Acid Ceramidase Deficiency in Mice Leads to Severe Ocular Pathology and Visual Impairment
2018; Elsevier BV; Volume: 189; Issue: 2 Linguagem: Inglês
10.1016/j.ajpath.2018.10.018
ISSN1525-2191
AutoresFabian P.S. Yu, Benjamin S. Sajdak, Jakub Sikora, Alexander E Salmon, Murtaza S. Nagree, Jiří Gurka, Iris S. Kassem, Daniel M. Lipinski, Joseph Carroll, Jeffrey A. Medin,
Tópico(s)Retinal Diseases and Treatments
ResumoFarber disease (FD) is a debilitating lysosomal storage disorder characterized by severe inflammation and neurodegeneration. FD is caused by mutations in the ASAH1 gene, resulting in deficient acid ceramidase (ACDase) activity. Patients with ACDase deficiency exhibit a broad clinical spectrum. In classic cases, patients develop hepatosplenomegaly, nervous system involvement, and childhood mortality. Ocular manifestations include decreased vision, a grayish appearance to the retina with a cherry red spot, and nystagmus. That said, the full effect of ACDase deficiency on the visual system has not been studied in detail. We previously developed a mouse model that is orthologous for a known patient mutation in Asah1 that recapitulates human FD. Herein, we report evidence of a severe ocular pathology in Asah1P361R/P361R mice. Asah1P361R/P361R mice exhibit progressive retinal and optic nerve pathology. Through noninvasive ocular imaging and histopathological analyses of these Asah1P361R/P361R animals, we revealed progressive inflammation, the presence of retinal dysplasia, and significant storage pathology in various cell types in both the retina and optic nerves. Lipidomic analyses of retinal tissues revealed an abnormal accumulation of ceramides and other sphingolipids. Electroretinograms and behavioral tests showed decreased retinal and visual responses. Taken together, these data suggest that ACDase deficiency leads to sphingolipid imbalance, inflammation, dysmorphic retinal and optic nerve pathology, and severe visual impairment. Farber disease (FD) is a debilitating lysosomal storage disorder characterized by severe inflammation and neurodegeneration. FD is caused by mutations in the ASAH1 gene, resulting in deficient acid ceramidase (ACDase) activity. Patients with ACDase deficiency exhibit a broad clinical spectrum. In classic cases, patients develop hepatosplenomegaly, nervous system involvement, and childhood mortality. Ocular manifestations include decreased vision, a grayish appearance to the retina with a cherry red spot, and nystagmus. That said, the full effect of ACDase deficiency on the visual system has not been studied in detail. We previously developed a mouse model that is orthologous for a known patient mutation in Asah1 that recapitulates human FD. Herein, we report evidence of a severe ocular pathology in Asah1P361R/P361R mice. Asah1P361R/P361R mice exhibit progressive retinal and optic nerve pathology. Through noninvasive ocular imaging and histopathological analyses of these Asah1P361R/P361R animals, we revealed progressive inflammation, the presence of retinal dysplasia, and significant storage pathology in various cell types in both the retina and optic nerves. Lipidomic analyses of retinal tissues revealed an abnormal accumulation of ceramides and other sphingolipids. Electroretinograms and behavioral tests showed decreased retinal and visual responses. Taken together, these data suggest that ACDase deficiency leads to sphingolipid imbalance, inflammation, dysmorphic retinal and optic nerve pathology, and severe visual impairment. Farber disease (FD; Online Mendelian Inheritance in Man number 228000) is an autosomal recessive lysosomal storage disorder caused by mutations in the ASAH1 gene, resulting in deficient acid ceramidase (ACDase) activity.1Levade T. Sandhoff K. Schulze H. Medin J.A. Valle D. Beaudet A.L. Vogelstein B. Kinzler K.W. Antonarakis S.E. Ballabio A. Acid Ceramidase Deficiency: Farber Lipogranulomatosis. Scriver's OMMBID (Online Metabolic and Molecular Bases of Inherited Diseases). McGraw-Hill, New York, NY2014Google Scholar ACDase is a key lysosomal enzyme that hydrolyzes the bioactive lipid ceramide into sphingosine (Sph) and a free fatty acid.2Schuchman E.H. Acid ceramidase and the treatment of ceramide diseases: the expanding role of enzyme replacement therapy.Biochim Biophys Acta. 2016; 1862: 1459-1471Crossref PubMed Scopus (25) Google Scholar Currently, there is no cure for FD, and with only 152 cases recorded in the literature to date, obtaining tissues and samples to study this disorder has been challenging.3Zielonka M. Garbade S.F. Kölker S. Hoffmann G.F. Ries M. A cross-sectional quantitative analysis of the natural history of Farber disease: an ultra-orphan condition with rheumatologic and neurological cardinal disease features.Genet Med. 2017; 20: 524-530Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar The clinical manifestations of FD are broad; patients with the classic variant die early in childhood.1Levade T. Sandhoff K. Schulze H. Medin J.A. Valle D. Beaudet A.L. Vogelstein B. Kinzler K.W. Antonarakis S.E. Ballabio A. Acid Ceramidase Deficiency: Farber Lipogranulomatosis. Scriver's OMMBID (Online Metabolic and Molecular Bases of Inherited Diseases). McGraw-Hill, New York, NY2014Google Scholar The cardinal features of FD are the presence of s.c. lipogranulomatous nodules, joint contractures, and aphonia.1Levade T. Sandhoff K. Schulze H. Medin J.A. Valle D. Beaudet A.L. Vogelstein B. Kinzler K.W. Antonarakis S.E. Ballabio A. Acid Ceramidase Deficiency: Farber Lipogranulomatosis. Scriver's OMMBID (Online Metabolic and Molecular Bases of Inherited Diseases). McGraw-Hill, New York, NY2014Google Scholar Patients with severe forms of FD will also develop respiratory complications, hepatosplenomegaly, and neurologic decline.4Ehlert K. Frosch M. Fehse N. Zander A. Roth J. Vormoor J. Farber disease: clinical presentation, pathogenesis and a new approach to treatment.Pediatr Rheumatol. 2007; 5: 15-22Crossref Scopus (82) Google Scholar, 5Bao X.H. Tian J.M. Ji T.Y. Chang X.Z. A case report of childhood Farber's disease and literature review.Zhonghua Er Ke Za Zhi. 2017; 55: 54-58PubMed Google Scholar Impaired ACDase activity leads to systemic ceramide accumulation in FD patients. Ceramide and other sphingolipids are key components of membranes and play a role in a variety of cellular functions, including inflammation, cell proliferation, and apoptosis.6Arana L. Gangoiti P. Ouro A. Trueba M. Gómez-Muñoz A. Ceramide and ceramide 1-phosphate in health and disease.Lipids Health Dis. 2010; 9: 15Crossref PubMed Scopus (145) Google Scholar The balance of ceramide and its metabolites are tightly regulated, and dysregulation results in disease and potential visual system defects.7Chen H. Tran J.A. Brush R.S. Saadi A. Rahman A.K. Yu M. Yasumura D. Matthes M.T. Ahern K. Yang H. Ceramide signaling in retinal degeneration.Retin Degenerative Dis. 2012; 723: 553-558Google Scholar, 8Hannun Y.A. Obeid L.M. Sphingolipids and their metabolism in physiology and disease.Nat Rev Mol Cell Biol. 2017; 19: 175-191Crossref PubMed Scopus (808) Google Scholar The most frequent ophthalmic manifestation that has been described in patients with FD is a cherry red spot in the macula.3Zielonka M. Garbade S.F. Kölker S. Hoffmann G.F. Ries M. A cross-sectional quantitative analysis of the natural history of Farber disease: an ultra-orphan condition with rheumatologic and neurological cardinal disease features.Genet Med. 2017; 20: 524-530Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar, 9Cogan D.G. Kuwabara T. Moser H. Hazard G.W. Retinopathy in a case of Farber's lipogranulomatosis.Arch Ophthalmol. 1966; 75: 752-757Crossref PubMed Scopus (18) Google Scholar, 10Moser H.W. Prensky A.L. Wolfe H.J. Rosman N.P. 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Acid sphingomyelinase (aSMase) deficiency leads to abnormal microglia behavior and disturbed retinal function.Biochem Biophys Res Commun. 2015; 464: 434-440Crossref PubMed Scopus (10) Google Scholar, 18Wu B.X. Fan J. Boyer N.P. Jenkins R.W. Koutalos Y. Hannun Y.A. Crosson C.E. Lack of acid sphingomyelinase induces age-related retinal degeneration.PLoS One. 2015; 10: e0133032PubMed Google Scholar, 19Grishchuk Y. Stember K.G. Matsunaga A. Olivares A.M. Cruz N.M. King V.E. Humphrey D.M. Wang S.L. Muzikansky A. Betensky R.A. Retinal dystrophy and optic nerve pathology in the mouse model of mucolipidosis IV.Am J Pathol. 2016; 186: 199-209Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar We previously reported the first viable model for ACDase deficiency, wherein a known human ASAH1 mutation, proline (P) 362 to arginine (R), was knocked in to the corresponding locus in murine Asah1 (P361R).20Alayoubi A.M. Wang J.C. Au B.C. Carpentier S. Garcia V. Dworski S. El-Ghamrasni S. Kirouac K.N. Exertier M.J. Xiong Z.J. Prive G.G. Simonaro C.M. Casas J. Fabrias G. Schuchman E.H. Turner P.V. Hakem R. Levade T. Medin J.A. Systemic ceramide accumulation leads to severe and varied pathological consequences.EMBO Mol Med. 2013; 5: 827-842Crossref PubMed Scopus (75) Google Scholar Mice homozygous for this mutation mirror many FD patient features, including heightened inflammation and pathology in the hematopoietic, respiratory, and neuroglial systems that leads to early mortality.20Alayoubi A.M. Wang J.C. Au B.C. Carpentier S. Garcia V. Dworski S. El-Ghamrasni S. Kirouac K.N. Exertier M.J. Xiong Z.J. Prive G.G. Simonaro C.M. Casas J. Fabrias G. Schuchman E.H. Turner P.V. Hakem R. Levade T. Medin J.A. Systemic ceramide accumulation leads to severe and varied pathological consequences.EMBO Mol Med. 2013; 5: 827-842Crossref PubMed Scopus (75) Google Scholar, 21Sikora J. Dworski S. Jones E.E. Kamani M.A. Micsenyi M.C. Sawada T. Le Faouder P. Bertrand-Michel J. Dupuy A. Dunn C.K. Yang Xuan Ingrid C. Casas J. Fabrias G. Hampson D.R. Levade T. Drake Richard R. Medin J.A. Walkley S.U. Acid ceramidase deficiency in mice results in a broad range of central nervous system abnormalities.Am J Pathol. 2017; 187: 864-883Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar, 22Yu F.P. Islam D. Sikora J. Dworski S. Gurka' J. Lopez-Vasquez L. Liu M. Kuebler W.M. Levade T. Zhang H. Medin J.A. Chronic lung injury and impaired pulmonary function in a mouse model of acid ceramidase deficiency.Am J Physiol Lung Cell Mol Physiol. 2017; 314: 406-420Crossref Scopus (18) Google Scholar In this study, we investigated the consequences of ACDase deficiency by completing a comprehensive investigation of ocular manifestations in the Asah1P361R/P361R mouse model of FD. Noninvasive ocular imaging was used to monitor disease progression and highlight the abnormal sphingolipids present in the retina. Furthermore, ACDase deficiency was found to reduce visual function that is, in part, due to progressive inflammation, neurologic involvement, and abnormal storage pathology in cells of the visual system. To generate homozygous Asah1P361R/P361R mice, Asah1+/P361R heterozygotes were crossed, as previously reported.20Alayoubi A.M. Wang J.C. Au B.C. Carpentier S. Garcia V. Dworski S. El-Ghamrasni S. Kirouac K.N. Exertier M.J. Xiong Z.J. Prive G.G. Simonaro C.M. Casas J. Fabrias G. Schuchman E.H. Turner P.V. Hakem R. Levade T. Medin J.A. Systemic ceramide accumulation leads to severe and varied pathological consequences.EMBO Mol Med. 2013; 5: 827-842Crossref PubMed Scopus (75) Google Scholar Genotypes were confirmed by PCR using genomic DNA isolated from ear notches. To detect the wild-type Asah1 allele, the following primers were used: 5′-CAGAAGGTATGCGGCATCGTCATAC-3′ (forward) and 5′-AGGGCCATACAGAGAAACCCTGTCTC-3′ (reverse). These primers yielded a 379-bp product. For the Asah1 knock-in allele, the following primers were used: 5′-TCAAGGCTTGACTTTGGGGCAC-3′ (forward) and 5′-GCTGGACGTAAACTCCTCTTCAGACC-3′ (reverse). These primers amplify a 469-bp product from the neomycin resistance cassette. All animal procedures were approved and performed in strict adherence to the policies of the Medical College of Wisconsin Institutional Animal Care and Use Committee. Animals used for this study were maintained in controlled ambient illumination on a 12-hour light/dark cycle, with an illumination level of 2 to 3 lux. Exposure to bright light was kept to a minimum for all study animals for the duration of this study. Mice were anesthetized with inhaled isoflurane (3% induction, 1% to 2% maintenance) in 0.6 L/minute oxygen flow. The cornea and the lens were evaluated and imaged with the Topcon SL-D81 slit-lamp biomicroscope (Topcon Medical Systems Inc., Oakland, NJ) with a digital camera (Nikon D810 36.3MP DSLR Camera; Nikon Inc., Melville, NY). The eye was then dilated and cyclopleged with one eye drop each of 2.5% phenylephrine hydrochloride and 1% tropicamide (Akron, Inc., Lake Forest, IL). The lens was then reevaluated and imaged after dilation. All examinations were performed by a board-certified ophthalmologist (I.S.K.) with experience in animal models of ocular disease. Mice were anesthetized and prepared for imaging as described above. Fundus images were taken with the Phoenix Micron IV (Phoenix Research Labs, Pleasanton, CA). Near-infrared (810-nm) reflectance imaging and blue autofluorescence (excitation, 486 nm; emission filter, 525/50 nm) imaging were performed with a customized Heidelberg Spectralis (Heidelberg Engineering, Heidelberg, Germany) confocal scanning laser ophthalmoscope (cSLO). The automatic real-time composite mode in the Spectralis software version 6.6.2.0 (Heidelberg Engineering, Heidelberg, Germany) was used to average 40 and 100 frames of the near-infrared and blue autofluorescence images, respectively. To perform optical coherence tomography (OCT) imaging, mice were anesthetized and prepared for imaging as described above. Imaging was performed with a Bioptigen Envisu R2200 spectral domain–OCT system (Leica Microsystems, Wetzlar, Germany) equipped with a Superlum Broadlighter T870 light source centered at 878.4 nm with a 186.3-nm bandwidth (Superlum, Cork, Ireland). InVivoVue control software version 2.4.33 (Leica Microsystems, Wetzlar, Germany) and the Bioptigen mouse objective were used for retinal imaging. A customized Bioptigen mouse stage was used to aim the imaging beam to the desired retinal location. GenTeal lubricant eye gel and Systane Ultra lubricating eye drops (Alcon, Fort Worth, TX) were used as needed to maintain corneal hydration. Dispersion, reference arm position, and light power of the sample arm were optimized iteratively for each animal at each time point. During acquisition, all scans were displayed and acquired in logarithmic intensity mode. Horizontal line scans (1 mm, 1000 A-scans/B-scan; 100 repeated scans) of the retina were acquired with the optic nerve head (ONH) centered for each scan. With our system and these scan parameters, the pixel size was calculated to be 1.00 × 1.61 μm (xz axes, respectively). B-scans (20 to 50) were registered to a manually selected template frame and averaged using custom software described previously.23Dubra A. Harvey Z. Registration of 2D images from fast scanning ophthalmic instruments.Biomed Image Registration. 2010; 6204: 60-71Google Scholar The registration was limited to a displacement of approximately 10 μm to exclude scans acquired at different retinal locations. The Duke OCT Retinal Analysis Program version 63.6 (Duke University, Durham, NC) was used to segment the inner limiting membrane and the retinal pigmented epithelium,24Chiu S.J. Li X.T. Nicholas P. Toth C.A. Izatt J.A. Farsiu S. Automatic segmentation of seven retinal layers in SDOCT images congruent with expert manual segmentation.Opt Express. 2010; 18: 19413-19428Crossref PubMed Scopus (558) Google Scholar which were clearly visible in all animals. Total retinal thickness was defined as the optical path length between these boundaries, assuming a group refractive index of 1.38. The order of the images was randomized, the boundaries were manually corrected, and thickness was analyzed by an observer (A.E.S.) masked to the genotype. Before testing herein, mice were dark adapted overnight. Apparatus setup and animal preparations were conducted under dim red illumination. Mice were anesthetized and prepared as they were for imaging. Mice were placed on a heated platform (38°C). A silver-coated nylon contact lens with a custom-made active electrode was positioned on the eye. To maintain electrical conductivity, two subdermal platinum needle electrodes were positioned in the scruff (reference) and base of the leg (ground). Prepared animals were then positioned inside the Ganzfeld dome of the Espion E2 system (Diagnosys LLC, Cambridge, UK). All recordings were completed in a custom-made Faraday cage. Signals from the electroretinogram (ERG) were differentially amplified and digitized at a rate of 5 kHz (bandpass filtered 0 to 100 Hz). Recording sessions started with the dark-adapted flash ERG, which consisted of a six-log intensity series (−4 to 1 log cd·second/m2). Twenty responses were collected and averaged for the −4 and −3 log cd·second/m2 stimulus with an interstimulus internal of 5 seconds, 10 responses were collected and averaged for the −2 and −1 log cd·second/m2 stimuli with an interstimulus interval of 10 seconds, and five responses were collected and averaged for the 0 and 1 log cd·second/m2 stimuli with an interstimulus interval of 20 seconds. For the light-adapted series, mice were first exposed to a 30 cd·second/m2 white light background for 10 minutes for rod saturation, then progressed to a light-adapted flash ERG over a two-log intensity series (0 to 1 log cd·second/m2). Twenty responses were collected and averaged for each condition. Recordings concluded with two flicker ERG tests performed with continuous 5- and 15-Hz flicker (30 cd·second/m2). Thirty responses were collected and averaged for each flicker condition. To evaluate visual perception, a mouse open-field setup was modified to replicate the presence of a visual cliff.25Fox M.W. The visual cliff test for the study of visual depth perception in the mouse.Anim Behav. 1965; 13: 232-233Crossref PubMed Scopus (54) Google Scholar In brief, a gray circular structure with a diameter of 49.5 cm was placed on top of a clear 60 × 60-cm plexiglass surface, half of which was hanging off a table. To enhance the edge effect, the portion of the plexiglass surface on the support table was covered with a checkered pattern. Lamps directed at the floor, which also had a checkered pattern, were used to enhance depth and add illumination. Mice were placed near the middle of the visual cliff and monitored for 5 minutes with a camera linked to the Any-Maze behavior tracking software version 3.9.6 (Any-Maze, Stoelting, IL). For data analysis, three zones were created: ground, air, and cliff zones. The cliff zone measured 3 cm in depth from the edge of both the ground and air side. This 3-cm area was excluded from data collection because normal animals frequently stretched their body over the cliff edge to assess the area. For data collection, total distance traveled and total movement time were used to measure activity. To reduce variability, circadian rhythms were taken into account and all experiments were performed between 7 and 11 am. All tests were performed on the same apparatus, in the same room, and by the same individual. For these studies, mice were euthanized via carbon dioxide inhalation and immediately perfused with ice-cold phosphate-buffered saline via cardiac puncture with a 24-gauge needle. Globes were enucleated with the optic nerve intact and fixed in 10% phosphate-buffered formalin or Davidson's fixative for 24 to 48 hours. Whole-eye globes and separated optic nerves were dehydrated and embedded in paraffin. Globes were sectioned sagittally at the midline through the optic nerve (ON) and stained for hematoxylin and eosin (H&E) and Luxol fast blue. Histology slides were scanned on the Aperio AT2 histology slide scanner (Leica Biosystems, Buffalo Grove, IL) or NanoZoomer 2.0-HT histology slide scanner (Hamamatsu Photonics, Ichinocho, Japan). All analyses and measurements were performed using Aperio ImageScope analysis software version 12.3.3 (Leica Biosystems, Buffalo Grove, IL). Morphometric analyses of the retinal layers were obtained approximately two- to three-disc diameters away from the optic nerve. Samples that contained retinal folding two- to three-disc diameters from the optic nerve were excluded from measurements. The anterior-to-posterior globe measurements were taken from the midline of the cornea to the base of the retinal epithelium (Bruch membrane). The remainder of the globe, lens, and corneal measurements were obtained at the anterior/posterior or dorsal/ventral midlines. H&E-stained retinal sections from Asah1+/+ and Asah1P361R/P361R mice at 3 to 4 and 8 to 9 weeks of age were evaluated for retinal dysplasia severity. Our retinal dysplasia scoring system contained three categories: normal, intermediate, and severe. Samples categorized as normal contained no overt folding but may have had minor ridges within the retina. The height of each ridge was ≤20 μm, and no more than two retinal layers were affected. Samples in the intermediate category consisted of one to two folds/whorls. Each fold/whorl affected up to three retinal layers, and the peak height of each fold/whorl was between 20 and 100 μm. Last, for the severe category, samples displayed more than two folds/whorls. The folds/whorls there also affected more than three retinal layers, and the peak height of each fold/whorl was >100 μm. Some cases of severe folding also showed signs of retinal detachment. Eyes were fixed in 10% formalin overnight for retinal sectioning, as described above. For immunohistochemistry (IHC), the following primary antibodies, secondary antibodies, and staining reagents were used: rat anti-mouse Mac-2 at 1:8000 (galectin-3 clone M3/38; Cedarlane, Burlington, ON, Canada); goat anti-mouse cathepsin D at 1:3000 (Santa Cruz Biotechnology, Dallas, TX); biotinylated rabbit anti-rat IgG antibody at 1:5000 (Vector Laboratories, Burlingame, CA); biotinylated donkey anti-rabbit IgG antibody at 1:500 (Vector Laboratories); avidin-biotin/horseradish peroxidase (Vector Laboratories); 3,3′-diaminobenzidine kit (Vector Laboratories); and Vectastain ABC Elite Standard kit (Vector Laboratories). For immunofluorescence staining, the following primary antibodies were used: rabbit anti-ionized, calcium-binding adapter molecule 1 (Iba1) at 1:2000 (Wako Chemicals USA, Cambridge, MA) and chicken anti–glial fibrillary acidic protein (GFAP) at 1:2000 (Aves Lab Inc., Tigard, OR). DAPI at 1:7000 (Sigma Aldrich, St. Louis, MO) was used for nuclear staining. The following secondary antibodies were used to detect the primary antibodies: goat anti-chicken fluorescein isothiocyanate 1:500 (Aves Lab Inc.) and donkey anti-rabbit Cy3 1:500 (Jackson ImmunoResearch USA, West Grove, PA). Immunofluorescence microscopy was performed on the Carl Zeiss LSM510 confocal microscope (Carl Zeiss Microscopy, LLC, Thornwood, NY) using the Zeiss Aim software version 4.2 (Carl Zeiss Microscopy, LLC). After euthanasia, mice were perfused with 4% paraformaldehyde. Eye globes with intact ONs were removed and placed in 4% paraformaldehyde for 24 to 48 hours. ONs were from the posterior pole of the eye globe; approximately 2-mm transverse sections were cut for transmission electron microscope processing. For analysis of the posterior chamber, the cornea was gently cut to expose and remove the lens. The remaining posterior segment structures were used for transmission electron microscope processing. In brief, samples were post-fixed in 3% glutaraldehyde, washed, and placed in 2% OsO4 in phosphate buffer overnight for contrasting. After dehydration, samples were embedded in Durcupan Epon (Fluka, Hatfield, PA) for polymerization. Ultrathin sections (60 nm thick) were cut from tissue blocks of ON and posterior eye samples and placed on copper grids. Ultrathin sections were further stained with uranyl acetate and lead citrate. Samples were analyzed with the JEOL 1400+ (JEOL, Tokyo, Japan) transmission electron microscope equipped with an Olympus Veleta charge-coupled device camera (Olympus Soft Imaging Solutions GMBH, Münster Germany) and Radius software version 1.3 (Olympus, Tokyo, Japan). To assess myelin sheath thickness, the G-ratio (axon diameter/total outer myelin sheath diameter) was measured on electron micrographs of ON cross sections from 8- to 9-week–old mice. Images of the ON were obtained at a magnification of ×2500 (xy axes image sampling density, 27.9 nm). The G-ratio was determined from 300 to 400 randomly chosen fibers per nerve cross section. Images were analyzed using ImageJ software version 1.51 (NIH, Bethesda, MD; https://imagej.nih.gov/ij/index.html), and G-ratio measurements were performed with the G-ratio plugin and online source code (http://gratio.efil.de, last accessed June 12, 2018). After mice were euthanized and perfused as described above, globes were enucleated. Retinas were then carefully separated from the globes under a dissection microscope. Retinal tissue was homogenized in 300 μL phosphate-buffered saline with the Omni Bead Raptor 24 tissue homogenizer (Omni International, Inc., Kennesaw, GA) using 2.8-mm ceramic beads. Lipids were extracted from 100 μL of retinal tissue lysate with 400 μL of methanol. The supernatant was reconstituted with 300 μL of water for mass spectrometry analyses, as previously described.26Yu F.P.S. Dworski S. Medin J.A. Deletion of MCP-1 impedes pathogenesis of acid ceramidase deficiency.Sci Rep. 2018; 8: 1808Crossref PubMed Scopus (11) Google Scholar The following internal standards were spiked in to each retina homogenate before extraction: 50 ng each of deuterated ceramide-1-phosphate [d18:1/16:0 or d18:1/24:0 (Matreya Inc., Pleasant Gap, PA) and d18:1/24:1 (Avanti Polar Lipids Inc., Alabaster, AL)]; 50 ng of deuterated ceramide (d18:1/22:0; Medical University of South Carolina Lipidomics Core, Charleston, SC); 50 ng of C17 analog of monohexosylceramide (d:18:1/17:0; Avanti Polar Lipids Inc.); 500 ng of C17 analog of sphingomyelin (d18:1/17:0; Avanti Polar Lipids Inc.); 100 ng of d7-Sph (Avanti Polar Lipids Inc.); and 100 ng of d7-sphingosine-1-phosphate (S1P; Avanti Polar Lipids Inc.). The samples were analyzed on the Shimadzu 20AD high-performance liquid chromatography system using reverse-phase C18 high-performance liquid chromatography columns (Agilent Co, Santa Clara, CA) and a Leap PAL autosampler coupled to a triple quadrupole mass spectrometer (API-4000; Applied Biosystems, Carlsbad, CA) operated in multiple reaction mode at the Medical University of South Carolina Lipidomics Core. Positive-ion electrospray ionization mode was used to detect all sphingolipids. Retinal extraction samples were injected in duplicate for data averaging. The Analyst software version 1.5.1 was used for data analysis (Applied Biosystems). Sphingolipid measurements were normalized for individual protein concentrations obtained via a bicinchoninic acid assay (Thermo Scientific Pierce, Waltham, MA) and expressed as fold-change over results from Asah1+/+ mice. Lipidomic analyses were conducted separately on retinal tissue from the two different age groups. Ceramide-1-phosphate (C1P) was analyzed in retinal samples from 8- to 9-week–old mice but could not be analyzed in 3- to 4-week–old mice because of insufficient tissue lysate and lipid standards. S1P levels were lower than the limit of detection in retinal samples from both 3- to 4- and 8- to 9-week–old mice. Data are expressed as means ± SEM and analyzed with a t-test unless otherwise stated. Statistics were performed using GraphPad Prism software version 5.0 (GraphPad Software Inc., La Jolla, CA) and MATLAB (MathWorks, Inc., Natick, MA). P < 0.05 was considered statistically significant. In comparison to control 8- to 9-week–old Asah1+/+ mice (Figure 1A), slit-lamp examination of the anterior chamber revealed signs of uveitis in 8- to 9-week–old Asah1P361R/P361R mouse eyes (Figure 1B). These included corneal endothelial granulomatous keratic precipitates, Busacca and Koeppe nodules on the iris, and occasional pigmented deposits along the anterior lens capsule (Figure 1B). No corneal epithelial or stromal defects were observed in the slit-lamp examination. En face infrared and bl
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