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.
See: https://www.metabolomicsworkbench.org/about/howtocite.php
| Study ID | ST000963 |
| 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 | University of Miami |
| Department | Ophthalmology, Bascom Palmer Eye Institute |
| Laboratory | Sanjoy K. Bhattacharya Lab |
| Last Name | Bhattacharya |
| First Name | Sanjoy |
| Address | 900 NW 17th St, Miami, FL 33136, USA |
| sbhattacharya@med.miami.edu | |
| Phone | 3054824103 |
| Submit Date | 2018-04-17 |
| Num Groups | 9 |
| Total Subjects | 28 |
| Num Males | 28 |
| Raw Data Available | Yes |
| Raw Data File Type(s) | raw(Thermo) |
| Analysis Type Detail | LC-MS |
| Release Date | 2018-09-27 |
| Release Version | 1 |
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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 |
|---|---|---|
| SA057645 | DS_30 | naïve control |
| SA057646 | DS_40 | naïve control |
| SA057647 | DS_12 | naïve control |
| SA057648 | DS_29 | naïve control |
| SA057649 | DS_19 | naïve control |
| SA057650 | DS_21 | naïve control |
| SA057651 | DS_20 | naïve control |
| SA057652 | DS_1 | naïve control |
| SA057653 | DS_28 | naïve control |
| SA057654 | DS_11 | naïve control |
| SA057655 | DS_2 | naïve control |
| SA057656 | DS_3 | naïve control |
| SA057657 | DS_38 | naïve control |
| SA057658 | DS_39 | naïve control |
| SA057659 | DS_10 | naïve control |
| SA057677 | DS_42 | optic nerve crush + vehicle |
| SA057678 | DS_43 | optic nerve crush + vehicle |
| SA057679 | DS_41 | optic nerve crush + vehicle |
| SA057680 | DS_24 | optic nerve crush + vehicle |
| SA057681 | DS_13 | optic nerve crush + vehicle |
| SA057682 | DS_6 | optic nerve crush + vehicle |
| SA057683 | DS_5 | optic nerve crush + vehicle |
| SA057684 | DS_4 | optic nerve crush + vehicle |
| SA057685 | DS_14 | optic nerve crush + vehicle |
| SA057686 | DS_15 | optic nerve crush + vehicle |
| SA057687 | DS_32 | optic nerve crush + vehicle |
| SA057688 | DS_31 | optic nerve crush + vehicle |
| SA057689 | DS_23 | optic nerve crush + vehicle |
| SA057690 | DS_22 | optic nerve crush + vehicle |
| SA057691 | DS_33 | optic nerve crush + vehicle |
| SA057660 | DS_25 | optic nerve crush + Zymosan + CPT-cAMP |
| SA057661 | DS_36 | optic nerve crush + Zymosan + CPT-cAMP |
| SA057662 | DS_27 | optic nerve crush + Zymosan + CPT-cAMP |
| SA057663 | DS_34 | optic nerve crush + Zymosan + CPT-cAMP |
| SA057664 | DS_35 | optic nerve crush + Zymosan + CPT-cAMP |
| SA057665 | DS_26 | optic nerve crush + Zymosan + CPT-cAMP |
| SA057666 | DS_18 | optic nerve crush + Zymosan + CPT-cAMP |
| SA057667 | DS_8 | optic nerve crush + Zymosan + CPT-cAMP |
| SA057668 | DS_9 | optic nerve crush + Zymosan + CPT-cAMP |
| SA057669 | DS_7 | optic nerve crush + Zymosan + CPT-cAMP |
| SA057670 | DS_46 | optic nerve crush + Zymosan + CPT-cAMP |
| SA057671 | DS_47 | optic nerve crush + Zymosan + CPT-cAMP |
| SA057672 | DS_45 | optic nerve crush + Zymosan + CPT-cAMP |
| SA057673 | DS_44 | optic nerve crush + Zymosan + CPT-cAMP |
| SA057674 | DS_37 | optic nerve crush + Zymosan + CPT-cAMP |
| SA057675 | DS_17 | optic nerve crush + Zymosan + CPT-cAMP |
| SA057676 | DS_16 | optic 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 |
