Mouse Optic Nerve Research Articles

Increased Tissue Stiffness in Tumors from Mice with Neurofibromatosis-1 Optic Glioma

Children with neurofibromatosis type 1 (NF1) cancer predisposition syndrome are prone to the development of low-grade brain tumors (gliomas) within the optic pathway (optic gliomas). One of the key obstacles to developing successful therapeutic strategies for these tumors is the striking lack of information about the mechanical properties that characterize these tumors relative to non-neoplastic optic nerve tissue. To study the physical changes that may occur when an optic nerve glioma is present, we employed atomic force microscopy to measure the stiffness of healthy versus tumor-bearing optic nerve tissue. We found that the average elastic moduli of non-neoplastic and tumor-bearing optic nerves were ∼3 and ∼6 kPa, respectively. Based on previous studies implicating changes in extracellular matrix remodeling in other, related optic nerve pathological states, we found decreased expression of one major metalloproteinase protein (MMP-2) and unchanged expression of lysyl oxidase and a second metalloproteinase, MMP-9, in murine optic gliomas relative to normal non-neoplastic optic nerve. Collectively, these observations suggest a productive interplay between physical properties of mouse optic nerve gliomas and the extracellular matrix.

Oligodendrocyte precursor cells in the mouse optic nerve originate in the preoptic area.

The present study aims to examine the origin of oligodendrocyte progenitor cells (OPCs) in the mouse optic nerve (ON) by labeling OPCs in the fetal forebrain. The labeling of OPCs in the ON was performed by injection of a retrovirus vector carrying the lacZ gene into the lateral ventricle, or by inducible Cre/loxP of Olig2-positive cells. The retrovirus labeling revealed that ventricular zone-derived cells of the fetal forebrain relocated to the ON and differentiated into oligodendrocytes. In addition, lineage tracing of Olig2-positive cells and whole-mount staining of PDGFRα-positive cells demonstrated that OPCs appeared by E12.5 in the preoptic area, and spread caudally to enter the ON. Our results also suggest that OPCs generated during the early stage are depleted from the ON after maturation.

Glioprotection of Retinal Astrocytes After Intravitreal Administration of Memantine in the Mouse Optic Nerve Crush Model.

BackgroundIn glaucoma, non-intraocular pressure (IOP)-related risk factors can result in increased levels of extracellular glutamate, which triggers a cascade of neurodegeneration characterized by the excessive activation of N-methyl-D-aspartate (NMDA). The purpose of our study was to evaluate the glioprotective effects of memantine as a prototypic uncompetitive NMDA blocker on retinal astrocytes in the optic nerve crush (ONC) mouse model for glaucoma.Material/MethodsOptic nerve crush was performed on all of the right eyes (n=8), whereas left eyes served as contralateral healthy controls (n=8) in Balb/c/Sca mice. Four randomly assigned mice received 2-μl intravitreal injections of memantine (1 mg/ml) after ONC in the experimental eye. One week after the experiment, optic nerves were dissected and stained with methylene blue. Retinae were detached from the sclera. The tissue was immunostained. Whole-mount retinae were investigated by fluorescent microscopy. Astrocyte counts for each image were performed manually.ResultsHistological sections of crushed optic nerves showed consistently moderate tissue damage in experimental groups. The mean number of astrocytes per image in the ONC group was significantly lower than in the healthy control group (7.13±1.5 and 10.47±1.9, respectively). Loss of astrocytes in the memantine-treated group was significantly lower (8.83±2.2) than in the ONC group. Assessment of inter-observer reliability showed excellent agreement among observations in control, ONC, and memantine groups.ConclusionsThe ONC is an effective method for investigation of astrocytic changes in mouse retina. Intravitreally administered memantine shows a promising glioprotective effect on mouse retinal astrocytes by preserving astrocyte count after ONC.

Store-operated calcium entry is essential for glial calcium signalling in CNS white matter

‘Calcium signalling’ is the ubiquitous response of glial cells to multiple extracellular stimuli. The primary mechanism of glial calcium signalling is by release of calcium from intracellular stores of the endoplasmic reticulum (ER). Replenishment of ER Ca2+ stores relies on store-operated calcium entry (SOCE). However, despite the importance of calcium signalling in glial cells, little is known about their mechanisms of SOCE. Here, we investigated SOCE in glia of the mouse optic nerve, a typical CNS white matter tract that comprises bundles of myelinated axons and the oligodendrocytes and astrocytes that support them. Using quantitative RT-PCR, we identified Orai1 channels, both Stim1 and Stim2, and the transient receptor potential M3 channel (TRPM3) as the primary channels for SOCE in the optic nerve, and their expression in both astrocytes and oligodendrocytes was demonstrated by immunolabelling of optic nerve sections and cultures. The functional importance of SOCE was demonstrated by fluo-4 calcium imaging on isolated intact optic nerves and optic nerve cultures. Removal of extracellular calcium ([Ca2+]o) resulted in a marked depletion of glial cytosolic calcium ([Ca2+]i), which recovered rapidly on restoration of [Ca2+]o via SOCE. 2-aminoethoxydiphenylborane (2APB) significantly decreased SOCE and severely attenuated ATP-mediated calcium signalling. The results provide evidence that Orai/Stim and TRPM3 are important components of the ‘calcium toolkit’ that underpins SOCE and the sustainability of calcium signalling in white matter glia.

Abstract TP90: Casein Kinase 2 Inhibition Preserves Axonal Function Against Ischemia

Axonal injury and dysfunction are responsible for much of the disability observed following a stroke. Human brain comprises equal proportions of gray matter and white matter and white matter is injured in most strokes. Casein kinase 2 (CK2) is a protein kinase expressed in brain, including white matter, and is regulated by ischemia. We therefore hypothesized that transient CK2 inhibition would protect white matter from ischemic injury. To assess the impact of CK2 inhibition on axonal electrical activity following oxygen glucose deprivation (OGD), mouse optic nerves (MONs), a pure white matter track, from C57BL/6J were subjected to OGD (1h) while eliciting compound action potentials (CAPs) and exposed to CX-4945, a selective CK2 inhibitor, or control artificial cerebrospinal fluid (ACSF). We observed that CX-4945 preserved CAPs when applied either before or after OGD. Then to determine the impact of CK2 inhibition on glial cell survival following OGD, MONs exposed to OGD that were treated with either CX-4945 or control ACSF were processed for immunohistochemistry. We observed that CX-4945 treatment protected oligodendrocytes from OGD. And finally, to determine if CK2 inhibition protected mitochondrial from OGD, MONs from Thy-1 mito-CFP mice were similarly subjected to OGD in the presence of either CX-4945 or control ACSF. We observed that CX-4945 maintained Thy-1 mito-CFP fluorescence following OGD. In conclusion, our results suggest that CK2 inhibition preserves axonal function by preserving oligodendrocytes and mitochondrial function following ischemic injury. We propose that CK2 inhibitors, which are currently in phase II-III clinical trials for cancer therapy could be repurposed and provide a novel therapeutic target to protect white matter against ischemic injury, reducing mortality and morbidity and improving recovery following stroke.

Measuring Deformation in the Mouse Optic Nerve Head and Peripapillary Sclera.

PurposeTo develop an ex vivo explant system using multiphoton microscopy and digital volume correlation to measure the full-field deformation response to intraocular pressure (IOP) change in the peripapillary sclera (PPS) and in the optic nerve head (ONH) astrocytic structure.MethodsGreen fluorescent protein (GFP)-glutamate transporter-GLT1 (GLT1/GFP) mouse eyes were explanted and imaged with a laser-scanning microscope under controlled inflation. Images were analyzed for regional strains and changes in astrocytic lamina and PPS shape. Astrocyte volume fraction in seven control GLT1/GFP mice was measured. The level of fluorescence of GFP fluorescent astrocytes was compared with glial fibrillary acidic protein (GFAP) labeled astrocytes using immunohistochemistry.ResultsThe ONH astrocytic structure remained stable during 3 hours in explants. Control strain—globally, in the central one-half or two-thirds of the astrocytic lamina—was significantly greater in the nasal-temporal direction than in the inferior-superior or anterior-posterior directions (each P ≤ 0.03, mixed models). The PPS opening (perimeter) in normal eye explants also became wider nasal-temporally than superior-inferiorly during inflation from 10 to 30 mm Hg (P = 0.0005). After 1 to 3 days of chronic IOP elevation, PPS area was larger than in control eyes (P = 0.035), perimeter elongation was 37% less than controls, and global nasal-temporal strain was significantly less than controls (P = 0.007). Astrocyte orientation was altered by chronic IOP elevation, with processes redirected toward the longitudinal axis of the optic nerve.ConclusionsThe explant inflation test measures the strain response of the mouse ONH to applied IOP. Initial studies indicate regional differences in response to both acute and chronic IOP elevation within the ONH region.

Overexpression of Brain-Derived Neurotrophic Factor Protects Large Retinal Ganglion Cells After Optic Nerve Crush in Mice.

Brain-derived neurotrophic factor (BDNF), a neurotrophin essential for neuron survival and function, plays an important role in neuroprotection during neurodegenerative diseases. In this study, we examined whether a modest increase of retinal BDNF promotes retinal ganglion cell (RGC) survival after acute injury of the optic nerve in mice. We adopted an inducible Cre-recombinase transgenic system to up-regulate BDNF in the mouse retina and then examined RGC survival after optic nerve crush by in vivo imaging. We focused on one subtype of RGC with large soma expressing yellow fluorescent protein transgene that accounts for ∼11% of the total SMI-32–positive RGCs. The median survival time of this subgroup of SMI-32 cells was 1 week after nerve injury in control mice but 2 weeks when BDNF was up-regulated. Interestingly, we found that the survival time for RGCs taken as a whole was 2 weeks, suggesting that these large-soma RGCs are especially vulnerable to optic nerve crush injury. We also studied changes in axon number using confocal imaging, confirming first the progressive loss reported previously for wild-type mice and demonstrating that BDNF up-regulation extended axon survival. Together, our results demonstrate that the time course of RGC loss induced by optic nerve injury is type specific and that overexpression of BDNF prolongs the survival of one subgroup of SMI-32–positive RGCs.

Evidence for Cooperative Selection of Axons for Myelination by Adjacent Oligodendrocytes in the Optic Nerve.

The cellular mechanisms that regulate the topographic arrangement of myelin internodes along axons remain largely uncharacterized. Recent clonal analysis of oligodendrocyte morphologies in the mouse optic nerve revealed that adjacent oligodendrocytes frequently formed adjacent internodes on one or more axons in common, whereas oligodendrocytes in the optic nerve were never observed to myelinate the same axon more than once. By modelling the process of axonal selection at the single cell level, we demonstrate that internode length and primary process length constrain the capacity of oligodendrocytes to myelinate the same axon more than once. On the other hand, probabilistic analysis reveals that the observed juxtaposition of myelin internodes among common sets of axons by adjacent oligodendrocytes is highly unlikely to occur by chance. Our analysis may reveal a hitherto unknown level of communication between adjacent oligodendrocytes in the selection of axons for myelination. Together, our analyses provide novel insights into the mechanisms that define the spatial organization of myelin internodes within white matter at the single cell level.

Age-Related Changes in Axonal and Mitochondrial Ultrastructure and Function in White Matter.

The impact of aging on CNS white matter (WM) is of general interest because the global effects of aging on myelinated nerve fibers are more complex and profound than those in cortical gray matter. It is important to distinguish between axonal changes created by normal aging and those caused by neurodegenerative diseases, including multiple sclerosis, stroke, glaucoma, Alzheimer's disease, and traumatic brain injury. Using three-dimensional electron microscopy, we show that in mouse optic nerve, which is a pure and fully myelinated WM tract, aging axons are larger, have thicker myelin, and are characterized by longer and thicker mitochondria, which are associated with altered levels of mitochondrial shaping proteins. These structural alterations in aging mitochondria correlate with lower ATP levels and increased generation of nitric oxide, protein nitration, and lipid peroxidation. Moreover, mitochondria-smooth endoplasmic reticulum interactions are compromised due to decreased associations and decreased levels of calnexin and calreticulin, suggesting a disruption in Ca(2+) homeostasis and defective unfolded protein responses in aging axons. Despite these age-related modifications, axon function is sustained in aging WM, which suggests that age-dependent changes do not lead to irreversible functional decline under normal conditions, as is observed in neurodegenerative diseases. Aging is a common risk factor for a number of neurodegenerative diseases, including stroke. Mitochondrial dysfunction and oxidative damage with age are hypothesized to increase risk for stroke. We compared axon-myelin-node-mitochondrion-smooth endoplasmic reticulum (SER) interactions in white matter obtained at 1 and 12 months. We show that aging axons have enlarged volume, thicker myelin, and elongated and thicker mitochondria. Furthermore, there are reduced SER connections to mitochondria that correlate with lower calnexin and calreticulin levels. Despite a prominent decrease in number, elongated aging mitochondria produce excessive stress markers with reduced ATP production. Because axons maintain function under these conditions, our study suggests that it is important to understand the process of normal brain aging to identify neurodegenerative changes.

Optic nerve energy metabolism: the role of astrocyte glycogen

SummaryGlycogen is a glucose storage molecule. We studied the physiology and functions of glycogen in CNS white matter using acutely isolated mouse optic nerve (MON), a typical CNS white matter tract. Glycogen is present in MON astrocytes. Aglycemia caused loss of the stimulus evoked compound action potential (CAP) after ~15 min. CAP decline coincided with exhaustion of usable tissue glycogen. Increasing glycogen content prolonged the latency to decline onset and, conversely, decreasing glycogen shortened this latency. Metabolic support provided by glycogen during aglycemia was abolished by an inhibitor of glycogen breakdown (DAB). The MON has a high resting level of extracellular lactate (~0.6 mM). The metabolic support provided by glycogen during aglycemia is abolished by an inhibitor of glycogen breakdown (DAB) in MON. Both tissues exhibit high levels of extracellular lactate ([Lactate−]o), up to 50% of which derives from glycogen. During aglycemia, glycogen in astrocytes is metabolized to lactate and ‘shuttled’ to axons (and possibly oligodendrocytes) to support oxidative energy metabolism. Glycogen breakdown and lactate transport to axons is also needed to sustain brief periods of intense axonal discharge.

Gas6 Promotes Oligodendrogenesis and Myelination in the Adult Central Nervous System and After Lysolecithin-Induced Demyelination.

A key aim of therapy for multiple sclerosis (MS) is to promote the regeneration of oligodendrocytes and remyelination in the central nervous system (CNS). The present study provides evidence that the vitamin K-dependent protein growth arrest specific 6 (Gas6) promotes such repair in in vitro cultures of mouse optic nerve and cerebellum. We first determined expression of Gas6 and TAM (Tyro3, Axl, Mer) receptors in the mouse CNS, with all three TAM receptors increasing in expression through postnatal development, reaching maximal levels in the adult. Treatment of cultured mouse optic nerves with Gas6 resulted in significant increases in oligodendrocyte numbers as well as expression of myelin basic protein (MBP). Gas6 stimulation also resulted in activation of STAT3 in optic nerves as well as downregulation of multiple genes involved in MS development, including matrix metalloproteinase-9 (MMP9), which may decrease the integrity of the blood–brain barrier and is found upregulated in MS lesions. The cytoprotective effects of Gas6 were examined in in vitro mouse cerebellar slice cultures, where lysolecithin was used to induce demyelination. Cotreatment of cerebellar slices with Gas6 significantly attenuated demyelination as determined by MBP immunostaining, and Gas6 activated Tyro3 receptor through its phosphorylation. In conclusion, these results demonstrate that Gas6/TAM signaling stimulates the generation of oligodendrocytes and increased myelin production via Tyro3 receptor in the adult CNS, including repair after demyelinating injury. Furthermore, the effects of Gas6 on STAT3 signaling and matrix MMP9 downregulation indicate potential glial cell repair and immunoregulatory roles for Gas6, indicating that Gas6-TAM signaling could be a potential therapeutic target in MS and other neuropathologies.

Preferential conduction block of myelinated axons by nitric oxide.

Conduction block by nitric oxide (NO) was examined in myelinated and unmyelinated axons from both the central nervous system and peripheral nervous system. In rat vagus nerves, mouse optic nerves at P12-P23, adult and developing mouse sciatic nerves, and mouse spinal cords, myelinated fibers were preferentially blocked reversibly by concentrations of NO similar to those encountered in inflammatory lesions. The possibility that these differences between myelinated and unmyelinated axons are due to the normal developmental substitution of Na+ channel subtype Nav 1.6 for Nav 1.2 at nodes of Ranvier was tested by repeating experiments on mice null for Nav 1.6. Results were unchanged in this mutant. In shiverer optic nerve, which has only scattered regions with nodes of Ranvier, only the fastest component of the compound action potential was reduced. NO was compared with three other methods of blocking conduction: low Na+ , high K+ , and tetrodotoxin (TTX). In each of these three cases, unmyelinated axons lost conduction simultaneously with myelinated fibers. From changes in conduction velocity in myelinated axons as they were blocked, it was ascertained that NO acted most similarly to TTX. It was concluded that NO likely interacts with axonal Na+ channels through an intermediate that is associated with myelin. © 2016 Wiley Periodicals, Inc.

Reassembly of Excitable Domains after CNS Axon Regeneration.

Action potential initiation and propagation in myelinated axons require ion channel clustering at axon initial segments (AIS) and nodes of Ranvier. Disruption of these domains after injury impairs nervous system function. Traditionally, injured CNS axons are considered refractory to regeneration, but some recent approaches challenge this view by showing robust long-distance regeneration. However, whether these approaches allow remyelination and promote the reestablishment of AIS and nodes of Ranvier is unknown. Using mouse optic nerve crush as a model for CNS traumatic injury, we performed a detailed analysis of AIS and node disruption after nerve crush. We found significant disruption of AIS and loss of nodes within days of the crush, and complete loss of nodes 1 week after injury. Genetic deletion of the tumor suppressor phosphatase and tensin homolog (Pten) in retinal ganglion cells (RGCs), coupled with stimulation of RGCs by inflammation and cAMP, dramatically enhanced regeneration. With this treatment, we found significant reestablishment of RGC AIS, remyelination, and even reassembly of nodes in regions proximal, within, and distal to the crush site. Remyelination began near the retina, progressed distally, and was confirmed by electron microscopy. Although axons grew rapidly, remyelination and nodal ion channel clustering was much slower. Finally, genetic deletion of ankyrinG from RGCs to block AIS reassembly did not affect axon regeneration, indicating that preservation of neuronal polarity is not required for axon regeneration. Together, our results demonstrate, for the first time, that regenerating CNS axons can be remyelinated and reassemble new AIS and nodes of Ranvier. We show, for the first time, that regenerated CNS axons have the capacity to both remyelinate and reassemble the axon initial segments and nodes of Ranvier necessary for rapid and efficient action potential propagation.

LG-52THEOPHYLLINE IN VISION IMPAIRED CHILDREN WITH OPTIC PATHWAY GLIOMA

LG-52. THEOPHYLLINE IN VISION IMPAIRED CHILDREN WITH OPTIC PATHWAY GLIOMA Gail Halliday, Peter Ling, Michal Zapotocky, Arun Reginald, Uri Tabori, Iris Fried, Eric Bouffet, Lee Dupuis, and Ute Bartels; The Hospital for Sick Children, Toronto, ON, Canada PURPOSE: Preclinical models suggest that phosphodiesterase inhibitors may slow or prevent axonal damage to the optic nerve in mice with visual pathway glioma likely through elevation of cAMP. A feasibility study was performed to evaluate the safety and efficacy of theophylline in vision impaired children with optic pathway glioma (OPG) at risk of visual loss. METHODS: Patients were treated with theophylline for a planned duration of 12 months. Eight of the eleven patients received concurrent standard OPG chemotherapies. The initial theophylline dose was calculated based on ideal body mass and subsequently adjusted according to trough serum levels. The primary end point was safety, defined by grade 3-4 toxicity using the National Cancer Institute Common Toxicity Criteria version 4.0. Secondary end points included visual acuity response rate. RESULTS: Preliminary data analysis of eleven patients, with a median age of 9.2 years, treated with theophylline at the Hospital for Sick Children, Toronto between 2009 and 2015 is detailed here. After a mean duration of 10.5 months no grade 3-4 treatment related toxicities were identified. Of the three patients who discontinued therapy prematurely two experienced grade 1 toxicities. Three of eleven patients studied showed improvements in binocular vision whilst on theophylline therapy with a further three patients showing stabilization of vision. CONCLUSIONS: Combining theophylline with standard chemotherapies for OPG in children is safe and feasible. In a proportion of patients with progressive visual decline despite prior chemotherapy theophylline enabled stabilization or improvement in visual acuity. Phase III proof of efficacy trials are warranted. Neuro-Oncology 18:iii78–iii96, 2016. doi:10.1093/neuonc/now075.52 #The Author(s) 2016. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

3-D imaging mass spectrometry of protein distributions in mouse Neurofibromatosis 1 (NF1)-associated optic glioma

Neurofibromatosis type 1 (NF1) is a common neurogenetic disorder, in which affected individuals develop tumors of the nervous system. Children with NF1 are particularly prone to brain tumors (gliomas) involving the optic pathway that can result in impaired vision. Since tumor formation and expansion requires a cooperative tumor microenvironment, it is important to identify the cellular and acellular components associated with glioma development and growth. In this study, we used 3-D matrix assisted laser desorption ionization imaging mass spectrometry (MALDI IMS) to measure the distributions of multiple molecular species throughout optic nerve tissue in mice with and without glioma, and to explore their spatial relationships within the 3-D volume of the optic nerve and chiasm. 3-D IMS studies often involve extensive workflows due to the high volume of sections required to generate high quality 3-D images. Herein, we present a workflow for 3-D data acquisition and volume reconstruction using mouse optic nerve tissue. The resulting 3-D IMS data yield both molecular similarities and differences between glioma-bearing and wild-type (WT) tissues, including protein distributions localizing to different anatomical subregions. Biological significanceThe current work addresses a number of challenges in 3-D MALDI IMS, driven by the small size of the mouse optic nerve and the need to maintain consistency across multiple 2-D IMS experiments. The 3-D IMS data yield both molecular similarities and differences between glioma-bearing and wild-type (WT) tissues, including protein distributions localizing to different anatomical subregions, which could then be targeted for identification and related back to the biology observed in gliomas of the optic nerve.

Abstract WP103: Mitochondrial/endoplasmic Reticulum Dysfunction Exacerbates Oxidative Damage in Aging White Matter Ischemia

White matter (WM) is frequently affected by stroke. Aging WM recovers less following stroke compared to younger WM. Free radicals accumulate with aging resulting in increased oxidative stress. Mitochondria/endoplasmic reticulum (ER) interactions are crucial for protection from oxidative stress. We hypothesized that aging WM is more susceptible to oxidative stress damage mediated by mitochondrial/ER dysfunction, leading to decreased ATP synthesis and impaired Ca2+ homeostasis. Mouse optic nerves (MON) obtained from C57BL/6J and Thy-1 CFP(+) mice at 1 (young) or 12 months (aging) of age were used. Axon function was quantified by recording evoked compound action potentials. Mitochondrial/ER structure and interactions were determined by 3D-electron microscopy, Western blotting, ATP assays, and CFP (+) fluorescent mitochondrial imaging. Oxidative stress was assessed by NOS and glutathione assays and by Western blotting for oxidative stress markers, 3-NT and 4-HNE. Oxygen-glucose deprivation (OGD) for 60 min resulted in decreased axon function recovery of aging WM. Aging WM showed elevated NOS activity and increased levels of by-products of lipid peroxidation (4-HNE) and protein nitration (3-NT), indicating aggravated oxidative stress. Structurally, aging axons were larger, with thicker myelin, and were characterized by longer and thicker mitochondria due to altered levels of mitochondrial shaping proteins. This was further confirmed by 3D-EM and CFP (+) imaging and may underlie the decreased ATP levels detected in aging WM. Moreover, mitochondrial-ER interactions were compromised due to decreased association between the organelles and due to decrease in levels of mitochondrial trafficking protein miro-2, which suggests defective Ca2+ homeostasis in aging axons. Calnexin, an ER stress response chaperone protein, was decreased under baseline conditions in aging axons, but did not show upregulation following OGD when compared to young axons, suggesting a defect in unfolded protein responses. We conclude that aging WM is increasingly vulnerable to stroke because of inherent structural changes in axons and mitochondria/ER interactions, leading to depletion of ATP and defective Ca2+ dynamics, resulting in increased oxidative stress.

MGluR5 protect astrocytes from ischemic damage in postnatal CNS white matter

Astrocytes perform essential neuron-supporting functions in the central nervous system (CNS) and their disruption has devastating effects on neuronal integrity in multiple neuropathologies. Although astrocytes are considered resistant to most pathological insults, ischemia can result in astrocyte injury and astrocytes in postnatal white matter are particularly vulnerable. Metabotropic glutamate receptors (mGluR) are neuroprotective in ischemia and are widely expressed by astrocytes throughout CNS grey matter, but their potential cytoprotective role in astrocytes had not been determined. Here, we identify functional expression of group I mGluR in white matter astrocytes and demonstrate their activation protects astrocytes from ischemic damage in the postnatal mouse optic nerve. Optic nerve astrocytes are shown to express mGluR5 using immunolabelling of sections and explant cultures from transgenic reporter mice in which GFAP drives expression of EGFP. In addition, using Fluo-4 calcium imaging in isolated intact optic nerves, we show that the group I/II mGluR agonist ACPD and the specific group I mGluR agonist DHPG evoke glial Ca2+ signals that were significantly inhibited by the group I mGluR antagonist AIDA. A key finding is that activation of group I mGluR protects astrocytes against oxygen-glucose deprivation (OGD) in situ, in isolated intact optic nerves from GFAP-EGFP mice. This study identifies a role for group I mGluR in protecting astrocytes against ischemia in postnatal white matter and suggests this may be a strategy for limiting damage in neuropathologies involving excitotoxity.

Isolation of intact astrocytes from the optic nerve head of adult mice

The astrocytes of the optic nerve head are a specialized subtype of white matter astrocytes that form the direct cellular environment of the unmyelinated ganglion cell axons. Due to their potential involvement in glaucoma, these astrocytes have become a target of research. Due to the heterogeneity of the optic nerve tissue, which also contains other cell types, in some cases it may be desirable to conduct gene expression studies on small numbers of well-characterized astrocytes or even individual cells. Here, we describe a simple method to isolate individual astrocytes. This method permits obtaining astrocytes with intact morphology from the adult mouse optic nerve and reduces contamination of the isolated astrocytes by other cell types. Individual astrocytes can be recognized by their morphology and collected under microscopic control. The whole procedure can be completed in 2–3 h. We also discuss downstream applications like multiplex single-cell PCR and quantitative PCR (qPCR).

Mitochondrial morphology differences and mitophagy deficit in murine glaucomatous optic nerve.

Decreased ATP correlates with intraocular pressure exposure in the optic nerves of mice with glaucoma. To understand what underlies this energy deficit, we examined mitochondria in the myelinated optic nerve axons of the DBA/2J mouse, a model of glaucoma secondary to iris pigment disease, and the DBA/2(wt-gpnmb) control strain. Mitochondrial length, width, surface area, and health status were measured in 30 electron microscopic fields within the myelinated portion of optic nerves from DBA/2J and DBA/2(wt-gpnmb) mice at 3, 6, and 10 months of age. Protein was isolated from optic nerve for analysis of PINK1, Parkin, LC3-I and -II, and lysosome-associated membrane protein 1 (LAMP1) by Western blot. The number of mitochondria in DBA/2J optic nerve was increased, and they had significantly smaller surface area. Mitochondria in DBA/2J were closer to the axolemma, more spatially isolated, and their cristae were more disrupted at every age group as compared to DBA/2(wt-gpnmb). Autophagosomes were significantly increased in DBA/2J optic nerve at all ages. Protein analysis showed higher LC3-II to LC3-I ratio in aged DBA/2J optic nerve than in DBA/2(wt-gpnmb). PINK1 and Parkin levels were not statistically different across age groups. LAMP1 was significantly decreased in the aged DBA/2J optic nerve. Decreased surface area, combined with reduced oxidative capacity in mitochondria from the aged DBA/2J axon, indicate that mitochondrial pathology may contribute to the energy deficit in glaucomatous optic nerve. Though autophagosomes were increased in DBA/2J optic nerve, the increased mitochondria and decreased LAMP1 suggest deteriorating mitochondria are not being efficiently recycled by mitophagy.

CLARITY法によるマウス網膜・視神経の透明化

CLARITY法によるマウス網膜・視神経の透明化