Myelination generates aberrant ultrastructure that is resolved by microglia.

To enable rapid propagation of action potentials, axons are ensheathed by myelin, a multilayered insulating membrane formed by oligodendrocytes. Most of the myelin is generated early in development, resulting in the generation of long-lasting stable membrane structures. Here, we explored structural and dynamic changes in central nervous system myelin during development. To achieve this, we performed an ultrastructural analysis of mouse optic nerves by serial block face scanning electron microscopy (SBF-SEM) and confocal time-lapse imaging in the zebrafish spinal cord. We found that myelin undergoes extensive ultrastructural changes during early postnatal development. Myelin degeneration profiles were engulfed and phagocytosed by microglia using exposed phosphatidylserine as one "eat me" signal. In contrast, retractions of entire myelin sheaths occurred independently of microglia and involved uptake of myelin by the oligodendrocyte itself. Our findings show that the generation of myelin early in development is an inaccurate process associated with aberrant ultrastructural features that require substantial refinement.

Real-time large-area imaging of the corneal subbasal nerve plexus.

The morphometric assessment of the corneal subbasal nerve plexus (SNP) by confocal microscopy holds great potential as a sensitive biomarker for various ocular and systemic conditions and diseases. Automated wide-field montages (or large-area mosaic images) of the SNP provide an opportunity to overcome the limited field of view of the available imaging systems without the need for manual, subjective image selection for morphometric characterization. However, current wide-field montaging solutions usually calculate the mosaic image after the examination session, without a reliable means for the clinician to predict or estimate the resulting mosaic image quality during the examination. This contribution describes a novel approach for a real-time creation and visualization of a mosaic image of the SNP that facilitates an informed evaluation of the quality of the acquired image data immediately at the time of recording. In cases of insufficient data quality, the examination can be aborted and repeated immediately, while the patient is still at the microscope. Online mosaicking also offers the chance to identify an overlap of the imaged tissue region with previous SNP mosaic images, which can be particularly advantageous for follow-up examinations.

Axonal Protection by Netarsudil, a ROCK Inhibitor, Is Linked to an AMPK-Autophagy Pathway in TNF-Induced Optic Nerve Degeneration.

Purpose: Netarsudil, a Rho kinase inhibitor with norepinephrine transport inhibitory effect, lowers intraocular pressure, however, its effect on axon damage remains to be elucidated. The aim of the current study was to investigate the effect of netarsudil on TNF-induced axon loss and to examine whether it affects phosphorylated-AMP-activated kinase (p-AMPK) and autophagy in the optic nerve. Methods: Intravitreal administration of TNF or TNF with netarsudil was carried out on rats and quantification of axon number was determined. Electron microscopy determined autophagosome numbers. Localization of p-AMPK expression was examined by immunohistochemistry. The changes in p62, LC3-II, and p-AMPK levels were estimated in the optic nerve by immunoblot analysis. The effect of an AMPK activator A769662 or an AMPK inhibitor dorsomorphin on axon number was evaluated. Results: Morphometric analysis revealed apparent protection by netarsudil against TNF-induced axon degeneration. Netarsudil increased autophagosome numbers inside axons. Netarsudil treatment significantly upregulated optic nerve LC3-II levels in both the TNF-treated eyes and the control eyes. Increased p62 protein level induced by TNF was significantly ameliorated by netarsudil. The netarsudil administration alone lessened p62 levels. Netarsudil significantly upregulated the optic nerve p-AMPK levels. A769662 exhibited obvious axonal protection against TNF-induced damage. A769662 treatment upregulated LC3-II levels and the increment of p62 level induced by TNF was significantly ameliorated by A769662. Immunohistochemical analysis revealed that p-AMPK is present in axons. Netarsudil-mediated axonal protection was significantly suppressed by dorsomorphin administration. Conclusions: Netarsudil upregulated p-AMPK and autophagy. Netarsudil-mediated axonal protection may be associated with upregulated p-AMPK.

Early Intervention and Lifelong Treatment with GLP1 Receptor Agonist Liraglutide in a Wolfram Syndrome Rat Model with an Emphasis on Visual Neurodegeneration, Sensorineural Hearing Loss and Diabetic Phenotype.

Wolfram syndrome (WS), also known as a DIDMOAD (diabetes insipidus, early-onset diabetes mellitus, optic nerve atrophy and deafness) is a rare autosomal disorder caused by mutations in the Wolframin1 (WFS1) gene. Previous studies have revealed that glucagon-like peptide-1 receptor agonist (GLP1 RA) are effective in delaying and restoring blood glucose control in WS animal models and patients. The GLP1 RA liraglutide has also been shown to have neuroprotective properties in aged WS rats. WS is an early-onset, chronic condition. Therefore, early diagnosis and lifelong pharmacological treatment is the best solution to control disease progression. Hence, the aim of this study was to evaluate the efficacy of the long-term liraglutide treatment on the progression of WS symptoms. For this purpose, 2-month-old WS rats were treated with liraglutide up to the age of 18 months and changes in diabetes markers, visual acuity, and hearing sensitivity were monitored over the course of the treatment period. We found that treatment with liraglutide delayed the onset of diabetes and protected against vision loss in a rat model of WS. Therefore, early diagnosis and prophylactic treatment with the liraglutide may also prove to be a promising treatment option for WS patients by increasing the quality of life.

Liraglutide, 7,8-DHF and their co-treatment prevents loss of vision and cognitive decline in a Wolfram syndrome rat model.

Wolfram syndrome (WS) is a monogenic progressive neurodegenerative disease and is characterized by various neurological symptoms, such as optic nerve atrophy, loss of vision, cognitive decline, memory impairment, and learning difficulties. GLP1 receptor agonist liraglutide and BDNF mimetic 7,8-dihydroxyflavone (7,8-DHF) have had protective effect to visual pathway and to learning and memory in different rat models of neurodegenerative disorders. Although synergistic co-treatment effect has not been reported before and therefore the aim of the current study was to investigate liraglutide, 7,8-DHF and most importantly for the first time their co-treatment effect on degenerative processes in WS rat model. We took 9 months old WS rats and their wild-type (WT) control animals and treated them daily with liraglutide, 7,8-DHF or with the combination of liraglutide and 7,8-DHF up to the age of 12.5 months (n = 47, 5-8 per group). We found that liraglutide, 7,8-DHF and their co-treatment all prevented lateral ventricle enlargement, improved learning in Morris Water maze, reduced neuronal inflammation, delayed the progression of optic nerve atrophy, had remyelinating effect on optic nerve and thereby improved visual acuity in WS rats compared to WT controls. Thus, the use of the liraglutide, 7,8-DHF and their co-treatment could potentially be used as a therapeutic intervention to induce neuroprotection or even neuronal regeneration.

Ex Vivo Studies of Optic Nerve Axon Electrophysiology.

The use of ex vivo compound action potential (CAP) recordings from intact optic nerves is an ideal model to study white matter function without the influence of gray matter. Here, we describe how freshly dissected optic nerves are placed in a humidified recording chamber and how evoked CAPs are recorded and monitored in real time for up to 10 h. Evoked CAP recordings allow for white matter to be studied under acute challenges such as anoxia, hypoxia, aglycemia, and ischemia.

Activation of adenosine A3 receptor protects retinal ganglion cells from degeneration induced by ocular hypertension.

Glaucoma is a progressive chronic retinal degenerative disease and a leading cause of global irreversible blindness. This disease is characterized by optic nerve damage and retinal ganglion cell (RGC) death. The current treatments available target the lowering of intraocular pressure (IOP), the main risk factor for disease onset and development. However, in some patients, vision loss progresses despite successful IOP control, indicating that new and effective treatments are needed, such as those targeting the neuroprotection of RGCs. Adenosine A3 receptor (A3R) activation confers protection to RGCs following an excitotoxic stimulus. In this work, we investigated whether the activation of A3R could also afford protection to RGCs in the laser-induced ocular hypertension (OHT) model, a well-characterized animal model of glaucoma. The intravitreal injection of 2-Cl-IB-MECA, a selective A3R agonist, abolished the alterations induced by OHT in the negative and positive components of scotopic threshold response (STR) without changing a- and b-wave amplitudes both in scotopic and photopic conditions. Moreover, the treatment of OHT eyes with the A3R agonist promoted the survival of RGCs, attenuated the impairment in retrograde axonal transport, and improved the structure of the optic nerve. Taking into consideration the beneficial effects afforded by 2-Cl-IB-MECA, we can envisage that A3R activation can be considered a good therapeutic strategy to protect RGCs from glaucomatous damage.

Gene Therapy with Single-Subunit Yeast NADH-Ubiquinone Oxidoreductase (NDI1) Improves the Visual Function in Experimental Autoimmune Encephalomyelitis (EAE) Mice Model of Multiple Sclerosis (MS).

Mitochondrial dysfunction mediated loss of respiration, oxidative stress, and loss of cellular homeostasis contributes to the neuronal and axonal degenerations permanent loss of function in experimental autoimmune encephalomyelitis model (EAE) of multiple sclerosis (MS). To address the mitochondrial dysfunction mediated visual loss in EAE mice, self-complementary adeno-associated virus (scAAV) containing the NADH-dehydrogenase type-2 (NDI1) complex I gene was intravitreally injected into the mice after the onset of visual defects. Visual function assessed by pattern electroretinogram (PERGs) showed progressive loss of function in EAE mice were improved significantly in NDI1 gene therapy-treated mice. Serial optical coherence tomography (OCT) revealed that progressive thinning of inner retinal layers in EAE mice was prevented upon NDI1 expression. The 45% optic nerve axonal and 33% retinal ganglion cell (RGC) loss contributed to the permanent loss of visual function in EAE mice were ameliorated by NDI1-mediated prevention of mitochondrial cristae dissolution and improved mitochondrial homeostasis. In conclusion, targeting the dysfunctional complex I using NDI1 gene can be an approach to address axonal and neuronal loss responsible for permanent disability in MS that is unaltered by current disease modifying drugs.

Optic nerve regeneration: A long view.

The optic nerve conveys information about the outside world from the retina to multiple subcortical relay centers. Until recently, the optic nerve was widely believed to be incapable of re-growing if injured, with dire consequences for victims of traumatic, ischemic, or neurodegenerative diseases of this pathway. Over the past 10-20 years, research from our lab and others has made considerable progress in defining factors that normally suppress axon regeneration and the ability of retinal ganglion cells, the projection neurons of the retina, to survive after nerve injury. Here we describe research from our lab on the role of inflammation-derived growth factors, suppression of inter-cellular signals among diverse retinal cell types, and combinatorial therapies, along with related studies from other labs, that enable animals with optic nerve injury to regenerate damaged retinal axons back to the brain. These studies raise the possibility that vision might one day be restored to people with optic nerve damage.

Role of ultrasonographic optic nerve sheath diameter in the diagnosis and follow-up of papilledema and its correlation with Frisén's severity grading.

Purpose: The aim of this study was to compare the ultrasonographic optic nerve sheath diameter (ONSD) in different grades of papilledema and in controls and to evaluate ONSD in atrophic papilledema/optic atrophy when raised ICP was suspected. Methods: Prospective cross-sectional case-control study. Following an ocular examination, papilledema was graded clinically using modified Frisén's grading. An ultrasonographic cross section of the retrobulbar optic nerve was obtained with a posterior transverse scan. Independent t-test and analysis of variance were the statistical tools used in the study. Results: The study included 55 cases and 55 age- and gender-matched controls; mean (± standard deviation) age was 37.17 (±11.25) years and male: female ratio was 49:61. There was a statistically significant difference in the mean ultrasonographic ONSD between cases [4.89 (±0.65) mm] and controls [3.12 (±0.22) mm] (P < 0.001). There was a significant difference in the mean ONSD across Frisén's grades of papilledema (P < 0.001). The mean ONSD in atrophic papilledema was 6.2 (±0.75) mm. Conclusion: In the presence of symptoms, ultrasonographic ONSD >4 mm is diagnostic of papilledema. Ultrasonographic ONSD correlates well with the severity of papilledema and can be used to follow-up patients with chronically elevated ICP. It is useful in detecting raised ICP in the presence of optic atrophy and to distinguish true papilledema from pseudopapilledema.

Increased Intracranial Pressure Damages Optic Nerve Structural Support.

Optic nerve sheath diameter (ONSD) is used clinically as a noninvasive measure for elevated intracranial pressure (ICP). This study had two purposes: to investigate the immediate effects optic nerve sheath (ONS) dilation post-ICP increase on trabecular fibers connecting the optic nerve to the ONS and to document any changes in these fibers 30 days post-increased ICP. In a swine model, ICP was increased by inflating a Foley catheter balloon in the epidural space. Three control pigs received the catheter insertion without inflation (no increase in ICP) and four experimental pigs received the catheter with inflation (increased ICP). The control and two randomly selected pigs with increased ICP were euthanized immediately after the procedure. The two other pigs were euthanized 30 days post-catheter inflation. For all pigs, the ONS was removed and imaged using a scanning electron microscope, calculating percent porosity values. Porosity values for the experimental groups (Immediately measured [IM] µ = 0.5749; Delayed measured [DM] µ = 0.5714) were larger than the control group (µ = 0.4336) and statistically significant (IM vs. Control, p = 0.0018; DM vs. Control, p = 0.0092). There was no significant difference (p = 0.9485) in porosity of the DM group when compared with the IM group. This study demonstrated that the trabecular fibers immediately post-increased ICP (ONS dilation) were more porous than the control and remained statistically different (more porous) after 30 days. These results suggest a structural change that occurs in the ONS with elevations in ICP.

Mouse Nr2f1 haploinsufficiency unveils new pathological mechanisms of a human optic atrophy syndrome.

Optic nerve atrophy represents the most common form of hereditary optic neuropathies leading to vision impairment. The recently described Bosch-Boonstra-Schaaf optic atrophy (BBSOA) syndrome denotes an autosomal dominant genetic form of neuropathy caused by mutations or deletions in the NR2F1 gene. Herein, we describe a mouse model recapitulating key features of BBSOA patients-optic nerve atrophy, optic disc anomalies, and visual deficits-thus representing the only available mouse model for this syndrome. Notably, Nr2f1-deficient optic nerves develop an imbalance between oligodendrocytes and astrocytes leading to postnatal hypomyelination and astrogliosis. Adult heterozygous mice display a slower optic axonal conduction velocity from the retina to high-order visual centers together with associative visual learning deficits. Importantly, some of these clinical features, such the optic nerve hypomyelination, could be rescued by chemical drug treatment in early postnatal life. Overall, our data shed new insights into the cellular mechanisms of optic nerve atrophy in BBSOA patients and open a promising avenue for future therapeutic approaches.

Hypothermic neuroprotection during reperfusion following exposure to aglycemia in central white matter is mediated by acidification.

Hypoglycemia is a common iatrogenic consequence of type 1 diabetes therapy that can lead to central nervous system injury and even death if untreated. In the absence of clinically effective neuroprotective drugs we sought to quantify the putative neuroprotective effects of imposing hypothermia during the reperfusion phase following aglycemic exposure to central white matter. Mouse optic nerves (MONs), central white matter tracts, were superfused with oxygenated artificial cerebrospinal fluid (aCSF) containing 10 mmol/L glucose at 37°C. The supramaximal compound action potential (CAP) was evoked and axon conduction was assessed as the CAP area. Extracellular lactate was measured using an enzyme biosensor. Exposure to aglycemia, simulated by omitting glucose from the aCSF, resulted in axon injury, quantified by electrophysiological recordings, electron microscopic analysis confirming axon damage, the extent of which was determined by the duration of aglycemia exposure. Hypothermia attenuated injury. Exposing MONs to hypothermia during reperfusion resulted in improved CAP recovery compared with control recovery measured at 37°C, an effect attenuated in alkaline aCSF. Hypothermia decreases pH implying that the hypothermic neuroprotection derives from interstitial acidification. These results have important clinical implications demonstrating that hypothermic intervention during reperfusion can improve recovery in central white matter following aglycemia.

Anatomy of the stemmata in the Photuris firefly larva.

Fireflies (Coleoptera: Lampyridae) have distinct visual systems at different stages of development. Larvae have stemmata and adults have compound eyes. Adults use compound eyes to mediate photic communication during courtship. Larvae do not manifest this behavior, yet they are bioluminescent. We investigated the structure of stemmata in Photuris firefly larvae to identify anatomical substrates (i.e., rhabdomeres) conferring visual function. Stemmata were located bilaterally on the antero-lateral surfaces of the head. Beneath the ~ 130 µm diameter lens, we identified a pigmented eye-cup. At its widest point, the eye-cup was ~ 150 µm in diameter. The optic nerve exited the eye-cup opposite the lens. Two distinct regions, asymmetric in size and devoid of pigmentation, were characterized in stemmata cross-sections. We refer to these regions as lobes. Each lobe contained a rhabdom of a radial network of rhabdomeres. Pairs of rhabdomeres formed interdigitating microvilli contributed from neighboring photoreceptor cell bodies. The optic nerve contained 88 axons separable into two populations based on size. The number of axons in the optic nerve together with distinct rhabdoms suggests these structures were formed from 'fusion stemmata.' This structural specialization provides an anatomical substrate for future studies of visually mediated behaviors in Photuris larvae.

Heterozygous Meg2 Ablation Causes Intraocular Pressure Elevation and Progressive Glaucomatous Neurodegeneration.

Glaucomatous neurodegeneration represents one of the major causes of irreversible blindness worldwide. Yet, the detailed molecular mechanisms that initiate optic nerve damage and retinal ganglion cell (RGC) loss are not fully understood. Members of the protein tyrosine phosphatase (PTP) superfamily are key players in numerous neurodegenerative diseases. In order to investigate the potential functional relevance of the PTP megakaryocyte 2 (Meg2) in retinal neurodegeneration, we analyzed Meg2 knockout (KO) and heterozygous (HET)-synonym protein-tyrosine phosphatase non-receptor type 9 (Ptpn9)-mice. Interestingly, via global microarray and quantitative real-time PCR (RT-qPCR) analyses of Meg2 KO and HET retinae, we observed a dysregulation of several candidate genes that are highly associated with retinal degeneration and intraocular pressure (IOP) elevation, the main risk factor for glaucoma. Subsequent IOP measurements in Meg2 HET mice verified progressive age-dependent IOP elevation. Ultrastructural analyses and immunohistochemistry showed severe optic nerve degeneration accompanied by a dramatic loss of RGCs. Additionally, HET mice displayed reactive micro-/macrogliosis and early activation of the classical complement cascade with pronounced deposition of the membrane attack complex (MAC) in the retina and optic nerve. When treated with latanoprost, significant IOP lowering prevented RGC loss and microglial invasion in HET mice. Finally, electroretinogram (ERG) recordings revealed reduced a- and b-wave amplitudes, indicating impaired retinal functionality in Meg2 HET mice. Collectively, our findings indicate that the heterozygous loss of Meg2 in mice is sufficient to cause IOP elevation and glaucomatous neurodegeneration. Thus, Meg2 HET mice may serve as a novel animal model to study the pathomechanism involved in the onset and progression of glaucoma.

Repulsive Environment Attenuation during Adult Mouse Optic Nerve Regeneration.

The regenerative capacity of CNS tracts has ever been a great hurdle to regenerative medicine. Although recent studies have described strategies to stimulate retinal ganglion cells (RGCs) to regenerate axons through the optic nerve, it still remains to be elucidated how these therapies modulate the inhibitory environment of CNS. Thus, the present work investigated the environmental content of the repulsive axon guidance cues, such as Sema3D and its receptors, myelin debris, and astrogliosis, within the regenerating optic nerve of mice submitted to intraocular inflammation + cAMP combined to conditional deletion of PTEN in RGC after optic nerve crush. We show here that treatment was able to promote axonal regeneration through the optic nerve and reach visual targets at twelve weeks after injury. The Regenerating group presented reduced MBP levels, increased microglia/macrophage number, and reduced astrocyte reactivity and CSPG content following optic nerve injury. In addition, Sema3D content and its receptors are reduced in the Regenerating group. Together, our results provide, for the first time, evidence that several regenerative repulsive signals are reduced in regenerating optic nerve fibers following a combined therapy. Therefore, the treatment used made the CNS microenvironment more permissive to regeneration.

Fundamental pharmacological expressions on ocular exposure to capsaicin, the principal constituent in pepper sprays.

Eye irritation assessment is compulsory to anticipate health risks in military personnel exposed to riot control agents such as capsaicin, the principal constituent of oleoresin capsicum, or pepper sprays. The present work investigates certain fundamental yet unaddressed pharmacological manifestations on ocular exposure to capsaicin. Ocular pharmacology of capsaicin was studied using acute eye irritation (AEI), bovine corneal opacity and permeability (BCOP) assay, corneal fluorescein staining and indirect ophthalmoscopy studies, transcorneal permeation, Schirmer tear secretion test, nerve conduction velocity study and enzyme-linked immunosorbent assay (ELISA). Additionally, histopathology and scanning electron microscopy (SEM) of bovine corneas and rat optic nerves were done to further estimate capsaicin induced morphological variations. Our findings demonstrated that AEI, BCOP, corneal fluorescein staining and indirect ophthalmoscopy were useful in assessing capsaicin induced ocular irritation; AEI and BCOP also contributed towards indicating the eye irritation potential of capsaicin as per the United Nations Globally Harmonized System of Classification and Labelling of Chemicals categorization. Additional experimental observations include considerable transcorneal permeation of capsaicin, capsaicin induced reduction in tear secretions and nerve conduction velocity and increased expression of proinflammatory cytokines by ELISA. Histopathology and SEM were favourable techniques for the detection of capsaicin induced ocular physiological modifications.

Three dimensional electron microscopy reveals changing axonal and myelin morphology along normal and partially injured optic nerves.

Following injury to the central nervous system, axons and myelin distinct from the initial injury site undergo changes associated with compromised function. Quantifying such changes is important to understanding the pathophysiology of neurotrauma; however, most studies to date used 2 dimensional (D) electron microscopy to analyse single sections, thereby failing to capture changes along individual axons. We used serial block face scanning electron microscopy (SBF SEM) to undertake 3D reconstruction of axons and myelin, analysing optic nerves from normal uninjured female rats and following partial optic nerve transection. Measures of axon and myelin dimensions were generated by examining 2D images at 5 µm intervals along the 100 µm segments. In both normal and injured animals, changes in axonal diameter, myelin thickness, fiber diameter, G-ratio and percentage myelin decompaction were apparent along the lengths of axons to varying degrees. The range of values for axon diameter along individual reconstructed axons in 3D was similar to the range from 2D datasets, encompassing reported variation in axonal diameter attributed to retinal ganglion cell diversity. 3D electron microscopy analyses have provided the means to demonstrate substantial variability in ultrastructure along the length of individual axons and to improve understanding of the pathophysiology of neurotrauma.

AAV2-Mediated Transduction of the Mouse Retina After Optic Nerve Injury.

Purpose: Gene therapy of retinal ganglion cells (RGCs) has promise as a powerful therapeutic for the rescue and regeneration of these cells after optic nerve damage. However, early after damage, RGCs undergo atrophic changes, including gene silencing. It is not known if these changes will deleteriously affect transduction and transgene expression, or if the therapeutic protein can influence reactivation of the endogenous genome. Methods: Double-transgenic mice carrying a Rosa26-(LoxP)-tdTomato reporter, and a mutant allele for the proapoptotic Bax gene were reared. The Bax mutant blocks apoptosis, but RGCs still exhibit nuclear atrophy and gene silencing. At times ranging from 1 hour to 4 weeks after optic nerve crush (ONC), eyes received an intravitreal injection of AAV2 virus carrying the Cre recombinase. Successful transduction was monitored by expression of the tdTomato reporter. Immunostaining was used to localize tdTomato expression in select cell types. Results: Successful transduction of RGCs was achieved at all time points after ONC using AAV2 expressing Cre from the phosphoglycerate kinase (Pgk) promoter, but not the CMV promoter. ONC promoted an increase in the transduction of cell types in the inner nuclear layer, including Müller cells and rod bipolar neurons. There was minimal evidence of transduction of amacrine cells and astrocytes in the inner retina or optic nerve. Conclusions: Damaged RGCs can be transduced and at least some endogenous genes can be subsequently activated. Optic nerve damage may change retinal architecture to allow greater penetration of an AAV2 virus to transduce several additional cell types in the inner nuclear layer.

In-vivo effects of intraocular and intracranial pressures on the lamina cribrosa microstructure.

There is increasing clinical evidence that the eye is not only affected by intraocular pressure (IOP), but also by intracranial pressure (ICP). Both pressures meet at the optic nerve head of the eye, specifically the lamina cribrosa (LC). The LC is a collagenous meshwork through which all retinal ganglion cell axons pass on their way to the brain. Distortion of the LC causes a biological cascade leading to neuropathy and impaired vision in situations such as glaucoma and idiopathic intracranial hypertension. While the effect of IOP on the LC has been studied extensively, the coupled effects of IOP and ICP on the LC remain poorly understood. We investigated in-vivo the effects of IOP and ICP, controlled via cannulation of the eye and lateral ventricle in the brain, on the LC microstructure of anesthetized rhesus monkeys eyes using the Bioptigen spectral-domain optical coherence tomography (OCT) device (Research Triangle, NC). The animals were imaged with their head upright and the rest of their body lying prone on a surgical table. The LC was imaged at a variety of IOP/ICP combinations, and microstructural parameters, such as the thickness of the LC collagenous beams and diameter of the pores were analyzed. LC microstructure was confirmed by histology. We determined that LC microstructure deformed in response to both IOP and ICP changes, with significant interaction between the two. These findings emphasize the importance of considering both IOP and ICP when assessing optic nerve health.