Summary of Study ST001073

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 PR000718. The data can be accessed directly via it's Project DOI: 10.21228/M8VT2P This work is supported by NIH grant, U2C- DK119886.

See: https://www.metabolomicsworkbench.org/about/howtocite.php

Study ID	ST001073
Study Title	Lipid profiling of Wnt3a-induced optic nerve regeneration
Study Type	untargeted lipid profiling
Study Summary	We analyzed lipid profiles of mouse retina and optic nerve for 2 time points - 7 and 15 days post-crush, and 3 conditions - intact control, optic nerve crush + vehicle (saline) intravitreal injection, optic nerve crush + 20 ng of Wnt3a injection.
Institute	University of Miami
Department	Ophthalmology, Bascom Palmer Eye Institute
Laboratory	Sanjoy K. Bhattacharya lab
Last Name	Bhattacharya
First Name	Sanjoy
Address	Bascom Palmer Eye Institute (McKnight Bldg.), 1638 NW 10th Avenue, Suite 707A, University of Miami, Miami, FL, 33136
Email	sbhattacharya@med.miami.edu
Phone	305-482-4103
Submit Date	2018-10-03
Num Groups	6
Total Subjects	12
Raw Data Available	Yes
Raw Data File Type(s)	raw(Thermo)
Analysis Type Detail	LC-MS
Release Date	2018-12-11
Release Version	1

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Project:

Project ID:	PR000718
Project DOI:	doi: 10.21228/M8VT2P
Project Title:	Lipid profiling of Wnt3a-induced optic nerve regeneration
Project Type:	untargeted lipid profiling
Project Summary:	We analyzed lipid profiles of mouse retina and optic nerve at 2 time points - 7 and 15 days post-crush, and 3 conditions - intact control, optic nerve crush + vehicle (saline) intravitreal injection, optic nerve crush + 20 ng of Wnt3a injection.
Institute:	University of Miami
Department:	Ophthalmology, Bascom Palmer Eye Institute
Laboratory:	Sanjoy K. Bhattacharya lab
Last Name:	Bhattacharya
First Name:	Sanjoy
Address:	Bascom Palmer Eye Institute (McKnight Bldg.), 1638 NW 10th Avenue, Suite 707A, University of Miami, Miami, FL, 33136
Email:	sbhattacharya@med.miami.edu
Phone:	305-482-4103
Contributors:	Abigail S. Hackam, Tal Carmy, Anna Trzeciecka

Subject:

Subject ID:	SU001117
Subject Type:	Mammal
Subject Species:	Mus musculus
Taxonomy ID:	10090
Genotype Strain:	Tcf/LacZ mice
Age Or Age Range:	6-8 weeks
Gender:	Male and female
Animal Inclusion Criteria:	The Tcf/LacZ strain was previously backcrossed onto the C3H background C3H background, and confirmed by PCR to lack the rd1 mutation

Factors:

Subject type: Mammal; Subject species: Mus musculus (Factor headings shown in green)

mb_sample_id	local_sample_id	Crush	Saline treatment	Wnt3a treatment	Day post-crush
SA072409	QC2	N/A	N/A	N/A	15
SA072410	QC1	N/A	N/A	N/A	15
SA072411	QC4	N/A	N/A	N/A	15
SA072412	QC3	N/A	N/A	N/A	15
SA072413	QC6	N/A	N/A	N/A	15
SA072414	QC5	N/A	N/A	N/A	15
SA072415	HA2_14	No	No	No	15
SA072416	HA2_5	No	No	No	15
SA072417	HA2_2	No	No	No	15
SA072418	HA2_15	No	No	No	15
SA072419	HA2_3	No	No	No	15
SA072420	HA2_24	No	No	No	15
SA072421	HA2_1	No	No	No	15
SA072422	HA2_23	No	No	No	15
SA072423	HA2_18	No	No	No	15
SA072424	HA2_17	No	No	No	15
SA072425	HA2_16	No	No	No	15
SA072426	HA2_4	No	No	No	15
SA072427	HA_16	No	No	No	7
SA072428	HA_11	No	No	No	7
SA072429	HA_12	No	No	No	7
SA072430	HA_15	No	No	No	7
SA072431	HA_20	No	No	No	7
SA072432	HA_8	No	No	No	7
SA072433	HA_7	No	No	No	7
SA072434	HA_3	No	No	No	7
SA072435	HA_4	No	No	No	7
SA072436	HA_23	No	No	No	7
SA072437	HA_19	No	No	No	7
SA072438	HA_24	No	No	No	7
SA072439	HA2_12	Yes	No	Yes	15
SA072440	HA2_11	Yes	No	Yes	15
SA072441	HA2_13	Yes	No	Yes	15
SA072442	HA2_7	Yes	No	Yes	15
SA072443	HA2_8	Yes	No	Yes	15
SA072444	HA2_9	Yes	No	Yes	15
SA072445	HA_9	Yes	No	Yes	7
SA072446	HA_10	Yes	No	Yes	7
SA072447	HA_1	Yes	No	Yes	7
SA072448	HA_2	Yes	No	Yes	7
SA072449	HA_6	Yes	No	Yes	7
SA072450	HA_5	Yes	No	Yes	7
SA072451	HA2_22	Yes	Yes	No	15
SA072452	HA2_21	Yes	Yes	No	15
SA072453	HA2_10	Yes	Yes	No	15
SA072454	HA2_6	Yes	Yes	No	15
SA072455	HA2_19	Yes	Yes	No	15
SA072456	HA2_20	Yes	Yes	No	15
SA072457	HA_21	Yes	Yes	No	7
SA072458	HA_14	Yes	Yes	No	7
SA072459	HA_22	Yes	Yes	No	7
SA072460	HA_18	Yes	Yes	No	7
SA072461	HA_17	Yes	Yes	No	7
SA072462	HA_13	Yes	Yes	No	7

Showing results 1 to 54 of 54

Showing results 1 to 54 of 54

Collection:

Collection ID:	CO001111
Collection Summary:	All procedures involving mice were performed in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and were approved by the Animal Care and Use Committee at the University of Miami. The optic nerve crush and associated treatments were done on Tcf/LacZ mice. As described previously1, the Tcf/LacZ mouse is a transgenic canonical Wnt reporter line that allows localization of active Wnt/β-catenin signaling. In these mice, binding of endogenous nuclear β-catenin to T-cell factor/lymphoid enhancer-binding factor (TCF/Lef) elements in a Wnt-responsive enhancer/promoter region upstream of the LacZ transgene leads to induction of LacZ/β-galactosidase (β-gal) expression wherever Wnt signaling is active. The Tcf/LacZ strain was previously backcrossed onto the C3H background C3H background, and confirmed by PCR to lack the rd1 mutation. Optic nerve crush (ONC) injury was performed as described previously. A total of twelve Tcf/LacZ mice were used for this study. Six mice were used for a 7 day period and six mice were used for a 14 day period. The mice were used at the age of 6-8 weeks and were anesthetized using a ketamine/xylazine cocktail which was injected intraperitoneally. Once the mice were under the anesthesia, the eyes were locally anesthetized with 0.5% proparacaine hydrochloride. The mice were placed on a heating pad to begin the optic nerve crush procedure. Mice of either sex were randomly selected to receive intravitreal injection of recombinant 20ngWnt3a ligand group or saline control group (saline was injected with equivalent volume as recombinant 20ngWnt3a ligand). A small diabetic needle was used to make an incision in the superior posterior area of the conjunctiva-sclera border of the left eye. After which, either recombinant 20ngWnt3a ligand or saline was injected intravitreally using a 1.5 cm 33-gauge Hamilton needle (Hamilton Company, Reno, NV). The injection needle was angled to avoid hitting the lens. The surface membrane of the left eye was cut around the conjunctiva-sclera border. Dumont #5 forceps were inserted between the membrane and the globe to move the surrounding tissues while searching for the optic nerve. Once the optic nerve was located, the forceps’ teeth surrounded the nerve 1 mm from the globe and crushed the nerve for 5 seconds. The crush is successful if there is very minimal or no damage to the surrounding blood supply. Affected left eyes were treated with topical erythromycin ointment and 1mg/ml of Buprenorphine-SR lab was injected subcutaneously. Any mouse with excessive bleeding around the eye was excluded from the study. Mouse’s overall health and left eye’s health were monitored daily from day 1 post crush until day 7 post crush. Mice portraying lethargy 24 hours after surgery or eye infection to the affected eye days after surgery were excluded from the study. OCT scans were obtained from left and right eyes of the mouse while anesthetized on the day of euthanasia. Nerves and retinas were removed immediately after OCT scan for mass spectrometry analysis.
Sample Type:	Eye tissue
Collection Frequency:	7 and 15 days post-crush
Volumeoramount Collected:	entire retina or optic nerve
Storage Conditions:	-80℃
Storage Vials:	low-binding 1.5 mL Eppendorf tubes

Treatment:

Treatment ID:	TR001131
Treatment Summary:	Mice of either sex were randomly selected to receive intravitreal injection of recombinant 20ng Wnt3a ligand group or saline control group (saline was injected with equivalent volume as recombinant 20ng Wnt3a ligand). A small diabetic needle was used to make an incision in the superior posterior area of the conjunctiva-sclera border of the left eye. After which, either recombinant 20ngWnt3a ligand or saline was injected intravitreally using a 1.5 cm 33-gauge Hamilton needle (Hamilton Company, Reno, NV). The injection needle was angled to avoid hitting the lens. The surface membrane of the left eye was cut around the conjunctiva-sclera border. Dumont #5 forceps were inserted between the membrane and the globe to move the surrounding tissues while searching for the optic nerve. Once the optic nerve was located, the forceps’ teeth surrounded the nerve 1 mm from the globe and crushed the nerve for 5 seconds. The crush is successful if there is very minimal or no damage to the surrounding blood supply. Affected left eyes were treated with topical erythromycin ointment and 1mg/ml of Buprenorphine-SR lab was injected subcutaneously. Animal Anesthesia: ketamine/xylazine cocktail intraperitoneally, eyes were locally anesthetized with 0.5% proparacaine hydrochloride.Any mouse with excessive bleeding around the eye was excluded from the study. Mouse’s overall health and left eye’s health were monitored daily from day 1 post crush until day 7 post crush. Animal Endpoint Clinical Signs: Mice portraying lethargy 24 hours after surgery or eye infection to the affected eye days after surgery were excluded from the study.
Treatment:	optic nerve crush, intravitreal injection
Treatment Compound:	Wnt3a or saline
Treatment Route:	intravitreal injection
Treatment Dosevolume:	20 ng of Wnt3a or equal volume of saline
Treatment Doseduration:	single injection
Animal Anesthesia:	See summary
Animal Endp Euthanasia:	CO2 + decapitation
Animal Endp Tissue Coll List:	retina, optic nerve
Animal Endp Clinical Signs:	See summary

Sample Preparation:

Sampleprep ID:	SP001124
Sampleprep Summary:	4 mL of methanol (LC-MS grade) and 2 mL of chloroform (LC-MS grade) were added to each sample. After 2 min vigorous vortexing and 2 min sonication in ultrasonic bath, the samples were incubated at 48˚C overnight (in borosilicate glass vials, PTFE-lined caps). The following day, 2 mL of water (LC-MS grade) and 1 mL of chloroform were added, samples vigorously vortexed for 2 min and centrifuged at 3000 RCF, 4˚C for 15 min to obtain phase separation. Lower phases were collected and dried in centrifugal vacuum concentrator. Samples were stored at -20˚C until reconstituted in 150 µL of chloroform:methanol (1:1) prior to mass spectrometric analysis. Lipid profiling was performed in 2 batches: 7 day and 15 day period. For the 15 day period, quality control (QC) pooled sample was prepared and run 6 times throughout the batch.
Processing Method:	lipid extraction
Processing Storage Conditions:	Described in summary
Extraction Method:	chloroform, methanol, water phase separation
Extract Storage:	-80℃
Sample Resuspension:	150 µL of chloroform:methanol (1:1)
Sample Spiking:	no internal standards added

Combined analysis:

Analysis ID	AN001755
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	relative abundance (main area)

Chromatography:

Chromatography ID:	CH001239
Chromatography Summary:	Reversed phase chromatographic separation utilized Accela Autosampler (Thermo), Accela 600 pump (Thermo) and Acclaim C30 column: 3 µm, 2.1x150 mm (Thermo). The column temperature was maintained at 30˚C and tray temperature at 20˚C. 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 (FA; LC-MS grade). Solvent B was composed of 10 mM ammonium acetate with 60:40 methanol:chloroform with 0.2% FA. The flow rate was 260 µL/min and injection volume was 10 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˚C
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:	10 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
Washing Buffer:	methanol
Chromatography Type:	Reversed phase

MS:

MS ID:	MS001623
Analysis ID:	AN001755
Instrument Name:	Thermo Q Exactive Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	positive and negative mode aligned together
Ion Mode:	UNSPECIFIED
Capillary Temperature:	310˚C (negative mode) or 350˚C (positive mode)
Collision Energy:	15, 30, 45, 60, 75, 90 (in positive and negative mode separately; total 12 runs per sample)
Ion Spray Voltage:	4.4 kV