Journal: Frontiers in Cellular Neuroscience

Article Title: The Endocannabinoid/Cannabinoid Receptor 2 System Protects Against Cisplatin-Induced Hearing Loss

doi: 10.3389/fncel.2018.00271

Figure Lengend Snippet: Distribution of the GFP-tagged CB2R knock-in mice. (A) The genetic background of the knock-in mice is shown where the GFP reporter gene was inserted within exon 3 of CB2R and the expression of GFP was driven by internal ribosomal entry (IRE) promoter. Cochleae isolated from these GFP-CB2R co-expressing knock-in mice were fixed in 4% paraformaldehyde followed by decalcification and then sectioned. (B) The representative mid-modiolar section shows endogenous GFP fluorescence (green) in different anatomical regions of the cochlea including organ of Corti (OC), stria vascularis (SV), spiral ligament (SL), and spiral ganglion (SG) neuron. (C) Immunolabeling of GFP-tagged CB2R (green) along with hair cell marker, Myo VIIa (red), show colocalization of CB2R in both, the OHCs and IHCs. (D) Co-labeling of these cochleae with Tuj1 (red), a neuronal marker, show the distribution of GFP-CB2R (green) in spiral ganglion neurons. (E) Whole mount sections of cochleae from GFP-CB2R co-expressing knock-in mice show co-localization of GFP-CB2R (green) and myosin VIIa (red) in the OHCs and IHCs of OC. (F) GFP-CB2R whole mounts sections were also co-stained with neuronal marker, Tuj1 (red), and pre-synaptic ribbon marker, CtBP2 (magenta), showing pre-synaptic localization of CB2R. Cell nuclei (blue) are stained with Hoechst stain in (B,E,F) . Studies reported here were repeated in at least three different animals and similar results were obtained.

Article Snippet: Two different myosin VIIa antibodies were used for different purposes: Mouse IgG1 anti-myosin-VIIa antibody was obtained from Developmental Studies Hybridoma Bank (#138-1), while rabbit anti-myosin-VIIa was purchased from Proteus Biosciences (#25-6790).

Techniques: Knock-In, Expressing, Isolation, Fluorescence, Immunolabeling, Marker, Labeling, Staining

Journal: Frontiers in Cellular Neuroscience

Article Title: The Endocannabinoid/Cannabinoid Receptor 2 System Protects Against Cisplatin-Induced Hearing Loss

doi: 10.3389/fncel.2018.00271

Figure Lengend Snippet: Distribution of the CB2R in the rat cochlea. (A) Immunolabeling of mid-modiolar cochlear sections with CB2R antibody shows labeling of cells in the organ of Corti (green). Labeling was observed in the outer hair cells (OHC), inner hair cells (IHC), inner pillar cell (IPC), and outer pillar cell (OPC). (B) Co-labeling of the organ of Corti with antibodies for CB2R (green) and myosin VIIa (red) showed co-localization of these proteins in the OHC and IHC. (C) Staining of CB2R was also observed in the intermediate and basal cells of the SV and type II fibrocytes in SL. CB2R (green) showed some overlap with Na + /K + -ATPase α1 subunit (red) in the SV. (D) CB2R was localized to the plasma membranes of SG. Approximately 60% of cells in this section were stained with both CB2R (green) and Hoechst (blue). (E) Co-labeling of CB2R and myosin VIIa in OHCs and IHCs in whole mount preparations of the organ of Corti shows that distribution of CB2R (green) is more widespread than that of myosin VIIa (red). Hoechst was used as a nuclear stain (blue) in (B–E) . Studies reported here were repeated in at least three different animals and similar results were obtained.

Article Snippet: Two different myosin VIIa antibodies were used for different purposes: Mouse IgG1 anti-myosin-VIIa antibody was obtained from Developmental Studies Hybridoma Bank (#138-1), while rabbit anti-myosin-VIIa was purchased from Proteus Biosciences (#25-6790).

Techniques: Immunolabeling, Labeling, Staining, Clinical Proteomics

Journal: Frontiers in Cellular Neuroscience

Article Title: The Endocannabinoid/Cannabinoid Receptor 2 System Protects Against Cisplatin-Induced Hearing Loss

doi: 10.3389/fncel.2018.00271

Figure Lengend Snippet: Trans -tympanic administration of JWH015 prevents cisplatin-induced hearing loss. (A) Baseline ABR thresholds were recorded in Wistar rats, which were then pre-treated with either trans -tympanic AM630 (2.5 nmoles) for 30 min followed by trans -tympanic JWH015 (2.5 nmoles) or individually with trans -tympanic AM630 or JWH015 for 24 h. Cisplatin (11 mg/kg) was then administered (i.p.) after day 1 and post-ABR was performed after 72 h. Pre-treatment with JWH015 diminished cisplatin-induce ABR threshold shift in all the three frequency regions. The protective action of JWH015 was completely blocked by AM630. Trans -tympanic administration of AM630 alone significantly elevated by ABR thresholds. Data (threshold shift) is plotted as mean ± SEM ( n = 12). (B) The animals were sacrificed at the end point and cochleae were collected, fixed by 4% PFA in PBS followed by decalcification for 3 weeks in 100 mM EDTA. The base of the cochlea were dissected out and stained with hair cell marker, myosin VIIa, to determine the hair cell loss. Representative images are shown. Scale bar is 20 μm. (C) The bar graph represents the average number of hair cells per 100 μm area shown in (B) ( n ≥ 3). Pre-treatment with JWH015 reduced cisplatin-induced outer hair cell loss. Administration of AM630 produced no hair cell loss. ∗ p < 0.05 vs. vehicle; ∗∗ p < 0.05 vs. cisplatin; # p < 0.05 vs. JWH015 + cisplatin; and ∗∗∗ p < 0.05 vs. JWH015 (one-way ANOVA).

Article Snippet: Two different myosin VIIa antibodies were used for different purposes: Mouse IgG1 anti-myosin-VIIa antibody was obtained from Developmental Studies Hybridoma Bank (#138-1), while rabbit anti-myosin-VIIa was purchased from Proteus Biosciences (#25-6790).

Techniques: Staining, Marker, Produced

Journal: Scientific Reports

Article Title: Fatty acid binding protein type 7 deficiency preserves auditory function in noise-exposed mice

doi: 10.1038/s41598-023-48702-4

Figure Lengend Snippet: Experimental design, body weights, and FABP7 expression in the cochlea. Schematic of the experiments using wild-type (WT) and Fabp7 knockout (KO) mice ( A ). The body weight (BW) of Fabp7 KO mice was not significantly different from that of WT mice ( B ). Immunohistochemistry of FABP7 in the cochlea of WT mice revealed that FABP7 was expressed throughout the spiral ganglion (SG), organ of Corti (OC), spiral limbus (SLim), and spiral ligament (SLig) ( C ). Scale bar, 200 μm ( C – D ). No expression of FABP7 was observed in the cochlea of Fabp7 KO mice ( D ). Maximum projection images of the OC showing high expression of FABP7 in Schwann cells and SOX2-positive supporting cells around the MYO7a-positive outer hair cells (OHCs) in the cochlea of WT mice ( E ). In the SG, FABP7 was expressed in satellite cells surrounding TUJ-1-positive neurons ( F ). Scale bars, 50 μm ( E – F ). Auditory brainstem response; ABR, weeks; W, post-noise exposure day; PNED, inner hair cell; IHC. Statistical significance was determined using two-way analysis of variance, followed by Šídák multiple comparison test. Error bars represent standard deviation.

Article Snippet: After rinsing with PBS, tissue sections were blocked with 3% bovine serum albumin/0.3% Triton X-100/ PBS for 30 min at room temperature, the unconjugated AffiniPure Fab fragment of anti-mouse IgG (1:10, Jackson ImmunoResearch, West Grove, PA) for 2 h at room temperature, and incubated overnight with the following primary antibodies: (1) rabbit IgG anti-FABP7 (1:1000, Merck, Darmstadt, Germany #PRS4259), (2) mouse IgG anti-TUJ-1 (1:500, Abcam, Cambridge, UK #ab78078), (3) mouse IgG2a anti-Myosin 7a (MYO7a) (1:200, Santa Cruz Biotechnology, Dallas, TX, USA #SC-74516), and (4) goat IgG anti-SOX2 (1:500, R&D Systems, Minneapolis, MN, USA #AF2018).

Techniques: Expressing, Knock-Out, Immunohistochemistry, Comparison, Standard Deviation