Differential SERCA Expression in Laryngeal Muscles

Animals In this study, male adult Wistar rats (Shimizu Laboratory Supplies Co., Kyoto, Japan), 8-12 weeks of age, weighing 280-350 g (N=10) were used as young rats. In addition, 24-month-old rats (N=10) were employed as aged models.

Animal tissue preparation Animals were anesthetized with an intraperitoneal injection of sodium pentobarbital (dose 30-60 mg/kg) and were fixed with 4% paraformaldehyde following cardiac perfusion with 0.01 M phosphate-buffered saline (PBS). The larynges were excised immediately and immersed in the same fixative for approximately 12 hours at (4°C). Tissues were processed in paraffin and sectioned using a microtome (8-10 μm thickness).

The intrinsic laryngeal muscles studied were: 1) medial and lateral thyroarytenoid (MTA, LTA), (2) lateral cricoarytenoid (LCA), (3) Superior cricoarytenoid (SCA), (4) posterior cricoarytenoid (PCA), and (5) cricothyroid (CT).

Immunohistochemistry (IHC) The investigators faced unsuccessful fast fiber type identification on frozen sections then we shifted to paraffin sections. Deparaffinized sections were incubated with 3% H2O2 in PBS for 10 minutes followed by microwave treatment (5 minutes ×3 times at 500 Watt in citrate buffer, pH 6). Then, sections were incubated in a blocking solution (0.3 M glycine, 50 mM ammonium chloride, and 1% BSA in PBS) for 30 minutes before incubation in primary antibodies. Sections were incubated with anti-SERCA1 or -SERCA2 and anti-MHC I or -MHCII (Table 1) antibodies for 1-2 days at 4 ◦C, then washed, and incubated with Alexa Fluor® 488 donkey anti-mouse IgG and Fluor® 594 donkey anti-goat IgG secondary Abs for 1 hour at room temperature. Double IHC was performed using (anti-SERCA1 + anti-SERCA2) and (anti-SERCA2 + anti-MHCII) combinations to check co-expression. In double IHC, the same steps were done with a mixture of primary or secondary antibodies with the same dilutions.

Immunostained tissue sections were examined using a FV-1000 laser confocal microscope (Olympus, Tokyo, Japan). ImageJ 1.46 software was used to manually count and calculate the percentage of positive cells for each antibody.

Immunohistochemistry for denervated rat In 8-12 weeks, aged rats, the larynx was approached through a midline incision using a standard operating microscope under anesthesia with sodium pentobarbital. After identification of the right recurrent and superior laryngeal nerves, a 1 cm segment was removed from the right recurrent laryngeal nerve and both ends were ligated. The right superior laryngeal nerve was also divided. The denervated rat groups were sacrificed after 2, 4, 8, and 12 weeks. The larynx was collected and prepared for IHC using the same procedure described above (n = 5 in each group).

MHC subtypes using multi-color fiber typing:

At the beginning of our research, we tried the frozen section. After trials of several techniques to decrease autofluorescence and background noise, the investigators failed to have good staining. Then, the investigators discontinued MHC subtyping and shifted to paraffin-embedded blocks Statistical analysis The relative SERCA and MHC composition for each muscle was calculated. The available data for the positive cell percentage of each rat antibody were statistically analyzed using SPSS statistical software, version 16. Statistical comparisons were performed using the unpaired two-tailed Student's t-test and were considered significant at P < 0.05.

One-way analysis of variance (ANOVA) followed by Scheffe's post hoc test was performed to compare the MHC and SERCA composition for each muscle in denervation with the contralateral side and young with age. Values presented are means ± SD (standard deviation), with SD being across rats.

• Immunohistochemistry (IHC) (Diagnostic test): We faced unsuccessful fast fiber type's identification on frozen sections then we shifted to paraffin sections. Deparaffinized sections were incubated with 3% H2O2 in PBS for 10 minutes followed by microwave treatment (5 minutes ×3 times at 500 Watt in citrate buffer, pH 6). Then, sections were incubated in blocking solution (0.3 M glycine, 50 mM ammonium chloride, and 1% BSA in PBS) for 30 minutes before incubation in primary antibodies. Sections were incubated with anti-SERCA1 or -SERCA2 and anti-MHC I or -MHCII (Table 1) antibodies for 1-2 days at 4 ◦C, then washed, and incubated with Alexa Fluor® 488 donkey anti-mouse IgG and Fluor® 594 donkey anti-goat IgG secondary Abs for 1 hour at room temperature. Double IHC was performed using (anti-SERCA1 + anti-SERCA2) and (anti-SERCA2 + anti-MHCII) combinations to check co-expression. In double IHC, the same steps were done as before with mixture of primary or secondary antibodies with same dilutions.

Primary outcomes

• Immuno-localization of SERCA and MHC isoform [6 months]: Immuno-localization of SERCA and MHC isoform (SERCA1, SERCA2, MHC I, and MHCII) expression and co-expression in the five ILMs

Sex: Male, ages: 8 Weeks to 24 Weeks

Inclusion Criteria:

* 8-12 weeks of age, weighing 280-350 g (N=10) were used as young rats. In addition, 24-month-old rats (N=10) were employed as aged models.

Exclusion Criteria:

\-

• Mao VH, Abaza M, Spiegel JR, Mandel S, Hawkshaw M, Heuer RJ, Sataloff RT. Laryngeal myasthenia gravis: report of 40 cases. J Voice. 2001 Mar;15(1):122-30. doi: 10.1016/S0892-1997(01)00012-1. PMID 12269627
• Marques MJ, Ferretti R, Vomero VU, Minatel E, Neto HS. Intrinsic laryngeal muscles are spared from myonecrosis in the mdx mouse model of Duchenne muscular dystrophy. Muscle Nerve. 2007 Mar;35(3):349-53. doi: 10.1002/mus.20697. PMID 17143878

• Mohammed E Ahmed, Study director, Sohag University