Acquiring BM Sca-1+ stem cells and culturing
All animal experiments had been accepted by the Institutional Animal Care and Use Committee of Harbin Medical College, and had been carried out in accordance with the Assertion for the Use of Animals in Ophthalmic and Imaginative and prescient Analysis by the Affiliation for Analysis in Imaginative and prescient and Ophthalmology, in addition to the Information for the Care and Use of Laboratory Animals from the Nationwide Institutes of Well being. Femurs and tibias from wild-type C57BL/6 mice [2–3 months, totally 90 mice] had been flushed with phosphate buffered saline (PBS) to acquire nucleated BM stem cells. These cells had been then sorted into 2 classes, Sca-1+ or Sca-1− stem cells [24], utilizing a magnetic affinity cell sorting package, in keeping with the producer’s directions (Stem Cell Expertise, Canada). After sorting, stem cells had been cultured in Iscove’s Modified Dulbecco’s Medium (Biosharp, China), with 10% (v/v) exosome-depleted fetal bovine serum (FBS; SBI, USA) and 1% antibiotic–antimycotic answer (Beyotime, China), and incubated for 48 h in an incubator, at 37 °C and with 5% CO2, for exosome enrichment.
Isolation and characterization of BM stem cell-derived exosomes
After 48 h tradition, cell supernatants had been obtained by way of centrifugation at 5000×g for 15 min, filtered by way of 0.45 μm filters, and concentrated by passing them by way of 100K ultrafiltration tubes (Millipore, USA). Exosome precipitation and purification was carried out utilizing Exo-spin™ exosome size-exclusion columns (Cell GS, UK). Their styles and sizes had been examined utilizing transmission electron microscopy (TEM) (Hitachi, HT-7700, Japan). Nanoparticle monitoring evaluation (NTA) was used to measure exosome diameter and portions (NanoFCM, N30E, China). Particular exosome markers cluster of differentiation (CD) 9 (1:1000, Cat # 92726, Abcam), CD81 (1:1000, Cat # ET1611-87, Huabio) and CD63 (1:1000, Cat # 217345, Abcam) [25], had been detected with Western blot.
Establishing the retinal I/R damage mouse mannequin and intravitreal injection of exosomes
To ascertain the retinal I/R damage animal mannequin, outdated mice (complete 48; 18–20 months) had been anaesthetized with 5% chloral hydrate, and each eyes had been subjected to retinal I/R damage, as beforehand described [26]. Briefly, a 500 mL IV bottle, containing sterile salt answer, comprised the traditional saline reservoir. This bottle was linked to a 32-gauge needle, and the needle was inserted into the anterior chamber of mouse eyes. The reservoir was then hung from an IV pole extension, and elevated at a top of 1.5 m, ensuing within the mouse eye being subjected to 110 mmHg of hydrostatic stress. The attention was then repeatedly infused with saline for 1 h, after which the needle was eliminated to permit for the reperfusion of retinal vasculature, leading to induction of retinal I/R damage. Mice who acquired retinal I/R in each eyes had been randomly assigned into the next teams: regular management with out I/R (Regular), I/R, I/R + Sca-1+, and I/R + Sca-1−; each eyes had been every injected intravitreally with 2 μL of exosomes proper after I/R damage.
Visible operate detection
To evaluate visible operate, we chosen mice who had a transparent refractive medium in each eyes, following modeling and intravitreal injection. Gentle/darkish field exploration and optomotor response duties had been used, as beforehand described [9]. For mild/darkish field exploration, briefly, after 2 h of darkish adaptation at nighttime field, mice (complete 9) had been positioned into the sunshine field (Fig. 2A), and visible operate was evaluated, primarily based on information collected for 10 min, by way of time spent within the mild field, in addition to the variety of transitions between the darkish and light-weight containers by way of passing by way of the doorway between them. For the optomotor response process, the mouse was positioned on a platform within the mild field, and revolving vertical stripes, at 3 totally different frequencies, had been projected on the encompassing LED display screen (Fig. 2D–E); the variety of mouse head actions elicited by the vertical stripe rotation, for five min, was recorded for every frequency. Visible acuity was primarily based on the very best spatial frequency eliciting this optomotor response. Every mouse was examined 3 instances per trial, and the information had been averaged for additional evaluation. Three animals from every experimental group had been examined.
Retinal thickness measurements
Histological evaluation was used to measure complete retinal thickness, in addition to for the 5 retinal layers. Mice had been anesthetized at 7 days post-retinal I/R, and acquired trans-cardiac perfusions of 4% paraformaldehyde (PFA). Eyeballs had been eliminated and stuck in 4% PFA in a single day at 4 °C, after limbal paracentesis with a sterile 32 G syringe. Mounted eyeballs had been dehydrated utilizing rising percentages of ethanol, from 50, to 70, to 95, to 100%, then paraffin-embedded and sectioned into 4 μm sections. The sections had been stained utilizing a hematoxylin and eosin (H&E)-staining package, following the producer’s directions (Beyotime, China). All retinal thickness measurements had been carried out 2 mm away from the optic disc edge.
Terminal deoxynucleotidyl transferase dUTP nick finish labeling (TUNEL) assay and immunofluorescence staining
For the TUNEL assay, mouse eyeballs had been harvested at 3 days post-retinal I/R, whereas they had been harvested at 7 days post-injury for immunofluorescence staining. This distinction in timepoints is as a result of totally different phases of the I/R damage response [27], through which neuronal apoptosis happens within the quick aftermath of the damage, adopted by inflammatory reactions, involving immune cell infiltration, in addition to subsequent differentiation and polarization into microglia for the clearance of apoptotic neurons. Eyeballs had been mounted in a single day in 4% PFA at 4 °C, after limbal paracentesis with a sterile 32 G syringe. Subsequently, they had been dehydrated utilizing rising percentages of sucrose, beginning at 10%, then 20%, for two h every, and at last at 30% for 30 min, all at 4 °C. Eyeballs had been then embedded in OCT compound (Sakura Finetek, Japan), and 4 µm transverse sections by way of the optic disc of the attention had been obtained. The ensuing tissue sections had been mounted with 4% PFA for 20 min, permeabilized with 0.1% Triton X-100 in PBS for 10 min, and blocked with 10% goat serum in PBS for two h. For TUNEL staining, the TUNEL assay package was used (Roche, Switzerland), following the producer’s directions.
As for immunofluorescence staining, the sections had been incubated in a single day, at 4 °C, with the next antibodies: NeuN (1:50; Cat # ab177487, Abcam), CD16/CD32 (1:50; Cat # ab223200, Abcam), and ionized calcium-binding adapter molecule 1 (Iba-1, 1:50; Cat # ab283319, Abcam). Sections had been then incubated with fluorescein (FITC) AffiniPure goat anti-rabbit (1:200; 111-095-003, Jackson) and Pink-X-AffiniPure goat anti-mouse immunoglobulin G (IgG) antibodies (1:200; 115-295-003, Jackson). Nuclei had been stained utilizing 4′,6-diamidino-2-phenylindole (DAPI; Beyotime, China) at room temperature for 3 min. Slides had been mounted utilizing an anti-fade fluorescence mounting medium (Dako; S3023, Denmark), and fluorescence photographs had been obtained by way of fluorescence microscopy (Leica, Germany). Quantifications of fluorescence depth, in addition to NeuN+ and CD16/CD32+ microglia, had been carried out utilizing ImageJ, for 3 mice from every remedy group.
Retinal flatmounts had been carried out by first harvesting and incubating mouse eyeballs for two h at 4 °C, adopted by dissection of their retinas. Retinas had been then positioned in 2% Triton X-100, diluted in PBS, for 40 min at − 80 °C, and transferred into blocking buffer (5% regular goat serum in 2% Triton X-100), to be incubated for two h at room temperature. Afterwards, they had been incubated with Iba-1 and CD16/CD32 major antibodies, diluted in 2% blocking buffer, in a single day at 4 °C, adopted by incubation with fluorescein (FITC) AffiniPure goat anti-rabbit (1:200; 111-095-003, Jackson) and Pink-X-AffiniPure goat anti-mouse immunoglobulin G (IgG) antibodies (1:200; 115-295-003, Jackson) at room temperature for two h. Nuclei had been stained utilizing 4′,6-diamidino-2-phenylindole (DAPI; Beyotime, China) at room temperature for 3 min. Retinal flatmounts had been then shaped by inserting the retinas on the slides, vitreous body-side down; the retinas had been flattened by making use of 4 symmetrical radial incisions, centered on the optic disc, and cover-slipped with Dako fluorescence mounting medium. CD16/CD32+ microglia had been quantified, utilizing ImageJ, in a blinded trend, inside a 0.01 mm2 (100 × 100 μm) rectangular area, from the peripheral fringe of the retina; 3 areas from every retinal pattern had been used.
BV2 cell tradition and uptake of exosomes
BV2, an immortalized murine microglial cell line, was maintained in Dulbecco’s minimal important medium, supplemented with 10% (v/v) FBS and penicillin/streptomycin (100 models and 100 μg/mL) at 37 °C in a 5% CO2 incubator. To ascertain the lipopolysaccharide (LPS)-induced inflammatory cell mannequin, BV2 at a density of 5 × 104 cells/effectively had been seeded into 6 effectively plates, cultured in a single day, then co-cultured with 100 μg/mL of exomes for 12 h, and uncovered to 1 μg/mL LPS (Sigma, USA) for twenty-four h.
To measure exosome uptake, BV2 cells had been seeded at a density of 5 × 103 cells/effectively right into a 24-well chamber slide and cultured in a single day. Cells had been then co-cultured for two h with 10 μM of DiI-labeled exosomes (DiI, Beyotime, China), mounted with 4% PFA for 20 min, permeabilized with 0.1% Triton X-100 in PBS for 10 min, and blocked with 10% goat serum in PBS for two h. Afterwards, cells had been incubated in a single day with major antibodies in opposition to Iba-1 at 4 °C, then incubated with FITC AffiniPure goat anti-rabbit IgG secondary antibody for 60 min in darkness at room temperature. Nuclei had been stained with DAPI, and cells imaged by fluorescence microscopy.
Reverse transcription quantitative real-time PCR (RT-qPCR)
Whole RNA from retinas, in addition to from BV2 cells, representing microglia, had been extracted utilizing TRIzol® Reagent (CWBIO, China), then reverse transcribed into cDNA utilizing the Transcriptor First Strand cDNA Synthesis Package, in accordance with the producer’s directions (Roche, Switzerland). qPCR was carried out utilizing NCSYB GREEN qPCR Grasp Combine (NCBIOTECH, China). As for exosomes, complete RNA was extracted utilizing the RNAsimple Whole RNA Package (Tiangen, DP419, China), reverse transcribed into cDNA utilizing the miRcute Plus miRNA First-Strand cDNA Package (Tiangen, KR211, China), and qPCR carried out utilizing the miRcute Plus miRNA qPCR Package (SYBR Inexperienced; Tiangen, FP411, China). Primer sequences used had been listed in Extra file 1: Desk S1.
Western blot
Radioimmunoprecipitation assay lysis buffer (Beyotime, China) was used to lyse retinas obtained after eradicating the lens and anterior parts of mouse eyes, in addition to exosomes, plus Sca-1+, Sca-1−, and BV2 cells, for protein extraction. Proteins had been quantified utilizing the BCA Protein Assay Package (Solarbio, China), and 15 µg of complete protein was separated on SDS-PAGE gels (Epizyme, China), then transferred to polyvinylidene difluoride membranes (Millipore, Germany). Major antibodies used included Sca-1 (1:1000, Cat # 0804-10, HUABIO), IL-6 (1:1000, Cat # 500286, ZENBIO), TNF-α (1:1000, Cat # 346654, ZENBIO), MEKK3 (1:1000, Cat # 381471, ZENBIO), phospho-SAPK (p-SAPK)/JNK (1:1000, Cat # 4668, CST), JNK (1:1000, Cat # 10023, Proteintech), p–c-Jun (1:1000, Cat # R22955, ZENBIO), c-Jun (1:1000, Cat # R23335, ZENBIO), and GAPDH (1:10000, Cat # 10494-1-AP, Proteintech), and protein ranges had been quantified by ImageJ.
Setting up exosomal RNA libraries and high-throughput sequencing
Whole RNA from exosomes had been extracted utilizing TRIzol (Invitrogen, CA, USA), handled with DNase I (Takara, Kusatsu, Japan) to take away any contaminating DNA, and monitored for degradation and contamination with 1% agarose gel electrophoresis. RNA focus and purity was measured, and 1 µg of complete RNA for every pattern was used to generate sequencing libraries, utilizing the NEBNext® Multiplex Small RNA Library Prep Set for Illumina® (NEB, USA). The libraries had been then sequenced utilizing Illumina Novaseq 6000, and 50 bp single-end reads had been generated. High quality management was carried out on uncooked reads to acquire clear reads for differential expression evaluation of miRNA between totally different samples, utilizing DEseq2 software program (P worth< 0.05, |log2foldchange|> 1).
Bioinformatics analyses
Goal genes for differentially-expressed miRNAs had been predicted utilizing miRDB (http://www.mirdb.org/), miRWalk (http://mirwalk.umm.uni-heidelberg.de/), microT (http://diana.cslab.ece.ntua.gr/microT/), miRanda (http://www.microrna.org/microrna/residence.do), and TargetScan databases (http://www.targetscan.org/). Gene Ontology (GO) useful annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses had been carried out utilizing the Database for Annotation, Visualization and Built-in Discovery (http://david.niaid.nih.gov).
Retrograde labeling of retinal ganglionic cells and quantification
Retrograde labeling of retinal ganglion cells was carried out 3 days previous to retinal I/R. Mice (18–20 month, completely 5 mice) had been deeply anesthetized and immobilized with a small stereotactic instrument, adopted by publicity of their skulls and identification of their bregma. A 22 G needle was then used to drill a gap above the superior colliculus of every hemisphere, and 1 μl of 4% hydroxystilbamidine in PBS (aka Fluoro-Gold; Cat # ab138870, Abcam, USA) was injected into each superior colliculi, utilizing a micro-injector, at 1 mm from the bony floor of the mind. Mice had been then sacrificed at days 3 and seven post-I/R, and retinal flat mounts had been ready. Fluoro-Gold-positive ganglion cells had been recognized below a fluorescent microscope, and quantitated by ImageJ.
Luciferase reporter assay
To find out the attainable goal binding sequences of miR-150-5p on MEKK3 (MAP3K3), targetScan (http://www.targetscan.org/) was used. Each wild-type and mutant pMIR-MAP3K3-3’UTR luciferase reporter plasmids (Guangzhou RiboBio Co., Ltd, China) had been obtained for the luciferase reporter assay, through which these plasmids had been co-transfected with both miR-150-5p mimic, or unfavourable management (NC) comprising of scrambled random miRNA, into 293T cells, utilizing Lipofectamine 3000 (Thermo Fisher, USA). Twin-luciferase exercise was then measured utilizing the Twin-Glo Luciferase Assay System (Promega, USA).
Statistical evaluation
All statistical analyses was carried out utilizing GraphPad Prism model 8.0, and all values had been introduced as imply ± commonplace error of the imply (SEM). Pupil’s t-test was used for comparisons between 2 teams, whereas for 3 or extra teams, a technique evaluation of variance (ANOVA) was used. Moreover, comparisons to “baseline” measurements was outlined by way of adjustments pre- and post-I/R damage, whereas comparisons to “Regular” was by way of variations between un-injured versus I/R-injured mice. p < 0.05 was thought-about statistically important.
