- Email: [email protected]
Tretinoin treatment before carbon-dioxide laser resurfacing: A clinical and biochemical analysis
Tretinoin treatment before carbon-dioxide laser resurfacing: A clinical and biochemical analysis Jeffrey S. Orringer, MD,a Sewon Kang, MD,a Timothy M. Johnson, MD,a,b,c Darius J. Karimipour, MD,a Ted Hamilton, MS,a Craig Hammerberg, PhD,a John J. Voorhees, MD, FRCP,a and Gary J. Fisher, PhDa Ann Arbor, Michigan Background: Tretinoin is often prescribed before laser resurfacing in an attempt to enhance results. Objective: We sought to assess the clinical and biochemical effects of preoperative tretinoin use before laser resurfacing. Methods: Patients were randomized to apply tretinoin to one forearm and placebo to the other for 3 weeks. Patients’ photodamaged forearms were focally treated by carbon-dioxide laser resurfacing. Biopsy specimens were obtained at baseline and various times posttreatment. Real-time polymerase chain reaction technology was used to quantify messenger RNA levels of types I and III procollagen and matrix metalloproteinases-1, 3, and 9. Wounds were assessed for degree of re-epithelialization using a computer graphics-generated template. A colorimeter was used to quantify postoperative erythema. Results: No substantial differences in either biochemical markers or clinical end points were identified between tretinoin and placebo pretreated forearms. Conclusions: We found no evidence of enhanced collagen formation, accelerated re-epithelialization, or quicker resolution of postoperative erythema with tretinoin pretreatment before laser resurfacing. ( J Am Acad Dermatol 2004;51:940-6.)
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arbon-dioxide (CO2) laser resurfacing is a highly efficacious treatment for improving the appearance of photodamaged skin.1-5 However, particularly given the significant risks associated with this treatment modality, optimization of perioperative therapy is vital.6-9 A course of topical tretinoin, a medication proven to enhance collagen production and impact wound remodeling, is frequently prescribed before ablative laser resurfacing. However, the rationale for the use of tretinoin in this setting is often based largely on anecdotal evidence From the Departments of Dermatology,a Surgery (Section of Plastic Surgery), b and Otorhinolaryngology,c University of Michigan Medical School. Supported by a research grant from the Dermatology Foundation and by the University of Michigan Department of Dermatology Laser Research Fund. Conflicts of interest: None identified. Accepted for publication April 28, 2004. Reprint requests: Jeffrey S. Orringer, MD, University of Michigan Medical School, 1500 E Medical Center Dr, Department of Dermatology, 1910 Taubman Center, Ann Arbor, MI 481090314. E-mail: [email protected]. 0190-9622/$30.00 ª 2004 by the American Academy of Dermatology, Inc. doi:10.1016/j.jaad.2004.04.040
940
and extrapolation from earlier reports involving its use before dermabrasion and chemical peeling.10-14 To clarify the use of this common clinical practice, we examined the effects of a regimen of tretinoin applied before CO2 laser resurfacing. The issue was prospectively and quantitatively analyzed from both biochemical and clinical perspectives in this study, as we sought to find a correlation between molecular changes and the clinical effects produced. It is known that CO2 laser resurfacing results in marked and characteristic changes in levels of collagen and enzymes involved in dermal remodeling known as matrix metalloproteinases (MMPs). The extent and time course of these changes has recently been described (Arch Dermatol, in press). Because tretinoin is known to inhibit MMP induction and enhance collagen production in photodamaged skin, we reasoned that any benefit of pretreatment with this medication before laser resurfacing would likely be mediated through altered collagen and/or MMP biochemistry in the healing wounds. We, therefore, specifically examined the impact of tretinoin therapy before laser resurfacing on levels of types I and III procollagen and MMPs-1, 3, and 9 at various time points postresurfacing.
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Clinically, the purported benefits of treating with tretinoin before ablative laser resurfacing include more rapid wound healing. Thus, the time to reepithelialization postresurfacing was the major clinical end point examined in this study. In addition, because postoperative erythema is one of the major disadvantages of ablative laser resurfacing, we also quantitatively assessed the impact of pretreatment with tretinoin on this aspect of wound healing.
MATERIALS AND METHODS This study was approved by our institutional review board, and written informed consent was obtained from all study patients. This was a prospective, randomized, double-blind clinical trial. In all, 11 patients aged 51 to 76 years with clinically evident moderate to severe photodamage of the forearms were enrolled. All 11 patients provided clinical data for this study, and a subset of 6 provided biochemical data. Potential patients were excluded for a history of topical retinoid use to the forearms within 6 months of study entry, the use of oral retinoids within 1 year, ongoing steroid use, a history of herpes simplex or herpes zoster of the forearms, a history of hypertrophic scarring, or a history of radiation therapy, laser therapy, or dermabrasion to the forearms. Patients each received two tubes that were identical in appearance, one of which contained topical tretinoin 0.05% in cream base, and the other cream base without the retinoid. The contents of the tubes were masked to the patients. High-performance liquid chromatography analysis of the tretinoin cream used in the study demonstrated that the tretinoin was stable for at least 2 months. In addition, biologic activity of the tretinoin cream was confirmed by demonstration of induction of retinoid receptor-dependent gene expression (CRABP II gene) after topical application in human skin in vivo.15 According to a randomized code, each patient was instructed to apply either tretinoin or vehicle cream to the designated forearm daily, as tolerated, for 3 weeks before laser resurfacing. Thus, patients treated one forearm with topical tretinoin 0.05% and the other forearm with vehicle cream without the retinoid. Patients kept a daily diary to record cream applications, and the tubes holding the creams were weighed before and after the application period to estimate compliance. Compliance appeared to be excellent with weights declining appropriately for both the tretinoin and vehicle cream tubes by means of 32.7 g and 32.5 g, respectively. Patients and the evaluating physician were blinded as to treatment assignment.
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Focal areas of each forearm were then injected with 1% lidocaine with epinephrine to achieve anesthesia and treated with the Coherent Ultrapulse CO2 laser (Coherent, Inc, Santa Clara, Calif) for two passes at 300 mJ and 60 W, and with computer pattern generator settings of 3/5/6. Serial 3-mm punch biopsy specimens were obtained from the treated areas at baseline and at various time points, ranging from 1 day to 6 months after the laser procedure. All biopsy specimens were separated by at least 5 mm. For quantification of alterations in messenger RNA (mRNA) levels, epidermis and dermis were separated by laser capture microscopy. Total RNA was extracted from laser capture microscopy—captured epidermis and dermis, and mRNA levels of specific genes (including types I and III procollagen and MMPs-1, 3, and 9) were quantified using reverse transcriptase real-time polymerase chain reaction technology. In addition, separate discrete areas of each forearm, set apart by at least 5 mm from the areas from which skin samples were obtained, were also anesthetized with 1% lidocaine with epinephrine and treated with the Coherent Ultrapulse CO2 laser with the following parameters: 300 mJ; 60 W; two passes; and computer pattern generator settings of 3/9/6. These separate areas were used for clinical assessments. On days 7, 14, 21, and 28 after the resurfacing procedure, visual evaluations of the wounds were made and the percentage of reepithelialization was graded using a semiquantitative scale of 0%, 25%, 50%, 75%, and 100%, choosing whichever value best approximated the actual degree of re-epithelialization. Grading was facilitated by the use of a transparent, computer graphics—generated template, illustrating the area remaining to undergo re-epithelialization at various stages of wound healing. That is, given the fixed size of the initial wound, the template delineated the size of wounds that were 0%, 25%, 50%, 75%, and 100% re-epithelialized for serial comparison with the healing postresurfacing sites. These areas were also evaluated with the use of a colorimeter (Minolta Chroma Meter, model CR-200; Konica Minolta Holdings, Inc, Tokyo, Japan) to quantitatively establish the levels of postoperative erythema on day 28 after resurfacing and again at 3 and 6 months posttreatment. Comparisons between tretinoin- and vehicle-pretreated skin, for all end points, were made with the 2-tailed paired t test. Statistical significance was attained for P values less than or equal to .05. Summary data are represented as means 6 1 SE. The data were analyzed with statistical software (SAS Institute Inc, Cary, NC).
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Fig 1. There is no statistically significant difference in mean peak messenger RNA (mRNA) levels of matrix metalloproteinase (MMP)-1 (A), MMP-3 (B), or MMP-9 (C) in skin pretreated with tretinoin (RA) as compared with vehicle pretreated skin 1 week after carbon-dioxide laser resurfacing of human skin in vivo. MMP mRNA levels were quantified by reverse transcriptase real-time polymerase chain reaction. N = 6.
RESULTS We have previously shown that mRNA levels of MMPs-1, 3, and 9 are markedly increased after CO2 laser resurfacing of photodamaged skin, peaking 1 week after treatment (Arch Dermatol, in press). We, therefore, compared mean peak levels of these enzymes in retinoic acid pretreated skin and placebo pretreated skin and found no statistically significant difference in peak mRNA levels for any of the MMPs examined (Fig 1). For example, mean peak induction of MMP-1 was found to be somewhat lower with tretinoin pretreatment as compared with placebo pretreated skin, but this was not statistically significant (Fig 1, A). Dermal MMP-3 mRNA levels peaked 7 days postlaser treatment in both tretinoin pretreated (191-fold increase) and placebo pretreated (240-fold increase) skin, but these mean peak levels were also found not to be significantly different when statistically analyzed (Fig 1, B). Similarly, MMP-9 mRNA levels peaked 1 week postresurfacing in both tretinoin pretreated (17-fold increase) and vehicle pretreated (19-fold increase) skin, but there was no statistically significant difference between these mean peak levels (Fig 1, C). Furthermore, there were no statistically significant differences in the mRNA levels of these MMPs demonstrated in tretinoin pretreated skin as compared with placebo pretreated skin at any of several other time points studied (data not shown). In CO2 laser treated skin, MMP induction is followed by an increase in the levels of types I and III procollagen, which peak approximately 3 weeks posttreatment (Arch Dermatol, in press). In this study, as expected, types I and III procollagen mRNA levels began to increase about 1 week posttreatment and peaked 3 weeks after resurfacing in both tretinoin pretreated and placebo pretreated
skin (data not shown). Type I procollagen mRNA levels were maximally increased 25-fold and 30-fold in tretinoin and placebo pretreated skin, respectively (Fig 2, A). These mean peak type I procollagen mRNA levels were not statistically significantly different. Type III procollagen mRNA levels, in both tretinoin and vehicle pretreated skin, also peaked 21 days postresurfacing. Type III procollagen mRNA levels peaked at 17 times baseline levels in retinoid pretreated skin and were elevated 22-fold in placebo pretreated skin (Fig 2, B). Again, a statistically significant difference was not demonstrated between these mean peak type III procollagen levels. The percent of wound re-epithelialization averaged 18% for both the tretinoin and vehicle pretreated skin on day 7 after resurfacing (Fig 3, A). By day 14 postresurfacing, percent re-epithelialization had increased to 75% on the treated side and 86% on control forearms. On day 21 after treatment, the degree of re-epithelialization was 98% for both the tretinoin pretreated side and the placebo pretreated side, and at day 28, those percentages were 100% and 98% for tretinoin and vehicle pretreated sides, respectively. By 3 months after laser treatment, all wounds were found to have completely re-epithelialized. Thus, no clinically or statistically significant differences were seen between re-epi-thelialization rates of the tretinoin pretreated and placebo pretreated laser wounds. The Minolta Chroma Meter is a device that measures the intensity of redness in the skin and assigns it a numeric value called the a* level. Colorimeter readings (a*) on day 28 postlaser treatment were 20.7 at the tretinoin pretreated laser wounds and 21.9 at the control side wounds (Fig 3, B). Erythema levels decreased at both tretinoin pretreated and placebo pretreated sites by 3 months
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Fig 2. There is no statistically significant difference in mean peak messenger RNA (mRNA) levels of procollagen I (A) or III (B) in skin pretreated with tretinoin (RA) as compared with vehicle pretreated skin 21 days after carbon-dioxide laser resurfacing of human skin in vivo. Procollagen mRNA levels were quantified by reverse transcriptase real-time polymerase chain reaction. N = 6.
after the treatment to 16.1 and 16.0, respectively. This gradual decline in redness continued bilaterally at month 6 postresurfacing where colorimeter readings were 14.0 for the tretinoin pretreated side and 13.4 for the control forearms. Although erythema clearly subsided after the laser procedure on both tretinoin pretreated and placebo pretreated forearms, there was no clinically or statistically significant difference in the intensity of redness when comparing the two sides.
DISCUSSION CO2 laser resurfacing is a commonly performed and highly efficacious treatment for achieving cutaneous rejuvenation.1-5 Many physicians advocate the preoperative use of various topical agents to speed healing of laser-induced wounds, minimize complications, and improve the results associated with ablative laser therapy. Tretinoin is one such topical medication used by many practitioners as a result of such purported effects. In fact, in a survey of physician members of the American Society for Laser Medicine and Surgery reported in 1998, 80% of respondents recommended a regimen of topical tretinoin before laser resurfacing.16 However, much of the evidence regarding the potential benefits of pre-CO2 laser tretinoin therapy is based on extrapolation from earlier animal and human studies regarding the use of this agent before dermabrasion, chemical peeling, and full-thickness wounding of the skin.10-14,17 The wounds created by ablative laser therapy may differ from those because of these other procedures, and we sought to examine the biochemical and clinical effects of pretreatment with tretinoin before CO2 laser therapy in photodamaged human skin in vivo.
We have recently reported that CO2 laser resurfacing is associated with marked elevations in the levels of various key enzymes called MMPs (Arch Dermatol, in press). It is also known that pretreatment with tretinoin inhibits the induction of MMPs (including collagenase, gelatinase, and stromelysin) in UV light—irradiated skin.18,19 We originally postulated that if tretinoin applied before laser resurfacing reduces the levels of these degradative enzymes, a positive impact on postlaser resurfacing wound healing might result. However, our data demonstrate no significant inhibition of MMP activity in tretinoin pretreated skin as compared with control skin pretreated with placebo cream only. Therefore, it is likely that retinoid-sensitive signaling pathways that lead to induction of MMPs after UV irradiation differ from those that induce MMPs after laser resurfacing. Furthermore, both CO2 laser therapy and tretinoin use have been linked to enhanced collagen production, which is thought to play an important role in achieving the cosmetic benefits associated with these therapies.20-25 Therefore, another proposed advantage of pretreating patients undergoing laser resurfacing with tretinoin is that priming collagen production might enhance the response to laser resurfacing, thereby improving cosmetic results. Our data indicate no such incremental increase in collagen production with tretinoin pretreatment beyond those increases seen with CO2 laser therapy alone. Taken together, the data discussed above suggest no benefit, on a biochemical basis, of pretreatment with tretinoin before ablative laser resurfacing. However, many practitioners cite the possibility that pretreatment with tretinoin speeds CO2 laser wound healing, and we, therefore, analyzed this issue in
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Fig 3. There is no statistically significant difference in rate of re-epithelialization (A) or degree of postresurfacing erythema (B) within 6 months after carbon-dioxide (CO2) laser resurfacing of human skin in vivo. N = 11. RA, Tretinoin pretreatment; Veh, vehicle pretreatment.
detail. Using a semiquantitative scale and assisted by a computer graphics—generated template to evaluate the degree of re-epithelialization after CO2 laser resurfacing, we found no statistically significant enhancement of wound healing rates in tretinoin pretreated skin. This is in contrast to the results of a previous study by McDonald et al26 using tretinoin and CO2 laser resurfacing in a guinea pig model that suggested more rapid healing in those animals pretreated with tretinoin.27 We can only postulate that interspecies differences in healing after laser resurfacing account for this discrepancy. One of the more common and troublesome sequela of ablative laser resurfacing is postoperative erythema that frequently persists for several months.28-31 Some physicians use tretinoin as part of a prelaser resurfacing regimen based on the concept that tretinoin therapy in some way hardens or primes the skin and, thus, among other things, protects against excessive postoperative redness. Our objective data examining the issue of postlaser
resurfacing erythema do not support this idea. The use of a colorimeter provided quantitative measurements of cutaneous erythema after CO2 laser resurfacing. No differences between skin pretreated with tretinoin and that pretreated with a placebo cream were observed. We recognize that there may potentially be differences in the ways in which forearm skin and facial skin respond to both CO2 laser treatment and topical tretinoin. We do know, however, that facial skin and forearm skin acutely respond to tretinoin in fundamentally similar ways. That is, in previous work, epidermal hyperplasia and retinoic acid target gene expression have been demonstrated after tretinoin application at each of these sites.20 The forearm was used as the treatment site in this study because of practical limitations in the number of biopsy specimens that could be taken from a more cosmetically sensitive area like the face. However, we did strive to mimic clinical practice in our selection of laser parameters and the in vivo use of
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photodamaged skin. Although we would not expect there to be fundamental differences in the biochemical or clinical changes seen after ablative laser resurfacing of the forearms as opposed to the face, the current study does not rule out this possibility. The selection of a 3-week tretinoin pretreatment course was intended to mimic clinical practice. Although there are no current clearly established clinical guidelines for the use of any topical agent before ablative laser therapy, a review of the literature indicated that an approximately 2- to 4week tretinoin pretreatment regimen was included in several prior studies of the clinical effects of laser resurfacing.32-35 It is possible that a longer application period might result in various benefits, but the complete lack of biochemical or clinical changes produced by a 3-week pretreatment regimen makes this unlikely. The clinical data presented here, along with a recent study by West and Alster36 that revealed no significant decrease in rates of post-CO2 laser resurfacing hyperpigmentation with the preoperative use of tretinoin, argue for a re-examination of the common clinical practice of pretreating patients undergoing laser resurfacing with this agent. In contrast, this study does not speak to the usefulness of postoperatively treating patients undergoing laser resurfacing with tretinoin. In fact, based on our working model of the biochemical events that follow CO2 laser therapy, there is reason to speculate that tretinoin in the postoperative period may be beneficial. That is, we believe that CO2 laser resurfacing is followed by an initial increase in MMP activity resulting in the clearance of photodamaged collagen that subsequently facilitates the up-regulation of new collagen production and remodeling (Arch Dermatol, in press). The ability of tretinoin to induce collagen formation may enhance this effect in skin that is actively undergoing neocollagenesis after ablative laser resurfacing. Once re-epithelialization occurs, tretinoin’s MMP-inhibiting effects may also be beneficial in limiting the breakdown of newly made collagen. Finally, there may be additional reasons to treat patients after CO2 laser resurfacing with tretinoin such as the decreased hyperpigmentation rates reported in patients thus treated.32,37 Future studies examining the biochemical and clinical impact of tretinoin in the postoperative period after CO2 laser resurfacing appear warranted. REFERENCES 1. Fitzpatrick R, Goldman M, Ruiz-Esparza J. Clinical advantage of the CO2 laser superpulsed mode: treatment of verruca vulgaris, seborrheic keratoses, lentigenes, and actinic cheilitis. J Dermatol Surg Oncol 1994;20:449-56.
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2. Alster T, Garg S. Treatment of facial rhytides with the ultrapulse high energy carbon dioxide laser. Plast Reconstr Surg 1996;98:791-4. 3. Fitzpatrick R. Facial resurfacing with the pulsed CO2 laser. Facial Plast Surg Clin 1996;4:231-40. 4. Schwartz R, Burns A, Rohrich R, Barton F, Byrd H. Longterm assessment of CO2 facial laser resurfacing: aesthetic results and complications. Plast Reconstr Surg 1999;103: 592-601. 5. David L, Lask G, Glassberg E, Jacoby R, Abergel R. Laser abrasion for cosmetic and medical treatment of facial actinic damage. Cutis 1989;43:583-7. 6. Fulton J. Complications of laser resurfacing. Dermatol Surg 1997;24:91-9. 7. Bernstein L, Kauvar A, Grossman M, Geronemus R. The short and long-term effects of carbon dioxide laser resurfacing. Dermatol Surg 1995;23:519-25. 8. Alster T. Cutaneous resurfacing with CO2 and erbium: YAG lasers; preoperative, intraoperative, and postoperative considerations. Plast Reconstr Surg 1999;103:242-6. 9. Nanni C, Alster T. Complications of cutaneous laser surgery. Dermatol Surg 1998;24:209-19. 10. Hevia O, Nemeth A, Taylor J. Tretinoin accelerates healing after trichloroacetic acid chemical peel. Arch Dermatol 1991;127: 678-82. 11. Mandy S. Tretinoin in the preoperative and postoperative management of dermabrasion. J Am Acad Dermatol 1986;15: 878-9. 12. Vagotis F, Brundage S. Histologic study of dermabrasion and chemical peel in an animal model after pretreatment with Retin A. Aesthetic Plast Surg 1995;19:243. 13. Mandy S. Dermabrasion. Semin Cutan Med Surg 1996;15: 162-9. 14. Drake L, Dinehart S, Goltz R. Guidelines of care for chemical peeling: guidelines/outcome committee; American Academy of Dermatology. J Am Acad Dermatol 1995;33: 497-503. 15. Elder J, Cromie M, Griffiths C, Chambon P, Voorhees J. Stimulus-selective induction of CRABP-II mRNA: a marker for retinoic acid action in human skin. J Invest Dermatol 1993;100: 356-9. 16. Duke D, Grevelink J. Care before and after laser skin resurfacing: a survey and review of the literature. Dermatol Surg 1998; 24:201-6. 17. Popp C, Kligman A, Stoudemayer T. Pretreatment of photoaged forearm skin with topical tretinoin accelerates healing of full-thickness wounds. Br J Dermatol 1995;132:46-53. 18. Fisher G, Talwar H, Lin J, Lin P, McPhillips F, Wang Z, et al. Retinoic acid inhibits induction of c-Jun protein by ultraviolet radiation that occurs subsequent to activation of mitogen-activated protein kinase pathways in human skin in vivo. J Clin Invest 1998;101:1432-40. 19. Fisher G, Voorhees J. Molecular mechanisms of photoaging and its prevention by retinoic acid: ultraviolet irradiation induces MAP kinase signal transduction cascades that induce AP-1-regulated matrix metalloproteinases that degrade human skin in vivo. J Invest Dermatol 1998;3(Suppl): S61-8. 20. Griffiths C, Russman A, Majmudar G, Singer R, Voorhees J. Restoration of collagen in photodamaged human skin by tretinoin (retinoic acid). N Engl J Med 1993;329:530-5. 21. Kim H, Bogdan N, D’Agostaro L, Gold L, Bryce G. Effect of topical retinoic acids on the levels of collagen mRNA during the repair of UV-B-induced dermal damage in the hairless mouse and the possible role of TGF-beta as a mediator. J Invest Dermatol 1992;98:359-63.
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22. Kirsch K, Zelickson B, Zachary C, Tope W. Ultrastructure of collagen thermally denatured by microsecond domain pulsed carbon dioxide laser. Arch Dermatol 1998;134:1255-9. 23. Ross E, McKinlay J, Anderson R. Why does carbon dioxide resurfacing work? A review. Arch Dermatol 1999;135:444-54. 24. Ross E, Grossman M, Duke D, Grevelink J. Long-term results after CO2 laser resurfacing: a comparison of scanned and pulsed systems. J Am Acad Dermatol 1997;37:709-18. 25. Rosenberg G, Brito MJ, Aportella R, Kapoor S. Long-term histologic effects of the CO2 laser. Plast Reconstr Surg 1999; 104:2239-44. 26. McDonald W, Beasley D, Jones C. Retinoic acid and CO2 laser resurfacing. Plast Reconstr Surg 1999;104:2229-35. 27. Alster T. Retinoic acid and CO2 laser resurfacing. Plast Reconstr Surg 1999;104:2236-8. 28. Trelles M, Mordon S, Svaasand L, Mellor T, Rigau J, Garcia L. The origin and role of erythema after carbon dioxide laser resurfacing: a clinical and histological study. Dermatol Surg 1998;24:25-9. 29. Ruiz-Esparza J, Gomez J, DeLaTorre O, David L. Erythema after laser skin resurfacing. Dermatol Surg 1998;24:31-4.
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30. Nanni C. Complications of laser surgery. Dermatol Clin 1997; 15:521-34. 31. Alster T, West T. Effect of topical vitamin C on postoperative carbon dioxide laser resurfacing erythema. Dermatol Surg 1998;24:331-4. 32. Lowe N, Lask G, Griffin M. Laser skin resurfacing: pre- and posttreatment guidelines. Dermatol Surg 1995;21:1017-9. 33. Apfelberg D. Ultrapulse carbon dioxide laser with CPG scanner for full-face resurfacing for rhytids, photoaging, and acne scars. Plast Reconstr Surg 1997;99:1817-25. 34. Manuskiatti W, Fitzpatrick R, Goldman M. Long-term effectiveness and side effects of carbon dioxide laser resurfacing for photoaged facial skin. J Am Acad Dermatol 1999;40: 401-11. 35. Ho C, Nguyen Q, Lowe N, Griffin M, Lask G. Laser resurfacing in pigmented skin. Dermatol Surg 1995;21:1035-7. 36. West T, Alster T. Effect of pretreatment on the incidence of hyperpigmentation following cutaneous CO2 laser resurfacing. Dermatol Surg 1999;25:15-7. 37. Weinstein C. Carbon dioxide laser resurfacing: long-term follow-up in 2123 patients. Clin Plast Surg 1998;25:109-30.
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arbon-dioxide (CO2) laser resurfacing is a highly efficacious treatment for improving the appearance of photodamaged skin.1-5 However, particularly given the significant risks associated with this treatment modality, optimization of perioperative therapy is vital.6-9 A course of topical tretinoin, a medication proven to enhance collagen production and impact wound remodeling, is frequently prescribed before ablative laser resurfacing. However, the rationale for the use of tretinoin in this setting is often based largely on anecdotal evidence From the Departments of Dermatology,a Surgery (Section of Plastic Surgery), b and Otorhinolaryngology,c University of Michigan Medical School. Supported by a research grant from the Dermatology Foundation and by the University of Michigan Department of Dermatology Laser Research Fund. Conflicts of interest: None identified. Accepted for publication April 28, 2004. Reprint requests: Jeffrey S. Orringer, MD, University of Michigan Medical School, 1500 E Medical Center Dr, Department of Dermatology, 1910 Taubman Center, Ann Arbor, MI 481090314. E-mail: [email protected]. 0190-9622/$30.00 ª 2004 by the American Academy of Dermatology, Inc. doi:10.1016/j.jaad.2004.04.040
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and extrapolation from earlier reports involving its use before dermabrasion and chemical peeling.10-14 To clarify the use of this common clinical practice, we examined the effects of a regimen of tretinoin applied before CO2 laser resurfacing. The issue was prospectively and quantitatively analyzed from both biochemical and clinical perspectives in this study, as we sought to find a correlation between molecular changes and the clinical effects produced. It is known that CO2 laser resurfacing results in marked and characteristic changes in levels of collagen and enzymes involved in dermal remodeling known as matrix metalloproteinases (MMPs). The extent and time course of these changes has recently been described (Arch Dermatol, in press). Because tretinoin is known to inhibit MMP induction and enhance collagen production in photodamaged skin, we reasoned that any benefit of pretreatment with this medication before laser resurfacing would likely be mediated through altered collagen and/or MMP biochemistry in the healing wounds. We, therefore, specifically examined the impact of tretinoin therapy before laser resurfacing on levels of types I and III procollagen and MMPs-1, 3, and 9 at various time points postresurfacing.
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Clinically, the purported benefits of treating with tretinoin before ablative laser resurfacing include more rapid wound healing. Thus, the time to reepithelialization postresurfacing was the major clinical end point examined in this study. In addition, because postoperative erythema is one of the major disadvantages of ablative laser resurfacing, we also quantitatively assessed the impact of pretreatment with tretinoin on this aspect of wound healing.
MATERIALS AND METHODS This study was approved by our institutional review board, and written informed consent was obtained from all study patients. This was a prospective, randomized, double-blind clinical trial. In all, 11 patients aged 51 to 76 years with clinically evident moderate to severe photodamage of the forearms were enrolled. All 11 patients provided clinical data for this study, and a subset of 6 provided biochemical data. Potential patients were excluded for a history of topical retinoid use to the forearms within 6 months of study entry, the use of oral retinoids within 1 year, ongoing steroid use, a history of herpes simplex or herpes zoster of the forearms, a history of hypertrophic scarring, or a history of radiation therapy, laser therapy, or dermabrasion to the forearms. Patients each received two tubes that were identical in appearance, one of which contained topical tretinoin 0.05% in cream base, and the other cream base without the retinoid. The contents of the tubes were masked to the patients. High-performance liquid chromatography analysis of the tretinoin cream used in the study demonstrated that the tretinoin was stable for at least 2 months. In addition, biologic activity of the tretinoin cream was confirmed by demonstration of induction of retinoid receptor-dependent gene expression (CRABP II gene) after topical application in human skin in vivo.15 According to a randomized code, each patient was instructed to apply either tretinoin or vehicle cream to the designated forearm daily, as tolerated, for 3 weeks before laser resurfacing. Thus, patients treated one forearm with topical tretinoin 0.05% and the other forearm with vehicle cream without the retinoid. Patients kept a daily diary to record cream applications, and the tubes holding the creams were weighed before and after the application period to estimate compliance. Compliance appeared to be excellent with weights declining appropriately for both the tretinoin and vehicle cream tubes by means of 32.7 g and 32.5 g, respectively. Patients and the evaluating physician were blinded as to treatment assignment.
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Focal areas of each forearm were then injected with 1% lidocaine with epinephrine to achieve anesthesia and treated with the Coherent Ultrapulse CO2 laser (Coherent, Inc, Santa Clara, Calif) for two passes at 300 mJ and 60 W, and with computer pattern generator settings of 3/5/6. Serial 3-mm punch biopsy specimens were obtained from the treated areas at baseline and at various time points, ranging from 1 day to 6 months after the laser procedure. All biopsy specimens were separated by at least 5 mm. For quantification of alterations in messenger RNA (mRNA) levels, epidermis and dermis were separated by laser capture microscopy. Total RNA was extracted from laser capture microscopy—captured epidermis and dermis, and mRNA levels of specific genes (including types I and III procollagen and MMPs-1, 3, and 9) were quantified using reverse transcriptase real-time polymerase chain reaction technology. In addition, separate discrete areas of each forearm, set apart by at least 5 mm from the areas from which skin samples were obtained, were also anesthetized with 1% lidocaine with epinephrine and treated with the Coherent Ultrapulse CO2 laser with the following parameters: 300 mJ; 60 W; two passes; and computer pattern generator settings of 3/9/6. These separate areas were used for clinical assessments. On days 7, 14, 21, and 28 after the resurfacing procedure, visual evaluations of the wounds were made and the percentage of reepithelialization was graded using a semiquantitative scale of 0%, 25%, 50%, 75%, and 100%, choosing whichever value best approximated the actual degree of re-epithelialization. Grading was facilitated by the use of a transparent, computer graphics—generated template, illustrating the area remaining to undergo re-epithelialization at various stages of wound healing. That is, given the fixed size of the initial wound, the template delineated the size of wounds that were 0%, 25%, 50%, 75%, and 100% re-epithelialized for serial comparison with the healing postresurfacing sites. These areas were also evaluated with the use of a colorimeter (Minolta Chroma Meter, model CR-200; Konica Minolta Holdings, Inc, Tokyo, Japan) to quantitatively establish the levels of postoperative erythema on day 28 after resurfacing and again at 3 and 6 months posttreatment. Comparisons between tretinoin- and vehicle-pretreated skin, for all end points, were made with the 2-tailed paired t test. Statistical significance was attained for P values less than or equal to .05. Summary data are represented as means 6 1 SE. The data were analyzed with statistical software (SAS Institute Inc, Cary, NC).
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Fig 1. There is no statistically significant difference in mean peak messenger RNA (mRNA) levels of matrix metalloproteinase (MMP)-1 (A), MMP-3 (B), or MMP-9 (C) in skin pretreated with tretinoin (RA) as compared with vehicle pretreated skin 1 week after carbon-dioxide laser resurfacing of human skin in vivo. MMP mRNA levels were quantified by reverse transcriptase real-time polymerase chain reaction. N = 6.
RESULTS We have previously shown that mRNA levels of MMPs-1, 3, and 9 are markedly increased after CO2 laser resurfacing of photodamaged skin, peaking 1 week after treatment (Arch Dermatol, in press). We, therefore, compared mean peak levels of these enzymes in retinoic acid pretreated skin and placebo pretreated skin and found no statistically significant difference in peak mRNA levels for any of the MMPs examined (Fig 1). For example, mean peak induction of MMP-1 was found to be somewhat lower with tretinoin pretreatment as compared with placebo pretreated skin, but this was not statistically significant (Fig 1, A). Dermal MMP-3 mRNA levels peaked 7 days postlaser treatment in both tretinoin pretreated (191-fold increase) and placebo pretreated (240-fold increase) skin, but these mean peak levels were also found not to be significantly different when statistically analyzed (Fig 1, B). Similarly, MMP-9 mRNA levels peaked 1 week postresurfacing in both tretinoin pretreated (17-fold increase) and vehicle pretreated (19-fold increase) skin, but there was no statistically significant difference between these mean peak levels (Fig 1, C). Furthermore, there were no statistically significant differences in the mRNA levels of these MMPs demonstrated in tretinoin pretreated skin as compared with placebo pretreated skin at any of several other time points studied (data not shown). In CO2 laser treated skin, MMP induction is followed by an increase in the levels of types I and III procollagen, which peak approximately 3 weeks posttreatment (Arch Dermatol, in press). In this study, as expected, types I and III procollagen mRNA levels began to increase about 1 week posttreatment and peaked 3 weeks after resurfacing in both tretinoin pretreated and placebo pretreated
skin (data not shown). Type I procollagen mRNA levels were maximally increased 25-fold and 30-fold in tretinoin and placebo pretreated skin, respectively (Fig 2, A). These mean peak type I procollagen mRNA levels were not statistically significantly different. Type III procollagen mRNA levels, in both tretinoin and vehicle pretreated skin, also peaked 21 days postresurfacing. Type III procollagen mRNA levels peaked at 17 times baseline levels in retinoid pretreated skin and were elevated 22-fold in placebo pretreated skin (Fig 2, B). Again, a statistically significant difference was not demonstrated between these mean peak type III procollagen levels. The percent of wound re-epithelialization averaged 18% for both the tretinoin and vehicle pretreated skin on day 7 after resurfacing (Fig 3, A). By day 14 postresurfacing, percent re-epithelialization had increased to 75% on the treated side and 86% on control forearms. On day 21 after treatment, the degree of re-epithelialization was 98% for both the tretinoin pretreated side and the placebo pretreated side, and at day 28, those percentages were 100% and 98% for tretinoin and vehicle pretreated sides, respectively. By 3 months after laser treatment, all wounds were found to have completely re-epithelialized. Thus, no clinically or statistically significant differences were seen between re-epi-thelialization rates of the tretinoin pretreated and placebo pretreated laser wounds. The Minolta Chroma Meter is a device that measures the intensity of redness in the skin and assigns it a numeric value called the a* level. Colorimeter readings (a*) on day 28 postlaser treatment were 20.7 at the tretinoin pretreated laser wounds and 21.9 at the control side wounds (Fig 3, B). Erythema levels decreased at both tretinoin pretreated and placebo pretreated sites by 3 months
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Fig 2. There is no statistically significant difference in mean peak messenger RNA (mRNA) levels of procollagen I (A) or III (B) in skin pretreated with tretinoin (RA) as compared with vehicle pretreated skin 21 days after carbon-dioxide laser resurfacing of human skin in vivo. Procollagen mRNA levels were quantified by reverse transcriptase real-time polymerase chain reaction. N = 6.
after the treatment to 16.1 and 16.0, respectively. This gradual decline in redness continued bilaterally at month 6 postresurfacing where colorimeter readings were 14.0 for the tretinoin pretreated side and 13.4 for the control forearms. Although erythema clearly subsided after the laser procedure on both tretinoin pretreated and placebo pretreated forearms, there was no clinically or statistically significant difference in the intensity of redness when comparing the two sides.
DISCUSSION CO2 laser resurfacing is a commonly performed and highly efficacious treatment for achieving cutaneous rejuvenation.1-5 Many physicians advocate the preoperative use of various topical agents to speed healing of laser-induced wounds, minimize complications, and improve the results associated with ablative laser therapy. Tretinoin is one such topical medication used by many practitioners as a result of such purported effects. In fact, in a survey of physician members of the American Society for Laser Medicine and Surgery reported in 1998, 80% of respondents recommended a regimen of topical tretinoin before laser resurfacing.16 However, much of the evidence regarding the potential benefits of pre-CO2 laser tretinoin therapy is based on extrapolation from earlier animal and human studies regarding the use of this agent before dermabrasion, chemical peeling, and full-thickness wounding of the skin.10-14,17 The wounds created by ablative laser therapy may differ from those because of these other procedures, and we sought to examine the biochemical and clinical effects of pretreatment with tretinoin before CO2 laser therapy in photodamaged human skin in vivo.
We have recently reported that CO2 laser resurfacing is associated with marked elevations in the levels of various key enzymes called MMPs (Arch Dermatol, in press). It is also known that pretreatment with tretinoin inhibits the induction of MMPs (including collagenase, gelatinase, and stromelysin) in UV light—irradiated skin.18,19 We originally postulated that if tretinoin applied before laser resurfacing reduces the levels of these degradative enzymes, a positive impact on postlaser resurfacing wound healing might result. However, our data demonstrate no significant inhibition of MMP activity in tretinoin pretreated skin as compared with control skin pretreated with placebo cream only. Therefore, it is likely that retinoid-sensitive signaling pathways that lead to induction of MMPs after UV irradiation differ from those that induce MMPs after laser resurfacing. Furthermore, both CO2 laser therapy and tretinoin use have been linked to enhanced collagen production, which is thought to play an important role in achieving the cosmetic benefits associated with these therapies.20-25 Therefore, another proposed advantage of pretreating patients undergoing laser resurfacing with tretinoin is that priming collagen production might enhance the response to laser resurfacing, thereby improving cosmetic results. Our data indicate no such incremental increase in collagen production with tretinoin pretreatment beyond those increases seen with CO2 laser therapy alone. Taken together, the data discussed above suggest no benefit, on a biochemical basis, of pretreatment with tretinoin before ablative laser resurfacing. However, many practitioners cite the possibility that pretreatment with tretinoin speeds CO2 laser wound healing, and we, therefore, analyzed this issue in
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Fig 3. There is no statistically significant difference in rate of re-epithelialization (A) or degree of postresurfacing erythema (B) within 6 months after carbon-dioxide (CO2) laser resurfacing of human skin in vivo. N = 11. RA, Tretinoin pretreatment; Veh, vehicle pretreatment.
detail. Using a semiquantitative scale and assisted by a computer graphics—generated template to evaluate the degree of re-epithelialization after CO2 laser resurfacing, we found no statistically significant enhancement of wound healing rates in tretinoin pretreated skin. This is in contrast to the results of a previous study by McDonald et al26 using tretinoin and CO2 laser resurfacing in a guinea pig model that suggested more rapid healing in those animals pretreated with tretinoin.27 We can only postulate that interspecies differences in healing after laser resurfacing account for this discrepancy. One of the more common and troublesome sequela of ablative laser resurfacing is postoperative erythema that frequently persists for several months.28-31 Some physicians use tretinoin as part of a prelaser resurfacing regimen based on the concept that tretinoin therapy in some way hardens or primes the skin and, thus, among other things, protects against excessive postoperative redness. Our objective data examining the issue of postlaser
resurfacing erythema do not support this idea. The use of a colorimeter provided quantitative measurements of cutaneous erythema after CO2 laser resurfacing. No differences between skin pretreated with tretinoin and that pretreated with a placebo cream were observed. We recognize that there may potentially be differences in the ways in which forearm skin and facial skin respond to both CO2 laser treatment and topical tretinoin. We do know, however, that facial skin and forearm skin acutely respond to tretinoin in fundamentally similar ways. That is, in previous work, epidermal hyperplasia and retinoic acid target gene expression have been demonstrated after tretinoin application at each of these sites.20 The forearm was used as the treatment site in this study because of practical limitations in the number of biopsy specimens that could be taken from a more cosmetically sensitive area like the face. However, we did strive to mimic clinical practice in our selection of laser parameters and the in vivo use of
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photodamaged skin. Although we would not expect there to be fundamental differences in the biochemical or clinical changes seen after ablative laser resurfacing of the forearms as opposed to the face, the current study does not rule out this possibility. The selection of a 3-week tretinoin pretreatment course was intended to mimic clinical practice. Although there are no current clearly established clinical guidelines for the use of any topical agent before ablative laser therapy, a review of the literature indicated that an approximately 2- to 4week tretinoin pretreatment regimen was included in several prior studies of the clinical effects of laser resurfacing.32-35 It is possible that a longer application period might result in various benefits, but the complete lack of biochemical or clinical changes produced by a 3-week pretreatment regimen makes this unlikely. The clinical data presented here, along with a recent study by West and Alster36 that revealed no significant decrease in rates of post-CO2 laser resurfacing hyperpigmentation with the preoperative use of tretinoin, argue for a re-examination of the common clinical practice of pretreating patients undergoing laser resurfacing with this agent. In contrast, this study does not speak to the usefulness of postoperatively treating patients undergoing laser resurfacing with tretinoin. In fact, based on our working model of the biochemical events that follow CO2 laser therapy, there is reason to speculate that tretinoin in the postoperative period may be beneficial. That is, we believe that CO2 laser resurfacing is followed by an initial increase in MMP activity resulting in the clearance of photodamaged collagen that subsequently facilitates the up-regulation of new collagen production and remodeling (Arch Dermatol, in press). The ability of tretinoin to induce collagen formation may enhance this effect in skin that is actively undergoing neocollagenesis after ablative laser resurfacing. Once re-epithelialization occurs, tretinoin’s MMP-inhibiting effects may also be beneficial in limiting the breakdown of newly made collagen. Finally, there may be additional reasons to treat patients after CO2 laser resurfacing with tretinoin such as the decreased hyperpigmentation rates reported in patients thus treated.32,37 Future studies examining the biochemical and clinical impact of tretinoin in the postoperative period after CO2 laser resurfacing appear warranted. REFERENCES 1. Fitzpatrick R, Goldman M, Ruiz-Esparza J. Clinical advantage of the CO2 laser superpulsed mode: treatment of verruca vulgaris, seborrheic keratoses, lentigenes, and actinic cheilitis. J Dermatol Surg Oncol 1994;20:449-56.
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2. Alster T, Garg S. Treatment of facial rhytides with the ultrapulse high energy carbon dioxide laser. Plast Reconstr Surg 1996;98:791-4. 3. Fitzpatrick R. Facial resurfacing with the pulsed CO2 laser. Facial Plast Surg Clin 1996;4:231-40. 4. Schwartz R, Burns A, Rohrich R, Barton F, Byrd H. Longterm assessment of CO2 facial laser resurfacing: aesthetic results and complications. Plast Reconstr Surg 1999;103: 592-601. 5. David L, Lask G, Glassberg E, Jacoby R, Abergel R. Laser abrasion for cosmetic and medical treatment of facial actinic damage. Cutis 1989;43:583-7. 6. Fulton J. Complications of laser resurfacing. Dermatol Surg 1997;24:91-9. 7. Bernstein L, Kauvar A, Grossman M, Geronemus R. The short and long-term effects of carbon dioxide laser resurfacing. Dermatol Surg 1995;23:519-25. 8. Alster T. Cutaneous resurfacing with CO2 and erbium: YAG lasers; preoperative, intraoperative, and postoperative considerations. Plast Reconstr Surg 1999;103:242-6. 9. Nanni C, Alster T. Complications of cutaneous laser surgery. Dermatol Surg 1998;24:209-19. 10. Hevia O, Nemeth A, Taylor J. Tretinoin accelerates healing after trichloroacetic acid chemical peel. Arch Dermatol 1991;127: 678-82. 11. Mandy S. Tretinoin in the preoperative and postoperative management of dermabrasion. J Am Acad Dermatol 1986;15: 878-9. 12. Vagotis F, Brundage S. Histologic study of dermabrasion and chemical peel in an animal model after pretreatment with Retin A. Aesthetic Plast Surg 1995;19:243. 13. Mandy S. Dermabrasion. Semin Cutan Med Surg 1996;15: 162-9. 14. Drake L, Dinehart S, Goltz R. Guidelines of care for chemical peeling: guidelines/outcome committee; American Academy of Dermatology. J Am Acad Dermatol 1995;33: 497-503. 15. Elder J, Cromie M, Griffiths C, Chambon P, Voorhees J. Stimulus-selective induction of CRABP-II mRNA: a marker for retinoic acid action in human skin. J Invest Dermatol 1993;100: 356-9. 16. Duke D, Grevelink J. Care before and after laser skin resurfacing: a survey and review of the literature. Dermatol Surg 1998; 24:201-6. 17. Popp C, Kligman A, Stoudemayer T. Pretreatment of photoaged forearm skin with topical tretinoin accelerates healing of full-thickness wounds. Br J Dermatol 1995;132:46-53. 18. Fisher G, Talwar H, Lin J, Lin P, McPhillips F, Wang Z, et al. Retinoic acid inhibits induction of c-Jun protein by ultraviolet radiation that occurs subsequent to activation of mitogen-activated protein kinase pathways in human skin in vivo. J Clin Invest 1998;101:1432-40. 19. Fisher G, Voorhees J. Molecular mechanisms of photoaging and its prevention by retinoic acid: ultraviolet irradiation induces MAP kinase signal transduction cascades that induce AP-1-regulated matrix metalloproteinases that degrade human skin in vivo. J Invest Dermatol 1998;3(Suppl): S61-8. 20. Griffiths C, Russman A, Majmudar G, Singer R, Voorhees J. Restoration of collagen in photodamaged human skin by tretinoin (retinoic acid). N Engl J Med 1993;329:530-5. 21. Kim H, Bogdan N, D’Agostaro L, Gold L, Bryce G. Effect of topical retinoic acids on the levels of collagen mRNA during the repair of UV-B-induced dermal damage in the hairless mouse and the possible role of TGF-beta as a mediator. J Invest Dermatol 1992;98:359-63.
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22. Kirsch K, Zelickson B, Zachary C, Tope W. Ultrastructure of collagen thermally denatured by microsecond domain pulsed carbon dioxide laser. Arch Dermatol 1998;134:1255-9. 23. Ross E, McKinlay J, Anderson R. Why does carbon dioxide resurfacing work? A review. Arch Dermatol 1999;135:444-54. 24. Ross E, Grossman M, Duke D, Grevelink J. Long-term results after CO2 laser resurfacing: a comparison of scanned and pulsed systems. J Am Acad Dermatol 1997;37:709-18. 25. Rosenberg G, Brito MJ, Aportella R, Kapoor S. Long-term histologic effects of the CO2 laser. Plast Reconstr Surg 1999; 104:2239-44. 26. McDonald W, Beasley D, Jones C. Retinoic acid and CO2 laser resurfacing. Plast Reconstr Surg 1999;104:2229-35. 27. Alster T. Retinoic acid and CO2 laser resurfacing. Plast Reconstr Surg 1999;104:2236-8. 28. Trelles M, Mordon S, Svaasand L, Mellor T, Rigau J, Garcia L. The origin and role of erythema after carbon dioxide laser resurfacing: a clinical and histological study. Dermatol Surg 1998;24:25-9. 29. Ruiz-Esparza J, Gomez J, DeLaTorre O, David L. Erythema after laser skin resurfacing. Dermatol Surg 1998;24:31-4.
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30. Nanni C. Complications of laser surgery. Dermatol Clin 1997; 15:521-34. 31. Alster T, West T. Effect of topical vitamin C on postoperative carbon dioxide laser resurfacing erythema. Dermatol Surg 1998;24:331-4. 32. Lowe N, Lask G, Griffin M. Laser skin resurfacing: pre- and posttreatment guidelines. Dermatol Surg 1995;21:1017-9. 33. Apfelberg D. Ultrapulse carbon dioxide laser with CPG scanner for full-face resurfacing for rhytids, photoaging, and acne scars. Plast Reconstr Surg 1997;99:1817-25. 34. Manuskiatti W, Fitzpatrick R, Goldman M. Long-term effectiveness and side effects of carbon dioxide laser resurfacing for photoaged facial skin. J Am Acad Dermatol 1999;40: 401-11. 35. Ho C, Nguyen Q, Lowe N, Griffin M, Lask G. Laser resurfacing in pigmented skin. Dermatol Surg 1995;21:1035-7. 36. West T, Alster T. Effect of pretreatment on the incidence of hyperpigmentation following cutaneous CO2 laser resurfacing. Dermatol Surg 1999;25:15-7. 37. Weinstein C. Carbon dioxide laser resurfacing: long-term follow-up in 2123 patients. Clin Plast Surg 1998;25:109-30.