median follow-up period was 3.93 years, and the median number of visits was 6.5. For VF, the Visual Field Index (VFI) showed statistically significantly better correlation with the common latent progression (=0.93; confidence interval (CI): 0.76-1.00) than both mean deviation (MD) and pattern standard deviation (PSD). For SD-OCT the parameters with the highest correlation with latent progression were cup area (=0.92; CI: 0.80-1.00) and rim area (=-0.92; CI: -1.00-- 0.80) and statistically significantly better than other measured parameters. SLO also showed cup area (=-0.85; CI: -1.00-- 0.73) and rim area (=-0.85; CI: 0.74-1.00) to be the parameters most correlated with latent progression. For all three devices the diagnosis had similar effect and was statistically significant. Using latent progression scores for each eye derived from the SEM, there was poor correlation between VF and SD-OCT progression scores (r=0.17), SD-OCT and HRT (r=0.36), and HRT and VF (r=0.03).
Conclusions: Cup and rim area were the most useful structural measurements of progression, while VFI was the most useful functional parameter. Even though SD-OCT and HRT quantify ONH structure there was a poor correlation between the devices in detecting progression.
Comparing Glaucoma Progression Detection in Various Disease Stages Using Macular and Peripapillary Scan Parameters of Spectral-Domain Optical Coherence Tomography
Authors: Jessica E. Nevins1, Gadi Wollstein1, Joel S. Schuman1, Richard A. Bilonick1,2, Hiroshi Ishikawa1,3, Yun Ling1,2, Advanced Imaging for Glaucoma Study Group4
Affiliations: 1UPMC Eye Center, Eye and Ear Institute, Ophthalmology and Visual Science Research Center, Department of Ophthalmology, University of Pittsburgh School of Medicine, Pittsburgh, PA; 2Department of Bioengineering, University of Pittsburgh, Pittsburgh, PA; 3Department of Biostatistics, University of Pittsburgh, Pittsburgh, PA.; 4www.AIGStudy.net
Purpose: Central vision is typically preserved until severe glaucoma stages, which might indicates that macular thickness is a better indicator than peripapillary measurements for detection of disease progression in advanced stages of the disease. This study compares the performance of Spectral-Domain Optical Coherence Tomography (SD-OCT) in detecting glaucoma progression in macula and peripapillary regions in various stages of glaucoma severity.
Methods: 487 eyes from healthy, glaucoma suspect, and glaucoma subjects had 4 visits with reliable visual field (VF) testing (Carl Zeiss Meditec, Dublin, CA) and SD-OCT (RTVue; Optovue, Fremont, CA) optic nerve head and macula cube scans. Retinal nerve fiber layer (RNFL) and ganglion cell complex (GCC) thickness measurements were used from peripapillary and macular regions, respectively. Rate of thickness change was computed using a linear mixed effect model.
Results: Individual eye RNFL and GCC slopes as a function of baseline RNFL and GCC thicknesses, respectively, are presented in the figure. Higher percentage of eyes showed a positive (thickening) rate of change with RNFL compared to GCC in all groups.
Discussion: Similar trends of longitudinal thickness change were observed for RNFL and GCC with more accelerated thinning at thicker baseline values and less accelerated thinning at thinner baseline values. RNFL and GCC similarly detect progression across all glaucoma stages.
IN VITRO EVALUATION OF THE INTERACTIONS BETWEEN HUMAN CORNEAL ENDOTHELIAL CELLS AND EXTRACELLULAR MATRIX PROTEINS
Jin San Choi14, Min Jeong Kim13, Eun Young Kim13, Matthew Giegengack2, Shay Soker1
1Wake Forest Institute for Regenerative Medicine, Wake Forest School of Medicine, 2Department of Ophthalmology, Wake Forest University School of Medicine, 3Department of BIN Fusion Technology, Polymer Fusion Research Center, Chonbuk National University, 4Ocular Systems, Inc., Piedmont Triad Research Park, Winston-Salem, NC
Purpose: Corneal endothelium is the innermost cell layer of cornea and rests on Descemet’s membrane containing of various extracellular matrix (ECM) proteins which can directly affect the cellular behaviors such as cell adhesion, proliferation, polarity, morphogenesis, and function. The objective of this study was to investigate the interactions between the ECM environment and human corneal endothelial cells (HCECs), with the ultimate goal to improve cell proliferation and function in vitro.
Methods: To evaluate the interaction of HCECs with ECM proteins, cells were seeded on ECM-coated tissue culture dishes, including collagen type I (COL I), collagen type IV (COL IV), fibronectin (FN), FNC coating mix (FNC), and laminin (LM). Cell adhesion and proliferation of HCECs on each substratum and expression of CEC markers were studied.
Results: We cultured HCEC on FN, LM, and ColIV-coated plates compared to cells cultured on untreated plates. The cell adhesion and proliferation of the HCEC on each substratum and expression of CEC markers were evaluated in each condition. There were no significant differences in cell numbers between the different protein-coated plate and untreated plate. However, the cells grown onto the FN, LM, and ColIV-coated plates showed more compact cellular morphology and expression of typical CEC markers.
Conclusions: The results showed that HCECs plated in ECM-coated tissue culture dishes had enhanced cell adhesion and growth and morphology. These results suggest that an adequate ECM protein combination can provide a long-term culture environment for HCECs for corneal endothelium transplantation.
Success Isolation of Human Corneal Endothelial Cells for Clinical Use
Jin San Choi14, Min Jeong Kim13, Eun Young Kim13, Matthew Giegengack2, Shay Soker1
1Wake Forest Institute for Regenerative Medicine, Wake Forest School of Medicine, 2Department of Ophthalmology, Wake Forest University School of Medicine, 3Department of BIN Fusion Technology, Polymer Fusion Research Center, Chonbuk National University, 4Ocular Systems, Inc., Piedmont Triad Research Park, Winston-Salem, NC
Purpose/Hypothesis: The inner layer of the cornea is a single layer of neural-crest derived cells that form a barrier between the cornea and the aqueous humor. The corneal endothelial cells (CEC) are essential for transport of water from the corneal stroma. Damaged or decomposition of the CEC pump results in corneal edema and loss of vision. CEC loss is most well documented as a result of accidental damage during cataract surgery or in an inherited condition known as Fuchs’ dystrophy. Corneal transplantation is a common transplant procedure to improve visual acuity by replacing the opaque or distorted host tissue by clear healthy donor tissue. However, its clinical utility is limited due to a lack of high quality donor corneas. Bioengineered neo-corneas, created using an expandable population of human donor-derived corneal endothelial cells (HCEC), could address this shortage. Thus, the objective this study were to establish HCEC sourcing with various isolation methods, including enzymatic digestion, culture medium components, and ECM proteins.
Methods: HCEC were obtained from 269 discarded corneas with various aged donors after endothelial keratoplasty. Under a dissection microscope, the Descemet’s membrane, including the attached corneal endothelium was stripped from the stroma and the cells were isolated and expanded by explant culture and the use of enzymatic digestion with enzyme such as collagenase II, dispase, and trypsin. The cultured HCEC were characterized by immunocytochemistry for ZO-1 and NA+/K+-ATPase.
Results: HCEC were successfully isolated from 32% (86/269) of donor corneas. HCEC were expanded to the numbers needed to re-endothelialize neo-corneal constructs, while expressing markers typical to HCEC. Of the parameters studied, the donor age and isolation method significantly affected HCEC in vitro culture results. Under all conditions tested, in vitro culture of HCEC on ECM protein-coated plates enhanced cell-to-cell interactions and proliferation of the cells.
Conclusions: The results indicate that both donor-derived and isolation technique factors significantly affect the success rate of obtaining HCEC cultures in vitro. These factors should guide the clinical expansion of HCEC for the generation of bioengineered corneas.
Optimization of Culture Medium for Human Corneal Endothelial Cells
Min Jeong Kim12, Jin San Choi14, Matthew Giegengack3, Gilson Khang2, and Shay Soker1
1Wake Forest Institute for Regenerative Medicine, Wake Forest School of Medicine, 2Department of BIN Fusion Technology, Polymer Fusion Research Center, Chonbuk National University, 3Department of Ophthalmology, Wake Forest University School of Medicine, 4Ocular Systems, Inc., Piedmont Triad Research Park, Winston-Salem, NC
Introduction: Corneal endothelium, located in the inner layer of cornea, is essential to maintain transparency and thickness of cornea by serving as a permeable barrier that controls stromal hydration. Damaged or diseased corneal endothelial cells (CECs) result in loss of vision, pain, and cornea edema. If it is damaged, corneal transplantation is commonly used as medical treatment. However, due to shortage of donor cornea for transplantation, bioengineered neo-cornea, using expandable population of CECs and a biocompatible material, would be expected to replace cornea for transplantation. In this study we attempted to optimize culture medium components for human CECs (HCECs) and evaluated by measuring cell proliferation, density, and phenotypic expression.
Methods: HCECs were obtained from eye donors by digestion in collagenase II and expanded on tissue culture plate at 37°C in a humidified atmosphere containing 5% CO2. To evaluate cell proliferation, HCECs (cell density: 50 cells/mm2) were tested in four different culture media: DMEM, DMEM/F-12 (1:1), α-MEM, and Medium 199 supplemented with 1 μg/ml L-glutamine, 1 μg/ml heparin, 2 μl/ml ITS (insulin, transferrin, and sodium selenite), and 0 or 10% fetal bovine serum (FBS). A complex mixture of basic fibroblast growth factor (bFGF, 2 or 10 ng/ml), epidermal growth factor (EGF, 5 ng/ml) and insulin-like growth factor (IGF-1, 20 ng/ml) was added. Endothelial growth medium-2 (EGM-2) with 10% FBS as control group was also tested. The cell proliferation was analyzed by NF CyQuant cell proliferation kit (Invitrogen) at 1, 4, and 8 days. The morphology of HCECs was observed using light microscope at 4 days.
Results and Conclusion: We successfully isolated and expanded HCECs from limbal Descemet’s membrane under different culture conditions. We investigated the optimal culture condition with various basal media with growth factors. Among those, HCECs cultured in DMEM/F-12 with bFGF showed best cell proliferation. However there were no significant differences in cellular morphology and expression of CEC markers. In conclusion, HCECs grow well in DMEM/F-12 containing bFGF and these results will utilize for optimization of in vitro cultivation of HCECs for producing a bioengineered neo-cornea for human transplantation.
Optimization of Culture Medium for Human Corneal Endothelial Cells
Min Jeong Kim12, Jin San Choi14, Matthew Giegengack3, Gilson Khang2, and Shay Soker1
1Wake Forest Institute for Regenerative Medicine, Wake Forest School of Medicine, 2Department of BIN Fusion Technology, Polymer Fusion Research Center, Chonbuk National University, 3Department of Ophthalmology, Wake Forest University School of Medicine, 4Ocular Systems, Inc., Piedmont Triad Research Park, Winston-Salem, NC
Introduction: Corneal endothelium, located in the inner layer of cornea, is essential to maintain transparency and thickness of cornea by serving as a permeable barrier that controls stromal hydration. Damaged or diseased corneal endothelial cells (CECs) result in loss of vision, pain, and cornea edema. If it is damaged, corneal transplantation is commonly used as medical treatment. However, due to shortage of donor cornea for transplantation, bioengineered neo-cornea, using expandable population of CECs and a biocompatible material, would be expected to replace cornea for transplantation. In this study we attempted to optimize culture medium components for human CECs (HCECs) and evaluated by measuring cell proliferation, density, and phenotypic expression.
Methods: HCECs were obtained from eye donors by digestion in collagenase II and expanded on tissue culture plate at 37°C in a humidified atmosphere containing 5% CO2. To evaluate cell proliferation, HCECs (cell density: 50 cells/mm2) were tested in four different culture media: DMEM, DMEM/F-12 (1:1), α-MEM, and Medium 199 supplemented with 1 μg/ml L-glutamine, 1 μg/ml heparin, 2 μl/ml ITS (insulin, transferrin, and sodium selenite), and 0 or 10% fetal bovine serum (FBS). A complex mixture of basic fibroblast growth factor (bFGF, 2 or 10 ng/ml), epidermal growth factor (EGF, 5 ng/ml) and insulin-like growth factor (IGF-1, 20 ng/ml) was added. Endothelial growth medium-2 (EGM-2) with 10% FBS as control group was also tested. The cell proliferation was analyzed by NF CyQuant cell proliferation kit (Invitrogen) at 1, 4, and 8 days. The morphology of HCECs was observed using light microscope at 4 days.
Results and Conclusion: We successfully isolated and expanded HCECs from limbal Descemet’s membrane under different culture conditions. We investigated the optimal culture condition with various basal media with growth factors. Among those, HCECs cultured in DMEM/F-12 with bFGF showed best cell proliferation. However there were no significant differences in cellular morphology and expression of CEC markers. In conclusion, HCECs grow well in DMEM/F-12 containing bFGF and these results will utilize for optimization of in vitro cultivation of HCECs for producing a bioengineered neo-cornea for human transplantation.
Title: In vivo MRI Monitoring of Secondary Degeneration in the Uninjured Visual Pathway upon Partial Optic Nerve Injury
Purpose: Glaucoma is a neurodegenerative disease involving the visual system in a topological manner. While the optic nerve (ON) head is a major site of injury, it is not clear if secondary degeneration occurs along the visual pathways that are not primarily injured in glaucoma. This study employed 3D T1-weighted Mn-enhanced MRI (MEMRI) at 7 Tesla to evaluate the uninjured nerves in the retinocollicular pathway longitudinally in an experimental model of partial ON injury.
Methods: The superior region of the right intraorbital ON in 10 adult Sprague-Dawley rats was partially transected at about 2mm from the eye. One and 6 weeks after surgery, MnCl2 solution (50mM, 3μL) was injected intravitreally into both eyes of the same animals. MEMRI was performed 1 day after Mn2+ administration. Throughout the experiments, the left ON was not transected and the visual pathway projected from left eye to right visual brain served as an internal control. After MRI experiments at Week 6, 4 rats were randomly chosen for histology using toluidine blue staining.
Results: At both Week 1 and Week 6, the right vitreous and retina, and the right ON exhibited significantly higher and lower signal intensities respectively than the contralateral eye (paired t-tests, p<0.05). In addition, a consistent hypointensity by about 28% was observed in left lateral superior colliculus (SC) relative to right lateral SC (paired t-tests, p<0.001). The left medial SC had a reduced signal intensity by 11% compared to right medial SC at Week 1 (paired t-test, p<0.001). Such reduction further increased to 16% at Week 6 (paired t-test, p<0.01). Histology confirmed degeneration in both superior and inferior ON, with the superior ON showng more pronounced myelin debris formation and atrophy.
Conclusions: As more than 90% of rat ON fibers cross to the contralateral hemisphere at the optic chiasm, the consistent hypointensity in left lateral SC at Week 1 and Week 6 reflected the primary loss of topological connections and Mn2+ anterograde transport from right superior retina and ON. The slightly weaker enhancement in left medial SC at Week 1 might be partly due to reduced Mn2+ transport upon spreading of oxidative stress through the inferior retina early after partial ON injury, whereas the increased signal reduction in left medial SC at Week 6 might indicate secondary loss of retinal ganglion cells and axons projecting through the uninjured, inferior ON. The current results demonstrated the primary and secondary degenerative events along the visual pathways in vivo after partial ON injury. Future MEMRI studies are envisioned that measure the integrity of topological connections longitudinally upon pharmacologic interventions against secondary degeneration.
Chromatin dynamics of the zebrafish retina during development and aging
Yi Wen, Jian Zou, Xiangyun Wei
Department of Ophthalmology, University of Pittsburgh School of Medicine
Abstract
During organogenesis, the retina develops from a single sheet of undifferentiated neuroepithelium into a layered mature tissue composed of Müller, photoreceptor, horizontal, bipolar, amacrine, interplexiform and ganglion cells. This differentiation of various retinal cell types requires the transcription of distinct sets of genes in precise temporal orders. It is unclear how chromatin organization and modifications are specifically regulated at global and regional levels to facilitate the generation of distinct transcriptional profiles required for different retinal cell types. Nor do we know to what extent chromatin structure and organization are altered during retinal aging, or how such alterations lead to unbalanced gene expression profiling and subsequent cell degeneration. In this study, we analyzed the dynamic characteristics of nuclear structures and chromatin organizations of zebrafish retinal cells during development and aging. We show that the degrees of chromatin condensation vary significantly among different retinal cell types, as suggested by varying nuclear volumes and heterochromatin/euchromatin organization. These structural features are altered during the aging process as well. For example, the nuclear volumes of cone photoreceptors increase significantly during aging. The distribution patterns of H3K9me3 and H4K20me3 heterochromatin modifications also vary dramatically among different retinal cell types and at different ages. The gross alterations in nuclear structures and chromatin condensation may suggest extensive changes in gene expression activities. These findings provide insight into how chromatin organization and modifications underlie balanced global transcriptional profiles and consequently, the overall fitness of retinal cells, as well as how such regulations might be altered during the aging process. Our study may have significant social impact because with increasing life expectancy, more and more older people will suffer from age-related macular degeneration and the identification of proper interventions to slow down the retinal aging process would dramatically enhance quality of the life for the elderly.
Crb Apical Polarity Proteins Maintain Zebrafish Retinal Cone Mosaics via Intercellular Binding of Their Extracellular Domains
Jian Zou
Cone photoreceptors are assembled by unknown mechanisms into geometrically regular mosaics in many vertebrate species. The formation and maintenance of photoreceptor mosaics are speculated to require differential cell-cell adhesion. However, the molecular basis for this theory is yet to be identified. The retina and many other tissues express the Crumbs (Crb) polarity proteins. The functions of the extracellular domains of the Crb proteins remain to be understood. Here we report cell-type specific expression of the crb2a and crb2b genes at the cell membranes of photoreceptor inner segments and Müller cell apical processes in the zebrafish retina. We demonstrate that the extracellular domains of Crb2a and Crb2b mediate a previously unrecognized cell-cell adhesion function, which plays an essential role in maintaining the integrity of photoreceptor layer and cone mosaics.
Abstract
Jian Wu
Recapitulating the microstructure of native human corneal stromal tissue is believed to be a key feature in successfully engineering corneal tissue. The stratified multilayered collagen-fibril lamellae with orthogonal orientation determine the robust biomechanical properties of this tissue, and the uniform collagen fibril size and inter-fibrillar spacing are critical to its optical transparency. The objective of this investigation was to develop a highly organized collagen-fibril construct to bio-mimic human corneal stromal tissue secreted by human corneal stromal stem cells (hCSSCs). Using a highly-aligned fibrous substrate made from poly(ester urethane) urea, basic fibroblast growth factor (bFGF, 10ng/ml) and transforming growth factor-beta3 (TGF-β3, 0.1 ng/ml) were shown to impact the organization of highly-aligned collagen-fibril matrix. hCSSCs were successfully differentiated into keratocytes with significant up-regulation of the typical gene markers including KERA, B3GnT7, and CHST6. Key components of human corneal stromal tissue, such as keratan sulfate, chondroitin sulfate, lumican, decorin, and keratocan, were detected in secreted extracellular matrix (ECM) and/or in culture media. Structurally, bFGF treatment stimulated hCSSCs to secrete collagen fibrils strongly aligned in one preferred direction, whereas TGF-β3 induced distinct orthogonal collagenous layers. The combination of bFGF and TGFβ3 induced multilayered lamellae with orthogonally-oriented collagen fibrils, in a pattern mimicking human corneal stromal tissue, and also produced thicker constructs (60~70 m), and increased expression of cornea-specific ECM components, including keratan sulfate, lumican, and keratocan. This approach of substrate cues combined with growth factor augmentation offer a new means to engineer well-organized, collagen-based constructs with appropriate nanoscale structure for corneal repair and regeneration.
Survival of Trabecular Meshwork Stem Cells
in the Presence of Glaucoma-Associated Factors
Yiqin Du, Martha L. Funderburgh, Hongmin Yun, Joel S. Schuman
Department of Ophthalmology, UMPC Eye Center, University of Pittsburgh, PA
Key Words: trabecular meshwork, glaucoma, cell survival, transplantation
Purpose: Previously we identified a cell population from human trabecular meshwork with properties of multipotent stem cells. These trabecular meshwork stem cells (TMSC) home to mouse trabecular meshwork (TM) in vivo adapting a TM phenotype. This study tests the hypothesis that TMSC resist stimulation by soluble factors associated with glaucoma better than TM cells.
Methods: DiO-labeled TMSC,were expanded two passages, stained with Hoechst dye, and for stem cell markers ABCG2, Notch1, MUC1 and TM marker CHI3L1. Passaged TMSC and TM cells were stimulated with glaucoma-associated factors dexamethasone, TGF2, or TNF. Apoptosis was detected by Annexin V staining and flow cytometry. qRT-PCT, immunoblotting, and immunofluorescent staining compared expression of myocilin, ELAM1, NF-kappa B, AQP1 and CHI3L1. TMSC, TM cells, and fibroblasts were transplanted into mouse anterior chamber for 4 weeks and 4 months. Viability of the injected cells was assessed using Calcein AM staining. Immunofluorescence examined inflammatory cells (CD45) and human-specific MUC1 and CHI3L1.
Results: Expanded TMSC cultures had heterogeneous DiO staining indicating cells with different rates of replication. DiO-bright cells had reduced Hoechst dye staining consistent with dye-efflux typical of stem cells. These cells stained for stem cell markers ABCG2, Notch1, and MUC1. DiO-dim, rapidly dividing cells, expressed TM marker CHI3L1. Stimulation with dexamethasone, TGF2 or TNF induced expression of myocilin, ELAM1, and NF-kappa B but reduced AQP1, CHI3L1 in both TMSC and TM cells. Changes in TMSC were quantitatively less than those of TM cells. After human TMSC transplantation into murine anterior chamber, no CD45 positive cells were seen in the TM of mice with TMSC. Injected TMSC remained viable 4 months after injection and expressed either MUC1 or CHI3L1. Fewer injected fibroblasts colonized the TM region. Apoptosis of injected fibroblasts was greater than that of injected TM or TMSC.
Conclusions: The slow-cycling and asymmetric replication of TMSC suggests a role in replacement of lost or damaged TM cells in vivo. The observation that TMSC tolerate stimulation with glaucoma-associated factors in vitro and survive in vivo for up to 4 months, supports the hypothesis that TMSC provide a source of healthy TM cells in vivo.
Support: NIH Grants EY016415, P30-EY008098, Eye & Ear Foundation of Pittsburgh, Research to Prevent Blindness, an anonymous philanthropic donation.
Slow-cycling stem cells in trabecular meshwork
Hongmin Yun, Yiqin Du.
UPMC Eye Center, Ophthalmology & Visual Science Research Center, Department of Ophthalmology, University of Pittsburgh School of Medicine, Pittsburgh, PA
Aim: To determine whether the stem cells in trabecular meshwork (TM) are slow cycling and can be chased as BrdU-retaining cells in vivo.
Methods: Passaged stem cells from human TM were labeled with DiO and after two more passages, the cells were detected with DiO retaining and stained with nuclear dye Hoechst 33342 and stem cell markers. C57BL/6 mice 3 days after birth were injected peritoneally with brumodeoxyuridine (BrdU) twice a day for three days. During chase periods of 4 hours, 1 day, 3 days, 1, 2, 3, 4 and 8 weeks, the mice were sacrificed and the anterior portions of the eyes including the TM were dissected and processed for BrdU and stem cell marker OCT4 staining. All samples were imaged using a confocal microscope.
Results: We have reported that the stem cells from TM (TMSC) can be isolated as side-population cell sorting with the ability to efflux DNA-binding dyes and expressed stem cell markers ABCG2, OCT4, AnkG and MUC1 (Du et al. IOVS 2012; 53: 1566-1575). In this study we found that after two passages, the DiO retaining cells can efflux Hoechst dye and were positive to ABCG2, Notch1 and MUC1. In BrdU in vivo labeling experiments, BrdU labeled cells were detected in the trabecular meshwork in the mice at every chasing period up to 8 weeks. The number of BrdU labeled cells decreased at longer chasing periods comparing to shorter chasing periods. There were still BrdU retaining cells in the TM eight weeks after labeling. Some BrdU labeling cells expressed stem cell marker OCT4.
Conclusions: The stem cells in trabecular meshwork are slow-cycling with the ability to retain DiO in culture and retain BrdU labeling in vivo for at least eight weeks in mice. The observation provides the possibility of using stem cells to restore the function of trabecular meshwork in glaucomatous eyes at any stage of the disease to decrease the intraocular pressure, which is the only determined risk factor of glaucoma.
Support: NIH Grants EY016415, P30-EY008098, Eye & Ear Foundation of Pittsburgh, Research to Prevent Blindness, an anonymous philanthropic donation.
Pluripotent Stem Cells in the Cornea? A New Approach to Isolation of Corneal Stromal Stem Cells.
Yiqin Du, James L. Funderburgh
UPMC Eye Center, Ophthalmology & Visual Science Research Center, Department of Ophthalmology, University of Pittsburgh School of Medicine, Pittsburgh, PA
We have identified human corneal stromal stem cells (hCSSCs) which can be isolated by side-population cell sorting in the presence of Hoechst 33342 (Du et al. Stem Cells. 2005, 23: 1266). These cells can differentiate into keratocytes, secrete corneal extracellular matrix such as lumican and keratocan, restore corneal thickness and collagen fibril defects after transplanting into lumican knockout mouse corneal stroma. However, isolation and initial expansion of the side-population cells remains problematic due to the influence of the DNA-binding Hoechst dye in the cells. In this study, we explored the potential for isolation of hCSSCs by clonal culture or by cell sorting using specific cell surface marker(s). Corneal limbal stromal cells isolated as clones were positive to CD73, CD90, CD166. Some of the cells also expressed OCT-3/4, ABCG2, Notch1, and CXCR4. CXCR4 is expressed in corneal limbal stromal niche cells (Xie H, et al. Stem Cells. 2011, 29: 1847). We isolated CXCR4+ cells from corneal stromal cells by fluorescence-activated cell sorting and compared the gene expression in cloned hCSSCs, CXCR4+ and CXCR4- cells. We found that the CXCR4+ cells were enriched in expression of ABCG2, OCT4, NaNog and KLF4. Our results provide a new method to isolate corneal stromal stem cells that can be used for cell-based therapy and for corneal bioengineering. Supported by NIH Grants EY016415, P30-EY008098, Eye & Ear Foundation of Pittsburgh, Research to Prevent Blindness, an anonymous philanthropic donation.
Name: Hertsenberg, Andrew
Advisor: Funderburgh, James
Differentiation of Human Embryonic Stem Cells Toward Neural Crest and Corneal Stroma Cell Fate
Body of Abstract:
Purpose: In this study we seek to determine if human embryonic stem cells can be differentiated into keratocytes and their potential application in corneal cell therapy.
Methods: Human embryonic stem cells (hESC) were differentiated into neural-crest cells using the stromal-derived inducing activity (SDIA) of PA6 cells. CD271+ cells were magnetically sorted and expanded in serum-free N2 medium before gene expression analysis and subsequent differentiation into keratocytes using a serum-free keratocyte differentiation medium (KDM). Each differentiated cell type was analyzed for cell-specific gene expression using RT-PCR and flow cytometry. CD271+ cells were labeled with DiO prior to intrastromal injection into healthy WT C57BL/6 corneas. At 1 month post-injection, corneas were harvested and cells isolated using FACS for RNA isolation and RT-PCR.
Results: hESC, induced to neural differentiation with SDIA, exhibit a transient phenotype in which neural crest genes are expressed. The neural crest cells were successfully isolated using magnetic capture with antibodies to low affinity neurotropin receptor (NGFR, CD271). The isolated cells expressed elevated levels of neural-crest genes: NGFR, SNAIL, NTRK3, SOX9, SLUG, and MSX1. After expanding CD271+ cells in culture they were incubated in KDM for 3 weeks, after which they expressed elevated levels of keratocyte specific markers: AQP1, CHST6, PTGDS, ALDH3A1, and KERA. The ES-derived neural crest cells were labeled with DiO and injected into mouse corneas. These remained in the stroma without rejection for one month, at which time they were harvested for phenotypic analysis.
Conclusions: Human ESC can be differentiated into CD271+ neural-crest cells which can be expanded and induced to express keratocyte specific genes. The differentiated hESC survive in vivo after intrastromal injection. In the future we plan to assess the ability of the injected cells to differentiate into keratocytes by looking at both gene and protein expression.
Supported by NIH EY016415.
Surface-Initiated Assembly of Monodisperse Protein Nanofibers for Tissue Engineering Applications
John Szymanski and Adam W. Feinberg
The extracellular matrix in tissues is a 3-D fibrillar network with tightly regulated fiber diameter, composition and orientation. It is challenging to achieve similar control over fibers within tissue engineered constructs. Traditional fabrication techniques such as phase separation and electrospinning enable the formation of highly porous networks of fibers with diameters as small as tens of nanometers but are limited in the regulation of their size distribution, orientation, and shape. Our bio-inspired approach mimics cell-mediated assembly using a process termed surface-initiated assembly. We hypothesized that this process would enable fabrication of protein nanofibers and nanostructures with enhanced control of uniformity, shape, and size. Here, we report that surface-initiated assembly results in the formation of protein fibers that are monodisperse both before and after release. Prior to release, nanofibers had initial widths of 20.30 + 0.44 μm and 20.32 + 0.41 with lengths of 50.51 + 0.47 μm and 199.95 + 0.65 μm, respectively. Upon thermally triggered release, the nanofibers contracted to final widths of 4.17 + 0.43 μm and 3.97 + 0.31 μm and lengths of 162.57 + 3.63 μm and 35.80 + 1.88 μm. We also show the ability to form fibers with complex shapes and orientations such as star and cross patterns, which cannot be created using other fabrication techniques. In the future, we hope to leverage surface-initiated assembly for precise control over the chemical composition, size distribution and orientation of protein fibers for applications such as the regeneration of the cornea.
Title: In Vitro Expansion of Corneal Endothelial Cells on Biomimetic Substrates
Purpose:
The corneal endothelium is responsible for maintaining the clarity of the cornea, and loss of endothelial cells leads to impaired vision and the need for corneal transplantation. Corneal endothelial cells (CECs) are non-proliferative in vivo with minimal proliferation in vitro, making expansion of these cells for therapeutic application difficult. Small molecules and defined culture media have been investigated, but with minimal effect on the proliferation of CECs. We hypothesized that culturing bovine CECs on a substrate that mimics the biomechanics and protein composition of the native Descemet’s membrane would enhance proliferation and maintain phenotype of CECs in vitro.
Methods:
CECs were isolated from the corneas of fresh whole bovine eyes and cultured on one of three different surfaces: standard tissue culture treated polystyrene (TCPS), collagen type IV coated TCPS (col4-TCPS), or collagen type IV coated polydimethylsiloxane (PDMS) (col4-PDMS). CECs were cultured in DMEM supplemented with 10% FBS and passaged 1:3 in order to expand the cells. Phase contrast microscopy was used to determine cell density at confluence up to passage 10. The CECs at passages 1, 5, 8 and 10 were immunofluorescently labeled for fibronectin, laminin, collagen IV, ZO-1 and F-actin and analyzed for changes in phenotype.
Results:
CECs cultured on the col4-PDMS and col4-TCPS reached confluence more rapidly than on TCPS over the course of the expansion. Cell density on col4-PDMS was approximately 1.5 times greater than on TCPS at each passage. CECs on Col4-PDMS maintained a cuboidal cellular morphology, and high cell density up to passage 8, while CECs on col4-TCPS and TCPS lost proliferative capacity and phenotypic markers at earlier passages.
Conclusions:
We have demonstrated that a biomimetic substrate that recapitulates the lower mechanical stiffness and collagen type IV composition of Descemet’s membrane is able to enhance the in vitro expansion of CECs. Preliminary estimates indicate an ~3000-fold expansion of bovine CECs in vitro while maintaining phenotypic markers. Current efforts are focused on using quantitative PCR to validate the maintenance of CEC gene expression throughout expansion and then extending this system to the expansion of human CECs. The ability to expand CECs is crucial to obtain the cell numbers necessary for bioengineering a corneal endothelium suitable for implantation, a future goal of this research project.
UBM-ECM Biomaterial Speeds the Early Reepithelialization of Corneal Abrasions
Q. Pan1, A.D. Janis2, G. Sun1, L. Tang1, K. Stuart2, H. Meng1, E.K. Akpek1, W.J. Stark1
1. Wilmer Eye Institute, Johns Hopkins University, Baltimore MD
2. ACell Inc., Columbia MD
Background. Urinary bladder matrix (UBM) is a minimally processed extracellular matrix (ECM)-derived biomaterial that has shown promise in a wide variety of regenerative applications, including soft tissue repair and epithelial wound healing. UBM has been shown to contain collagens I, III, IV, and VII, as well as glycosaminoglycans and growth factors. Anecdotal reports of success using a lyophilized sheet form of UBM in veterinary corneal abrasion and ulceration cases support the further investigation of UBM in a controlled feasibility study of corneal injury using a well-characterized rabbit model. A particulate form of UBM biomaterial (MatriStem MicroMatrix; ACell Inc.) was investigated due to the wider clinical applicability of this form of the material.
Methods. Ten rabbits received a central, 8mm-diameter injury to the corneal epithelium of both eyes under anesthesia. Ethanol (20%) was delivered via trephine to the surface of each eye to lift the epithelium, which was gently dissected. One eye was treated with UBM (~10mg), the contralateral eye served as an untreated control. Treatment eyes were randomized. Contact lenses were placed over the UBM and control injuries for the first 24 hours. Re-epithelialization was assessed with slit lamp and image analysis of blinded microscopy images with fluorescein at 1, 2, 3, & 4 days post-injury (Image J v1.45s). One rabbit was assessed for corneal haze on Day 22 using confocal microscopy.
Results. The largest observable effect in healing occurred during the first 24 hours, with complete epithelialization occurring within 4 days for all eyes. Of the ten rabbits treated, five had increased reepithelialization after 24 hours when treated with UBM (mean increase of 23.0 ± 9% for treatment over control) and two had similar amounts of healing after 24 hours. Three rabbits showed greater reductions in wounded area in the control eye, however the decrease in wounded area was less in these animals than in those animals which had increased healing with UBM treatment. It was noted that the dose of the powder UBM treatment was difficult to control, and that the contact lens did not always remain in place during the first 24 hours. Additionally, as the study progressed there was increased efficacy with UBM treatment, suggesting the potential that the injury and the delivery of the treatment became better controlled. Confocal microscopy in a single rabbit showed greater haze in the cornea of the control than in the treatment eye at 22 days post-injury.
Conclusions. The results of this preliminary study warrant further investigation of the effects of UBM in corneal epithelial healing after injury. UBM treatment accelerated healing in more than half of the rabbits treated. Future studies will include improved methods to ensure the biomaterial treatment is retained in the injured area during the first 24 hours of healing, will investigate dosing and timing effects, and will include longer term assessment of corneal haze.