Controlled Elevation of Intraocular Pressure in Anesthetized Mice.

Our purpose was to develop a protocol for prolonged anesthesia in mice and evaluate optic nerve axon injury in response to 4 hours of controlled elevation of intraocular pressure (CEI). During CEI, C57BL/6 male mice (3-5 months old) were anesthetized with 1.5% isoflurane with 100% oxygen for 4 hours and placed on a warm platform, with expired gas and anesthetic actively evacuated. Lactated ringers (0.5 ml) with 5% dextrose was administered subcutaneously at the start and end of CEI. Physiological parameters (oxygen saturation = O2, heart rate = HR, systolic blood pressure = SBP, and temperature) were monitored throughout the 4-hour CEI. One eye was cannulated with polyurethane tubing connected to a balanced salt solution reservoir and IOP elevated to 20 (N = 18), 30 (N = 13), 50 (N = 14), and 60 mmHg (N = 16). An additional group of 22 female mice was exposed to CEI of 60 mmHg. Fourteen days after CEI, optic nerves were assessed for axonal injury by masked observers that assigned a grade on a scale from 1 (normal) to 5 (>50% of axons degenerating). CEI optic nerve injury was compared to injury assessed in contralateral optic nerves (N = 84) and naïve optic nerves (N = 18) using a one-way ANOVA followed by Kruskal-Wallis test for multiple comparisons. The relationship between optic nerve injury, physiological parameters, and IOP were assessed by linear regression analyses. Physiologic parameters remained stable throughout CEI (O2 = 95 ± 9%; HR = 450 ± 39; SBP = 102 ± 15 mmHg, and temperature = 38 ± 0.7°C) and were not statistically different between groups (all comparisons had P > 0.5). Mean optic nerve injury grades (±SD) for naïve optic nerves (1.01 ± 0.02) were not significantly different from fellow/contralateral optic nerves (1.03 ± 0.07, P>0.99), or from CEI of 20 mmHg (1.04 ± 0.08, P>0.99) or 30 mmHg (1.05 ± 0.06, P=0.6). However, animals exposed to CEI of 50 mmHg (2.09 ± 1.43, P=0.0005) and 60 mmHg (male: 2.86 ± 1.30, P<0.0001, female: 1.63 ± 1.00, P = 0.0006) developed significant optic nerve injury relative to their fellow/contralateral optic nerves. Axonal injury grades following a CEI of 60 mmHg were not significantly different between male and female mice (P=0.19). Optic nerve injury positively correlated (P < 0.0001) with IOP and not with physiological parameters, indicating that the optic nerve injury is IOP-related. In conclusion, prolonged anesthesia in mice requires careful attention to animal physiology. With this, a 4-hour exposure to elevated IOP can produce significant optic nerve injury with IOPs equal to or greater than 50 mmHg. We provide detailed descriptions of methods and materials for producing prolonged elevations of IOP in mice while maintaining and monitoring their physiology, as well as a unique, cost-effective transducer system for monitoring pressure delivery.

A method describing the microdissection of trabecular meshwork tissue from Brown Norway rat eyes.

Glaucoma is often associated with elevated intraocular pressure (IOP), generally due to obstruction of aqueous humor outflow within the trabecular meshwork (TM). Despite many decades of research, the molecular cause of this obstruction remains elusive. To study IOP regulation, several in vitro models, such as perfusion of anterior segments or mechanical stretching of TM cells, have identified several IOP-responsive genes and proteins. While these studies have proved informative, they do not fully recapitulate the in vivo environment where IOP is subject to additional factors, such as circadian rhythms. Thus, rodent animal models are now commonly used to study IOP-responsive genes in vivo. Several single-cell RNAseq studies have been performed where angle tissue, containing cornea, iris, ciliary body tissue in addition to TM, is dissected. However, it is advantageous to physically separate TM from other tissues because the ratio of TM cells is relatively low compared to the other cell types. In this report, we describe a new technique for rat TM microdissection. Evaluating tissue post-dissection by histology and immunostaining clearly shows successful removal of the TM. In addition, TaqMan PCR primers targeting biomarkers of trabecular meshwork (Myoc, Mgp, Chi3l1) or ciliary body (Myh11, Des) genes showed little contamination of TM tissue by the ciliary body. Finally, pitfalls encountered during TM microdissection are discussed to enable others to successfully perform this microsurgical technique in the rat eye.

Retinal capillary oximetry with visible light optical coherence tomography.

Assessing oxygen saturation (sO2) remains challenging but is nonetheless necessary for understanding retinal metabolism. We and others previously achieved oximetry on major retinal vessels and measured the total retinal oxygen metabolic rate in rats using visible-light optical coherence tomography. Here we extend oximetry measurements to capillaries and investigate all three retinal vascular plexuses by amplifying and extracting the spectroscopic signal from each capillary segment under the guidance of optical coherence tomography (OCT) angiography. Using this approach, we measured capillary sO2 in the retinal circulation in rats, demonstrated reproducibility of the results, validated the measurements in superficial capillaries with known perfusion pathways, and determined sO2 responses to hypoxia and hyperoxia in the different retinal capillary beds. OCT capillary oximetry has the potential to provide new insights into the retinal circulation in the normal eye as well as in retinal vascular diseases.

Wide-field sensorless adaptive optics swept-source optical coherence tomographic angiography in rodents.

In this study, we present a sensorless adaptive optics swept-source optical coherence tomographic angiography (sAO-SS-OCTA) imaging system for mice. Real-time graphics processing unit (GPU)-based OCTA image acquisition and processing software were applied to guide wavefront correction using a deformable mirror based on signal strength index (SSI) from both OCT and OCTA images. High-resolution OCTA images with aberrations corrected and contrast enhanced were successfully acquired. Fifty-degree field of view high-resolution montaged OCTA images were also acquired.

Individual-Specific Modeling of Rat Optic Nerve Head Biomechanics in Glaucoma.

Glaucoma is the second leading cause of blindness worldwide and is characterized by the death of retinal ganglion cells (RGCs), the cells that send vision information to the brain. Their axons exit the eye at the optic nerve head (ONH), the main site of damage in glaucoma. The importance of biomechanics in glaucoma is indicated by the fact that elevated intraocular pressure (IOP) is a causative risk factor for the disease. However, exactly how biomechanical insult leads to RGC death is not understood. Although rat models are widely used to study glaucoma, their ONH biomechanics have not been characterized in depth. Therefore, we aimed to do so through finite element (FE) modeling. Utilizing our previously described method, we constructed and analyzed ONH models with individual-specific geometry in which the sclera was modeled as a matrix reinforced with collagen fibers. We developed eight sets of scleral material parameters based on results from our previous inverse FE study and used them to simulate the effects of elevated IOP in eight model variants of each of seven rat ONHs. Within the optic nerve, highest strains were seen inferiorly, a pattern that was consistent across model geometries and model variants. In addition, changing the collagen fiber direction to be circumferential within the peripapillary sclera resulted in more pronounced decreases in strain than changing scleral stiffness. The results from this study can be used to interpret data from rat glaucoma studies to learn more about how biomechanics affects RGC pathogenesis in glaucoma.

Evidence for Prophylactic Transfusion during Pregnancy for Women with Sickle Cell Disease.

The objective of this study was to examine prior studies on maternal and neonatal outcomes with prophylactic compared with emergent blood transfusion in pregnant women with sickle cell disease. A review of the literature was performed. Twenty-one articles were identified and included in the analysis. A generalized linear mixed-effects model was used to analyze the outcomes. Pregnancy outcomes assessed were preeclampsia, pneumonia, pyelonephritis, pain crises, intrauterine growth restriction, neonatal death, perinatal death, and maternal mortality. Women who underwent emergent transfusion were more likely than women who underwent prophylactic transfusion to have the following adverse perinatal outcomes: preterm delivery (adjusted odds ratio [aOR 2.04], 95% confidence interval [CI] 1.14-3.63), pneumonia (aOR 2.98, 95% CI 1.44-6.15), pain crises (aOR 1.67, 95% CI 1.18-2.38), and perinatal death (aOR 1.84, 95% CI 1.06-3.07). Prophylactic transfusion should be reexamined as a potentially beneficial approach to the management of sickle cell disease in pregnancy.

Measuring Glaucomatous Focal Perfusion Loss in the Peripapillary Retina Using OCT Angiography.

PURPOSE: To measure low perfusion areas (LPAs) and focal perfusion loss (FPL) in the peripapillary retina using OCT angiography (OCTA) in glaucoma. DESIGN: Prospective, observational study. PARTICIPANTS: A total of 47 patients with primary open-angle glaucoma (POAG) and 36 normal participants were analyzed. METHODS: One eye of each subject was scanned using an AngioVue (Optovue, Fremont, CA) 4.5-mm OCTA scan centered on the disc. En face nerve fiber layer (NFL) plexus angiogram was generated. With the use of custom software, a capillary density map was obtained by computing the fraction of area occupied by flow pixels after low-pass filtering by local averaging 21×21 pixels. The low-perfusion map is defined by local capillary density below 0.5 percentile over a contiguous area above 98.5 percentile of the normal reference population. The LPA parameter is the cumulative area, and the FPL is the percent capillary density loss (relative to normal mean) integrated over the LPA. MAIN OUTCOME MEASURES: Peripapillary retinal LPA and FPL. RESULTS: Among patients with POAG, 3 had preperimetric glaucoma and 44 had perimetric glaucoma, with visual field (VF) mean deviation (MD) of -5.14±4.25 decibels (dB). The LPA was 3.40±2.29 mm2 in those with POAG and 0.11±0.18 mm2 in normal subjects (P < 0.001). The FPL was 21.8%±17.0% in those with POAG and 0.3%±0.7% in normal subjects (P < 0.001). The diagnostic accuracy as measured by the area under the receiver operating curve was 0.965 for both LPA and FPL, with a sensitivity of 93.7% at 95% specificity. The repeatability as measured by intraclass correlation coefficient was 0.977 for LPA and 0.958 for FPL. The FPL had excellent correlation with VF MD (Spearman's rho = -0.843), which was significantly (P = 0.008) better than the correlation between NFL thickness and VF MD (rho = 0.760). The hemispheric difference correlation between FPL and VF (Spearman's rho = 0.770) was significantly (P < 0.001) higher than the hemispheric difference correlation between LPA and VF (rho = 0.595). CONCLUSIONS: The low-perfusion map and LPA and FPL parameters are able to assess the location and severity of focal glaucoma damage with good agreement with VF.

Imaging retinal structures at cellular-level resolution by visible-light optical coherence tomography.

In vivo high-resolution images are the most direct way to understand retinal function and diseases. Here we report the use of visible-light optical coherence tomography with volumetric registration and averaging to achieve cellular-level retinal structural imaging in a rat eye, covering the entire depth of the retina. Vitreous fibers, nerve fiber bundles, and vasculature were clearly revealed, as well as at least three laminar sublayers in the inner plexiform layer. We also successfully visualized ganglion cell somas in the ganglion cell layer, cells in the inner nuclear layer, and photoreceptors in the outer nuclear layer and ellipsoid zone. This technique provides, to the best of our knowledge, a new means to visualize the retina in vivo at a cellular resolution and may enable detection or discovery of cellular neuronal biomarkers to help better diagnose ocular disease.

Real-time cross-sectional and en face OCT angiography guiding high-quality scan acquisition.

Defocusing, vignetting, and bulk motion degrade the image quality of optical coherence tomography angiography (OCTA) more significantly than structural OCT. The assessment of focus, alignment conditions, and stability of imaging subjects in commercially available OCTA systems are currently based on OCT signal quality alone, without knowledge of OCTA signal quality. This results in low yield rates for further quantification. In this Letter, we developed a novel OCTA platform based on a graphics processing unit (GPU) for a real-time, high refresh rate, B-san-by-B-scan split-spectrum amplitude-decorrelation angiography. The GPU provides a real-time display of both cross-sectional and en face images to assist operators during scan acquisition and ensure OCTA scan quality.

Fast and robust standard-deviation-based method for bulk motion compensation in phase-based functional OCT.

Phase-based optical coherence tomography (OCT), such as OCT angiography (OCTA) and Doppler OCT, is sensitive to the confounding phase shift introduced by subject bulk motion. Traditional bulk motion compensation methods are limited by their accuracy and computing cost-effectiveness. In this Letter, to the best of our knowledge, we present a novel bulk motion compensation method for phase-based functional OCT. Bulk motion associated phase shift can be directly derived by solving its equation using a standard deviation of phase-based OCTA and Doppler OCT flow signals. This method was evaluated on rodent retinal images acquired by a prototype visible light OCT and human retinal images acquired by a commercial system. The image quality and computational speed were significantly improved, compared to two conventional phase compensation methods.

A Methodology for Individual-Specific Modeling of Rat Optic Nerve Head Biomechanics in Glaucoma.

Glaucoma is the leading cause of irreversible blindness and involves the death of retinal ganglion cells (RGCs). Although biomechanics likely contributes to axonal injury within the optic nerve head (ONH), leading to RGC death, the pathways by which this occurs are not well understood. While rat models of glaucoma are well-suited for mechanistic studies, the anatomy of the rat ONH is different from the human, and the resulting differences in biomechanics have not been characterized. The aim of this study is to describe a methodology for building individual-specific finite element (FE) models of rat ONHs. This method was used to build three rat ONH FE models and compute the biomechanical environment within these ONHs. Initial results show that rat ONH strains are larger and more asymmetric than those seen in human ONH modeling studies. This method provides a framework for building additional models of normotensive and glaucomatous rat ONHs. Comparing model strain patterns with patterns of cellular response seen in studies using rat glaucoma models will help us to learn more about the link between biomechanics and glaucomatous cell death, which in turn may drive the development of novel therapies for glaucoma.

Maternal/Perinatal Outcome in Women with Sickle Cell Disease: A Comparison of Two Time Periods.

OBJECTIVE: To compare pregnancy outcomes in women with sickle cell disease from recent deliveries with a similar group delivered earlier. METHODS: During a 12-year period (2005-2016), data from pregnant women with hemoglobin SS or SC were collected from three university medical centers and compared with earlier studies (1979-2003) involving similar patients. The primary endpoints were maternal complications during pregnancy and newborn outcomes. RESULTS: There were 278 patients in the control group (1979-2003) compared with 150 patients in the study group (2005-2016). Women in the study group were older (P < 0.0001) and of less parity (P =0.0001), and complications of preterm delivery, preeclampsia, and having a transfusion were similar between the two groups (P = 0.45, 0.95, and 0.49, respectively). Pain crises were more common in the study group (P = 0.02) as was cesarean section (P < 0.0001), but there was a reduction in pulmonary complications (P = 0.0002). Maternal mortality was uncommon (control group [N=4] vs study group [N=3], P = 0.40). Newborn statistics revealed a similar gestational age at delivery (37 weeks), and the incidence of intrauterine growth restriction, as well as 5-minute Apgar score <7 did not differ by group (P = 0. 91, 0.85, and 0.16, respectively). Infants in the study group were heavier on average by approximately 220 g (P = 0.02), whereas the neonatal death rate was low (control group [N=1], study group [N=2] P = 0.60). CONCLUSIONS: Recent pregnancy outcome statistics in women with sickle cell disease have not changed through the years. Innovative strategies to improve maternal and newborn outcomes among such patients are needed.

Projection-Resolved Optical Coherence Tomography Angiography of Macular Retinal Circulation in Glaucoma.

PURPOSE: To detect macular perfusion defects in glaucoma using projection-resolved optical coherence tomography (OCT) angiography. DESIGN: Prospective observation study. PARTICIPANTS: A total of 30 perimetric glaucoma and 30 age-matched normal participants were included. METHODS: One eye of each participant was imaged using 6×6-mm macular OCT angiography (OCTA) scan pattern by 70-kHz 840-nm spectral-domain OCT. Flow signal was calculated by the split-spectrum amplitude-decorrelation angiography algorithm. A projection-resolved OCTA (PR-OCTA) algorithm was used to remove flow projection artifacts. Four en face OCTA slabs were analyzed: the superficial vascular complex (SVC), intermediate capillary plexus (ICP), deep capillary plexus (DCP), and all-plexus retina (SVC + ICP + DCP). The vessel density (VD), defined as the percentage area occupied by flow pixels, was calculated from en face OCTA. A novel algorithm was used to adjust the vessel density to compensate for local variations in OCT signal strength. MAIN OUTCOME MEASURES: Macular retinal VD, ganglion cell complex (GCC) thickness, and visual field (VF) sensitivity. RESULTS: Focal capillary dropout could be visualized in the SVC, but not the ICP and DVP, in glaucomatous eyes. In the glaucoma group, the SVC and all-plexus retinal VD (mean ± standard deviation: 47.2%±7.1% and 73.5%±6.6%) were lower than in the normal group (60.5%±4.0% and 83.2%±4.2%, both P < 0.001, t test). The ICP and DCP VD were not significantly lower in the glaucoma group. Among the overall macular VD parameters, the SVC VD had the best diagnostic accuracy as measured by the area under the receiver operating characteristic curve (AROC). The accuracy was even better when the worse hemisphere (inferior or superior) was used, achieving an AROC of 0.983 and a sensitivity of 96.7% at a specificity of 95%. Among the glaucoma participants, the hemispheric SVC VD values were highly correlated with the corresponding GCC thickness and VF sensitivity (P < 0.003). The reflectance compensation step in VD calculation significantly improved repeatability, normal population variation, and correlation with VF and GCC thickness. CONCLUSIONS: On the basis of PR-OCTA, glaucoma preferentially affects perfusion in the SVC in the macula more than the deeper plexuses. Reflectance-compensated SVC VD measurement by PR-OCTA detected glaucoma with high accuracy and could be useful in the clinical evaluation of glaucoma.

Use of antenatal corticosteroids in special circumstances: a comprehensive review.

The aim of this study was to determine, in pregnancies complicated by preterm premature rupture of membranes (PPROM), hypertension, intrauterine growth restriction, multi-fetal gestations and pregnancies 23-26 weeks and ≥34 weeks' gestation, whether antenatal corticosteroids benefit the fetus. Literature review using PubMed, Web of Science, Clinical trials.gov, Cochrane Database of Systematic Reviews (1990-2015). Search terms linked special circumstances with corticosteroids. Randomized clinical trials, retrospective and prospective cohort studies, and case control studies were reviewed. In all, 468 abstracts were identified and 84 added from selected article bibliographies; of these, 503 abstracts were excluded, leaving 49 articles as the basis of review. The literature supports corticosteroids for PPROM up to 32-34 weeks without chorioamnionitis. Antenatal corticosteroids are beneficial for preterm infants of women with HELLP (hemolysis, elevated liver enzymes, low platelets) syndrome/severe preeclampsia. Postpartum corticosteroid treatment of HELLP patients results in more rapid correction of liver enzymes, platelet counts, and urinary output, which can reduce maternal morbidity. Corticosteroids in twin pregnancies decrease respiratory distress syndrome but the effect is less than in singleton pregnancies. Corticosteroid effects on intrauterine growth restriction pregnancies are conflicting and steroid use should be individualized. Corticosteroid use for 23-26 weeks reduces neonatal mortality but not morbidity. In pregnancies of 34 weeks to 366/7  weeks, corticosteroids reduce respiratory distress syndrome, but follow-up studies are unavailable. No long-term adverse child developmental effects have been observed with one or two courses of corticosteroids, but three or more steroid courses lowers birthweight and organ weight, and there may be an increase in neurodevelopmental abnormalities. Longer follow up of corticosteroid-treated fetuses is required.

Tachysystole Following Cervical Ripening and Induction of Labor Is Not Associated with Adverse Outcomes.

PURPOSE: This study was aimed at determining if significant uterine tachysystole was associated with adverse fetal or neonatal outcomes during cervical ripening and induction of labor. METHODS: Women undergoing cervical ripening and subsequent labor induction (n = 905) were assessed for tachysystole, defined as ≥6 contractions in each of 2 consecutive 10-minute windows. Women with ≥3 episodes of tachysystole were compared to women with no tachysystole. RESULTS: Over a 5-year period, 70% of the 905 participants (n = 631) had no tachysystole, 143 had 1 or 2 episodes whereas 131 or 15% had ≥3 episodes (p = 0.991). The cesarean delivery rate was lower among those with tachysystole (28.2 vs. 34.1%), but the difference was not significant (p = 0.197). Non-reassuring fetal tracings were more common in the tachysystole group (14.4 vs. 21.4%, p = 0.017), but the Apgar scores at 5 min and the umbilical cord pH and base excess were similar between the 2 groups (p = 0.502, p = 0.435, and p = 0.535, respectively). CONCLUSIONS: Tachysystole was not associated with adverse perinatal outcomes when compared to women with no tachysystole during cervical ripening and induction of labor.

Expansions of the neurovascular scleral canal and contained optic nerve occur early in the hypertonic saline rat experimental glaucoma model.

PURPOSE: To characterize early optic nerve head (ONH) structural change in rat experimental glaucoma (EG). METHODS: Unilateral intraocular pressure (IOP) elevation was induced in Brown Norway rats by hypertonic saline injection into the episcleral veins and animals were sacrificed 4 weeks later by perfusion fixation. Optic nerve cross-sections were graded from 1 (normal) to 5 (extensive injury) by 5 masked observers. ONHs with peripapillary retina and sclera were embedded, serial sectioned, 3-D reconstructed, delineated, and quantified. Overall and animal-specific EG versus Control eye ONH parameter differences were assessed globally and regionally by linear mixed effect models with significance criteria adjusted for multiple comparisons. RESULTS: Expansions of the optic nerve and surrounding anterior scleral canal opening achieved statistical significance overall (p < 0.0022), and in 7 of 8 EG eyes (p < 0.005). In at least 5 EG eyes, significant expansions (p < 0.005) in Bruch's membrane opening (BMO) (range 3-10%), the anterior and posterior scleral canal openings (8-21% and 5-21%, respectively), and the optic nerve at the anterior and posterior scleral canal openings (11-30% and 8-41%, respectively) were detected. Optic nerve expansion was greatest within the superior and inferior quadrants. Optic nerve expansion at the posterior scleral canal opening was significantly correlated to optic nerve damage (R = 0.768, p = 0.042). CONCLUSION: In the rat ONH, the optic nerve and surrounding BMO and neurovascular scleral canal expand early in their response to chronic experimental IOP elevation. These findings provide phenotypic landmarks and imaging targets for detecting the development of experimental glaucomatous optic neuropathy in the rat eye.

Induction of autophagy in rats upon overexpression of wild-type and mutant optineurin gene.

BACKGROUND: Optineurin is a gene associated with normal tension glaucoma and amyotrophic lateral sclerosis. It has been reported previously that in cultured RGC5 cells, the turnover of endogenous optineurin involves mainly the ubiquitin-proteasome pathway (UPP). When optineurin is upregulated or mutated, the UPP function is compromised as evidenced by a decreased proteasome ß5 subunit (PSMB5) level and autophagy is induced for clearance of the optineurin protein. RESULTS: Adeno-associated type 2 viral (AAV2) vectors for green fluorescence protein (GFP) only, GFP-tagged wild-type and Glu50Lys (E50K) mutated optineurin were intravitreally injected into rats for expression in retinal ganglion cells (RGCs). Following intravitreal injections, eyes that received optineurin vectors exhibited retinal thinning, as well as RGC and axonal loss compared to GFP controls. By immunostaining and Western blotting, the level of PSMB5 and autophagic substrate degradation marker p62 was reduced, and the level of autophagic marker microtubule associated protein 1 light chain 3 (LC3) was enhanced. The UPP impairment and autophagy induction evidently occurred in vivo as in vitro. The optineurin level, RGC and axonal counts, and apoptosis in AAV2-E50K-GFP-injected rat eyes were averted to closer to normal limits after treatment with rapamycin, an autophagic enhancer. CONCLUSIONS: The UPP function was reduced and autophagy was induced when wild-type and E50K optineurin was overexpressed in rat eyes. This study validates the in vitro findings, confirming that UPP impairment and autophagy induction also occur in vivo. In addition, rapamycin is demonstrated to clear the accumulated mutant optineurin. This agent may potentially be useful for rescuing of the adverse optineurin phenotypes in vivo.

Modeling glaucoma in rats by sclerosing aqueous outflow pathways to elevate intraocular pressure.

Injection of hypertonic saline via episcleral veins toward the limbus in laboratory rats can produce elevated intraocular pressure (IOP) by sclerosis of aqueous humor outflow pathways. This article describes important anatomic characteristics of the rat optic nerve head (ONH) that make it an attractive animal model for human glaucoma, along with the anatomy of rat aqueous humor outflow on which this technique is based. The injection technique itself is also described, with the aid of a supplemental movie, including necessary equipment and specific tips to acquire this skill. Outcomes of a successful injection are presented, including IOP elevation and patterns of optic nerve injury. These concepts are then specifically considered in light of the use of this model to assess potential neuroprotective therapies. Advantages of the hypertonic saline model include a delayed and relatively gradual IOP elevation, likely reproduction of scleral and ONH stresses and strains that may be important in producing axonal injury, and its ability to be applied to any rat (and potentially mouse) strain, leaving the unmanipulated fellow eye as an internal control. Challenges include the demanding surgical skill required by the technique itself, a wide range of IOP response, and mild corneal clouding in some animals. However, meticulous application of the principles detailed in this article and practice will allow most researchers to attain this useful skill for studying cellular events of glaucomatous optic nerve damage.

Rat optic nerve head anatomy within 3D histomorphometric reconstructions of normal control eyes.

The purpose of this study is to three-dimensionally (3D) characterize the principal macroscopic and microscopic relationships within the rat optic nerve head (ONH) and quantify them in normal control eyes. Perfusion-fixed, trephinated ONH from 8 normal control eyes of 8 Brown Norway Rats were 3D histomorphometrically reconstructed, visualized, delineated and parameterized. The rat ONH consists of 2 scleral openings, (a superior neurovascular and inferior arterial) separated by a thin connective tissue strip we have termed the "scleral sling". Within the superior opening, the nerve abuts a prominent extension of Bruch's Membrane (BM) superiorly and is surrounded by a vascular plexus, as it passes through the sclera, that is a continuous from the choroid into and through the dural sheath and contains the central retinal vein (CRV), (inferiorly). The inferior scleral opening contains the central retinal artery and three long posterior ciliary arteries which obliquely pass through the sclera to obtain the choroid. Bruch's Membrane Opening (BMO) is irregular and vertically elongated, enclosing the nerve (superiorly) and CRV and CRA (inferiorly). Overall mean BMO Depth, BMO Area, Choroidal Thickness and peripapillary Scleral Thickness were 29 µm, 56.5 × 10(3) µm(2), 57 µm and 104 µm respectively. Mean anterior scleral canal opening (ASCO) and posterior scleral canal opening (PSCO) radii were 201 ± 15 µm and 204 ± 16 µm, respectively. Mean optic nerve area at the ASCO and PSCO were 46.3 × 10(3)±4.4 × 10(3) µm(2) and 44.1 × 10(3)±4.5 × 10(3) µm(2) respectively. In conclusion, the 3D complexity of the rat ONH and the extent to which it differs from the primate have been under-appreciated within previous 2D studies. Properly understood, these anatomic differences may provide new insights into the relative susceptibilities of the rat and primate ONH to elevated intraocular pressure.

An acute intraocular pressure challenge to assess retinal ganglion cell injury and recovery in the mouse.

We describe a model of acute intraocular pressure (IOP) elevation in the mouse eye that induces reversible loss of inner retinal function associated with oxidative stress, glial cell activation and minimal loss of retinal ganglion cell (RGC) number. Young healthy mouse eyes recover inner retinal function within 7-days but more persistent functional loss is seen in older mice. Manipulation of diet and exercise further modify RGC recovery demonstrating the utility of this injury model for investigating lifestyle and therapeutic interventions. We believe that systematic investigation into the characteristics and determinants of RGC recovery following an IOP challenge will shed light on processes that govern RGC vulnerability in the early stages of glaucoma.