Differential Expression of SERCA and Myosin Heavy Chain Isoforms in Rat Laryngeal Muscles
试验速览
- 阶段
- 不适用
- 状态
- 已完成
- 入组人数
- 40
- 试验地点
- 1
- 主要终点
- Immuno-localization of SERCA and MHC isoform
研究概览
简要总结
Immuno-localization of SERCA and MHC isoform (SERCA1, SERCA2, MHC I, and MHCII) expression and co-expression in the five ILMs were investigated in both young and aged rats with immunohistochemistry. Special concern has been placed to the denervation effect on ILMS.
详细描述
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.
研究设计
- 研究类型
- Interventional
- 分配方式
- Randomized
- 干预模型
- Parallel
- 主要目的
- Diagnostic
- 盲法
- Single (Participant)
入排标准
- 年龄范围
- 8 Weeks 至 24 Weeks（Child）
- 性别
- Male
- 接受健康志愿者
- 否
入选标准
- •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.
排除标准
- 未提供
研究组 & 干预措施
Young rat
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 Immunohistochemistry (IHC) 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.
干预措施: Immunohistochemistry (IHC) (Diagnostic Test)
old rats
24-month-old rats (N=10) were employed as aged models. Immunohistochemistry (IHC) 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.
干预措施: Immunohistochemistry (IHC) (Diagnostic Test)
Denervated rats
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).
Immunohistochemistry (IHC) 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.
干预措施: Immunohistochemistry (IHC) (Diagnostic Test)
结局指标
主要结局
Immuno-localization of SERCA and MHC isoform
时间窗: 6 months
Immuno-localization of SERCA and MHC isoform (SERCA1, SERCA2, MHC I, and MHCII) expression and co-expression in the five ILMs
次要结局
未报告次要终点
研究者
Mohammed Elrabie Ahmed
Clinical Professor
Sohag University