Reuss et al., supplementary material

Supplementary material

Ngb mRNA Transcription in Mouse Cochlea

Quantitative assays for Ngb mRNA expression levels in mouse cochleae and brains were run in duplicate using quantitative RT-PCR. cDNA was synthesized from 1 g of total RNA extracted from three individual brains, and two cochleae pooled from two separate mice. Methods are described in materials and methods (chapter 2.6) in the manuscript. The Ngb mRNA transcription levels in each of the three mouse brains were quite similar, and the amount of Ngb mRNA expressed in the cochlea was in the same range (≈85%) as that of the brain #1 calibrator (suppl. fig. 1).

Ngb Protein Expression in Rat and Mouse Cochlea

Two different polyclonal anti-Ngb antibodies, raised in rabbits and purified by affinity chromatography, were used in Western blot assays to detect Ngb in total protein extracts from murine brains and cochleae (25 g/sample). The specificity and selectivity of the two Ngb antibodies for detecting Ngb have been established earlier by ELISA, Western Blot and preabsorption tests [1-3]. Both anti-Ngb antibodies detected Ngb in protein extracts from adult C.B-17 mouse brains and cochleae at the predicted molecular mass of ~17 kDa (suppl. fig. 2). The intensity of the Western blot signals (IOD: Integrated Optical Density) was quantified and the amount of Ngb in the two brain samples was estimated to be ~7-fold higher than in the total cochlea extracts.

Western blots

Cochleae and brains were homogenized in lysis buffer (1% SDS, 5% β-mercaptoethanol, 10% glycerol, 65 mmol Tris, pH 6.8) and clarified by centrifugation (10,000 x g for 10 min). Protein concentrations were determined by Bradford Protein Assays (Bio-Rad, Hercules, CA. USA), and 25 g of total protein was analyzed by 12% SDS-polyacrylamide gel electrophoresis and transferred to Immuno-Blot poly(vinylidene difluoride) membranes (Bio-Rad). Membranes were incubated overnight at 40C with rabbit polyclonal antibody against Ngb (1:2000) and stained with horseradish peroxidase-conjugated anti-rabbit secondary antibody (Santa Cruz Biotechnology, Santa Cruz, CA). The signals were detected using an enhanced chemiluminescence substrate system (PerkinElmer, Torrence, CA), scanned with a Hewlett Packard ScanJet 4c scanner (Palo Alto, CA) and quantified using GelPro version 4.0 software (Media Cybernetics, Silver Spring, MD).

Characterization of spiral ganglion neurons

Digital images were collected for all 1350 spiral ganglion cells with well-defined nuclei that immunostained with anti-Ngb and/or anti-peripherin antisera in 21 cochlear sections sampled from four different mice. Since the relative intensity of the Ngb immunolabeling within the SgnI (peripherin-negative: n=1260) and SgnII (peripherin-positive: n=90) cell populations did not vary along the length of the cochlea from base-to-apex, data were pooled across cochlear turns. For each turn of the cochlea that contained one or more SgnII cells, 15 SgnI cells [i.e., ratio=(90/1350)=(1/15)] were selected from the same Rosenthal’s canal using a statistical random number generator. First, the area of each SgnI (peripherin-negative: N=330) and SgnII (peripherin-positive: N=90) cell body was calculated (suppl. figure 3): the average area (mean+SE) of the perikarya of the peripherin-negative SgnI cells (98.50+1.02 μm2) was 15% larger than that of the peripherin-positive SgnII cells (83.26+1.75 μm2; P<0.001, Student’s t-test). Second, the fluorescence intensity of each selected spiral ganglion cell was quantified using ImagePro analysis (Media Cybernetics). The mean Ngb fluorescence intensity per pixel was determined by dividing the sum of all pixel intensities by the area of the cell perikaryon [4]. The average fluorescence intensity (mean+SE) of Ngb immunostaining of the SgnI (peripherin-negative) cells (62.28+0.96) was significantly greater than that for the SgnII (peripherin-positive) cells (22.21+1.38) (P<0.01, Student’s t-test, see suppl. fig. 4). However, because the cross-sectional somatic areas of SgnII cells were smaller on average than those for the SgnI cells (suppl. fig. 3), we considered the possibility that the Ngb fluorescence intensity measures could reflect, in part, the amount of cytoplasm in the cell soma. Correlation coefficients between somatic cross-sectional area and the mean Ngb fluorescence for SgnI and SgnII cells (suppl. fig. 5) were R=0.204 and R=0.037, respectively (both non-significant: P>0.05, Pearson’s correlation coefficient tests). Consequently, the relative amount of cytoplasm in the spiral ganglion cell soma did not significantly influence the measures of cellular Ngb fluorescence intensity.

References

1. Sun Y, Jin K, Mao XO, Zhu Y, Greenberg DA (2001) Neuroglobin is up-regulated by and protects neurons from hypoxic-ischemic injury. ProcNat Acad Sci USA 98:15306-15311

2. Sun YJ, Jin KL, Peel A, Mao XO, Xie L, Greenberg DA (2003) Neuroglobin protects the brain from experimental stroke in vivo. Proc Nat Acad Sci USA 100:3497-3500

3. Schmidt M, Giessl A, Laufs T, Hankeln T, Wolfrum U, Burmester T (2003) How does the eye breathe? - Evidence for neuroglobin-mediated oxygen supply in the mammalian retina. J Biol Chem 278:1932-1935

4. Nishimura T, Fukata Y, Kato K, Yamaguchi T, Matsuura Y, Kamiguchi H, Kaibuchi K (2003) CRMP-2 regulates polarized Numb-mediated endocytosis for axon growth. Nature Cell Biol 5:819-826.

5. Riemann R, Reuss S (1999) Nitric oxide synthase in identified olivocochlear projection neurons in rat and guinea pig. Hear Res 135:181-189

Suppl. fig. 1. Real-time RT-PCR quantification of Ngb mRNA gene expression relative to that of GAPDH assayed from three individual brains, and two cochleae combined from two separate mice.

Suppl. fig. 2. Example of a Western blot assay showing Ngb protein expression in mouse total brain and cochlea at the predicted relative molecular mass of approximately 17,000 Dalton.

Suppl. fig. 3. Quantitative distribution of somatic cross-sectional areas of SgnI (peripherin-negative) and SgnII (peripherin-positive) cells in adult mouse cochleae (see text for details).

Suppl. fig. 4. Quantitative distribution of cellular mean Ngb fluorescence intensity per pixel for SgnI (peripherin-negative) and SgnII (peripherin-positive) cells in adult mouse cochleae. Mean Ngb fluorescence was determined for each cell by dividing the sum of all pixel intensities by the area of the cell perikarya (see text for details).

Suppl. fig. 5. Distribution and cross-correlation coefficients for Ngb mean fluorescence density versus cell size (i.e., cross sectional area of cell perikarya) computed for (A) SgnI and (B) SgnII neuronal populations.

Suppl. fig. 6. Frontal section of the human brainstem. A Nissl-stain showing the location of LSO and MSO between the abducens nerve (N VI) and the facial nerve (N VII) and its nucleus (NcN VII). B: corresponding section stained for neuroglobin (NGB). Higher magnifications from MSO and LSO are shown in fig.7 of the manuscript.

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