CHONDROITIN SULFATE IN NORMAL AND SCARRED ......scarring and the healing process.1 A chondroitin...
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INTRODUCTION DISCUSSION RESULTS ABSTRACT METHODS AND MATERIALS CONCLUSIONS REFERENCES CONTACT CHONDROITIN SULFATE IN NORMAL AND SCARRED RAT VOCAL FOLDS PATNARIN MAHATTANASAKUL, MD 1,2 ; ICHIRO TATEYA, MD, PhD 2 ; NAO HIWATASHI, MD 2 ; RYO SUZUKI, MD 2 ; YOSHITAKA KAWAI, MD 2 ; YO KISHIMOTO, MD, PhD 2 ; SHIGERU HIRANO, MD, PhD 2 1 King Chulalongkorn Memorial Hospital, Thai Red Cross Society, Chulalongkorn University, 2 Kyoto University Introduction: Chondroitin sulfate (CS) is a glycosaminoglycan(GAG) known to work in various signal transduction pathways. Recent studies showed that CS is upregulated in keloid tissue, and it was suggested that CS acts in wound healing by regulating cell adhesion and cell proliferation. We hypothesized that CS plays an important role in the wound healing process of vocal folds. Here we examined the expression of CS in normal and scarred rat vocal folds. Materials and methods: The vocal folds of 24 female 13-wk-old Sprague-Dawley rats were unilaterally stripped by micro-scissors. After sacrifice, the rats’ larynges were harvested at 1 day, 3 days, 1 week, 1 month and 3 months after stripping. The CS expressions in the untouched and scarred vocal folds were examined by immunohistochemistry, and a quantitative measurement was performed by image analysis. Results: CS was expressed in both the scarred and normal vocal folds. The ratio of the areas expressing CS in the scarred vocal folds were significantly lower than those in the normal vocal folds at day 3 (p<0.01), day 7 (p<0.01), 1 month(p<0.01) and 3 months (p<0.05), and the ratio was lower but not significantly on day 1. Conclusions: The expression pattern of CS in the scarred rat vocal folds was different from that in human keloid tissue. The results suggest that the wound healing process of the vocal folds is different from that of keloid tissue. Patnarin Mahattanasakul King Chulalongkorn Memorial Hospital, Thai Red Cross Society, Chulalongkorn University Kyoto University Email: [email protected] Vocal fold scar is one of the challenging diseases. Several prior studies have focused on the mechanisms of the development of vocal fold scarring and the healing process. 1 A chondroitin sulfate(CS) is a sulfated glycosaminoglycan(GAG).It can be found in intracellular organelles, on the cell surface, and in the extracellular matrix(ECM). 2 For neuroscientists, CSs were increased after CNS injury. They acted in the inhibition of neurite outgrowth. 3 In dermatology, A keloid is one of the dermal fibrotic growths. It lack plasticity and contain an excessive accumulation of ECM and a decreased amount of elastic fiber. The level of CS in the keloids was found to be 6.9-fold higher than in normal skin. 4 We hypothesized that CS may play one or more important roles in the healing process of vocal fold scars, and we conducted this study to identify the expression of CS in normal and scarred rat vocal folds. CS was expressed in both scarred and normal vocal folds. It was found in the lamina propria. (Fig.1) The ratio of the areas expressing CS in the scarred vocal folds were significantly lower than those in the normal vocal folds at day 3(p<0.005), day 7(p<0.01), 1 month(p<0.01) and 3 months(p<0.05), and the ratio was lower but not significantly on day 1(p=0.1)(Chart.1) 24 female 13-wk-old Sprague-Dawley rats was anesthetized. Scar models were performed at 5 time-points 1 day, 3 days, 1 week, 1 month, and 3 months with 4-5 rats at each time point.Rat videolaryngoscopic surgery was visualized. Unilateral vocal fold stripping was performed using micro-scissors. The other side was left intact.The larynges were harvested. Coronal sections were sliced and stained. Immunohistochemistry The sections were blocked by 5% skim milk The primary antibody : mouse anti-CS56 (1:200; Abcam, Cambridge, MA) The secondary antibody : goat anti-mouse Alexa 488 (1:500; Invitrogen, Carlsbad, CA) Images were captured and analysed by software CS immunopositive areas and pixels was measured and calculated Ratio of CS positive : CS positive area Total vocal fold area Statistical analysis used paired t-tests to compare the differences between the normal side and the scarred sides within each group Ratio of CS (%) 0 20 40 60 80 100 1 day 3 days 7 days 1 month 3 months normal scar CS is present in both normal and scarred vocal folds. The expression pattern of CS in the scarred rat vocal folds was different from that reported in keloid and normal skin tissue. Our present findings suggest that the wound healing process of vocal folds is different from that of normal skin and keloid tissue. Figure 1. Expression of CS in control and scarred rat vocal folds (original 10×). The CS is stained green and the nuclei are stained blue. 1.Hirano S, Minamiguchi S, Yamashita M, Ohno T, Kanemaru S, Kitamura M.Histologic characterization of human scarred vocal folds. J Voice. 2009:23(4):399-407. 2.Hook M, Kjellen L, Johansson S. Cell-surface glycosaminoglycans. Ann Rev Biochem. 1984;53:847-869. 3.Li HP, Komuta Y, Kimura-Kuroda J, Kuppevelt TH, Kawano H. Roles of chondroitin sulfate and dermatan sulfate in the formation at a lesion scar and axonal regeneration after traumatic injury of the mouse brain. J Neurotrauma. 2013;30(5):413-425. 4.Ikeda M, Naitoh M, Kubota H, Ishiko T, Yoshikawa K, Yamawaki S, Kurokawa M. Utani A, Nakamura T, Nagata K, Suzuki S. Elastic fiber assembly is disrupted by excessive accumulation of chondroitin sulfate in the human dermal fibrotic disease, keloid. Biochem Biophys Res Commun. 2009;390(4):1221-1228. 5.Hahn M, Jao CY, Faquin W, Grande-Allen KJ. Glycosaminoglycan composition of the vocal fold lamina propria in the relation to function. Ann Otol Rhinol Laryngol. 2008;117(5):371-381. ACKNOWLEDGEMENT This study was supported in part by grants-in-aid for research from the Ministry of Health, Labor, and Welfare of Japan and the Ministry of Education, Culture, Sports, Science, and Technology of Japan. P. Mahattanasakul received funding from King Chulalongkorn Memorial Hospital, Thai Red Cross, Faculty of medicine, Chulalongkorn University which supported the grant for Research Fellowship Program at the Department of Otolaryngology- Head and Neck Surgery, Kyoto University, Japan normal 1 day 3 days 7 days 1 month 3 months Chart 1. Mean scores of the ratio of CS in the normal and scarred vocal folds at the different time points.*p<0.05 VS normal This is the first report describing the immunological staining of CS in vocal folds. After the injury, a significant decrease in CS with scarring was observed. This reduction consistent with previous study that measured CS by fluorophore- assisted carbohydrate electrophoresis(FACE) 5 but different from in normal skin tissue and keloids that the level of CS was increased after the injury. 4 We hypothesized that vocal folds may undergo a unique scar remodelling process.
Transcript of CHONDROITIN SULFATE IN NORMAL AND SCARRED ......scarring and the healing process.1 A chondroitin...
INTRODUCTION
DISCUSSION
RESULTSABSTRACT
METHODS AND MATERIALS
CONCLUSIONS
REFERENCES
CONTACT
CHONDROITIN SULFATE IN NORMAL AND SCARRED RAT VOCAL FOLDSPATNARIN MAHATTANASAKUL, MD1,2; ICHIRO TATEYA, MD, PhD2; NAO HIWATASHI, MD2; RYO SUZUKI, MD2;
YOSHITAKA KAWAI, MD2; YO KISHIMOTO, MD, PhD2; SHIGERU HIRANO, MD, PhD2 1King Chulalongkorn Memorial Hospital, Thai Red Cross Society, Chulalongkorn University, 2Kyoto University
Introduction: Chondroitin sulfate (CS) is a glycosaminoglycan(GAG) known to work in various signal transduction pathways. Recent studies showed that CS is upregulated in keloid tissue, and it was suggested that CS acts in wound healing by regulating cell adhesion and cell proliferation. We hypothesized that CS plays an important role in the wound healing process of vocal folds. Here we examined the expression of CS in normal and scarred rat vocal folds.
Materials and methods: The vocal folds of 24 female 13-wk-old Sprague-Dawley rats were unilaterally stripped by micro-scissors. After sacrifice, the rats’ larynges were harvested at 1 day, 3 days, 1 week, 1 month and 3 months after stripping. The CS expressions in the untouched and scarred vocal folds w e r e e x a m i n e d b y i m m u n o h i s t o c h e m i s t r y, a n d a quant i ta t i ve measurement was performed by image analysis.
Results: CS was expressed in both the scarred and normal vocal folds. The ratio of the areas expressing CS in the scarred vocal folds were significantly lower than those in the normal vocal folds at day 3 (p<0.01), day 7 (p<0.01), 1 month(p<0.01) and 3 months (p<0.05), and the ratio was lower but not significantly on day 1.
Conclusions: The expression pattern of CS in the scarred rat vocal folds was different from that in human keloid tissue. The results suggest that the wound healing process of the vocal folds is different from that of keloid tissue.
Patnarin Mahattanasakul King Chulalongkorn Memorial Hospital, Thai Red Cross Society, Chulalongkorn University Kyoto University Email: [email protected]
Vocal fold scar is one of the challenging diseases. Several prior studies have focused on the mechanisms of the development of vocal fold scarring and the healing process.1
A chondroitin sulfate(CS) is a sulfated glycosaminoglycan(GAG).It can be found in intracellular organelles, on the cell surface, and in the extracellular matrix(ECM).2
For neuroscientists, CSs were increased after CNS injury. They acted in the inhibition of neurite outgrowth.3
In dermatology, A keloid is one of the dermal fibrotic growths. It lack plasticity and contain an excessive accumulation of ECM and a decreased amount of elastic fiber. The level of CS in the keloids was found to be 6.9-fold higher than in normal skin.4
We hypothesized that CS may play one or more important roles in the healing process of vocal fold scars, and we conducted this study to identify the expression of CS in normal and scarred rat vocal folds.
CS was expressed in both scarred and normal vocal folds. It was found in the lamina propria.(Fig.1)
The ratio of the areas expressing CS in the scarred vocal folds were significantly lower than those in the normal vocal folds at day 3(p<0.005), day 7(p<0.01), 1 month(p<0.01) and 3 months(p<0.05), and the ratio was lower but not significantly on day 1(p=0.1)(Chart.1)
24 female 13-wk-old Sprague-Dawley rats was anesthetized. Scar models were performed at 5 time-points 1 day, 3 days, 1 week, 1 month, and 3 months with 4-5 rats at each time point.Rat videolaryngoscopic surgery was visualized. Unilateral vocal fold stripping was performed using micro-scissors. The other side was left intact.The larynges were harvested. Coronal sections were sliced and stained.
Immunohistochemistry The sections were blocked by 5% skim milk The primary antibody : mouse anti-CS56 (1:200; Abcam, Cambridge, MA) The secondary antibody : goat anti-mouse Alexa 488 (1:500; Invitrogen, Carlsbad, CA)
Images were captured and analysed by software CS immunopositive areas and pixels was measured and calculated
Ratio of CS positive : CS positive area Total vocal fold area
Statistical analysis used paired t-tests to compare the differences between the normal side and the scarred sides within each group
Rati
o of
CS
(%)
0
20
40
60
80
100
1 day 3 days 7 days 1 month 3 months
normalscar
CS is present in both normal and scarred vocal folds. The expression pattern of CS in the scarred rat vocal folds was different from that reported in keloid and normal skin tissue.
Our present findings suggest that the wound healing process of vocal folds is different from that of normal skin and keloid tissue.
Figure 1. Expression of CS in control and scarred rat vocal folds (original 10×).
The CS is stained green and the nuclei are stained blue.
1.Hirano S, Minamiguchi S, Yamashita M, Ohno T, Kanemaru S, Kitamura M.Histologic characterization of human scarred vocal folds. J Voice. 2009:23(4):399-407.
2.Hook M, Kjellen L, Johansson S. Cell-surface glycosaminoglycans. Ann Rev Biochem. 1984;53:847-869.
3.Li HP, Komuta Y, Kimura-Kuroda J, Kuppevelt TH, Kawano H. Roles of chondroitin sulfate and dermatan sulfate in the formation at a lesion scar and axonal regeneration after traumatic injury of the mouse brain. J Neurotrauma. 2013;30(5):413-425.
4.Ikeda M, Naitoh M, Kubota H, Ishiko T, Yoshikawa K, Yamawaki S, Kurokawa M. Utani A, Nakamura T, Nagata K, Suzuki S. Elastic fiber assembly is disrupted by excessive accumulation of chondroitin sulfate in the human dermal fibrotic disease, keloid. Biochem Biophys Res Commun. 2009;390(4):1221-1228.
5.Hahn M, Jao CY, Faquin W, Grande-Allen KJ. Glycosaminoglycan composition of the vocal fold lamina propria in the relation to function. Ann Otol Rhinol Laryngol. 2008;117(5):371-381.
ACKNOWLEDGEMENTThis study was supported in part by grants-in-aid for research from the Ministry of Health, Labor, and Welfare of Japan and the Ministry of Education, Culture, Sports, Science, and Technology of Japan. P. Mahattanasakul received funding from King Chulalongkorn Memorial Hospital, Thai Red Cross, Faculty of medicine, Chulalongkorn University which supported the grant for Research Fellowship Program at the Department of Otolaryngology-Head and Neck Surgery, Kyoto University, Japan
normal
1 day
3 days
7 days
1 month
3 months
Chart 1. Mean scores of the ratio of CS in the normal and scarred vocal folds
at the different time points.*p<0.05 VS normal
This is the first report describing the immunological staining of CS in vocal folds. After the injury, a significant decrease in CS with scarring was observed. This reduction consistent with previous study that measured CS by fluorophore-assisted carbohydrate electrophoresis(FACE)5 but different from in normal skin tissue and keloids that the level of CS was increased after the injury.4 We hypothesized that vocal folds may undergo a unique scar remodelling process.
