Lipid profiling dataset of the Wnt3a-induced optic nerve regeneration

We present lipid profiling data from mouse retina and optic nerve after optic nerve crush and during Wnt3a-induced axonal regeneration at 7 and 15 days post-crush. This data is available at the Metabolomics Workbench, http://www.metabolomicsworkbench.org (Project ID: PR000718).


Data
We performed lipid profiling of the mouse retina and optic nerve after optic nerve crush followed by a single injection of saline (vehicle) or Wnt3a to induce retinal ganglion cells regeneration (Fig. 1). Specimens were collected 7 and 15 days post-crush, followed by chloroform-methanol based lipid extraction. Extracted lipid samples were analyzed using reversed-phase high-performance liquid chromatography (HPLC) with C30 column coupled to a Q Exactive mass spectrometer. Lipid identification and relative quantification were performed in LipidSearch software. Distributions of the CV % values for the experimental groups including intact controls are presented in Fig. 2 a-b. Each timepoint groups of samples were clearly distinguished from quality control (QC) group and from each other with 75.7e80.2% of variance accounted for by PC1 and PC2 (Fig. 2 c-g). Biological replicates showed Pearson correlation coefficients ranging from 0.92 to 0.98 (Fig. 2 h). To identify features undergoing significant changes between experimental groups, we used one-way ANOVA analysis with Tukey's post-hoc test. We examined a number of significant features at different FDR adjusted p values for lipid species (Fig. 2 i). Next, we performed hierarchical clustering and heatmap visualization of the dysregulated species (FDR adjusted p values < 0.05). The heatmaps of significant species in the post-crush 7-day retina, 15-day retina, 7-day ON and 15-day ON are presented in Figs. 3e6 respectively.

ON crush and intravitreal injections
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 previously [1], theTcf/LacZ mouse is a transgenic canonical Wnt reporter line that allows localization of active Wnt/b-catenin signaling [2]. In these mice, binding of endogenous nuclear b-catenin to T-cell factor/lymphoid enhancer-binding factor (TCF/Lef) elements in a Wntresponsive enhancer/promoter region upstream of the LacZ transgene leads to induction of LacZ/

Value of the Data
Dataset explores retina and optic nerve lipid profiles after optic nerve crush and during Wnt3a-promoted axonal regeneration. This data adds to the repertoire of axonal regeneration omics profiling datasets from different animal models and may be useful for integrative analysis and interpretation. This dataset will be useful for the community of researchers studying optic neuropathies and optic nerve regeneration.
Specifications b-galactosidase (b-gal) expression wherever Wnt signaling is active. The Tcf/LacZ strain was previously backcrossed onto the C3H background [3], and confirmed by PCR to lack the rd1 mutation. Optic nerve crush (ONC) injury was performed as described previously [4]. 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 6e8 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 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 20ng Wnt3a 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.
Regenerating RGC axons were anterogradely labeled using cholera toxin b subunit (CTB) with a conjugated Alexafluor 546 (Thermo Fisher Scientific, Waltham, MA) that was intravitreously injected in a volume of 2 mL at two days prior to animal euthanasia. The animals were then perfused using 4% paraformaldehyde and eyes and optic nerves were dissected and processed through a 5e20% sucrose gradient and then embedded in OCT Compound (Tissue Tek) for cyrosectioning. Longitudinal optic nerve sections were cut at a thickness of 8 mm and mounted onto slides and then imaged using a fluorescent microscope (Zeiss).      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 a centrifugal vacuum concentrator. Samples were stored at À20 C until reconstituted in 150 mL of chloroform:methanol (1:1) prior to mass spectrometric analysis. Samples list is in Table 1. 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.

Mass spectrometry
The Q Exactive (Thermo) mass spectrometer was operated under heated electrospray ionization (HESI) in positive and negative mode separately. The spray voltage was 4.4 kV, the heated capillary was held at 310 C (negative mode) or 350 C (positive mode) and heater at 275 C (positive mode). The S-lens radio frequency (RF) level was 70. The sheath gas flow rate was 30 (negative mode) or 45 units (positive mode) and auxiliary gas was 14 (negative mode) or 15 units (positive mode). Full scan used resolution 70,000 with automatic gain control (AGC) target of 1 Â 10 6 ions and maximum ion injection time (IT) of 100 ms. Data-dependent MS/MS (top10) were acquired using the following parameters: resolution 17,500; AGC 1 Â 10 5 ; maximum IT 75 ms; 1.3 m/z isolation window; underfill ratio 0.1%; intensity threshold 1 Â 10 3 ; dynamic exclusion time 3 s. Normalized collision energy (NCE) settings were: 15, 30, 45, 60, 75, 90 (in positive and negative mode separately; total 12 runs per sample). Total instrument time~216 h. External mass calibration was performed using the standard calibration mixture. Each sample was preceded and followed by a blank run (1:1 chloroform:methanol).

Data processing
Positive and negative mode identifications at different NCE were aligned in LipidSearch, allowing calculation of unassigned peaks. The following settings were applied: product search; alignment method max; retention time tolerance 0.1 min; filters: toprank, main isomer peak; M-score 5; molecular lipid identification grade: A-B (A: lipid class and fatty acid completely identified or B: lipid class and some fatty acid identified). Peaks of the following lipid classes: BisMePA, SQMG, MGDG and MGMG, were considered false positives and were removed. Only peaks appearing in all replicates were accepted. Peaks with the same annotated lipid species were merged. Lists of species and their main areas were uploaded to Metaboanalyst 4.0 [5] statistical analysis module. Missing values were replaced with a small number (half of the minimum positive value in the original data). Normalization to median and log2 transformation were applied.

Transparency document
Transparency document associated with this article can be found in the online version at https:// doi.org/10.1016/j.dib.2019.103966.