Lipidomics dataset of PTEN deletion-induced optic nerve regeneration mouse model

The optic nerve is part of the mammalian adult central nervous system (CNS) and has limited capability to regenerate after injury. Deletion of phosphatase and tensin homolog (PTEN), a negative regulator of the PI3 kinase/Akt pathway, has been shown to promote regeneration in retinal ganglion cells (RGCs) after optic nerve injury [1]. We present the lipidome of adult PTENloxP/loxP mice subjected to intravitreal injection of adeno-associated viruses expressing Cre (AAV-Cre) as a model of CNS neuroregeneration. At 4 weeks old, PTENloxP/loxP mice were intravitreally-injected with 2–3 μl of either AAV-Cre (KO) or AAV-PLAP (control), and two weeks later optic nerve crush was performed. At indicated time-points after crush (0 days, 7 days, 14 days), mice were euthanized and optic nerves were immediately dissected out, and then flash frozen on dry ice. A modified Bligh and Dyer [2] method was used for lipid extraction from the optic nerves, followed by liquid chromatography-mass spectrometry (LC MS-MS) lipid profiling using a Q-Exactive Orbitrap instrument coupled with Accela 600 HPLC. The raw scans were analysed with LipidSearch 4.2 and the statistical analysis was conducted through Metaboanalyst 4.0. This data is available at Metabolomics Workbench, study ID ST001477.


a b s t r a c t
The optic nerve is part of the mammalian adult central nervous system (CNS) and has limited capability to regenerate after injury. Deletion of phosphatase and tensin homolog (PTEN), a negative regulator of the PI3 kinase/Akt pathway, has been shown to promote regeneration in retinal ganglion cells (RGCs) after optic nerve injury [1] . We present the lipidome of adult PTEN loxP/loxP mice subjected to intravitreal injection of adeno-associated viruses expressing Cre (AAV-Cre) as a model of CNS neuroregeneration. At 4 weeks old, PTEN loxP/loxP mice were intravitreally-injected with 2-3 μl of either AAV-Cre (KO) or AAV-PLAP (control), and two weeks later optic nerve crush was performed. At indicated timepoints after crush (0 days, 7 days, 14 days), mice were euthanized and optic nerves were immediately dissected out, and then flash frozen on dry ice. A modified Bligh and Dyer [2] method was used for lipid extraction from the optic nerves, followed by liquid chromatography-mass spectrometry (LC MS-MS) lipid profiling using a Q-Exactive Orbitrap instrument coupled with Accela 600 HPLC. The raw scans were analysed with LipidSearch 4.2 and the statistical analysis was

Value of the Data
• The data provided pertain to the lipid changes that occur in the optic nerves of Pten loxP/loxP (KO and control) mice after optic nerve crush injury and subsequent axon regeneration. • The data is useful for the future studies of central nervous system (CNS) nerve injury and regeneration promoted using different genetic and pharmacological treatments. • The data can be employed for the development of databases for future targeted lipidomics experiments for CNS neuronal regeneration.

Data Description
Lipid profiling was performed on PTEN deletion-induced optic nerve regeneration mouse model and controls. At postnatal day 28, PTEN loxP/loxP mice were subjected to intravitreal injection of adeno-associated viruses expressing Cre (AAV2-Cre) or control placenta alkaline phosphatase (AAV2-PLAP). Optic nerve crush (ONC) was performed 2 weeks after the AAV injections and at three different time points (0, 7, 14 days post crush) the nerves were collected for lipidomics ( Fig. 1 ). Cholera toxin subunit B conjugated with Alexfluor555 (CTB-555) was used  for anterograde labelling of the RGCs on crush day and 14 days post AVV injections. The images were taken with a Zeiss LSM 710 multiphoton confocal microscope ( Fig. 2 ). Lipids were extracted using a modified Bligh and Dyer [2] method with an EquiSplash standard and analyzed using reversed-phase high-performance liquid chromatography (HPLC) coupled to a Q Exactive mass spectrometer. Lipid identification and relative quantification were performed in LipidSearch software 4.2. Metaboanalyst 4.0 was used for statistical analysis ( Fig. 3 and Fig. 4 ).

Animals
All surgical procedures were performed in compliance with animal protocols approved by the IACUC at Boston Children's Hospital. Mice were anaesthetized with ketamine and xylazine and received buprenorphine as a postoperative analgesic. For AAV injection, 4-week-old Pten loxP/loxP mice were intravitreally-injected with 2-3 μl of either AAV-Cre (KO) or AAV-PLAP (control) with a pulled glass micropipette attached to a Hamilton syringe (Hamilton, Reno, NV). For intravitreal injections, the pulled-glass micropipette was inserted near the peripheral retina behind the ora serrata and deliberately angled to avoid damage to the lens. Optic nerve crush (ONC) injury was performed two weeks after AAV injection, as per previously described [1]. Briefly, the optic nerve was exposed at the intra orbital space and crushed with fine forceps (Dumont #5, Fine Science Tools, Foster City, CA) for 5 s approximately 500 mm behind the optic disc. Eye ointment was applied post-operatively to protect the cornea. At indicated time-points, mice were euthanized, and optic nerves were immediately dissected out, and then flash frozen on dry ice.

Optic nerve regeneration visualization
For anterograde labeling of retinal ganglion cell axons within the optic nerve, 1 μl of cholera toxin subunit B with a conjugated Alexfluor555 (1 μg/μl in sterile saline, Thermo Fisher Scientific, Waltham, MA) was injected intravitreally two days before perfusion. Dissected optic nerves were cleared using Visikol clearing solutions (per manufacturer's instructions). Cleared optic nerves anterogradely-labelled with CTB were then imaged using a Zeiss LSM 710 multiphoton confocal microscope.

Lipid extraction
Lipids were isolated from optic nerves subjected to an alternating cycle of freeze at -80 °C and thaw at 37 °C, followed by the Bligh and Dyer [2] method. The lower organic phase containing the lipids was removed and dried in a Speed-Vac (Model 7810014; Labconco, Kansas City, MO). To prevent lipid oxidation, the tubes were flushed with argon gas for storage. The upper phase was used to measure the sample protein concentration with a BCA Protein Assay Kit (ab102536).

High performance liquid chromatography and mass spectrometry
The dried lipid samples were resuspended in 50 μl of chloroform: methanol 1:1 (v/v), placed in an ultrasonic water bath for 20 minutes. From each sample, 24 μl were added to two vials, of which one contained 2 ug/ml of EquiSPLASH TM LIPIDOMIX® Quantitative Mass Spec Internal Standard (330731). An Accela Autosampler and Accela 600 pump (Thermo) was used for reverse phase separation with an Acclaim column (120 C18 3 μm). The Q Exactive (Thermo) mass spectrometer was operated under heated electrospray ionization (HESI) and samples were ran in both positive and negative mode separately. The solvents were LC-MS grade methanol: water 60:40 (v/v) with 10 mM ammonium acetate and methanol chloroform 60:40 (v/v) with 10 mM ammonium acetate.

Lipid identification and statistical analysis
The * .RAW scans were analysed with LipidSearch 4.2 software (Thermo). The parameters were set to M-score 2 and product search. The quantification was carried by the internal standard classes identified and protein concentration from upper phase of extraction. In Metaboanalyst 4.0, the autoscale feature was employed (mean-centered and divided by standard deviation of each variable).

Ethics Statement
Studies in humans and animals. This study utilized animals (mouse, encompassing both genders) only. All animal experiments were performed in compliance with the US National Institutes of Health guide for the care and use of Laboratory animals. The sex of the mouse is not known to influence or have an association with optic nerve neuron regeneration.