J Korean Med Sci.  2011 Jun;26(6):814-823. 10.3346/jkms.2011.26.6.814.

Spinal Cord Injury Markedly Altered Protein Expression Patterns in the Affected Rat Urinary Bladder during Healing Stages

• Lee JY
• Kim BJ
• Sim G
• Kim GT
• Kang D
• Jung JH
• Hwa JS
• Kwak YJ
• Choi YJ
• Park YS
• Han J
• Lee CS
• Kang KR

• KMID: 1785970
• DOI: http://doi.org/10.3346/jkms.2011.26.6.814

Abstract

The influence of spinal cord injury (SCI) on protein expression in the rat urinary bladder was assessed by proteomic analysis at different time intervals post-injury. After contusion SCI between T9 and T10, bladder tissues were processed by 2-DE and MALDI-TOF/MS at 6 hr to 28 days after SCI to identify proteins involved in the healing process of SCI-induced neurogenic bladder. Approximately 1,000 spots from the bladder of SCI and sham groups were visualized and identified. At one day after SCI, the expression levels of three protein were increased, and seven spots were down-regulated, including heat shock protein 27 (Hsp27) and heat shock protein 20 (Hsp20). Fifteen spots such as S100-A11 were differentially expressed seven days post-injury, and seven proteins including transgelin had altered expression patterns 28 days after injury. Of the proteins with altered expression levels, transgelin, S100-A11, Hsp27 and Hsp20 were continuously and variably expressed throughout the entire post-SCI recovery of the bladder. The identified proteins at each time point belong to eight functional categories. The altered expression patterns identified by 2-DE of transgelin and S100-A11 were verified by Western blot. Transgelin and protein S100-A11 may be candidates for protein biomarkers in the bladder healing process after SCI.

Keyword

Spinal Cord Injuries; Urinary Bladder, Neurogenic; Proteomic Analysis; Biological Markers; S100-A11; Transgelin

MeSH Terms

Animals
Biological Markers/metabolism
Electrophoresis, Gel, Two-Dimensional
Female
HSP20 Heat-Shock Proteins/metabolism
HSP27 Heat-Shock Proteins/metabolism
Microfilament Proteins/metabolism
Muscle Proteins/metabolism
Proteome/*biosynthesis
Proteomics
Rats
Rats, Sprague-Dawley
S100 Proteins/metabolism
Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
Spinal Cord Injuries/*metabolism/pathology
Urinary Bladder/*metabolism
*Wound Healing

Figure

• 

  Fig. 1 Evaluation of spinal cord injury by locomotor behavior scoring. (A) A photograph of spinal cord injured rat. Sutured muscles and skin in the back after surgery are shown, and hindlimbs are drooped and paralyzed. (B) Basso Beattie Bresnahan (BBB) open field locomotor rating scale is scored in the course of healing process after contusion SCI. Adult rats (n = 5-6 animals per group) were contused at T-9 exposed spinal cord with a 250 gram weight drop device from a 25 mm height using the New York University (NYU) Impactor. Locomotor behavior was evaluated before injury and then for up to 28 days with different time intervals. The graph exhibits functional hindlimb recovery of rats in contusion group compared with those of sham control group. Data were presented as mean ± SD and P < 0.05 in both groups.

• 

  Fig. 2 Comparison of protein expression patterns of rat urinary bladder after contusion SCI at different time points. The bladder tissue proteins (50 µg) from each stage (sham control [A], 6 hr [B], 1 day [C], 2 days [D], 7 days [E], 9 days [F], 21 days [G], and 28 days [H] post-injury) were separated on pH 4?? IPG strips (17 cm, Bio-Rad) in the first dimension and on 7.5%-17.5% linear-gradient SDS-PAGE in the second dimension, and visualized by silver nitrate staining. Protein names specified by Arabic numerals (A and H) are listed in Table 1.

• 

  Fig. 3 Expression profiles of selected protein spots in the bladder post-injury at different time points. Representative spots specified at each stage (spot intensity from PDQuest software), identified as transgelin, protein S100-A11, Hsp27, and Hsp20 are shown in the healing course of rat bladder after contusion spinal cord injury. The bar graphs exhibited the expression level of each protein at indicated time points compared with the sham control. Data are presented mean ± SD (n = 5 per group), P < 0.05 (ANOVA).

• 

  Fig. 4 Validation of differentially expressed proteins in the rat bladder after contusion injury by Western blot analysis. Selected proteins in rat bladder tissues after SCI and sham control were analyzed and confirmed by Western blot at each time points. The expression levels of transgelin and S100-A11 in the protein blot are well agreement with quantitative 2-DE results (Fig. 3), respectively. β-actin was used as a loading control.

• 

  Fig. 5 Functional manifestations of differentially expressed proteins in rat bladder tissues after SCI. The expression profiles and functions of the proteins were categorized into eight different subgroups. Values exhibited the number of identified proteins in each category. (A) to (D) shows total proteins expressed at indicated time points: (A) 1 day post-injury, (B) 2 days post-injury, (C) 9 days post-injury, and (D) 28 days post-injury.

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