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Effects of Hepatocyte Growth Factor Injection and Reinjection on Healing in the Rabbit Vocal Fold
Effects of Hepatocyte Growth Factor Injection and Reinjection on Healing in the Rabbit Vocal Fold *,†Roberta Ismael Dias Garcia, †Domingos Hiroshi Tsuji, †Rui Imamura, ‡Thais Mauad, and ‡Luiz Fernando Ferraz da Silva, *yzS~ao Paulo, Brazil Summary: Objectives/Hypothesis. Hepatocyte growth factor (HGF) is a multifunctional polypeptide that plays various roles in embryogenesis and tissue regeneration and exhibits marked antifibrotic activity. The present study sought to assess the effects of HGF injection and reinjection coinciding with its peak of activity on collagen density, vessel density, inflammatory reaction in the lamina propria, and mean epithelial thickness in the injured rabbit vocal fold. Study Design. Prospective, controlled, experimental animal study. Methods. Fourteen rabbits were subdivided into two groups and underwent injury of the vocal folds. Immediately after injury, animals in group 1 received HGF injections into the right vocal fold (RVF), whereas those in group 2 received bilateral HGF injections and a single reinjection into the RVF 10 days after the first, to coincide with the peak of HGF activity. The left vocal folds (LVFs) served as controls in both groups. Histological assessment of laryngeal specimens was performed at 30 and 40 days, respectively. Results. In both groups, collagen density was lower in the right (treated) vocal folds than in the left (control) folds (P ¼ 0.018). Vessel density was higher in the RVFs in group 2 (P ¼ 0.018). Differences were found in mean epithelial thickness and inflammatory reaction in the lamina propria but did not reach statistical significance. Conclusions. In the scarred rabbit vocal fold, HGF injection is associated with decreased collagen density in the lamina propria, whereas reinjection after 10 days produces decreased collagen density and higher vessel density. Key Words: Wound healing–Vocal folds–Oryctolagus cuniculus–Hepatocyte growth factor. INTRODUCTION Treatment of vocal fold fibrosis is one of the most challenging issues in laryngology practice.1,2 Scar tissue formation is associated with changes in the lamina propria, leading to increased mucosal rigidity, reduced mucosal vibration, and, possibly, difficult-to-control dysphonia3; indeed, scarring has been described as the number one cause of postoperative dysphonia in patients undergoing laryngeal surgery.4 Development of methods that minimize the laryngeal changes brought about by scar tissue formation is thus a subject of great research interest5–7 because proper viscoelasticity is difficult to restore when the lamina propria of the vocal fold is replaced by fibrotic tissue.8 Hepatocyte growth factor (HGF) is a multifunctional polypeptide that plays roles in embryogenesis, organogenesis, and tissue regeneration. HGF has marked antifibrotic activity, and potential therapeutic applications in acute and chronic liver, kidney, and lung conditions have been demonstrated in animal models.9 HGF receptors are present in the vocal folds,10 and Hirano et al11 proposed that HGF, which modulates the synthesis and expression of profibrotic substances, may be a useful agent for the prevention of vocal fold scarring.
Accepted for publication August 16, 2011. Study funded by the S~ao Paulo Research Foundation (FAPESP). From the *Oropharynx and Larynx Service, Department of Otorhinolaryngology, Otorhinolaryngology Clinic, University of S~ao Paulo School of Medicine, S~ao Paulo, Brazil; yDepartment of Otorhinolaryngology, University of S~ao Paulo School of Medicine, S~ao Paulo, Brazil; and the zDepartment of Pathology, University of S~ao Paulo School of Medicine, S~ao Paulo, Brazil. Address correspondence and reprint requests to Roberta Ismael Dias Garcia, Rua Tuim, 444, apartamento 11 – Moema, S~ao Paulo, SP 04514-101, Brazil. E-mail: robertaismael@ uol.com.br Journal of Voice, Vol. 26, No. 5, pp. 667.e7-667.e12 0892-1997/$36.00 Ó 2012 The Voice Foundation doi:10.1016/j.jvoice.2011.08.007
Another study by the same team8 notes that concentration and timing of administration are always a concern when seeking satisfactory outcomes with growth factor treatment. The authors describe several options for administration of HGF as a treatment for vocal fold disorders—topical injection, topical injection with normal fibroblasts, systemic administration, and gene therapy—and later note3 that several other questions remain unanswered, such as the optimal timing and dosage of administration. Ohno et al12 obtained satisfactory results with a hydrogelbased sustained HFG release system in the canine vocal fold but failed to define the optimal dosage and frequency of administration for best regeneration of the vocal fold; the authors later conducted a study13 assessing the effects of HGF on procollagen expression during the acute phase of vocal fold injury and suggested that further research was required. Gilbert et al14 assessed the efficacy of porcine liver extracellular matrix, a known reservoir of HGF, as a scaffold for vocal fold healing in dogs; again, the authors stressed the importance of further research. Other several studies have demonstrated effects of HGF on vocal fold scarring in animal models.15–20 However, the optimal mode and timing of administration have yet to be determined. In light of current uncertainty surrounding HGF administration, the aim of this study was to assess the effects of HGF injection and reinjection coinciding with its peak activity10 on collagen density, vessel density, inflammatory reaction in the lamina propria, and mean epithelial thickness in a rabbit model of vocal fold scarring.
MATERIALS AND METHODS All animals received humane care in compliance with the ethical standards of the Brazilian Society for Laboratory Animal
667.e8 Science (SBCAL/COBEA), an affiliate of the International Council for Laboratory Animal Science. Fourteen female albino New Zealand rabbits weighing 2.5–3.5 kg were anesthetized with xylazine 5 mg/kg and ketamine 50 mg/kg intramuscularly (Vetbrands, Paulınia, Brazil) and kept under spontaneous ventilation. The entire length of both vocal folds was injured by cutting under direct laryngoscopy, performed with a suspension laryngoscope (Ferrari Medical, S~ao Paulo, Brazil). Animals were subdivided into two groups, G1 and G2. The seven animals in G1 received an injection of 0.1 mL commercially available human HGF (R&D Systems, Minneapolis, MN), 100 ng/100 mL in phosphate-buffered saline,3 into the middle third of the right vocal fold (RVF) immediately after the cutting injury. In the seven animals in G2, HGF was injected bilaterally after injury and reinjected into the RVF, in the same site of the original injection, 10 days after the first procedure, to coincide with peak activity of the first dose8 (Figure 1). The left vocal fold (LVF) served as a control in all animals. Animals in G1 were euthanized 30 days postprocedure, whereas those in G2 were euthanized 30 days after the second injection. Anesthesia was induced as noted above, and a lethal dose of potassium chloride 20% was administered intravenously. A cervical incision was made, and the larynx was excised en bloc. Specimens were dissected, and the RVF and LVF were isolated, placed in 10% formalin for 48 hours, and embedded in paraffin. Serial slices 3-mm thick (coronal plane) were cut with a microtome along the anteroposterior axis. A variable number of slices were obtained from each specimen until optimal visualization of the structures of interest was achieved.
Journal of Voice, Vol. 26, No. 5, 2012
After deparaffinization, slides were stained with Sirius red (for analysis of collagen density in the lamina propria) and hematoxylin and eosin (for assessment of vessel density and inflammatory reaction in the lamina propria and mean epithelial thickness). Slides were viewed under 4003 magnification with a Leica DMR microscope (Leica Microsystems, Wetzler, Germany) and captured with a Zeiss MRc5 digital camera (Carl Zeiss Microimaging GmbH, G€ottingen, Germany) connected to a desktop computer running the Image-Pro Plus 4.1 for Windows software (Media Cybernetics, Silver Spring, MD). Ten fields of view from each slide were selected randomly for assessment. Collagen content, vessel density, and mean epithelial thickness were quantified by computer image analysis methods. Collagen density was calculated by dividing the total collagencontaining area (in mm2) by the total visible area (also in mm2). Vessel density was calculated by division of a manual count of the number of visible vessels by the total visible area (in mm2). Mean epithelial thickness was expressed in micrometers (mm). In each field of view, two lines were manually drawn to delimit the vocal fold epithelium, and the mean thickness between all points laid on these two lines was calculated. Inflammatory reaction in the lamina propria was assessed semiquantitatively. Each slide was analyzed independently by two pathologists who were blind to group allocation. In the event of disagreement between the observers, the slide was reviewed by both and a consensus was reached. Assessment was graded on a scale of 0–3, with 0 ¼ no inflammatory reaction, 1 25% inflammatory cells in all fields of view, 2 ¼ 25–50% inflammatory cells, and 3 50% inflammatory cells.
FIGURE 1. Instrumentation and stripping procedure. A. Laryngoscopic image showing stripping of the LVF. B. Knife used for stripping. Note transverse stop on the blade (insets) to prevent insertion beyond the desired depth of 2 mm. C. Injection of HGF into the LVF. D. Tonsil-type infiltration needle. Note ridge on the shaft (inset) to prevent insertion beyond the desired depth of 2 mm.
Roberta Ismael Dias Garcia, et al
HGF Injection and Reinjection in the Rabbit Vocal Fold
Statistical analyses were performed in the SPSS 17.0 software environment (SPSS Inc., Chicago, IL). The Wilcoxon test was used for intrasubject comparison between the RVF and LVF in animals in G1 and G2, and the Mann-Whitney U test was used for between-group comparison, considering the RVF in animals in G1 and the LVF in animals in G2. The significance level was set at 5% (P < 0.050). The G-Power 3.0.10 software (Kiel University, Germany, 2008) was used to calculate statistical power. The chosen variable was —COL+ (collagen-positive), and calculation was based on intrasubject comparison between the RVF and LVF. RESULTS There were no deaths or infections in either group. Statistical analysis showed that collagen density was lower in the RVF (treatment) than in LVF (control) in both groups (P ¼ 0.018). Comparison of the RVF in G1 animals and LVF in G2 animals showed nonsignificant differences in collagen density (P ¼ 0.565) (Figures 2 and 3). Intrasubject comparison of vessel density (in the RVF vs LVF) in animals showed nonsignificant differences (P ¼ 0.063) in G1 and significant differences (P ¼ 0.018) in G2. Differences between the RVFs of animals in G1 and LVFs of animals in G2 did not reach statistical significance (P ¼ 0.085, Figure 4). Figure 5 shows mean epithelial thickness in the RVF and LVF of animals in G1 and G2 (P > 0.999 for comparisons between RVF and LVF in animals in G1 and G2; P ¼ 0.565 for comparisons between RVF of animals in G1 and LVF of animals in G2). Comparative analysis of inflammatory reaction in the lamina propria showed P ¼ 0.564 for differences between RVF and LVF in G1, P > 0.999 for differences between RVF and LVF
667.e9
in G2, and P ¼ 0.298 for differences between the RVF in G1 animals and LVF in G2 animals (Figure 6). The power of the study was determined as 80.41% for the sample used. DISCUSSION The results of this study showed that HGF reinjection timed to coincide with peak action of the first dose10 promotes further decrease in collagen density and increases vessel density. In our sample, vocal fold collagen density was significantly lower in animals that received HGF injection immediately after vocal fold injury than in controls (P ¼ 0.018). Hirano et al3 conclude that HGF injection immediately after injury prevents excessive collagen deposition in the vocal fold, which corroborates our findings. In this study of 20 animals, the authors randomly selected one vocal fold for injury, with the contralateral fold kept as an uninjured control. Immediately after injury, 10 animals underwent injection of HGF, whereas the other 10 received an injection of saline solution. The authors found that HGF-treated vocal folds exhibited less collagen deposition as compared with normal vocal folds (controls); in the saline solution group, collagen density was not statistically different in the experimental and uninjured (control) groups. In the present study, the decision was made to injure both vocal folds and inject HGF on only one side, with the contralateral fold used as a control. Normal vocal folds were not included, as the purpose of injuring both folds was to ascertain whether injection might induce any further injury that could compromise the end effect of HGF. Furthermore, one may infer from the findings of Hirano et al3 that HGF has a positive effect on the vocal fold scarring process when compared with injection of saline solution; therefore, we saw no rationale for injection of saline solution in the control group of our sample and did not perform it.
FIGURE 2. Photomicrographs showing collagen density in G1 and G2 specimens. Picrosirius red stain. Original magnification, 4003. Scale, 100 m. A. LVF, G1 specimen. B. RVF, G1 specimen. C. LVF, G2 specimen. D. RVF, G2 specimen. E, epithelium; LP, lamina propria.
667.e10
FIGURE 3. Collagen density in the RVF (gray) and LVF (white)
Journal of Voice, Vol. 26, No. 5, 2012
lamina propria in each study group.
FIGURE 5. Mean epithelial thickness in the RVF (gray) and LVF (white) in each study group.
In response to the many uncertainties surrounding clinical use of HGF, the present study was designed to assess a novel regimen of administration: reinjection timed to coincide with peak action of the first dose.10 Collagen density was lower in reinjected vocal folds than in controls (P ¼ 0.018), which suggests that repeat administration of HGF promotes improved outcomes in terms of decreasing buildup of compounds involved in vocal fold fibrosis, as excess collagen increases mucosal rigidity.21 In an in vitro study, Kishimoto et al18 assessed the effects of exogenous HGF on endogenous HGF expression and synthesis of extracellular matrix components by fibroblasts in the rat vocal fold and found that exogenous HGF is capable of modulating endogenous HGF expression in vocal fold fibroblasts
without affecting expression of type I collagen, type III collagen, or c-Met at any of the assayed concentrations. We did not assess the expression of endogenous HGF or c-Met receptor in the present study, but our finding of lower collagen densities in HGF-treated vocal folds suggests that exogenous HGF injection interferes with collagen synthesis by fibroblasts in the lamina propria. Disagreement between our findings and those of Kishimoto et al18 as they pertain to collagen expression may be explained by the fact that the authors only assessed animals up to day 7; Hirano et al10 reported that HGF only becomes active after c-Met binding, and Ohno et al19 found that c-Met expression declined on the first, third, and fifth day after vocal fold injury in the rat and rises significantly from day 28 onward. Therefore, HGF activity may not have had time to peak before
FIGURE 4. Vessel density in the RVF (gray) and LVF (white) lamina propria in each study group.
FIGURE 6. Inflammatory reaction in the RVF and LVF lamina propria in each study group.
Roberta Ismael Dias Garcia, et al
HGF Injection and Reinjection in the Rabbit Vocal Fold
the end of this experiment; conversely, in the present study, animals were euthanized at 30 and 40 days postinjury. Our results also showed higher RVF vessel density in animals in G2 (P ¼ 0.018). Ding et al22 report that the exact role of HGF on the neovascularization process has yet to be fully established but describe HGF as a potent stimulant of angiogenesis; furthermore, Ozeki et al23 found that HGF has an effect on angiogenesis in subcutaneous tissues in the rat. Umeno et al20 assessed the efficacy of fat injection with an added adenoviral vector expressing HGF to minimize resorption of fatty tissue and found increased vascularization surrounding adipocytes in the HGF-treated group. In our study, reinjection of HGF likely increased vocal fold vascularization, but further research is required to clarify these aspects. We did not find differences in mean epithelial thickness across all comparisons. Ohno et al19 reported epithelial migration and hypertrophy three days after injury in the rat vocal fold, with reepithelialization on day 7 and complete epithelialization between days 10 and 14, corresponding to the peak of HGF activity. Hirano et al10 also found HGF activity to peak between 10 and 15 days after administration. HGF thus appears to play a role in reepithelialization of the vocal fold. Our findings may be explained by the fact that animals were euthanized relatively late after HGF administration—at 30 and 40 days in G1 and G2, respectively, at which time the vocal fold epithelium was completely formed, as previously reported.7,24,25 We conducted an analysis of the inflammatory process to ascertain whether HGF injection would delay inflammation during healing but did not find differences between vocal folds that received one HGF injection, those that received a second injection on day 10, and their respective controls. In the present study, the surgical intervention on the RVF (experimental) in animals in G1 was performed in the exact same manner as that performed on the LVF (control) in animals in G2; we thus chose to compare both groups and found no significant differences. This finding suggests that the second surgical procedure performed in G2 animals did not interfere with any measured parameters of the scar formation process, nor did postoperative time, as animals in G1 were euthanized 30 days after the initial procedure and those in G2 after 40 days. Our findings preclude any inference regarding systemic absorption of HGF after topical injection into the rabbit vocal fold, but we believe that even if absorption does occur, the effects of HGF are more intense at the site of injection. This belief is corroborated by our finding of differences between collagen and vessel density in the lamina propria of the RVF and that of the LVF in a single animal. Further studies are required for indepth assessment of this aspect. Hirano et al3 note that as the early phase of scarring is most important to the end result of the process, HGF should probably be administered as soon as possible; nevertheless, the authors did not determine the duration of the effects of HGF. In the present study, the vocal fold scarring process was not yet completed at 30 and 40 days.6 Therefore, further studies are needed to clarify how this early effect during the acute phase of scarring affects scarring in the chronic stage.
667.e11
CONCLUSIONS In the present study, HGF injection promoted lower collagen density in the lamina propria of scarred rabbit vocal folds as compared with control vocal folds that received no HGF. There were no differences in vessel density or inflammatory reaction in the lamina propria or in epithelial thickness. Reinjection of HGF timed to coincide with its peak of action10 produced further decrease in collagen densities in the lamina propria, and induced higher vessel density as well. Again, there were no significant differences in inflammatory reaction in the lamina propria or in mean epithelial thickness. This was a pioneering study in that it assessed the effects of HGF reinjection into the rabbit vocal fold timed to coincide with the peak of action of a previous dose.10 The study design did not, however, include an assessment of the effects of HGF on individual collagen subtypes found in the lamina propria. We thus propose that further studies be conducted to assess these effects by means of immunohistochemical assays and that a rheological assessment of HGF-treated vocal fold be conducted. Evaluating the effects of systemic HGF administration on the vocal folds should also be an interesting research subject. Acknowledgments The authors would like to thank the S~ao Paulo Research Foundation (FAPESP) for its financial support. REFERENCES 1. Benninger MS, Alessi D, Archer S, et al. Vocal fold scarring: current concepts and management. Otolaryngol Head Neck Surg. 1996;115:474–482. 2. Rosen CA. Vocal fold scar: evaluation and treatment. Otolaryngol Clin North Am. 2000;33:1081–1086. 3. Hirano S, Bless DM, Rousseau B, Welham N, Montequin D, Chan RW, Ford CN. Prevention of vocal fold scarring by topical injection of hepatocyte growth factor in a rabbit model. Laryngoscope. 2004;114:548–556. 4. Woo P, Casper J, Colton R, Brewer D. Diagnosis and treatment of persistent dysphonia after laryngeal surgery: a retrospective analysis of 62 patientes. Laryngoscope. 1994;104:1084–1091. 5. Rousseau B, Hirano S, Scheidt TD, Welham NV, Thibeault SL, Chan RW, Bless DM. Characterization of vocal fold scarring in a canine model. Laryngoscope. 2003;113:620–627. 6. Rousseau B, Hirano S, Chan RW, Welham NV, Thibeault SL, Ford C, Bless DM. Characterization of chronic vocal fold scarring in a rabbit model. J Voice. 2004;18:116–124. 7. Branski RC, Verdolini K, Rosen CA, Hebda PA. Acute vocal fold wound healing in a rabbit model. Ann Otol Rhinol Laryngol. 2005;114:19–24. 8. Hirano S, Bless D, Heisey D, Ford C. Roles of hepatocyte growth factor and transforming growth factor beta 1 in production of extracellular matrix by canine vocal fold fibroblasts. Laryngoscope. 2003;113:144–148. 9. Matsumoto K, Nakamura T. Hepatocyte growth factor as a tissue organizer for organogenesis and regeneration. Biochem Biophys Res Commun. 1997; 239:639–644. 10. Hirano S, Thibeault S, Ford CN, Bless DM, Kanemaru S. Hepatocyte growth factor and its receptor c-met in rat and rabbit vocal folds. Ann Otol Rhinol Laryngol. 2002;111:661–666. 11. Hirano S, Bless DM, Massey RJ, Hartig GK, Ford CN. Morphological and functional changes of human vocal fold fibroblasts with hepatocyte growth factor. Ann Otol Rhinol Laryngol. 2003;112:1026–1033. 12. Ohno T, Hirano S, Kanemaru S, et al. Drug delivery system of hepatocyte growth factor for the treatment of vocal fold scarring in a canine model. Ann Otol Rhinol Laryngol. 2007;116:762–769.
667.e12 13. Ohno T, Yoo MJ, Swanson ER, Hirano S, Ossoff RH, Rousseau B. Regeneration of aged rat vocal folds using hepatocyte growth factor therapy. Laryngoscope. 2009;119:1424–1430. 14. Gilbert TW, Agrawal V, Gilbert MR, Povirk KM, Badylak SF, Rosen CA. Liver-derived extracellular matrix as a biologic scaffold for acute vocal fold repair in a canine model. Laryngoscope. 2009;119:1856–1863. 15. Hirano S, Bless DM, Welham NV, Nagai H, Montequin DW, Rousseau B, Ford CN. Growth factor therapy for vocal fold scarring in a canine model. Ann Otol Rhinol Laryngol. 2004;113:777–785. 16. Hirano S, Bless DM, Mu~noz Del Rio A, Connor NP, Ford CN. Therapeutic potential of growth factors for aging voice. Laryngoscope. 2004;114: 2161–2167. 17. Ohno T, French LC, Hirano S, Ossof R, Rousseau B. Effect of hepatocyte growth factor on gene expression of extracellular matrix during wound healing of the injured rat vocal fold. Ann Otol Rhinol Laryngol. 2008; 117:696–702. 18. Kishimoto Y, Hirano S, Suehiro A, Tateya I, Kanemaru S, Nakamura T, Ito J. Effect of exogenous hepatocyte growth factor on vocal fold fibroblasts. Ann Otol Rhinol Laryngol. 2009;118:606–611.
Journal of Voice, Vol. 26, No. 5, 2012 19. Ohno T, Hirano S, Rousseau B. Gene expression of transforming growth factor b1 and hepatocyte growth factor during wound healing of injured rat vocal fold. Laryngoscope. 2009;119:806–810. 20. Umeno H, Chitose S, Murofushi Y, Kosai K, Sato K, Kawahara A, Nakashima T. Efficacy of autologous fat injection laryngoplasty with an adenoviral vector expressing hepatocyte growth factor in a canine model. J Laryngol Otol. 2009;123(suppl 31):24–29. 21. Gray SD, Titze IR, Chan R, Hammond TH. Vocal fold proteoglycans and their influence on biomechanics. Laryngoscope. 1999;109:845–854. 22. Ding S, Merkulova-Rainon T, Han ZC, Tobelem G. HGF receptor upregulation contributes to the angiogenic phenotype of human endothelial cells and promotes angiogenesis in vitro. Blood. 2003;101:4816–4822. 23. Ozeki M, Ishii T, Hirano Y, Tabata Y. Controlled release of hepatocyte growth factor from gelatin hydrogels based on hydrogel degradation. J Drug Target. 2001;9:461–471. 24. Tateya T, Tateya I, Sohn JH, Bless DM. Histological characterization of rat vocal fold scarring. Ann Otol Rhinol Laryngol. 2005;114:183–191. 25. Tateya T, Tateya I, Sohn JH, Bless DM. Histological study of acute vocal fold injury in a rat model. Ann Otol Rhinol Laryngol. 2006;115:285–292.
Accepted for publication August 16, 2011. Study funded by the S~ao Paulo Research Foundation (FAPESP). From the *Oropharynx and Larynx Service, Department of Otorhinolaryngology, Otorhinolaryngology Clinic, University of S~ao Paulo School of Medicine, S~ao Paulo, Brazil; yDepartment of Otorhinolaryngology, University of S~ao Paulo School of Medicine, S~ao Paulo, Brazil; and the zDepartment of Pathology, University of S~ao Paulo School of Medicine, S~ao Paulo, Brazil. Address correspondence and reprint requests to Roberta Ismael Dias Garcia, Rua Tuim, 444, apartamento 11 – Moema, S~ao Paulo, SP 04514-101, Brazil. E-mail: robertaismael@ uol.com.br Journal of Voice, Vol. 26, No. 5, pp. 667.e7-667.e12 0892-1997/$36.00 Ó 2012 The Voice Foundation doi:10.1016/j.jvoice.2011.08.007
Another study by the same team8 notes that concentration and timing of administration are always a concern when seeking satisfactory outcomes with growth factor treatment. The authors describe several options for administration of HGF as a treatment for vocal fold disorders—topical injection, topical injection with normal fibroblasts, systemic administration, and gene therapy—and later note3 that several other questions remain unanswered, such as the optimal timing and dosage of administration. Ohno et al12 obtained satisfactory results with a hydrogelbased sustained HFG release system in the canine vocal fold but failed to define the optimal dosage and frequency of administration for best regeneration of the vocal fold; the authors later conducted a study13 assessing the effects of HGF on procollagen expression during the acute phase of vocal fold injury and suggested that further research was required. Gilbert et al14 assessed the efficacy of porcine liver extracellular matrix, a known reservoir of HGF, as a scaffold for vocal fold healing in dogs; again, the authors stressed the importance of further research. Other several studies have demonstrated effects of HGF on vocal fold scarring in animal models.15–20 However, the optimal mode and timing of administration have yet to be determined. In light of current uncertainty surrounding HGF administration, the aim of this study was to assess the effects of HGF injection and reinjection coinciding with its peak activity10 on collagen density, vessel density, inflammatory reaction in the lamina propria, and mean epithelial thickness in a rabbit model of vocal fold scarring.
MATERIALS AND METHODS All animals received humane care in compliance with the ethical standards of the Brazilian Society for Laboratory Animal
667.e8 Science (SBCAL/COBEA), an affiliate of the International Council for Laboratory Animal Science. Fourteen female albino New Zealand rabbits weighing 2.5–3.5 kg were anesthetized with xylazine 5 mg/kg and ketamine 50 mg/kg intramuscularly (Vetbrands, Paulınia, Brazil) and kept under spontaneous ventilation. The entire length of both vocal folds was injured by cutting under direct laryngoscopy, performed with a suspension laryngoscope (Ferrari Medical, S~ao Paulo, Brazil). Animals were subdivided into two groups, G1 and G2. The seven animals in G1 received an injection of 0.1 mL commercially available human HGF (R&D Systems, Minneapolis, MN), 100 ng/100 mL in phosphate-buffered saline,3 into the middle third of the right vocal fold (RVF) immediately after the cutting injury. In the seven animals in G2, HGF was injected bilaterally after injury and reinjected into the RVF, in the same site of the original injection, 10 days after the first procedure, to coincide with peak activity of the first dose8 (Figure 1). The left vocal fold (LVF) served as a control in all animals. Animals in G1 were euthanized 30 days postprocedure, whereas those in G2 were euthanized 30 days after the second injection. Anesthesia was induced as noted above, and a lethal dose of potassium chloride 20% was administered intravenously. A cervical incision was made, and the larynx was excised en bloc. Specimens were dissected, and the RVF and LVF were isolated, placed in 10% formalin for 48 hours, and embedded in paraffin. Serial slices 3-mm thick (coronal plane) were cut with a microtome along the anteroposterior axis. A variable number of slices were obtained from each specimen until optimal visualization of the structures of interest was achieved.
Journal of Voice, Vol. 26, No. 5, 2012
After deparaffinization, slides were stained with Sirius red (for analysis of collagen density in the lamina propria) and hematoxylin and eosin (for assessment of vessel density and inflammatory reaction in the lamina propria and mean epithelial thickness). Slides were viewed under 4003 magnification with a Leica DMR microscope (Leica Microsystems, Wetzler, Germany) and captured with a Zeiss MRc5 digital camera (Carl Zeiss Microimaging GmbH, G€ottingen, Germany) connected to a desktop computer running the Image-Pro Plus 4.1 for Windows software (Media Cybernetics, Silver Spring, MD). Ten fields of view from each slide were selected randomly for assessment. Collagen content, vessel density, and mean epithelial thickness were quantified by computer image analysis methods. Collagen density was calculated by dividing the total collagencontaining area (in mm2) by the total visible area (also in mm2). Vessel density was calculated by division of a manual count of the number of visible vessels by the total visible area (in mm2). Mean epithelial thickness was expressed in micrometers (mm). In each field of view, two lines were manually drawn to delimit the vocal fold epithelium, and the mean thickness between all points laid on these two lines was calculated. Inflammatory reaction in the lamina propria was assessed semiquantitatively. Each slide was analyzed independently by two pathologists who were blind to group allocation. In the event of disagreement between the observers, the slide was reviewed by both and a consensus was reached. Assessment was graded on a scale of 0–3, with 0 ¼ no inflammatory reaction, 1 25% inflammatory cells in all fields of view, 2 ¼ 25–50% inflammatory cells, and 3 50% inflammatory cells.
FIGURE 1. Instrumentation and stripping procedure. A. Laryngoscopic image showing stripping of the LVF. B. Knife used for stripping. Note transverse stop on the blade (insets) to prevent insertion beyond the desired depth of 2 mm. C. Injection of HGF into the LVF. D. Tonsil-type infiltration needle. Note ridge on the shaft (inset) to prevent insertion beyond the desired depth of 2 mm.
Roberta Ismael Dias Garcia, et al
HGF Injection and Reinjection in the Rabbit Vocal Fold
Statistical analyses were performed in the SPSS 17.0 software environment (SPSS Inc., Chicago, IL). The Wilcoxon test was used for intrasubject comparison between the RVF and LVF in animals in G1 and G2, and the Mann-Whitney U test was used for between-group comparison, considering the RVF in animals in G1 and the LVF in animals in G2. The significance level was set at 5% (P < 0.050). The G-Power 3.0.10 software (Kiel University, Germany, 2008) was used to calculate statistical power. The chosen variable was —COL+ (collagen-positive), and calculation was based on intrasubject comparison between the RVF and LVF. RESULTS There were no deaths or infections in either group. Statistical analysis showed that collagen density was lower in the RVF (treatment) than in LVF (control) in both groups (P ¼ 0.018). Comparison of the RVF in G1 animals and LVF in G2 animals showed nonsignificant differences in collagen density (P ¼ 0.565) (Figures 2 and 3). Intrasubject comparison of vessel density (in the RVF vs LVF) in animals showed nonsignificant differences (P ¼ 0.063) in G1 and significant differences (P ¼ 0.018) in G2. Differences between the RVFs of animals in G1 and LVFs of animals in G2 did not reach statistical significance (P ¼ 0.085, Figure 4). Figure 5 shows mean epithelial thickness in the RVF and LVF of animals in G1 and G2 (P > 0.999 for comparisons between RVF and LVF in animals in G1 and G2; P ¼ 0.565 for comparisons between RVF of animals in G1 and LVF of animals in G2). Comparative analysis of inflammatory reaction in the lamina propria showed P ¼ 0.564 for differences between RVF and LVF in G1, P > 0.999 for differences between RVF and LVF
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in G2, and P ¼ 0.298 for differences between the RVF in G1 animals and LVF in G2 animals (Figure 6). The power of the study was determined as 80.41% for the sample used. DISCUSSION The results of this study showed that HGF reinjection timed to coincide with peak action of the first dose10 promotes further decrease in collagen density and increases vessel density. In our sample, vocal fold collagen density was significantly lower in animals that received HGF injection immediately after vocal fold injury than in controls (P ¼ 0.018). Hirano et al3 conclude that HGF injection immediately after injury prevents excessive collagen deposition in the vocal fold, which corroborates our findings. In this study of 20 animals, the authors randomly selected one vocal fold for injury, with the contralateral fold kept as an uninjured control. Immediately after injury, 10 animals underwent injection of HGF, whereas the other 10 received an injection of saline solution. The authors found that HGF-treated vocal folds exhibited less collagen deposition as compared with normal vocal folds (controls); in the saline solution group, collagen density was not statistically different in the experimental and uninjured (control) groups. In the present study, the decision was made to injure both vocal folds and inject HGF on only one side, with the contralateral fold used as a control. Normal vocal folds were not included, as the purpose of injuring both folds was to ascertain whether injection might induce any further injury that could compromise the end effect of HGF. Furthermore, one may infer from the findings of Hirano et al3 that HGF has a positive effect on the vocal fold scarring process when compared with injection of saline solution; therefore, we saw no rationale for injection of saline solution in the control group of our sample and did not perform it.
FIGURE 2. Photomicrographs showing collagen density in G1 and G2 specimens. Picrosirius red stain. Original magnification, 4003. Scale, 100 m. A. LVF, G1 specimen. B. RVF, G1 specimen. C. LVF, G2 specimen. D. RVF, G2 specimen. E, epithelium; LP, lamina propria.
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FIGURE 3. Collagen density in the RVF (gray) and LVF (white)
Journal of Voice, Vol. 26, No. 5, 2012
lamina propria in each study group.
FIGURE 5. Mean epithelial thickness in the RVF (gray) and LVF (white) in each study group.
In response to the many uncertainties surrounding clinical use of HGF, the present study was designed to assess a novel regimen of administration: reinjection timed to coincide with peak action of the first dose.10 Collagen density was lower in reinjected vocal folds than in controls (P ¼ 0.018), which suggests that repeat administration of HGF promotes improved outcomes in terms of decreasing buildup of compounds involved in vocal fold fibrosis, as excess collagen increases mucosal rigidity.21 In an in vitro study, Kishimoto et al18 assessed the effects of exogenous HGF on endogenous HGF expression and synthesis of extracellular matrix components by fibroblasts in the rat vocal fold and found that exogenous HGF is capable of modulating endogenous HGF expression in vocal fold fibroblasts
without affecting expression of type I collagen, type III collagen, or c-Met at any of the assayed concentrations. We did not assess the expression of endogenous HGF or c-Met receptor in the present study, but our finding of lower collagen densities in HGF-treated vocal folds suggests that exogenous HGF injection interferes with collagen synthesis by fibroblasts in the lamina propria. Disagreement between our findings and those of Kishimoto et al18 as they pertain to collagen expression may be explained by the fact that the authors only assessed animals up to day 7; Hirano et al10 reported that HGF only becomes active after c-Met binding, and Ohno et al19 found that c-Met expression declined on the first, third, and fifth day after vocal fold injury in the rat and rises significantly from day 28 onward. Therefore, HGF activity may not have had time to peak before
FIGURE 4. Vessel density in the RVF (gray) and LVF (white) lamina propria in each study group.
FIGURE 6. Inflammatory reaction in the RVF and LVF lamina propria in each study group.
Roberta Ismael Dias Garcia, et al
HGF Injection and Reinjection in the Rabbit Vocal Fold
the end of this experiment; conversely, in the present study, animals were euthanized at 30 and 40 days postinjury. Our results also showed higher RVF vessel density in animals in G2 (P ¼ 0.018). Ding et al22 report that the exact role of HGF on the neovascularization process has yet to be fully established but describe HGF as a potent stimulant of angiogenesis; furthermore, Ozeki et al23 found that HGF has an effect on angiogenesis in subcutaneous tissues in the rat. Umeno et al20 assessed the efficacy of fat injection with an added adenoviral vector expressing HGF to minimize resorption of fatty tissue and found increased vascularization surrounding adipocytes in the HGF-treated group. In our study, reinjection of HGF likely increased vocal fold vascularization, but further research is required to clarify these aspects. We did not find differences in mean epithelial thickness across all comparisons. Ohno et al19 reported epithelial migration and hypertrophy three days after injury in the rat vocal fold, with reepithelialization on day 7 and complete epithelialization between days 10 and 14, corresponding to the peak of HGF activity. Hirano et al10 also found HGF activity to peak between 10 and 15 days after administration. HGF thus appears to play a role in reepithelialization of the vocal fold. Our findings may be explained by the fact that animals were euthanized relatively late after HGF administration—at 30 and 40 days in G1 and G2, respectively, at which time the vocal fold epithelium was completely formed, as previously reported.7,24,25 We conducted an analysis of the inflammatory process to ascertain whether HGF injection would delay inflammation during healing but did not find differences between vocal folds that received one HGF injection, those that received a second injection on day 10, and their respective controls. In the present study, the surgical intervention on the RVF (experimental) in animals in G1 was performed in the exact same manner as that performed on the LVF (control) in animals in G2; we thus chose to compare both groups and found no significant differences. This finding suggests that the second surgical procedure performed in G2 animals did not interfere with any measured parameters of the scar formation process, nor did postoperative time, as animals in G1 were euthanized 30 days after the initial procedure and those in G2 after 40 days. Our findings preclude any inference regarding systemic absorption of HGF after topical injection into the rabbit vocal fold, but we believe that even if absorption does occur, the effects of HGF are more intense at the site of injection. This belief is corroborated by our finding of differences between collagen and vessel density in the lamina propria of the RVF and that of the LVF in a single animal. Further studies are required for indepth assessment of this aspect. Hirano et al3 note that as the early phase of scarring is most important to the end result of the process, HGF should probably be administered as soon as possible; nevertheless, the authors did not determine the duration of the effects of HGF. In the present study, the vocal fold scarring process was not yet completed at 30 and 40 days.6 Therefore, further studies are needed to clarify how this early effect during the acute phase of scarring affects scarring in the chronic stage.
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CONCLUSIONS In the present study, HGF injection promoted lower collagen density in the lamina propria of scarred rabbit vocal folds as compared with control vocal folds that received no HGF. There were no differences in vessel density or inflammatory reaction in the lamina propria or in epithelial thickness. Reinjection of HGF timed to coincide with its peak of action10 produced further decrease in collagen densities in the lamina propria, and induced higher vessel density as well. Again, there were no significant differences in inflammatory reaction in the lamina propria or in mean epithelial thickness. This was a pioneering study in that it assessed the effects of HGF reinjection into the rabbit vocal fold timed to coincide with the peak of action of a previous dose.10 The study design did not, however, include an assessment of the effects of HGF on individual collagen subtypes found in the lamina propria. We thus propose that further studies be conducted to assess these effects by means of immunohistochemical assays and that a rheological assessment of HGF-treated vocal fold be conducted. Evaluating the effects of systemic HGF administration on the vocal folds should also be an interesting research subject. Acknowledgments The authors would like to thank the S~ao Paulo Research Foundation (FAPESP) for its financial support. REFERENCES 1. Benninger MS, Alessi D, Archer S, et al. Vocal fold scarring: current concepts and management. Otolaryngol Head Neck Surg. 1996;115:474–482. 2. Rosen CA. Vocal fold scar: evaluation and treatment. Otolaryngol Clin North Am. 2000;33:1081–1086. 3. Hirano S, Bless DM, Rousseau B, Welham N, Montequin D, Chan RW, Ford CN. Prevention of vocal fold scarring by topical injection of hepatocyte growth factor in a rabbit model. Laryngoscope. 2004;114:548–556. 4. Woo P, Casper J, Colton R, Brewer D. Diagnosis and treatment of persistent dysphonia after laryngeal surgery: a retrospective analysis of 62 patientes. Laryngoscope. 1994;104:1084–1091. 5. Rousseau B, Hirano S, Scheidt TD, Welham NV, Thibeault SL, Chan RW, Bless DM. Characterization of vocal fold scarring in a canine model. Laryngoscope. 2003;113:620–627. 6. Rousseau B, Hirano S, Chan RW, Welham NV, Thibeault SL, Ford C, Bless DM. Characterization of chronic vocal fold scarring in a rabbit model. J Voice. 2004;18:116–124. 7. Branski RC, Verdolini K, Rosen CA, Hebda PA. Acute vocal fold wound healing in a rabbit model. Ann Otol Rhinol Laryngol. 2005;114:19–24. 8. Hirano S, Bless D, Heisey D, Ford C. Roles of hepatocyte growth factor and transforming growth factor beta 1 in production of extracellular matrix by canine vocal fold fibroblasts. Laryngoscope. 2003;113:144–148. 9. Matsumoto K, Nakamura T. Hepatocyte growth factor as a tissue organizer for organogenesis and regeneration. Biochem Biophys Res Commun. 1997; 239:639–644. 10. Hirano S, Thibeault S, Ford CN, Bless DM, Kanemaru S. Hepatocyte growth factor and its receptor c-met in rat and rabbit vocal folds. Ann Otol Rhinol Laryngol. 2002;111:661–666. 11. Hirano S, Bless DM, Massey RJ, Hartig GK, Ford CN. Morphological and functional changes of human vocal fold fibroblasts with hepatocyte growth factor. Ann Otol Rhinol Laryngol. 2003;112:1026–1033. 12. Ohno T, Hirano S, Kanemaru S, et al. Drug delivery system of hepatocyte growth factor for the treatment of vocal fold scarring in a canine model. Ann Otol Rhinol Laryngol. 2007;116:762–769.
667.e12 13. Ohno T, Yoo MJ, Swanson ER, Hirano S, Ossoff RH, Rousseau B. Regeneration of aged rat vocal folds using hepatocyte growth factor therapy. Laryngoscope. 2009;119:1424–1430. 14. Gilbert TW, Agrawal V, Gilbert MR, Povirk KM, Badylak SF, Rosen CA. Liver-derived extracellular matrix as a biologic scaffold for acute vocal fold repair in a canine model. Laryngoscope. 2009;119:1856–1863. 15. Hirano S, Bless DM, Welham NV, Nagai H, Montequin DW, Rousseau B, Ford CN. Growth factor therapy for vocal fold scarring in a canine model. Ann Otol Rhinol Laryngol. 2004;113:777–785. 16. Hirano S, Bless DM, Mu~noz Del Rio A, Connor NP, Ford CN. Therapeutic potential of growth factors for aging voice. Laryngoscope. 2004;114: 2161–2167. 17. Ohno T, French LC, Hirano S, Ossof R, Rousseau B. Effect of hepatocyte growth factor on gene expression of extracellular matrix during wound healing of the injured rat vocal fold. Ann Otol Rhinol Laryngol. 2008; 117:696–702. 18. Kishimoto Y, Hirano S, Suehiro A, Tateya I, Kanemaru S, Nakamura T, Ito J. Effect of exogenous hepatocyte growth factor on vocal fold fibroblasts. Ann Otol Rhinol Laryngol. 2009;118:606–611.
Journal of Voice, Vol. 26, No. 5, 2012 19. Ohno T, Hirano S, Rousseau B. Gene expression of transforming growth factor b1 and hepatocyte growth factor during wound healing of injured rat vocal fold. Laryngoscope. 2009;119:806–810. 20. Umeno H, Chitose S, Murofushi Y, Kosai K, Sato K, Kawahara A, Nakashima T. Efficacy of autologous fat injection laryngoplasty with an adenoviral vector expressing hepatocyte growth factor in a canine model. J Laryngol Otol. 2009;123(suppl 31):24–29. 21. Gray SD, Titze IR, Chan R, Hammond TH. Vocal fold proteoglycans and their influence on biomechanics. Laryngoscope. 1999;109:845–854. 22. Ding S, Merkulova-Rainon T, Han ZC, Tobelem G. HGF receptor upregulation contributes to the angiogenic phenotype of human endothelial cells and promotes angiogenesis in vitro. Blood. 2003;101:4816–4822. 23. Ozeki M, Ishii T, Hirano Y, Tabata Y. Controlled release of hepatocyte growth factor from gelatin hydrogels based on hydrogel degradation. J Drug Target. 2001;9:461–471. 24. Tateya T, Tateya I, Sohn JH, Bless DM. Histological characterization of rat vocal fold scarring. Ann Otol Rhinol Laryngol. 2005;114:183–191. 25. Tateya T, Tateya I, Sohn JH, Bless DM. Histological study of acute vocal fold injury in a rat model. Ann Otol Rhinol Laryngol. 2006;115:285–292.