Abstract
Purpose
The purpose of this study was to determine the more important growth factor expression between basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) in the healing of acute tympanic membrane (TM) perforation.
Materials and methods
Bilateral perforations of the TM were created in 12 rats. The TM perforations in the right ears were treated with dexamethasone, and left ears were designated as the control group. The TM was examined for the growth factor expression immunohistochemically in the epithelial and fibrous layers according to the rate of TM perforation healing.
Results
The mean spontaneous healing time of the TM perforations was 11.0 ± 2.0 days. However, dexamethasone-treated group showed no evidence of closure. The bFGF and VEGF expression were significantly correlated with the rate of healing of acute TM perforations. The VEGF expression was decreased both in the epithelial and fibrous layers, but bFGF expression was decreased only in the epithelial layer in the dexamethasone-treated group. The VEGF was expressed to a lesser degree than bFGF in the dexamethasone-treated group.
Conclusion
Vascular endothelial growth factor is the more specific and important growth factor than bFGF in the healing of acute TM perforation,
1
Introduction
The regeneration of the tympanic membrane (TM) after traumatic perforation is a complex biological process that involves epithelial proliferation, epithelial migration, fibroblast proliferation, neoangiogenesis, and tissue remodeling . Several growth factors and cytokines have been postulated to be involved in the healing process of TM perforation. Local application of epidermal growth factor (EGF) or basic fibroblast growth factor (bFGF) have recently been reported to accelerate the closure of acute TM perforation . In addition, immunohistochemical reactivity for endogenous bFGF, EGF, and transforming growth factor- α (TGF- α ) is induced during TM perforation healing .
Epidermal growth factor and TGF- α play a major role in epithelial reconstruction; bFGF and vascular endothelial growth factor (VEGF) are mainly involved in the angiogenesis and reconstruction of connective tissue . Although VEGF is the most specific known growth factor for neovascularization , the relation between the expression of VEGF and the healing of TM perforation remains poorly understood.
In this study, we have examined the expression of bFGF and VEGF in the epithelial and fibrous layers of acute traumatic TM perforations and investigated the more important growth factor in the healing of acute TM perforation.
2
Materials and methods
2.1
Animals
The protocol for this animal study was approved by the Institutional Animal Care and Use Committee of the Pusan National University School of Medicine. Twelve healthy, pathogen-free female Sprague-Dawley rats (200–250 g) were used in this study.
2.2
Operative procedures
During all procedures, including ear drop instillation, the animals were sedated with sevoflurane. Tympanic membrane perforations were performed with a sterile pick from the anterior malleus handle to the anterior annulus in both ears of each animal using a 0°, 4-mm rigid endoscope (Karl Storz, Tuttlingen, Germany). The right TM was treated with dexamethasone, and the left one was treated with isotonic sodium chloride solution as the control ear. Beginning on the day of surgery, dexamethasone (5 mg/mL) and isotonic sodium chloride solution were applied in a volume of 50 μ L in the external auditory canals of the animals using a pipette once a day for 30 days. This volume filled the ear canals, and appropriate delivery of the drops was confirmed by inspection with an endoscope. All animals were observed in separate cages with free access to food and water, and 150 mg/kg of ampicillin was administrated intramuscularly during the first 5 days to prevent infection.
2.3
Inspection of perforated TM closure
Inspection for both TMs of each animal was continued until complete closure of the TM, or postoperative day (POD) 30 in nonhealed cases. Photographs were taken with a digital camera (Coolpix 950, Nikon, Japan) with an attached 0°, 4-mm rigid endoscope. Photographs were taken preoperatively, immediately postoperatively, and on POD 2, 4, and 6. After the seventh day, photographs were taken every other day up to POD 30 to determine the duration of perforation closure.
2.4
Histologic analysis
Each animal was sacrificed after the closure of the TM perforation, or on POD 30 in nonhealed cases. After intraperitoneal injection of penobarbital (80 mg/kg), the animals were decapitated and their bilateral external ears were separated at the osteocartilaginous junctions. The bullae were opened, and the temporal bones were kept overnight in 10% formalin solution and then decalcified in 10% formic acid solution for 7 to 10 days. The cortex of the temporal bones was removed and embedded in paraffin. Tissue slices were cut into 2 pieces through a line bisecting the perforation and running perpendicular to the manubrium of the malleus, with a thickness of 4 to 6 μ m. Each section was stained with hematoxylin and eosin and examined under light microscopy (Bx51, Olympus Inc, Japan). Histologically, the thickness of the TM was quantitatively measured in the epithelial and fibrous layers.
2.5
Immunohistochemical staining
For analysis of the expression of bFGF and VEGF, sections were stained with anti–fibroblast growth factor (FGF) and anti-VEGF IgG and evaluated semiquantitatively in the epithelial and fibrous layers. For immunohistochemical staining, sections were washed with phosphate-buffered saline (PBS) and blocked with 2% bovine serum albumin (Sigma, St. Louis, MO)/PBS to remove nonspecific binding. The sections were incubated with purified anti-FGF IgG (Santa Cruz Biotechnology, Santa Cruz, CA) and anti-VEGF IgG (Santa Cruz Biotechnology) primary antibody for overnight at 4°C. After incubation, sections were washed with PBS and then incubated with biotin-conjugated antirabbit Ig (Santa Cruz Biotechnology) for 1 hour at room temperature. Cells with bound antibodies were detected with a peroxidase substrate kit (Vectastain ABC avidin/biotin Complex Kit, Vector Laboratories, Burlingame, CA). The sections were washed, counterstained with hematoxylin, and examined by light microscopy (Bx51, Olympus Inc). Cytoplasmic staining was recorded by the semiquantitative grading system that considered both the intensity of the staining and the proportion of stained cells. The intensity was recorded as 0 (no staining), 1 (light staining), 2 (moderate staining), or 3 (intense staining). The proportion of tissue sample with a positively stained cytoplasmic area was recorded as 0 (no cells were positive), 1 (positive staining in <10% of cells), 2 (positive staining in 10%–50% of cells), or 3 (positive staining in >50% of cells). A staining index was calculated as the sum of the staining intensity and the proportion of stained cells .
2.6
Statistical analysis
Data are presented as mean ± SEM. Statistical significance was assessed by an independent t test or a paired t test using SPSS, version 12.0 (SPSS Inc, Chicago, IL). A value of P < .05 was considered to be significant.
2
Materials and methods
2.1
Animals
The protocol for this animal study was approved by the Institutional Animal Care and Use Committee of the Pusan National University School of Medicine. Twelve healthy, pathogen-free female Sprague-Dawley rats (200–250 g) were used in this study.
2.2
Operative procedures
During all procedures, including ear drop instillation, the animals were sedated with sevoflurane. Tympanic membrane perforations were performed with a sterile pick from the anterior malleus handle to the anterior annulus in both ears of each animal using a 0°, 4-mm rigid endoscope (Karl Storz, Tuttlingen, Germany). The right TM was treated with dexamethasone, and the left one was treated with isotonic sodium chloride solution as the control ear. Beginning on the day of surgery, dexamethasone (5 mg/mL) and isotonic sodium chloride solution were applied in a volume of 50 μ L in the external auditory canals of the animals using a pipette once a day for 30 days. This volume filled the ear canals, and appropriate delivery of the drops was confirmed by inspection with an endoscope. All animals were observed in separate cages with free access to food and water, and 150 mg/kg of ampicillin was administrated intramuscularly during the first 5 days to prevent infection.
2.3
Inspection of perforated TM closure
Inspection for both TMs of each animal was continued until complete closure of the TM, or postoperative day (POD) 30 in nonhealed cases. Photographs were taken with a digital camera (Coolpix 950, Nikon, Japan) with an attached 0°, 4-mm rigid endoscope. Photographs were taken preoperatively, immediately postoperatively, and on POD 2, 4, and 6. After the seventh day, photographs were taken every other day up to POD 30 to determine the duration of perforation closure.
2.4
Histologic analysis
Each animal was sacrificed after the closure of the TM perforation, or on POD 30 in nonhealed cases. After intraperitoneal injection of penobarbital (80 mg/kg), the animals were decapitated and their bilateral external ears were separated at the osteocartilaginous junctions. The bullae were opened, and the temporal bones were kept overnight in 10% formalin solution and then decalcified in 10% formic acid solution for 7 to 10 days. The cortex of the temporal bones was removed and embedded in paraffin. Tissue slices were cut into 2 pieces through a line bisecting the perforation and running perpendicular to the manubrium of the malleus, with a thickness of 4 to 6 μ m. Each section was stained with hematoxylin and eosin and examined under light microscopy (Bx51, Olympus Inc, Japan). Histologically, the thickness of the TM was quantitatively measured in the epithelial and fibrous layers.
2.5
Immunohistochemical staining
For analysis of the expression of bFGF and VEGF, sections were stained with anti–fibroblast growth factor (FGF) and anti-VEGF IgG and evaluated semiquantitatively in the epithelial and fibrous layers. For immunohistochemical staining, sections were washed with phosphate-buffered saline (PBS) and blocked with 2% bovine serum albumin (Sigma, St. Louis, MO)/PBS to remove nonspecific binding. The sections were incubated with purified anti-FGF IgG (Santa Cruz Biotechnology, Santa Cruz, CA) and anti-VEGF IgG (Santa Cruz Biotechnology) primary antibody for overnight at 4°C. After incubation, sections were washed with PBS and then incubated with biotin-conjugated antirabbit Ig (Santa Cruz Biotechnology) for 1 hour at room temperature. Cells with bound antibodies were detected with a peroxidase substrate kit (Vectastain ABC avidin/biotin Complex Kit, Vector Laboratories, Burlingame, CA). The sections were washed, counterstained with hematoxylin, and examined by light microscopy (Bx51, Olympus Inc). Cytoplasmic staining was recorded by the semiquantitative grading system that considered both the intensity of the staining and the proportion of stained cells. The intensity was recorded as 0 (no staining), 1 (light staining), 2 (moderate staining), or 3 (intense staining). The proportion of tissue sample with a positively stained cytoplasmic area was recorded as 0 (no cells were positive), 1 (positive staining in <10% of cells), 2 (positive staining in 10%–50% of cells), or 3 (positive staining in >50% of cells). A staining index was calculated as the sum of the staining intensity and the proportion of stained cells .
2.6
Statistical analysis
Data are presented as mean ± SEM. Statistical significance was assessed by an independent t test or a paired t test using SPSS, version 12.0 (SPSS Inc, Chicago, IL). A value of P < .05 was considered to be significant.
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