Differential SERCA Expression in Laryngeal Muscles

NCT06837467 | Completed DMC

• 1 other identifier: sohag-5-24-2023-01
• Study Type: interventional
• Target: 40
• Locations: 1 country
• Sites: 1

Brief Summary

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.

Conditions

Myopathy

Keywords

SERCA and MHC isoform

Outcome Measures

Primary Outcomes (1)

• Immuno-localization of SERCA and MHC isoform

  Immuno-localization of SERCA and MHC isoform (SERCA1, SERCA2, MHC I, and MHCII) expression and co-expression in the five ILMs

  6 months

Study Arms (3)

Young rat

EXPERIMENTAL

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.

Diagnostic Test: Immunohistochemistry (IHC)

old rats

EXPERIMENTAL

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.

Diagnostic Test: Immunohistochemistry (IHC)

Denervated rats

ACTIVE COMPARATOR

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.

Diagnostic Test: Immunohistochemistry (IHC)

Interventions

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.

Denervated rats; Young rat; old rats

Eligibility Criteria

• Age: 8 Weeks - 24 Weeks
• Sex: male
• Healthy Volunteers: No
• Age Groups: Child (0-17)

You may qualify if:

• 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.

Sponsors & Collaborators

• Sohag University: lead

Study Sites (1)

Sohag University

Sohag, 82524, Egypt

Location

Related Publications (2)

• 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 BACKGROUND

• 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 BACKGROUND

MeSH Terms

Conditions

Muscular Diseases

Interventions

Immunohistochemistry

Condition Hierarchy (Ancestors)

Musculoskeletal Diseases; Neuromuscular Diseases; Nervous System Diseases

Intervention Hierarchy (Ancestors)

Histocytochemistry; Cytological Techniques; Clinical Laboratory Techniques; Diagnostic Techniques and Procedures; Diagnosis; Histological Techniques; Investigative Techniques; Immunologic Techniques

Study Officials

• Mohammed E Ahmed

  Sohag University

  STUDY DIRECTOR

Study Design

• Study Type: interventional
• Phase: not applicable
• Allocation: RANDOMIZED
• Masking: SINGLE
• Who Masked: PARTICIPANT
• Purpose: DIAGNOSTIC
• Intervention Model: PARALLEL
• Sponsor Type: OTHER
• Responsible Party: PRINCIPAL INVESTIGATOR
• PI Title: Clinical Professor

Model Details: 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. 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).

Study Record Dates

• First Submitted: February 7, 2025
• First Posted: February 20, 2025
• Study Start: January 25, 2024
• Primary Completion: January 25, 2025
• Study Completion: February 1, 2025
• Last Updated: February 20, 2025

Record last verified: 2025-02

Data Sharing

• IPD Sharing: Will not share

Locations

EG (1)