Optic Nerve Head Tissue Research Articles

The Impact of Intraocular Pressure Elevation on Optic Nerve Head and Choroidal Blood Flow.

To use laser speckle flowgraphy (LSFG) to assess blood flow (BF) in the optic nerve head (ONH) tissue and choroid during elevated intraocular pressure (IOP). This prospective study included 20 eyes of 20 healthy volunteers. The testing protocol had a baseline phase, two elevated IOP phases (+10 and +20 mm Hg), and a recovery phase. IOP was elevated by pushing against the eyelid with a novel tubular device attached to the LSFG apparatus. Measurement parameters in each phase included: LSFG-derived mean blur rate (MBR) and flow acceleration index (FAI); systemic parameters, and IOP. The % change against baseline was calculated for each phase. The protocol was repeated five times to calculate the coefficient of variation (CV) for % change MBR and to determine the effect of mydriasis on % change MBR. We compared % change MBR and FAI and evaluated the relationship between % change ocular perfusion pressure (OPP) and MBR in the choroid and ONH tissue. The % change MBR was highly reproducible (CV: 6.1-8.7%) and not affected by mydriasis (P = 0.57-0.96). The % change MBR and FAI were higher in the ONH tissue than choroid during IOP elevation (P = 0.04). The % change OPP and MBR showed positive linear correlations and two-segmental linear correlations in the choroid and ONH tissue, respectively (P < 0.01). Hemodynamics during IOP elevation differ in the choroid and ONH tissue. LSFG enables highly reproducible assessment of the dynamic autoregulation of ocular BF in the ONH tissue.

DRUNET: a dilated-residual U-Net deep learning network to segment optic nerve head tissues in optical coherence tomography images.

Given that the neural and connective tissues of the optic nerve head (ONH) exhibit complex morphological changes with the development and progression of glaucoma, their simultaneous isolation from optical coherence tomography (OCT) images may be of great interest for the clinical diagnosis and management of this pathology. A deep learning algorithm (custom U-NET) was designed and trained to segment 6 ONH tissue layers by capturing both the local (tissue texture) and contextual information (spatial arrangement of tissues). The overall Dice coefficient (mean of all tissues) was 0.91 ± 0.05 when assessed against manual segmentations performed by an expert observer. Further, we automatically extracted six clinically relevant neural and connective tissue structural parameters from the segmented tissues. We offer here a robust segmentation framework that could also be extended to the 3D segmentation of the ONH tissues.

Optic disc drusen: understanding an old problem from a new perspective.

Optic disc drusen (ODD) are acellular deposits located in the optic nerve head of up to 2.4% of the population. They may develop as by-products of impaired axonal metabolism in genetically predisposed individuals, in whom a narrow scleral canal is hypothesized to play a role. Although ODD are often considered as benign innocent bystanders, recognized as part of a routine ophthalmological examination, the vast majority of patients with ODD have visual field defects. Optic disc drusen (ODD)-associated complications with severe visual loss, most often due to anterior ischaemic optic neuropathy, are also known to occur. There are no treatments available to prevent or ameliorate the vision loss caused by ODD. In children, the ODD are usually uncalcified and buried within the optic nerve head tissue. In these cases, the condition can be difficult to diagnose, as it often resembles a papilloedema with optic nerve head swelling caused by raised intracranial pressure. During the teenage years, the ODD progressively become more calcified and probably also larger, which allow them to be visible on ophthalmoscopy. With the advent and proper utilization of high-resolution modalities of optical coherence tomography (OCT), it has now become possible to detect even the smallest and most deeply located ODD. This allows for ODD detection at a much earlier developmental stage than has previously been possible and enhances the possibilities of research in underlying mechanisms. A review of the literature on ODD was conducted using the PUBMED database. The review focuses on the current knowledge regarding pathogenesis, diagnostics, clinical disease-tracking methodologies, structure-function relationships and treatment strategies of ODD.

3D Histomorphometric Reconstruction and Quantification of the Optic Nerve Head Connective Tissues.

Accurately characterizing the 3D geometry of the optic nerve head neural and connective tissues has been the goal of a large and important body of scientific work. In the present report, we summarize our methods for the high-resolution, digital, 3D histomorphometric reconstruction of the optic nerve head tissues, including their visualization, parameterization, and quantification. In addition, we present our methods for between-eye comparisons of this anatomy, and their use to determine animal-specific and experiment-wide experimental glaucoma versus Control eye differences in the unilateral, monkey experimental glaucoma model. Finally, we demonstrate its application to finite element modeling, 3D optic nerve head reconstruction of other species, and 3D optic nerve head reconstructions using other imaging modalities.

Development of a Platform for Studying 3D Astrocyte Mechanobiology: Compression of Astrocytes in Collagen Gels.

Glaucoma is a common optic neuropathy characterized by retinal ganglion cell death. Elevated intraocular pressure (IOP), a key risk factor for glaucoma, leads to significant biomechanical deformation of optic nerve head (ONH) cells and tissues. ONH astrocytes respond to this deformation by transforming to a reactive, proliferative phenotype, which has been implicated in the progression of glaucomatous vision loss. However, little is known about the mechanisms of this transformation. In this study, we developed a 3D collagen gel culture system to mimic features of ONH deformation due to elevated IOP. Compressive loading of astrocyte-seeded collagen gels led to cell alignment perpendicular to the direction of strain, and increased astrocyte activation, as assayed by GFAP, vimentin, and s100β levels, as well as MMP activity. This proof-of-concept study shows that this system has potential for studying mechanisms of astrocyte mechanobiology as related to the pathogenesis of glaucoma. Further work is needed to establish the possible interplay of mechanical stimulation, matrix properties, and hypoxia on the observed response of astrocytes.

Morphology of the optic nerve head in glaucomatous eyes with visual field defects in superior or inferior hemifield

To evaluate the morphology of optic nerve head (ONH) and border tissue (BT) of Elschnig in glaucomatous eyes with visual field defects in superior or inferior hemifield. In a case-control study, we included 25 patients with superior arcuate scotoma, 25 patients with inferior arcuate scotoma, and 25 healthy controls. They received visual field testing, measurement of peripapillary retinal nerve fiber layer (RNFL) thickness, and ONH examination in a radial pattern with spectral-domain optical coherence tomography. In each ONH scan, the length of Bruch membrane opening (BMO) and BT were measured. Pattern deviation of 6 areas of the visual field and RNFL thickness in corresponding sectors was calculated. Mean BMO length did not differ between groups. Compared with controls, glaucomatous eyes with superior scotoma had a greater BT length in inferior sectors (p<0.001), and eyes with inferior scotoma had a greater BT length in superotemporal sectors (p = 0.006). In both groups, a significant correlation was found between BT length and pattern deviation and RNFL thickness of corresponding sectors of superior and inferior hemifields. In patients with arcuate scotoma in one hemifield, the length of the BT correlates with glaucomatous anatomical and functional damage.

A Novel Method for Assessing Lamina Cribrosa Structure Ex Vivo Using Anterior Segment Enhanced Depth Imaging Optical Coherence Tomography.

The purpose of this study is to investigate the use of anterior segment enhanced depth imaging (EDI) optical coherence tomography (OCT) for ex vivo lamina cribrosa (LC) imaging. After removing anterior segment and vitreous, the optic nerve head (ONH) tissue of porcine eyes was placed on a customized eye holder for imaging. Serial EDI OCT B-scans (interval, ∼35 μm) of the ONH were obtained using anterior segment module of spectral-domain OCT. Various conditions were tested for better quality LC images. After EDI OCT, serial histologic sections were obtained (distance between sections, ∼5 μm). LC structures in OCT scans were compared with those in histologic sections. Three-dimensional LC reconstructions created using serial OCT scans were compared with LC structures in disc photographs. ONHs of 3 enucleated eyes were examined. The LC was more clearly imaged when the retina and part of the prelaminar tissue were removed (quality score, 39.01±3.30 vs. 26.40±5.85; P<0.001) and when the tissue was kept moist during imaging (quality score, 38.70±2.11 vs. 36.18±5.98; P<0.001). LC image quality was similar before and after fixation (quality score, 38.84±6.57 vs. 39.21±9.69; P=0.79). LC beams and part of retrolaminar glial columns identified in OCT scans matched those in histologic sections. LC beams and pores in 3-dimensional reconstructions matched those in disc photographs. High-resolution cross-sectional images of the LC, comparable to histologic sections, can be obtained using anterior segment EDI OCT in ex vivo eyes with proper tissue preparation.

Relationship between laser speckle flowgraphy and optical coherence tomography angiography measurements of ocular microcirculation.

The purpose of this study was to investigate the relationship between laser speckle flowgraphy (LSFG) and optical coherence tomography angiography (OCTA) measurements of ocular microcirculation in normal and open-angle glaucoma (OAG) subjects. This study included 18 eyes of 18 OAG patients and ten eyes of ten age-matched healthy controls. LSFG was used to measure mean blur rate (MBR) in the optic nerve head (ONH) vessel area (MV) and tissue area (MT). OCTA was used to measure a new parameter, peripapillary relative intensity (PRI), in the superficial retina, superficial choroid, and deep choroid. Statistical associations were then determined. MV, MT, superficial-retinal PRI, and superficial-choroidal PRI were lower in the OAG subjects than the controls (P = 0.02, P < 0.001, P = 0.02 and P = 0.008, respectively). Superficial-retinal PRI was correlated with MV and MT (R = 0.68, P < 0.001 and R = 0.63, P < 0.001, respectively). Superficial-choroidal PRI was also correlated with MV and MT (R = 0.45, P = 0.02 and R = 0.57, P = 0.002, respectively). Multiple regression analysis revealed that MV and MT independently contributed to superficial-retinal PRI (P = 0.008 and P = 0.04, respectively), while only MT contributed to superficial-choroidal PRI (P = 0.03). Our finding that OCTA-measured PRI was related to LSFG-measured MBR was reasonable, considering the vascular anatomy of the eye. Thus, PRI, like MBR, may be a promising biomarker of ocular microcirculation that can reveal the presence of ocular diseases such as OAG.

Predictions of Optic Nerve Traction Forces and Peripapillary Tissue Stresses Following Horizontal Eye Movements.

To use finite element (FE) analysis to predict the optic nerve sheath traction forces that act on the optic nerve head (ONH) following horizontal eye movements, and the resulting stress levels in the peripapillary connective tissues of the ONH (Bruch's membrane [BM] and sclera). An FE model of a healthy eye was reconstructed in primary gaze position that included details from the orbital and ONH tissues. Optic nerve sheath traction forces and peripapillary tissue stresses in both adduction and abduction (13°) were computed using FE analysis. Our models predicted that, following eye movements, the ONH was sheared in the transverse plane due to the pulling action of the optic nerve. The optic nerve sheath traction forces were 90 mN in abduction and 150 mN in adduction. Peripapillary tissue stresses were concentrated in the nasal and temporal quadrants. In adduction, scleral stresses were highest in the temporal region, and BM stresses were slightly higher in the nasal region. This trend was reversed in abduction. Following eye movements, our models predicted high optic nerve sheath traction forces of the same order of magnitude as extraocular muscle forces. Optic nerve traction resulted in significant peripapillary stresses, and thus may have a role to play in the development of peripapillary zones, glaucoma, and myopia.

A Digital Staining Algorithm for Optical Coherence Tomography Images of the Optic Nerve Head.

PurposeTo digitally stain spectral-domain optical coherence tomography (OCT) images of the optic nerve head (ONH), and highlight either connective or neural tissues.MethodsOCT volumes of the ONH were acquired from one eye of 10 healthy subjects. We processed all volumes with adaptive compensation to remove shadows and enhance deep tissue visibility. For each ONH, we identified the four most dissimilar pixel-intensity histograms, each of which was assumed to represent a tissue group. These four histograms formed a vector basis on which we ‘projected' each OCT volume in order to generate four digitally stained volumes P1 to P4. Digital staining was also verified using a digital phantom, and compared with k-means clustering for three and four clusters.ResultsDigital staining was able to isolate three regions of interest from the proposed phantom. For the ONH, the digitally stained images P1 highlighted mostly connective tissues, as demonstrated through an excellent contrast increase across the anterior lamina cribrosa boundary (3.6 ± 0.6 times). P2 highlighted the nerve fiber layer and the prelamina, P3 the remaining layers of the retina, and P4 the image background. Further, digital staining was able to separate ONH tissue layers that were not well separated by k-means clustering.ConclusionWe have described an algorithm that can digitally stain connective and neural tissues in OCT images of the ONH.Translational RelevanceBecause connective and neural tissues are considerably altered in glaucoma, digital staining of the ONH tissues may be of interest in the clinical management of this pathology.

Verification of a virtual fields method to extract the mechanical properties of human optic nerve head tissues in vivo.

We aimed to verify a custom virtual fields method (VFM) to estimate the patient-specific biomechanical properties of human optic nerve head (ONH) tissues, given their full-field deformations induced by intraocular pressure (IOP). To verify the accuracy of VFM, we first generated 'artificial' ONH displacements from predetermined (known) ONH tissue biomechanical properties using finite element analysis. Using such deformations, if we are able to match back the known biomechanical properties, it would indicate that our VFM technique is accurate. The peripapillary sclera was assumed anisotropic hyperelastic, while all other ONH tissues were considered isotropic. The simulated ONH displacements were fed into the VFM algorithm to extract back the biomechanical properties. The robustness of VFM was also tested against rigid body motions and noise added to the simulated displacements. Then, the computational speed of VFM was compared to that of a gold-standard stiffness measurement method (inverse finite element method or IFEM). Finally, as proof of principle, VFM was applied to IOP-induced ONH deformation data (obtained from one subject's eye imaged with OCT), and the biomechanical properties of the prelamina and lamina cribrosa (LC) were extracted. From given ONH displacements, VFM successfully matched back the biomechanical properties of ONH tissues with high accuracy and efficiency. For all parameters, the percentage errors were less than 0.05%. Our method was insensitive to rigid body motions and was also able to recover the material parameters in the presence of noise. VFM was also found 125 times faster than the gold-standard IFEM. Finally, the estimated shear modulus for the prelamina and the LC of the studied subject's eye were 33.7 and 63.5 kPa, respectively. VFM may be capable of measuring the biomechanical properties of ONH tissues with high speed and accuracy. It has potential in identifying patient-specific ONH biomechanical properties in the clinic if combined with optical coherence tomography.

EFFECT OF MECHANICAL PROPERTIES OF THE SUBSTRATE TISSUES ON THE DETERMINATION OF ELASTIC MODULUS OF THE SCLERA USING INDENTATION TEST

The sclera is an important connective tissue that protects the sensitive layers within the eyeball. Identifying the mechanical properties of the sclera near the posterior pole is necessary to analyze the deformation of the sclera and stresses changing in the optic nerve head tissues. We propose a method to determine the mechanical properties of the sclera using dimensional analysis, finite element method and the indentation test. The elastic moduli of the sclera for different indentation depths and positions were identified. We found that the elastic moduli of the sclera varied with indentation depth. This was due to the effect of the mechanical properties of the substrate tissues inside the sclera. The elastic modulus of the choroid had the biggest effect on the determination of elastic modulus of the sclera, whereas that of the vitreous body could be ignored when the ratio of the indentation depth to the thickness of the sclera was less than 0.5. The effects of mechanical properties of the substrate tissues become more pronounced at greater indentation depths.

Deformation of Optic Nerve Head and Peripapillary Tissues by Horizontal Duction

To ascertain deformation of the optic nerve head (ONH) and peripapillary tissues caused by horizontal duction. Prospective, experimental study. Optical coherence tomography of the ONH region was performed in 23 eyes of 12 normal volunteers in central gaze and increasing (10, 20, and 30 degrees) adduction and abduction. Main outcome measures were changes from central gaze in the configuration of the ONH and peripapillary tissues in eccentric gazes. Adduction but not abduction was associated with significant, progressive relative posterior displacement of the temporal peripapillary retinal pigment epithelium (tRPE) from its position in central gaze reaching 49 ± 10μm in 30-degree adduction (standard error of mean, P < .0001). Absolute (anterior or posterior) optic cup displacement (OCD) averaged 41 ± 7μm in 30-degree adduction. Linear regression showed significant effect of adduction on absolute OCD (slope 1.09 ± 0.36 μm/degree, P= .0037). In 20-degree and 30-degree adduction, all eyes exhibited significant progressive temporal ONH tilting reaching 3.1 ± 0.4 degrees in 30-degree adduction (P < .0001). Abduction was not associated with significant peripapillary RPE displacement, OCD, or ONH tilt. Both nasal and temporal peripapillary choroid averaged 9-19μm thinner in adduction and abduction than in central gaze (P < .02). Adduction temporally tilts and displaces the prelaminar ONH and peripapillary tissues. Both adduction and abduction compress the peripapillary choroid. These effects support magnetic resonance imaging and biomechanical evidence that adduction imposes strain on the ONH and peripapillary tissues. Repetitive strain from eye movements over decades might in susceptible individuals lead to optic neuropathies such as normal tension glaucoma.

Optic Nerve Head Blood Flow, as Measured by Laser Speckle Flowgraphy, Is Significantly Reduced in Preperimetric Glaucoma

ABSTRACTPurpose: To compare optic nerve head (ONH) blood flow in healthy eyes, eyes with preperimetric glaucoma (PPG), and eyes with mild normal-tension glaucoma (NTG) using laser speckle flowgraphy (LSFG).Methods: In 172 eyes (normal: 44 eyes; PPG: 62 eyes; mild NTG: 66 eyes), LSFG was used to measure mean blur rate in the ONH tissue area (MBRT), an index of capillary blood flow. Multiple regression analysis was performed to determine factors affecting circumpapillary retinal nerve fiber layer thickness (cpRNFLT), mean deviation (MD) and pattern standard deviation (PSD) of the visual field.Results: Despite similar characteristics in age, refractive error and systemic variables among the study groups, MBRT in the normal eyes differed significantly from both the PPG and mild NTG eyes (P = 0.001, P < 0.001, respectively). Multiple regression analysis revealed that MBRT was an independent factor affecting cpRNFLT, MD and PSD (P < 0.001, P = 0.001, P = 0.003, respectively).Conclusion: ONH blood flow was detectibly reduced in eyes with PPG, in close association with structural and visual field damage. This suggests that measuring ONH tissue-area blood flow with LSFG may be a useful way of monitoring glaucoma severity, even in the early stages of glaucoma.

Finite Element Analysis Predicts Large Optic Nerve Head Strains During Horizontal Eye Movements.

We combined finite element (FE) analysis and dynamic magnetic resonance imaging (MRI) to estimate optic nerve head (ONH) strains during horizontal eye movements, and identified factors influencing such strains. We also compared ONH strains (prelamina, lamina cribrosa, and retrolamina strains) induced by eye movements to those induced by IOP. The ocular globes and orbits of a healthy subject were visualized during horizontal eye movements (up to 13°), using dynamic MRI. A baseline FE model of one eye was reconstructed in the primary gaze position, including details from the orbital and ONH tissues. Finite element-derived ONH strains induced by eye movements were compared to those resulting from an IOP of 50 mm Hg. Finally, a FE sensitivity study was performed, in which we varied the stiffness of all ONH connective tissues, to understand their influence on ONH strains. Our models predicted that, during horizontal eye movements, the optic nerve pulled the ONH posteriorly. Optic nerve head strains following a lateral eye movement of 13° were large and higher than those resulting from an IOP of 50 mm Hg. These results held true even with variations in connective tissue stiffness. We also found that stiff sclerae reduced lamina cribrosa and prelamina strains during eye movements, but stiff optic nerve sheaths significantly increased those strains. Our models predicted high ONH strains during eye movements, which were aggravated with stiffer optic nerve sheaths. Further studies are needed to explore links between ONH strains induced by eye movements and axonal loss in glaucoma.

Finite Element Modeling of Factors Influencing Optic Nerve Head Deformation Due to Intracranial Pressure.

Visual impairment and intracranial pressure (VIIP) syndrome is a health concern for long-duration spaceflight, and a proposed risk factor is elevation of intracranial pressure (ICP). Our goal was to use finite element modeling to simulate how elevated ICP and interindividual differences affect tissue deformation within the optic nerve head (ONH). We considered three ICP conditions: the upright and supine position on earth and an elevated ICP assumed to occur in chronic microgravity. Within each condition we used Latin hypercube sampling to consider a range of pressures and ONH tissue mechanical properties, determining the influence of each input on the following outcome measures: peak strains in the prelaminar tissue, lamina cribrosa, and retrolaminar optic nerve. Elevated strains can alter cell phenotype and induce tissue remodeling. Elevating ICP increased the strains in the retrolaminar optic nerve. Variations in IOP, ICP, and in optic nerve and lamina cribrosa stiffness had the strongest influence on strains within the ONH. We predicted that 5% to 47% of individuals in microgravity would experience peak strains in the retrolaminar optic nerve larger than expected on earth. Having a soft optic nerve or pia mater and elevated ICP were identified as risk factors for these "extreme" strains. Intracranial pressure and mechanical properties of the ONH influence the risk for experiencing extreme strains in the retrolaminar optic nerve. These extreme strains may activate mechanosensitive cells that induce tissue remodeling and are a risk factor for the development of VIIP. Future studies must also consider variations in ONH anatomy.

Laser speckle and hydrogen gas clearance measurements of optic nerve circulation in albino and pigmented rabbits with or without optic disc atrophy.

The purpose of this study was to evaluate the correlation between laser speckle flowgraphy measurements of mean blur rate (MBR) and hydrogen gas clearance measurements of capillary blood flow (CBF) in the optic nerve head (ONH) of albino and pigmented rabbits, with or without chronic ischemia-induced ONH atrophy. The ONH MBR and ONH CBF were measured at baseline, 30 and 60 minutes after the intravenous administration of endothelin-1 (ET-1) (10(-10) mol/kg) in six albino and six pigmented rabbit eyes. The ONH MBR and ONH CBF were also measured in nine pigmented rabbit eyes that underwent the intravitreal administration of ET-1 (20 pmol) twice per week for 4 weeks to provoke chronic ischemia-induced ONH atrophy. In the group that received intravenous ET-1, average measurements of ONH MBR and ONH CBF at all time points were correlated in both the albino (r = 0.88, P < 0.001, n = 18) and pigmented rabbits (r = 0.85, P < 0.001, n = 18), with no intrarabbit correlations (P = 0.524). The ONH MBR and ONH CBF were also correlated in the model of chronic ischemia-induced ONH atrophy (r = 0.78, P = 0.013, n = 9). Pooled ONH MBR and ONH CBF measurements in both the intravenous and intravitreal groups were also highly correlated (r = 0.87, P < 0.001, n = 45), with no significant intergroup differences in the relationship between ONH MBR and ONH CBF (P = 0.138). Regardless of the presence of fundus pigmentation or ONH atrophy, ONH MBR and ONH CBF were highly correlated, suggesting that MBR in the ONH tissue is usable for interindividual and intergroup comparisons.

BDNF impairment is associated with age-related changes in the inner retina and exacerbates experimental glaucoma

Brain-derived neurotrophic factor (BDNF) stimulation of its high-affinity receptor TrkB results in activation of pro-survival cell-signalling pathways that can afford neuroprotection to the retina. Reduction in retrograde axonal transport of neurotrophic factors such as BDNF from the brain to the neuronal cell bodies in the retina has been suggested as a critical factor underlying progressive and selective degeneration of ganglion cell layer and optic nerve in glaucoma. We investigated the role of BDNF in preserving inner retinal homeostasis in normal and glaucoma states using BDNF+/− mice and compared it with wild type controls. This study demonstrated that BDNF+/− animals were more susceptible to functional, morphological and molecular degenerative changes in the inner retina caused by age as well as upon exposure to experimental glaucoma caused by increased intraocular pressure. Glaucoma induced a down regulation of BDNF/TrkB signalling and an increase in levels of neurotoxic amyloid β 1–42 in the optic nerve head which were exacerbated in BDNF+/− mice. Similar results were obtained upon analysing the human optic nerve head tissues. Our data highlighted the role of BDNF in maintaining the inner retinal integrity under normal conditions and the detrimental effects of its insufficiency on the retina and optic nerve in glaucoma.

Mechanosensitive release of adenosine 5'-triphosphate through pannexin channels and mechanosensitive upregulation of pannexin channels in optic nerve head astrocytes: a mechanism for purinergic involvement in chronic strain.

As adenosine 5'-triphosphate (ATP) released from astrocytes can modulate many neural signaling systems, the triggers and pathways for this ATP release are important. Here, the ability of mechanical strain to trigger ATP release through pannexin channels and the effects of sustained strain on pannexin expression were examined in rat optic nerve head astrocytes. Astrocytes released ATP when subjected to 5% of equibiaxial strain or to hypotonic swelling. Although astrocytes expressed mRNA for pannexins 1-3, connexin 43, and VNUT, pharmacological analysis suggested a predominant role for pannexins in mechanosensitive ATP release, with Rho kinase contribution. Astrocytes from panx1(-/-) mice had reduced baseline and stimulated levels of extracellular ATP, confirming the role for pannexins. Swelling astrocytes triggered a regulatory volume decrease that was inhibited by apyrase or probenecid. The swelling-induced rise in calcium was inhibited by P2X7 receptor antagonists A438079 and AZ10606120, in addition to apyrase and carbenoxolone. Extended stretch of astrocytes in vitro upregulated expression of panx1 and panx2 mRNA. A similar upregulation was observed in vivo in optic nerve head tissue from the Tg-MYOC(Y437H) mouse model of chronic glaucoma; genes for panx1, panx2, and panx3 were increased, whereas immunohistochemistry confirmed increased expression of pannexin 1 protein. In summary, astrocytes released ATP in response to mechanical strain, with pannexin 1 the predominant efflux pathway. Sustained strain upregulated pannexins in vitro and in vivo. Together, these findings provide a mechanism by which extracellular ATP remains elevated under chronic mechanical strain, as found in the optic nerve head of patients with glaucoma.

Indentation Testing of the Optic Nerve Head and Posterior Sclera

The mechanical behavior of the optic nerve head (ONH) and the surrounding sclera play important roles in the development of optic neuropathy. We assessed the indentation behavior of the ONH and the surrounding sclera in unfixed human autopsy eyes from individuals between 27 and 87 years of age. Our testing device utilized a round-tipped 250 µm diameter stainless steel probe to measure the force applied in discrete 50 µm steps from the surface of the tissues to a depth of 400 µm. Thirteen eyes from eight individuals were indented in various locations within and around the ONH and the posterior sclera. Tissue thickness, force and depth were recorded at each position tested. Data was analyzed by evaluating the recorded force as a function of the depth of indentation. The data showed that, for both the sclera and the ONH, the force rose exponentially with increased depth of indentation. Apparent stiffness values were estimated using 2 different equations that assess soft biologic tissues. Results showed that significantly higher levels of force were required with aging to indent the posterior sclera, with a corresponding increase in stiffness values. However, no significant increase in indentation force as a function of age was noted for the ONH. Interestingly, the data suggests that some individuals have relatively large differences in the stiffness between the ONH and the posterior sclera, while others show less difference regardless of age. This data supports the notion that some individuals have relatively softer ONH tissue, and as the sclera becomes stiffer with age, this difference is magnified. This difference may be important in understanding the biomechanical contribution to the individually different susceptibility to glaucoma.