{
"METABOLOMICS WORKBENCH":{"STUDY_ID":"ST000963","ANALYSIS_ID":"AN001577","VERSION":"1","CREATED_ON":"April 24, 2018, 12:43 pm"},

"PROJECT":{"PROJECT_TITLE":"Lipidomics of inflammation-induced optic nerve regeneration","PROJECT_TYPE":"untargeted LC-MS/MS lipid profiling","PROJECT_SUMMARY":"In adult mammals, retinal ganglion cells (RGCs) fail to regenerate their axons when damaged. As a result, RGCs die after acute injury and in progressive degenerative diseases such as glaucoma; such damage can lead to permanent vision loss and blindness. Little is known about the roles of lipids in axon injury and repair despite their fundamental importance in composition of cell membranes, myelin sheaths and mediation of signaling pathways. Study of the lipidome in the biology of optic nerve (ON) regeneration has been largely neglected. A better understanding of the roles that lipids play in RGC biology may enhance understanding of RGC-related diseases and point to novel treatments. Established experimental models of ON regeneration allow exploration of molecular determinants of RGC axon regenerative success and failure. In this study, we used high-resolution liquid chromatography-tandem mass spectrometry to analyze lipidomic profiles of the ON and retina in an ON crush model with and without intravitreal Zymosan injections to enhance regeneration. Our results reveal profound remodeling of retina and ON lipidomes that occur after injury. In the retina, Zymosan treatment largely abrogates widespread lipidome alterations. In the ON, Zymosan induces lipid profiles that are distinct from those observed in naïve and vehicle-injected crush controls. We have identified a number of lipid species, classes and fatty acids that may be involved in the mechanisms of axon damage and repair. Lipids upregulated during RGC regeneration may be interesting candidates for further functional studies.","INSTITUTE":"Bascom Palmer Eye Insitute, University of Miami","DEPARTMENT":"Ophthalmology","LABORATORY":"Sanjoy K. Bhattacharya lab","LAST_NAME":"Bhattacharya","FIRST_NAME":"Sanjoy","ADDRESS":"McKnight Vision Research Center, 900 NW 17th St, Miami, FL 33136, USA","EMAIL":"sbhattacharya@med.miami.edu","PHONE":"3054824103","FUNDING_SOURCE":"This work was partly supported by a grant from Glaucoma Research Foundation, Payden Glaucoma Research Fund (UCLA), NIH grants U01 EY027257, EY14801, Department of Defense grant number W81XWH-15-1-0079 and an unrestricted grant each from Research to Prevent Blindness to the University of Miami and UCLA.","CONTRIBUTORS":"Anna M. Trzeciecka, David T. Stark, Jacky M. K. Kwong, Maria C. Piqueras, Sanjoy K. Bhattacharya and Joseph Caprioli"},

"STUDY":{"STUDY_TITLE":"Lipidomics of inflammation-induced optic nerve regeneration","STUDY_TYPE":"untargeted LC-MS/MS profiling","STUDY_SUMMARY":"In adult mammals, retinal ganglion cells (RGCs) fail to regenerate their axons when damaged. As a result, RGCs die after acute injury and in progressive degenerative diseases such as glaucoma; such damage can lead to permanent vision loss and blindness. Little is known about the roles of lipids in axon injury and repair despite their fundamental importance in composition of cell membranes, myelin sheaths and mediation of signaling pathways. Study of the lipidome in the biology of optic nerve (ON) regeneration has been largely neglected. A better understanding of the roles that lipids play in RGC biology may enhance understanding of RGC-related diseases and point to novel treatments. Established experimental models of ON regeneration allow exploration of molecular determinants of RGC axon regenerative success and failure. In this study, we used high-resolution liquid chromatography-tandem mass spectrometry to analyze lipidomic profiles of the ON and retina in an ON crush model with and without intravitreal Zymosan injections to enhance regeneration. Our results reveal profound remodeling of retina and ON lipidomes that occur after injury. In the retina, Zymosan treatment largely abrogates widespread lipidome alterations. In the ON, Zymosan induces lipid profiles that are distinct from those observed in naïve and vehicle-injected crush controls. We have identified a number of lipid species, classes and fatty acids that may be involved in the mechanisms of axon damage and repair. Lipids upregulated during RGC regeneration may be interesting candidates for further functional studies.","INSTITUTE":"Bascom Palmer Eye Institute, University of Miami","DEPARTMENT":"Ophthalmology","LABORATORY":"Sanjoy K. Bhattacharya Lab","LAST_NAME":"Bhattacharya","FIRST_NAME":"Sanjoy","ADDRESS":"900 NW 17th St, Miami, FL 33136, USA","EMAIL":"sbhattacharya@med.miami.edu","PHONE":"3054824103","NUM_GROUPS":"9","TOTAL_SUBJECTS":"28","NUM_MALES":"28"},

"SUBJECT":{"SUBJECT_TYPE":"Mammal","SUBJECT_SPECIES":"Rattus norvegicus","TAXONOMY_ID":"10116","GENOTYPE_STRAIN":"Fisher rat","AGE_OR_AGE_RANGE":"10-week-old","WEIGHT_OR_WEIGHT_RANGE":"not measured","HEIGHT_OR_HEIGHT_RANGE":"not measured","GENDER":"Male","ANIMAL_ANIMAL_SUPPLIER":"Charles River","ANIMAL_HOUSING":"standard","ANIMAL_LIGHT_CYCLE":"standard 12 h","ANIMAL_FEED":"standard grain-based","ANIMAL_WATER":"standard unlimited","ANIMAL_INCLUSION_CRITERIA":"random","SPECIES_GROUP":"-"},
"SUBJECT_SAMPLE_FACTORS":[
{
"Subject ID":"-",
"Sample ID":"DS_1",
"Factors":{"Treatment":"naïve control"},
"Additional sample data":{"MS method":"positive (pos) & negative (neg) mode","Sample":"optic nerve","Day post-crush":"3"}
},
{
"Subject ID":"-",
"Sample ID":"DS_2",
"Factors":{"Treatment":"naïve control"},
"Additional sample data":{"MS method":"positive (pos) & negative (neg) mode","Sample":"optic nerve","Day post-crush":"3"}
},
{
"Subject ID":"-",
"Sample ID":"DS_3",
"Factors":{"Treatment":"naïve control"},
"Additional sample data":{"MS method":"positive (pos) & negative (neg) mode","Sample":"optic nerve","Day post-crush":"3"}
},
{
"Subject ID":"-",
"Sample ID":"DS_4",
"Factors":{"Treatment":"optic nerve crush +  vehicle"},
"Additional sample data":{"MS method":"positive (pos) & negative (neg) mode","Sample":"optic nerve","Day post-crush":"3"}
},
{
"Subject ID":"-",
"Sample ID":"DS_5",
"Factors":{"Treatment":"optic nerve crush +  vehicle"},
"Additional sample data":{"MS method":"positive (pos) & negative (neg) mode","Sample":"optic nerve","Day post-crush":"3"}
},
{
"Subject ID":"-",
"Sample ID":"DS_6",
"Factors":{"Treatment":"optic nerve crush +  vehicle"},
"Additional sample data":{"MS method":"positive (pos) & negative (neg) mode","Sample":"optic nerve","Day post-crush":"3"}
},
{
"Subject ID":"-",
"Sample ID":"DS_7",
"Factors":{"Treatment":"optic nerve crush +  Zymosan + CPT-cAMP"},
"Additional sample data":{"MS method":"positive (pos) & negative (neg) mode","Sample":"optic nerve","Day post-crush":"3"}
},
{
"Subject ID":"-",
"Sample ID":"DS_8",
"Factors":{"Treatment":"optic nerve crush +  Zymosan + CPT-cAMP"},
"Additional sample data":{"MS method":"positive (pos) & negative (neg) mode","Sample":"optic nerve","Day post-crush":"3"}
},
{
"Subject ID":"-",
"Sample ID":"DS_9",
"Factors":{"Treatment":"optic nerve crush +  Zymosan + CPT-cAMP"},
"Additional sample data":{"MS method":"positive (pos) & negative (neg) mode","Sample":"optic nerve","Day post-crush":"3"}
},
{
"Subject ID":"-",
"Sample ID":"DS_10",
"Factors":{"Treatment":"naïve control"},
"Additional sample data":{"MS method":"positive (pos) & negative (neg) mode","Sample":"optic nerve","Day post-crush":"7"}
},
{
"Subject ID":"-",
"Sample ID":"DS_11",
"Factors":{"Treatment":"naïve control"},
"Additional sample data":{"MS method":"positive (pos) & negative (neg) mode","Sample":"optic nerve","Day post-crush":"7"}
},
{
"Subject ID":"-",
"Sample ID":"DS_12",
"Factors":{"Treatment":"naïve control"},
"Additional sample data":{"MS method":"positive (pos) & negative (neg) mode","Sample":"optic nerve","Day post-crush":"7"}
},
{
"Subject ID":"-",
"Sample ID":"DS_13",
"Factors":{"Treatment":"optic nerve crush +  vehicle"},
"Additional sample data":{"MS method":"positive (pos) & negative (neg) mode","Sample":"optic nerve","Day post-crush":"7"}
},
{
"Subject ID":"-",
"Sample ID":"DS_14",
"Factors":{"Treatment":"optic nerve crush +  vehicle"},
"Additional sample data":{"MS method":"positive (pos) & negative (neg) mode","Sample":"optic nerve","Day post-crush":"7"}
},
{
"Subject ID":"-",
"Sample ID":"DS_15",
"Factors":{"Treatment":"optic nerve crush +  vehicle"},
"Additional sample data":{"MS method":"positive (pos) & negative (neg) mode","Sample":"optic nerve","Day post-crush":"7"}
},
{
"Subject ID":"-",
"Sample ID":"DS_16",
"Factors":{"Treatment":"optic nerve crush +  Zymosan + CPT-cAMP"},
"Additional sample data":{"MS method":"positive (pos) & negative (neg) mode","Sample":"optic nerve","Day post-crush":"7"}
},
{
"Subject ID":"-",
"Sample ID":"DS_17",
"Factors":{"Treatment":"optic nerve crush +  Zymosan + CPT-cAMP"},
"Additional sample data":{"MS method":"positive (pos) & negative (neg) mode","Sample":"optic nerve","Day post-crush":"7"}
},
{
"Subject ID":"-",
"Sample ID":"DS_18",
"Factors":{"Treatment":"optic nerve crush +  Zymosan + CPT-cAMP"},
"Additional sample data":{"MS method":"positive (pos) & negative (neg) mode","Sample":"optic nerve","Day post-crush":"7"}
},
{
"Subject ID":"-",
"Sample ID":"DS_19",
"Factors":{"Treatment":"naïve control"},
"Additional sample data":{"MS method":"positive (pos) & negative (neg) mode","Sample":"retina","Day post-crush":"7"}
},
{
"Subject ID":"-",
"Sample ID":"DS_20",
"Factors":{"Treatment":"naïve control"},
"Additional sample data":{"MS method":"positive (pos) & negative (neg) mode","Sample":"retina","Day post-crush":"7"}
},
{
"Subject ID":"-",
"Sample ID":"DS_21",
"Factors":{"Treatment":"naïve control"},
"Additional sample data":{"MS method":"positive (pos) & negative (neg) mode","Sample":"retina","Day post-crush":"7"}
},
{
"Subject ID":"-",
"Sample ID":"DS_22",
"Factors":{"Treatment":"optic nerve crush +  vehicle"},
"Additional sample data":{"MS method":"positive (pos) & negative (neg) mode","Sample":"retina","Day post-crush":"7"}
},
{
"Subject ID":"-",
"Sample ID":"DS_23",
"Factors":{"Treatment":"optic nerve crush +  vehicle"},
"Additional sample data":{"MS method":"positive (pos) & negative (neg) mode","Sample":"retina","Day post-crush":"7"}
},
{
"Subject ID":"-",
"Sample ID":"DS_24",
"Factors":{"Treatment":"optic nerve crush +  vehicle"},
"Additional sample data":{"MS method":"positive (pos) & negative (neg) mode","Sample":"retina","Day post-crush":"7"}
},
{
"Subject ID":"-",
"Sample ID":"DS_25",
"Factors":{"Treatment":"optic nerve crush +  Zymosan + CPT-cAMP"},
"Additional sample data":{"MS method":"positive (pos) & negative (neg) mode","Sample":"retina","Day post-crush":"7"}
},
{
"Subject ID":"-",
"Sample ID":"DS_26",
"Factors":{"Treatment":"optic nerve crush +  Zymosan + CPT-cAMP"},
"Additional sample data":{"MS method":"positive (pos) & negative (neg) mode","Sample":"retina","Day post-crush":"7"}
},
{
"Subject ID":"-",
"Sample ID":"DS_27",
"Factors":{"Treatment":"optic nerve crush +  Zymosan + CPT-cAMP"},
"Additional sample data":{"MS method":"positive (pos) & negative (neg) mode","Sample":"retina","Day post-crush":"7"}
},
{
"Subject ID":"-",
"Sample ID":"DS_28",
"Factors":{"Treatment":"naïve control"},
"Additional sample data":{"MS method":"positive (pos) & negative (neg) mode","Sample":"optic nerve","Day post-crush":"14"}
},
{
"Subject ID":"-",
"Sample ID":"DS_29",
"Factors":{"Treatment":"naïve control"},
"Additional sample data":{"MS method":"positive (pos) & negative (neg) mode","Sample":"optic nerve","Day post-crush":"14"}
},
{
"Subject ID":"-",
"Sample ID":"DS_30",
"Factors":{"Treatment":"naïve control"},
"Additional sample data":{"MS method":"positive (pos) & negative (neg) mode","Sample":"optic nerve","Day post-crush":"14"}
},
{
"Subject ID":"-",
"Sample ID":"DS_31",
"Factors":{"Treatment":"optic nerve crush +  vehicle"},
"Additional sample data":{"MS method":"positive (pos) & negative (neg) mode","Sample":"optic nerve","Day post-crush":"14"}
},
{
"Subject ID":"-",
"Sample ID":"DS_32",
"Factors":{"Treatment":"optic nerve crush +  vehicle"},
"Additional sample data":{"MS method":"positive (pos) & negative (neg) mode","Sample":"optic nerve","Day post-crush":"14"}
},
{
"Subject ID":"-",
"Sample ID":"DS_33",
"Factors":{"Treatment":"optic nerve crush +  vehicle"},
"Additional sample data":{"MS method":"positive (pos) & negative (neg) mode","Sample":"optic nerve","Day post-crush":"14"}
},
{
"Subject ID":"-",
"Sample ID":"DS_34",
"Factors":{"Treatment":"optic nerve crush +  Zymosan + CPT-cAMP"},
"Additional sample data":{"MS method":"positive (pos) & negative (neg) mode","Sample":"optic nerve","Day post-crush":"14"}
},
{
"Subject ID":"-",
"Sample ID":"DS_35",
"Factors":{"Treatment":"optic nerve crush +  Zymosan + CPT-cAMP"},
"Additional sample data":{"MS method":"positive (pos) & negative (neg) mode","Sample":"optic nerve","Day post-crush":"14"}
},
{
"Subject ID":"-",
"Sample ID":"DS_36",
"Factors":{"Treatment":"optic nerve crush +  Zymosan + CPT-cAMP"},
"Additional sample data":{"MS method":"positive (pos) & negative (neg) mode","Sample":"optic nerve","Day post-crush":"14"}
},
{
"Subject ID":"-",
"Sample ID":"DS_37",
"Factors":{"Treatment":"optic nerve crush +  Zymosan + CPT-cAMP"},
"Additional sample data":{"MS method":"positive (pos) & negative (neg) mode","Sample":"optic nerve","Day post-crush":"14"}
},
{
"Subject ID":"-",
"Sample ID":"DS_38",
"Factors":{"Treatment":"naïve control"},
"Additional sample data":{"MS method":"positive (pos) & negative (neg) mode","Sample":"retina","Day post-crush":"14"}
},
{
"Subject ID":"-",
"Sample ID":"DS_39",
"Factors":{"Treatment":"naïve control"},
"Additional sample data":{"MS method":"positive (pos) & negative (neg) mode","Sample":"retina","Day post-crush":"14"}
},
{
"Subject ID":"-",
"Sample ID":"DS_40",
"Factors":{"Treatment":"naïve control"},
"Additional sample data":{"MS method":"positive (pos) & negative (neg) mode","Sample":"retina","Day post-crush":"14"}
},
{
"Subject ID":"-",
"Sample ID":"DS_41",
"Factors":{"Treatment":"optic nerve crush +  vehicle"},
"Additional sample data":{"MS method":"positive (pos) & negative (neg) mode","Sample":"retina","Day post-crush":"14"}
},
{
"Subject ID":"-",
"Sample ID":"DS_42",
"Factors":{"Treatment":"optic nerve crush +  vehicle"},
"Additional sample data":{"MS method":"positive (pos) & negative (neg) mode","Sample":"retina","Day post-crush":"14"}
},
{
"Subject ID":"-",
"Sample ID":"DS_43",
"Factors":{"Treatment":"optic nerve crush +  vehicle"},
"Additional sample data":{"MS method":"positive (pos) & negative (neg) mode","Sample":"retina","Day post-crush":"14"}
},
{
"Subject ID":"-",
"Sample ID":"DS_44",
"Factors":{"Treatment":"optic nerve crush +  Zymosan + CPT-cAMP"},
"Additional sample data":{"MS method":"positive (pos) & negative (neg) mode","Sample":"retina","Day post-crush":"14"}
},
{
"Subject ID":"-",
"Sample ID":"DS_45",
"Factors":{"Treatment":"optic nerve crush +  Zymosan + CPT-cAMP"},
"Additional sample data":{"MS method":"positive (pos) & negative (neg) mode","Sample":"retina","Day post-crush":"14"}
},
{
"Subject ID":"-",
"Sample ID":"DS_46",
"Factors":{"Treatment":"optic nerve crush +  Zymosan + CPT-cAMP"},
"Additional sample data":{"MS method":"positive (pos) & negative (neg) mode","Sample":"retina","Day post-crush":"14"}
},
{
"Subject ID":"-",
"Sample ID":"DS_47",
"Factors":{"Treatment":"optic nerve crush +  Zymosan + CPT-cAMP"},
"Additional sample data":{"MS method":"positive (pos) & negative (neg) mode","Sample":"retina","Day post-crush":"14"}
}
],
"COLLECTION":{"COLLECTION_SUMMARY":"Experimental groups consisted of naïve controls (control), ON crush + intravitreal Zymosan + CPT-cAMP (regeneration), and ON crush + intravitreal vehicle (crush). Retinas were harvested on day 7 and 14 and ON were harvested on day 3, 7 and 14 post-crush.","COLLECTION_PROTOCOL_COMMENTS":"retina and optic nerves collected","SAMPLE_TYPE":"Eye tissue","COLLECTION_FREQUENCY":"day 3, 7 and 14 post-crush","STORAGE_CONDITIONS":"-80℃","COLLECTION_VIALS":"1.5 mL polypropylene tubes","STORAGE_VIALS":"1.5 mL polypropylene tubes"},

"TREATMENT":{"TREATMENT_SUMMARY":"A rat model of inflammation-induced ON regeneration was established by intravitreal injection of a yeast cell wall preparation (Zymosan A) and a cell permeant CPT-cAMP, immediately after ON crush. Ten-week-old male Fischer rats were deeply anesthetized with inhaled isoflurane, and the eyes were treated with topical anesthetic (proparacaine HCl 0.5% ophthalmic) and a cycloplegic (tropicamide 0.5% ophthalmic) to reduce pain and assist with visualization of intravitreal injections. The left ON was exposed by blunt dissection through a temporal, fornix-based conjunctival incision and crushed for 10 seconds with Dumoxel #N5 self-closing forceps (Dumont, Montignez, Switzerland). Absence of injury to the retinal vascular supply was confirmed by post-crush funduscopic examination. Intravitreal injections (5 µL) of PBS vehicle or a suspension of finely ground, sterilized Zymosan A (Z4250; Sigma-Aldrich, St. Louis, MO, USA; 12.5 mg/mL) plus CPT-cAMP (C3912; Sigma-Aldrich, St. Louis, MO, USA; 100 µM) were performed with a pulled glass pipette attached to a Hamilton syringe on a manual micromanipulator. Injections were made 2 mm posterior to the limbus, and care was taken to prevent lens injury, choroidal hemorrhage, or retinal detachment. Absence of these intraocular adverse events was confirmed by fundoscopic examination. Conjunctival incisions were closed with 8-0 vicryl sutures and petrolatum ophthalmic ointment was applied to the ocular surface."},

"SAMPLEPREP":{"SAMPLEPREP_SUMMARY":"Specimens were stored at -80ºC. Before extraction, ON and retinas were cut into ~1 mm3 pieces. 6 mL of methanol (LC-MS grade) and 3 mL of chloroform (LC-MS grade) were added to each sample. For this high throughput approach, no internal standards were added to samples. After 2 min vigorous vortexing and 2 min sonication in ultrasonic bath, the samples were incubated at 48ºC overnight (in borosilicate glass vials, PTFE-lined caps). The following day, 3 mL of water (LC-MS grade) and 1.5 mL of chloroform were added, samples vigorously vortexed for 2 min and centrifuged at 3000 RCF, 4ºC for 15 min to obtain phase separation. Lower phases were collected and dried in a centrifugal vacuum concentrator. Samples were stored at -20ºC until reconstituted in 60 µL of chloroform:methanol (1:1) prior to mass spectrometric analysis.","PROCESSING_METHOD":"lipid extraction","PROCESSING_STORAGE_CONDITIONS":"Described in summary","EXTRACT_STORAGE":"-20℃","SAMPLE_SPIKING":"no internal standards added"},

"CHROMATOGRAPHY":{"CHROMATOGRAPHY_SUMMARY":"Reversed phase chromatographic separation was achieved using Accela Autosampler, Accela 600 pump and Acclaim C30 column: 3 µm, 2.1x150 mm (Thermo Fisher Scientific, Waltham, MA). The column temperature was maintained at 30 ºC (negative mode) or 45ºC (positive mode) and tray temperature at 20ºC. Solvent A was composed of 10 mM ammonium acetate (LC-MS grade) in 60:40 methanol:water (LC-MS grade) with 0.2% formic acid (LC-MS grade). Solvent B was composed of 10 mM ammonium acetate with 60:40 methanol:chloroform with 0.2% formic acid. The flow rate was 260 µL/min and injection volume was 5 µL. The gradient was 35-100% solvent B over 13.0 min, 100% solvent B over 13.0-13.8 min, 100-35% solvent B over 13.8-14.5 min, 35% solvent B over 14.5-18.0 min, 0% solvent B over 18.0-20.0 min.","CHROMATOGRAPHY_TYPE":"Reversed phase","INSTRUMENT_NAME":"Thermo Accela 600","COLUMN_NAME":"Acclaim C30 column: 3 µm, 2.1x150 mm","FLOW_GRADIENT":"The gradient was 35-100% solvent B over 13.0 min, 100% solvent B over 13.0-13.8 min, 100-35% solvent B over 13.8-14.5 min, 35% solvent B over 14.5-18.0 min, 0% solvent B over 18.0-20.0 min.","FLOW_RATE":"260 µL/min","COLUMN_TEMPERATURE":"30 (negative mode)/45 (positive mode)","SOLVENT_A":"10 mM ammonium acetate in 60:40 methanol:water with 0.2% formic acid","SOLVENT_B":"10 mM ammonium acetate in 60:40 methanol:chloroform with 0.2% formic acid","SAMPLE_INJECTION":"5 µL","ANALYTICAL_TIME":"20 min","OVEN_TEMPERATURE":"20ºC"},

"ANALYSIS":{"ANALYSIS_TYPE":"MS","DATA_FORMAT":".RAW"},

"MS":{"MS_COMMENTS":"-","INSTRUMENT_NAME":"Thermo Q Exactive Orbitrap","INSTRUMENT_TYPE":"Orbitrap","MS_TYPE":"ESI","ION_MODE":"UNSPECIFIED","CAPILLARY_TEMPERATURE":"310ºC (negative mode) or 350ºC (positive mode)","COLLISION_ENERGY":"40±30% for the negative mode and 30, parallel with 19±5% for the positive mode","IONIZATION":"+/-","SPRAY_VOLTAGE":"4.4 kV","AUTOMATIC_GAIN_CONTROL":"MS: 1x106; MS/MS: 2x105 (negative mode) or 1x105 (positive mode)","DATAFORMAT":".RAW","RESOLUTION_SETTING":"MS: 70,000; MS/MS: 17,500"},

"MS_METABOLITE_DATA":{
"Units":"peak area",

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}