Ethics Statement All procedures involving animals complied with the ARRIVE guidelines and adhered to the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research and were approved by the Institutional Animal Care and Use Committee of University of Miami, Miller School of Medicine.
Animals Wildtype mice (C57Bl/6) were purchased from Jackson Labs (Bar Harbor, ME), and TMEM97−/− mice (Tmem97tm1(KOMP)Vlcg, stock # 050147-UCD) were obtained from the Mutant Mouse Resource & Research Centers (MMRRC, https://www.mmrrc.org/). TMEM97−/− mice were backcrossed and maintained on a C57BL/6 background. Genotypes were confirmed by PCR according to MMRRC instructions. Animals were kept on a 12:12 light-dark cycle. Adult male mice (2–3 months old) were used in all experiments.
Histology For retinal histology, animals were killed by CO2 overdose and immediately followed by vascular perfusion with mixed aldehydes 50,51. Eyes were collected and embedded in an Epon/Araldite mixture. Semi-thin sections (1 µm) were cut to display the entire retina along the vertical meridian 50,51. Retinal sections were stained with toluidine blue and examined by light microscopy.
Immunocytochemistry For TMEM97 immunostaining, eyes were removed from animals after vascular perfusion with 4% paraformaldehyde, cryoprotected with 20% sucrose, and embedded in Tissue-Tek OCT compound (Miles Inc., Elkhart, IN). Sections (10 µm) along the vertical meridian were cut on a Cryostat at -20°C and thaw-mounted onto Super Frost Plus glass slides (Fisher Scientific, Pittsburgh, PA). Retinal sections were incubated with anti-TMEM97 antibodies (PA5-23003, Thermo Fisher Scientific Technology, Waltham, MA) at 4 ºC overnight. TMEM97 immunoactivity was visualized by staining with Cy2 conjugated secondary antibodies (Jackson ImmunoResearch Labs, West Grove, PA) for 1 h at room temperature (RT). Cell nuclei were stained with DAPI (4',6-diamidino-2-phenylindole). Fluorescent signals in the retinal sections were examined by confocal microscopy (LSM700; Carl Zeiss, Jena, Germany).
X-gal stain in the retina TMEM97−/− mice were generated by replacing 8169 base pairs of the TMEM97 gene in chromosome 11 (positions 78355984–78364152) with a Velocigene cassette ZEN-Ub1 that has a lacZ reporter gene under the control of the TMEM97 promoter (https://www.mmrrc.org/catalog/sds.php?mmrrc_id=50147) 26. The expression of lacZ (encoding β-galactosidase) thus reflects the expression of TMEM97. To characterize the activity of β-galactosidase in the retinas of TMEM97−/− mice, eyes were collected and fixed in 2% paraformaldehyde and 0.2% glutaraldehyde. Retinal cryo-sections (20 µm) were cut and stained with a phosphate-buffered saline (PBS) solution containing 0.5 mg/mL X-gal (5-bromo-4-chloro-3-indolyl β-D-galactopyranoside), 5 mM potassium ferricyanide, 5 mM potassium ferrocyanide, 2 mM MgCl2, 0.01% sodium deoxycholate, and 0.02% Nonidet P-40 (NP-40) at 37 ºC overnight. Stained retinal sections were examined by DIC (differential interference contrast) microscopy.
TMEM97 RNA expression TMEM97 mRNA expression in wildtype and TMEM97−/− mice was examined by reverse transcription PCR (RT-PCR). Total RNA was extracted from 4 retinas of TMEM97−/− or wildtype mice with the RNeasy kit (Qiagen, Germantown, MD). cDNA was synthesized using 2 µg total RNA as the template and poly d(T) (deoxythymidine) primer with the ProtoScript II cDNA synthesis kit in a 20 µl reaction (New England Biolabs, Ipswich, MA). TMEM97 expression was examined by PCR with forward primer 5’-TCTACTTCGTCTCGCACATCCC-3’ and reverse primer 5’-CCGGCAGCTTCCTTTGAAGAAGG-3’. The expression of the housekeeping gene Gapdh (Glyceraldehyde-3-phosphate dehydrogenase, forward primer 5’-AGGTTGTCTCCTGCGACTTC-3’ and reverse primer 5’-GGGTGGTCCAGGGTTTCTTAC-3’) was used as a reference. PCR was carried out with 2 µl cDNA reaction as the template using OneTaq DNA polymerase (New England Biolabs). The total volume of PCR reaction (30 µl) was electrophoresed on a 2% agarose gel.
Retinal ischemia-reperfusion Retinal ischemia was created by elevating the intraocular pressure (IOP) of the eye to 120 mm Hg. An animal was anesthetized with isoflurane through a nose cone and body temperature was kept at 37°C with a heating pad. The anterior chamber of the left eyes was cannulated with a 33-gauge needle connected to a reservoir of saline (0.9% NaCl) via a one-way stop valve. The reservoir was placed at 163 cm above the eye to create a pressure of 163 cm H2O (equivalent to 120 mm Hg). IOP was elevated to 120 mm Hg (163 cm H2O) by turning the valve on to connect the needle to the reservoir. Ischemia was visually confirmed as the eye turned pale. Elevated IOP was maintained for 45 min and then perfusion to the eye was resumed by turning the valve off to disconnect the needle from the reservoir followed by needle withdrawal. The right eye was untouched to serve as the naïve control.
Quantification of RGC survival RGCs were identified by immunostaining of RBPMS (RNA binding protein with multiple splicing, an RGC marker) 27. Eyes were collected 7 days after ischemia. The anterior segments were removed, and the eyecups were incubated with anti-RBPMS antibodies (GTX118619, GeneTex, Irvine, CA) at 4 ºC overnight. The eyecups were then washed and stained with Cy2 conjugated secondary antibodies (Jackson ImmunoResearch Labs, West Grove, PA) for 1 h at RT to visualize RBPMS immunoreactivity. After staining, retinas were cut into four quadrants (superior, inferior, nasal, and temporal), flat-mounted onto glass slides, and examined by confocal microscopy (LSM700; Carl Zeiss, Jena, Germany).
RBPMS positive cells in each quadrant were counted in five fields (319 X 319 µm each) at 0.5 mm (1 field), 1 mm (2 fields), and 1.5 mm (2 fields) from the optic nerve head and RGC densities were calculated. RGC survival rate is the ratio of RBPMS-positive cells in the left eye vs those in the right eye.
Pattern electroretinogram (PERG) recording PERG was recorded as previously described 52,53. Mice were anesthetized with ketamine/xylazine (80/10 mg/kg) and gently restrained in an animal holder. Animals were kept at a constant body temperature of 37°C using a feedback-controlled heating pad (TCAT-2LV; Physitemp Instruments, Inc. Clifton, NJ). Pupils were undilated and small (< 1 mm) to ensure a large depth of focus. PERG was recorded with a JÖRVEC system (JÖRVEC, Miami, FL) using a stainless-steel subcutaneous needle electrode (Grass, West Warwick, RI) placed in the snout. The reference and ground electrodes were identical needles placed in the medial portion of the scalp and at the root of the tail, respectively. Visual stimuli were contrast-reversing bars (0.05 cycles/deg, 98% contrast, 800 cd/m2 mean luminance) generated on a light-emitting diode tablet (15×15 cm size) and presented to the eye at a 10 cm distance. PERG responses were analyzed by automated detection of the main positive peak (P1) around 100 ms, and the subsequent negative trough (N2) around 250 ms. PERG amplitude was defined as the amplitude difference between the P1 peak and the N2 trough. PERG latency was defined as the time-to-peak of the P1 wave.
Intravitreal injections The selective σ2R/TMEM97 ligand DKR-1677 (Fig. 5f) was prepared as previously described 54 and dissolved in DMSO (dimethylsulfoxide) to a concentration of 20 µg/µL. A 33-gauge needle connected to a 10-µL microsyringe (Hamilton, Reno, NV) was used for intravitreal injections as described 55. The left eye of a mouse in the DKR-1677 group was injected with DKR-1677 (20 µg/µL, 2 µL/eye) and the right eyes received no injection. In the vehicle control group, the left eyes were injected with DMSO (2 µL/eye), and the right eyes were not injected.
Statistics. Data are presented as mean ± standard error of mean (SEM). Student’s t test was used to determine the statistical significance of the difference between two groups of data.