Summary of Study ST000963

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR000661. The data can be accessed directly via it's Project DOI: 10.21228/M87D53 This work is supported by NIH grant, U2C- DK119886.

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Study IDST000963
Study TitleLipidomics of inflammation-induced optic nerve regeneration
Study Typeuntargeted LC-MS/MS profiling
Study SummaryIn 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
University of Miami
DepartmentOphthalmology, Bascom Palmer Eye Institute
LaboratorySanjoy K. Bhattacharya Lab
Last NameBhattacharya
First NameSanjoy
Address900 NW 17th St, Miami, FL 33136, USA
Emailsbhattacharya@med.miami.edu
Phone3054824103
Submit Date2018-04-17
Num Groups9
Total Subjects28
Num Males28
Raw Data AvailableYes
Raw Data File Type(s)raw(Thermo)
Analysis Type DetailLC-MS
Release Date2018-09-27
Release Version1
Sanjoy Bhattacharya Sanjoy Bhattacharya
https://dx.doi.org/10.21228/M87D53
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

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Project:

Project ID:PR000661
Project DOI:doi: 10.21228/M87D53
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:University of Miami
Department:Bascom Palmer Eye Insitute, 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

Subject:

Subject ID:SU001002
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:-

Factors:

Subject type: Mammal; Subject species: Rattus norvegicus (Factor headings shown in green)

mb_sample_id local_sample_id Treatment
SA057645DS_30naïve control
SA057646DS_40naïve control
SA057647DS_12naïve control
SA057648DS_29naïve control
SA057649DS_19naïve control
SA057650DS_21naïve control
SA057651DS_20naïve control
SA057652DS_1naïve control
SA057653DS_28naïve control
SA057654DS_11naïve control
SA057655DS_2naïve control
SA057656DS_3naïve control
SA057657DS_38naïve control
SA057658DS_39naïve control
SA057659DS_10naïve control
SA057677DS_42optic nerve crush + vehicle
SA057678DS_43optic nerve crush + vehicle
SA057679DS_41optic nerve crush + vehicle
SA057680DS_24optic nerve crush + vehicle
SA057681DS_13optic nerve crush + vehicle
SA057682DS_6optic nerve crush + vehicle
SA057683DS_5optic nerve crush + vehicle
SA057684DS_4optic nerve crush + vehicle
SA057685DS_14optic nerve crush + vehicle
SA057686DS_15optic nerve crush + vehicle
SA057687DS_32optic nerve crush + vehicle
SA057688DS_31optic nerve crush + vehicle
SA057689DS_23optic nerve crush + vehicle
SA057690DS_22optic nerve crush + vehicle
SA057691DS_33optic nerve crush + vehicle
SA057660DS_25optic nerve crush + Zymosan + CPT-cAMP
SA057661DS_36optic nerve crush + Zymosan + CPT-cAMP
SA057662DS_27optic nerve crush + Zymosan + CPT-cAMP
SA057663DS_34optic nerve crush + Zymosan + CPT-cAMP
SA057664DS_35optic nerve crush + Zymosan + CPT-cAMP
SA057665DS_26optic nerve crush + Zymosan + CPT-cAMP
SA057666DS_18optic nerve crush + Zymosan + CPT-cAMP
SA057667DS_8optic nerve crush + Zymosan + CPT-cAMP
SA057668DS_9optic nerve crush + Zymosan + CPT-cAMP
SA057669DS_7optic nerve crush + Zymosan + CPT-cAMP
SA057670DS_46optic nerve crush + Zymosan + CPT-cAMP
SA057671DS_47optic nerve crush + Zymosan + CPT-cAMP
SA057672DS_45optic nerve crush + Zymosan + CPT-cAMP
SA057673DS_44optic nerve crush + Zymosan + CPT-cAMP
SA057674DS_37optic nerve crush + Zymosan + CPT-cAMP
SA057675DS_17optic nerve crush + Zymosan + CPT-cAMP
SA057676DS_16optic nerve crush + Zymosan + CPT-cAMP
Showing results 1 to 47 of 47

Collection:

Collection ID:CO000996
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 ID:TR001016
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.

Sample Preparation:

Sampleprep ID:SP001009
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.
Sampleprep Protocol Filename:method.docx
Processing Method:lipid extraction
Processing Storage Conditions:Described in summary
Extract Storage:-20℃
Sample Spiking:no internal standards added

Combined analysis:

Analysis ID AN001577
Analysis type MS
Chromatography type Reversed phase
Chromatography system Thermo Accela 600
Column Thermo Acclaim C30 (150 x 2.1mm,3um)
MS Type ESI
MS instrument type Orbitrap
MS instrument name Thermo Q Exactive Orbitrap
Ion Mode UNSPECIFIED
Units peak area

Chromatography:

Chromatography ID:CH001106
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.
Instrument Name:Thermo Accela 600
Column Name:Thermo Acclaim C30 (150 x 2.1mm,3um)
Column Temperature:30 (negative mode)/45 (positive mode)
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
Sample Injection:5 µL
Solvent A:60% methanol/40% water; 0.2% formic acid; 10 mM ammonium acetate
Solvent B:60% methanol/40% chloroform; 0.2% formic acid; 10 mM ammonium acetate
Analytical Time:20 min
Oven Temperature:20ºC
Chromatography Type:Reversed phase

MS:

MS ID:MS001455
Analysis ID:AN001577
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
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