Michael P. Fautsch

Intracranial Pressure in Primary Open Angle Glaucoma, Normal Tension Glaucoma, and Ocular Hypertension: A Case–Control Study

PURPOSE To compare intracranial pressure (ICP) in subjects with primary open-angle glaucoma (POAG), normal-tension glaucoma (NTG; subset of POAG), and ocular hypertension (OHT) with that in subjects with no glaucoma. METHODS The study was a retrospective review of medical records of 62,468 subjects who had lumbar puncture between 1985 and 2007 at the Mayo Clinic. Of these, 57 POAG subjects, 11 NTG subjects (subset of POAG), 27 OHT subjects, and 105 control subjects met the criteria and were analyzed. A masked comparison of the relationship between ICP and other ocular and nonocular variables was performed by using univariate and multivariate analyses. RESULTS ICP was significantly lower in POAG compared with age-matched control subjects with no glaucoma (9.1 +/- 0.77 mm Hg vs. 11.8 +/- 0.71 mm Hg; P < 0.0001). Subjects with NTG also had reduced ICP compared with the control subjects (8.7 +/- 1.16 mm Hg vs. 11.8 +/- 0.71 mm Hg; P < 0.01). ICP was higher in OHT than in age-matched control subjects (12.6 +/- 0.85 mm Hg vs. 10.6 +/- 0.81 mm Hg; P < 0.05). CONCLUSIONS ICP is lower in POAG and NTG and elevated in OHT. ICP may play an important role in the development of POAG and NTG and in preventing the progression of OHT to POAG. Further prospective and experimental studies are warranted to determine whether ICP has a fundamental role in the pathogenesis of glaucoma.

Elastic Modulus Determination of Normal and Glaucomatous Human Trabecular Meshwork

PURPOSE Elevated intraocular pressure (IOP) is a risk factor for glaucoma. The principal outflow pathway for aqueous humor in the human eye is through the trabecular meshwork (HTM) and Schlemms canal (SC). The junction between the HTM and SC is thought to have a significant role in the regulation of IOP. A possible mechanism for the increased resistance to flow in glaucomatous eyes is an increase in stiffness (increased elastic modulus) of the HTM. In this study, the stiffness of the HTM in normal and glaucomatous tissue was compared, and a mathematical model was developed to predict the impact of changes in stiffness of the juxtacanalicular layer of HTM on flow dynamics through this region. METHODS Atomic force microscopy (AFM) was used to measure the elastic modulus of normal and glaucomatous HTM. According to these results, a model was developed that simulated the juxtacanalicular layer of the HTM as a flexible membrane with embedded pores. RESULTS The mean elastic modulus increased substantially in the glaucomatous HTM (mean = 80.8 kPa) compared with that in the normal HTM (mean = 4.0 kPa). Regional variation was identified across the glaucomatous HTM, possibly corresponding to the disease state. Mathematical modeling suggested an increased flow resistance with increasing HTM modulus. CONCLUSIONS The data indicate that the stiffness of glaucomatous HTM is significantly increased compared with that of normal HTM. Modeling exercises support substantial impairment in outflow facility with increased HTM stiffness. Alterations in the biophysical attributes of the HTM may participate directly in the onset and progression of glaucoma.

Proteome analysis of human aqueous humor.

PURPOSE Human aqueous humor (hAH) provides nutrition and immunity within the anterior chamber of the eye. Characterization of the protein composition of hAH will identify molecules involved in maintaining a homeostatic environment for anterior segment tissues. The present study was conducted to analyze the proteome of hAH. METHODS hAH samples obtained during elective cataract surgery were divided into three matched groups and immunodepleted of albumin, IgG, IgA, haploglobin, antitrypsin, and transferrin. Reduced and denatured proteins (20 μg) from each group were separated by gel electrophoresis. Thirty-three gel slices were excised from each of three gel lanes (n = 99), digested with trypsin, and subjected to nanoflow liquid chromatography electrospray ionization tandem mass spectrometry (nano-LC-ESI-MS/MS). The protein component of hAH was also analyzed by antibody-based protein arrays, and selected proteins were quantified. RESULTS A total of 676 proteins were identified in hAH. Of the 355 proteins identified by nano-LC-ESI-MS/MS, 206 were found in all three groups. Most of the proteins identified by nano-LC-ESI-MS/MS had catalytic, enzymatic, and structural properties. Using antibody-based protein arrays, 328 cytokines, chemokines, and receptors were identified. Most of the quantified proteins had concentrations that ranged between 0.1 and 2.5 ng/mL. Ten proteins were identified by both nano-LC-ESI-MS/MS and antibody protein arrays. CONCLUSIONS Proteomic analysis of hAH identified 676 nonredundant proteins. More than 80% of these proteins are novel identifications. The elucidation of the aqueous proteome will establish a foundation for protein function analysis and identification of differentially expressed markers associated with diseases of the anterior segment.

Glaucoma-associated myocilin: a better understanding but much more to learn.

Over a decade has passed since myocilin was identified as the first gene linked to early and late-onset primary open-angle glaucoma. During this time, considerable effort has been put forth to understand the functional role myocilin has in normal and glaucomatous eyes. Myocilin is expressed in many ocular and non-ocular tissues, is found in both intracellular and extracellular spaces, and has been linked to elevations in intraocular pressure. Mutations in the myocilin gene that have been associated with glaucoma appear to confer a gain-of-functional activity rather than loss of function. Unfortunately, what the normal function of myocilin is and how alterations in the function can confer a glaucoma phenotype have yet to be elucidated. We will review the current understanding of myocilin with special emphasis on the structural makeup of the myocilin gene and protein, its possible physiological roles internal and external to ocular cells, the regulation of intraocular pressure as evidenced through the use of perfusion culture systems and animal models, and as a causative agent in some forms of glaucoma.

Genetic Modification of Human Trabecular Meshwork with Lentiviral Vectors

Glaucoma, a group of optic neuropathies, is the leading cause of irreversible blindness. Neuronal apoptosis in glaucoma is primarily associated with high intraocular pressure caused by chronically impaired outflow of aqueous humor through the trabecular meshwork, a reticulum of mitotically inactive endothelial-like cells located in the angle of the anterior chamber. Anatomic, genetic, and expression profiling data suggest the possibility of using gene transfer to treat glaucomatous intraocular pressure dysregulation, but this approach will require stable genetic modification of the differentiated aqueous outflow tract. We injected transducing unit-normalized preparations of either of two lentiviral vectors or an oncoretroviral vector as a single bolus into the aqueous circulation of cultured human donor eyes, under perfusion conditions that mimicked natural anterior chamber flow and maintained viability ex vivo. Reporter gene expression was assessed in trabecular meshwork from 3 to 16 days after infusion of 1.0 x 10(8) transducing units of each vector. The oncoretroviral vector failed to transduce the trabecular meshwork. In contrast, feline immunodeficiency virus and human immunodeficiency virus vectors produced efficient, localized transduction of the trabecular meshwork in situ. The results demonstrate that lentiviral vectors permit efficient genetic modification of the human trabecular meshwork when delivered via the afferent aqueous circulation, a clinically accessible route. In addition, controlled comparisons in this study establish that feline and human immunodeficiency virus vectors are equivalently efficacious in delivering genes to this terminally differentiated human tissue.

Second-generation trabecular meshwork bypass stent (istent inject) increases outflow facility in cultured human anterior segments

PURPOSE To determine whether a second-generation trabecular meshwork (TM) bypass stent (iStent inject) influences outflow facility in cultured human anterior segments. DESIGN Prospective laboratory investigation using normal human donor eyes. METHODS Human anterior segments (n = 7) were placed in perfusion organ culture. One or 2 iStent inject stents were inserted into the TM within the nasal and/or superior quadrants using a specially designed injector. Anterior segments were returned to culture and perfused for an additional 24 hours. Morphology of the TM and Schlemm canal (SC) was assessed by scanning electron microscopy (SEM) and 3-dimensional micro-computed tomography (3D micro-CT). RESULTS Insertion of 1 iStent inject into the nasal or superior quadrant of the TM increased outflow facility from 0.16 ± 0.05 μL/min/mm Hg to 0.38 ± 0.23 μL/min/mm Hg (P < .03, n = 7), with concurrent pressure reduction from 16.7 ± 5.4 mm Hg to 8.6 ± 4.4 mm Hg. Addition of a second iStent inject further increased outflow facility to 0.78 ± 0.66 μL/min/mm Hg (n = 2). SEM showed the iStent inject flange compressed against the uveal region of the TM, the thorax securely inserted within the TM, and the head located in the lumen of SC. Dilation of SC was noted around the iStent inject head and SC cell disruption was observed at the iStent inject insertion site. 3D micro-CT confirmed iStent inject placement. CONCLUSION iStent inject, a second-generation bypass stent, increased outflow facility in human anterior segment culture. The iStent inject is a promising new device to lower intraocular pressure via TM bypass.

HUMAN CORNEAL ENDOTHELIAL CELL TRANSPLANTATION IN A HUMAN EX VIVO MODEL

PURPOSE To determine the effects of incorporating superparamagnetic microspheres (SPMs) into cultured human corneal endothelial cells (HCECs) and to describe preliminary experiments of HCEC transplantation, facilitated by SPMs and an external magnetic field, in a human anterior segment ex vivo model. METHODS HCECs were cultured as monolayers and incorporated with magnetite oxide SPMs (900, 300, and 100 nm) at different concentrations. Cell viability, migration toward a magnetic field, and light transmittance were measured after incorporation of the SPMs. HCEC transplantation into the eyes of human recipients was investigated by subjecting anterior segments in organ culture to an external magnetic field. Light and electron microscopy were used to assess HCEC attachment to corneal stroma. RESULTS SPMs were incorporated into the cytoplasm of HCECs after overnight incubation. None of the SPMs affected the short-term viability of cultured HCECs (P > 0.14, n = 6) or their light transmittance (P > 0.06, n = 5), although there was a trend toward decreased transmittance with the higher concentration of 900-nm SPMs. Cell migration toward a magnetic field was higher for HCECs with incorporated SPMs than for HCECs without SPMs (P < or = 0.01, n = 6), with dose-response relationships evident for the 300- and 100-nm SPMs. SPMs facilitated the attachment of HCECs to the corneal stroma in the human anterior segment model with minimal change in intracameral (intraocular) pressure. CONCLUSIONS SPMs facilitate migration of HCECs toward a magnetic source and attachment of cells to the corneal stroma without affecting cell viability or light transmittance. The human anterior segment model can be used to study HCEC transplantation.

Preferential fluid flow in the human trabecular meshwork near collector channels

PURPOSE To determine whether preferential pathways exist within the human trabecular meshwork, pigmented and nonpigmented regions adjacent to and between collector channels were examined, and the configuration of the juxtacanalicular tissue (JCT) was analyzed. METHODS Healthy whole human eyes were perfused at 10 or 25 mm Hg with 0.5 mum fluorescent beads. Tissue wedges of pigmented and nonpigmented meshwork (with and without collector channels) were dissected from each eye and examined by confocal microscopy. Bead concentration adjacent to and between collector channels was quantified. The configuration of the JCT adjacent to collector channels from whole eyes perfused at 20 mm Hg was analyzed by light microscopy. RESULTS Eyes perfused at 25 mm Hg had more beads adjacent to collector channels in pigmented than in nonpigmented regions (4.9%+/-3.5% vs. 1.1%+/-0.9%; P=0.02). In pigmented regions without collector channels, bead concentration was decreased by fivefold (4.9%+/-3.5% vs. 0.96%+/-0.88%; P=0.04). Perfusion at 25 mm Hg increased beads by threefold under pigmented collector channels compared with the same regions in eyes perfused at 10 mm Hg. Expansion of the JCT occurred more often under collector channels at 25 mm Hg than at 10 mm Hg (44% vs. 17%; P=0.01). The JCT region under collector channels was expanded compared with JCT regions between adjacent collector channels (1053+/-424 microm(2) vs. 571+/-216 microm(2); P<0.001). CONCLUSIONS Increased levels of beads in pigmented trabecular meshwork adjacent to collector channels suggest preferential flow pathways are present in human trabecular meshwork. At elevated pressure, the JCT region under collector channels is expanded, possibly because of increased fluid flow.

Body mass index has a linear relationship with cerebrospinal fluid pressure.

PURPOSE To examine the relationship between body mass index (BMI) and cerebrospinal fluid pressure (CSFP), as low BMI and low CSFP have recently been described as risk factors for primary open-angle glaucoma (POAG). METHODS This was a retrospective review of the electronic medical records of patients who had CSFP measured by lumbar puncture and data to calculate BMI at the Mayo Clinic (Rochester, MN). Exclusion criteria included diagnoses, surgical procedures and medications known to affect CSFP. Mean CSFP for each unit BMI was calculated. The probabilities were two-tailed, and the α level was set at P < 0.05. Patients with documented BMI, CSFP, and intraocular pressure (IOP) were analyzed for the relationship between IOP and BMI. RESULTS A total of 4235 patients, primarily of Caucasian descent, met the entry criteria. Median BMI was 26 and the mean CSFP was 10.9 ± 2.6 mm Hg. The increase in CSFP with increasing BMI was linear with an r(2) = 0.20 (P < 0.001). CSFP increased by 37.7% from BMI 18 (8.6 ± 2.1 mm Hg) to BMI 39 (14.1 ± 2.5 mm Hg). The r(2) (0.21) of the model of BMI and sex was similar to the r(2) of a BMI-only model (0.20). There was no relation between IOP and BMI within a subgroup of the study population (r (2) = 0.005; P = 0.14). CONCLUSIONS CSFP has a positive, linear relationship with BMI. IOP is not influenced by BMI. If CSFP influences the risk for POAG, then individuals with a lower BMI may have an increased risk for developing POAG. Similarly, a higher BMI may be protective.

Cerebrospinal Fluid Pressure Decreases with Older Age

Purpose Clinical studies implicate low cerebrospinal fluid pressure (CSFP) or a high translaminar pressure difference in the pathogenesis of primary open angle glaucoma (POAG) and normal tension glaucoma (NTG). This study was performed to examine the effect of age, sex, race and body mass index (BMI) on CSFP. Methods Electronic medical records from all patients who had a lumbar puncture (LP) performed at the Mayo Clinic from 1996–2009 were reviewed. Information including age, sex, race, height and weight, ocular and medical diagnoses, intraocular pressure (IOP) and LP opening pressure was obtained. Patients using medications or with medical diagnoses known to affect CSFP, and those who underwent neurosurgical procedures or where more than one LP was performed were excluded from analysis. Results Electronic medical records of 33,922 patients with a history of having an LP during a 13-year period (1996–2009) were extracted. Of these, 12,118 patients met all entry criteria. Relative to mean CSFP at age group 20–49 (mean 11.5±2.8 mmHg), mean CSFP declined steadily after age 50, with percent reduction of 2.5% for the 50–54 age group (mean 11.2±2.7 mmHg, p<0.002) to 26.9% for the 90–95 group (mean 8.4±2.4 mmHg, p<0.001). Females had lower CSFP than males throughout all age groups. BMI was positively and independently associated with CSFP within all age groups. Conclusion There is a sustained and significant reduction of CSFP with age that begins in the 6th decade. CSFP is consistently lower in females. BMI is positively and independently associated with CSFP in all age groups. The age where CSFP begins to decline coincides with the age where the prevalence of POAG increases. These data support the hypothesis that reduced CSFP may be a risk factor for POAG and may provide an explanation for the mechanism that underlies the age-related increase in the prevalence of POAG and NTG.