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MB Sample ID: SA072445
| Local Sample ID: | HA_9 |
| 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 |
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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:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| HA_9 | SA072445 | FL011356 | Yes | Crush |
| HA_9 | SA072445 | FL011356 | No | Saline treatment |
| HA_9 | SA072445 | FL011356 | Yes | Wnt3a treatment |
| HA_9 | SA072445 | FL011356 | 7 | Day post-crush |
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 |
