Application of Umbilical Cord Serum Eyedrops for the Treatment of Neurotrophic Keratitis

Transcription

1 Application of Umbilical Cord Serum Eyedrops for the Treatment of Neurotrophic Keratitis Kyung-Chul Yoon, MD, PhD, 1 In-Cheon You, MD, 1 Seong-Kyu Im, MD, 1 Tae-Sun Jeong, MD, 1 Yeoung-Geol Park, MD, PhD, 1 Jin Choi, MD, PhD 2 Purpose: To investigate the efficacy of umbilical cord serum eyedrops for the treatment of neurotrophic keratitis. Design: Prospective noncomparative case series. Participants: Twenty-eight eyes of 28 patients with neurotrophic keratitis who were refractory to conventional treatment. Methods: The patients with neurotrophic keraitis were treated with 20% umbilical cord serum eyedrops 6 to 10 times a day. Ophthalmic examinations including best-corrected visual acuity (VA) measurement, corneal sensitivity test, corneal fluorescein staining, and anterior segment photography were performed before and after the treatment. Concentrations of substance P, insulinlike growth factor 1 (IGF-1), and nerve growth factor (NGF) in umbilical cord serum, normal peripheral blood serum, and tears were measured. Main Outcome Measures: Epithelial healing time; changes of VA and corneal sensitivity after treatment; and levels of substance P, IGF-1, and NGF in umbilical cord serum, normal peripheral blood serum, and tears. Results: The epithelial defect healed completely in all eyes, with a mean healing time of weeks. The epithelial defect healed within 2 weeks in 8 eyes (28.6%), between 2 and 4 weeks in 14 eyes (50.0%), and after 4 weeks in 6 eyes (21.4%). After treatment, VA improved by 2 lines in 17 eyes (60.7%). Mean pretreatment corneal sensitivity was mm, and mean posttreatment corneal sensitivity was mm (P 0.01). Mean concentrations of substance P, IGF-1, and NGF were pg/ml, ng/ml, and pg/ml in umbilical cord serum; pg/ml, ng/ml, and pg/ml in peripheral blood serum; and pg/ml, ng/ml, and pg/ml in tears, respectively. Conclusions: Umbilical cord serum contains many neurotrophic factors, and umbilical cord serum eyedrops appeared to be effective for the treatment of neurotrophic keratitis. Ophthalmology 2007;114: by the American Academy of Ophthalmology. Originally received: July 29, Final revision: December 7, Accepted: December 7, Available online: March 22, Manuscript no Department of Ophthalmology, Chonnam National University Medical School and Hospital, Gwang-Ju, South Korea. 2 Department of Obstetrics, Eun Hospital, Gwang-Ju, South Korea. Correspondence to Kyung-Chul Yoon, MD, Department of Ophthalmology, Chonnam National University Hospital, 8 Hak-Dong, Dong-Gu, Gwang-Ju, , South Korea. kcyoon@chonnam.ac.kr. Neurotrophic keratitis is a degenerative corneal disease characterized by impaired healing of the corneal epithelium due to damage of trigeminal corneal innervation. The depletion of trophic mediators such as acetylcholine and substance P as well as an aqueous tear deficiency have been reported to contribute to the pathogenesis of this disease. 1,2 It is caused by herpes simplex and herpes zoster infection of the ocular surface; chemical, physical, and surgical injuries; neurosurgical procedures such as acoustic neuroma, meningioma, and aneurysm; and systemic disease such as diabetes, multiple sclerosis, and leprosy. 3 The progression of this disease can lead to a corneal ulcer, melting, and perforation. The treatment of neurotrophic keraitis is difficult. Conventional treatments, including application of therapeutic contact lenses, artificial tears, cyanoacrylate glue, conjunctival flap, tarsorrhaphy, and amniotic membrane transplantation, have therapeutic limitations. 3 6 The topical application of neurotrophic substances such as substance P, insulinlike growth factor 1 (IGF-1), and nerve growth factor (NGF) has been tried to promote corneal wound healing However, these substances may induce side effects such as contact hypersensitivity, conjunctival hyperemia, photophobia, and ocular pain Autologous serum contains many growth factors and neuropeptides like epidermal growth factor (EGF), vitamin A, transforming growth factor (TGF- ), fibronectin, substance P, IGF-1, NGF, and other cytokines that are essential for the proliferation, differentiation, and maturation of the 2007 by the American Academy of Ophthalmology ISSN /07/$ see front matter Published by Elsevier Inc. doi: /j.ophtha

2 Ophthalmology Volume 114, Number 9, September 2007 normal ocular surface epithelium. 13,14 On the basis of these properties, autologous serum eyedrops have been effectively applied to treat neurotrophic keratitis as well as severe dry eye in Sjögren s syndrome, persistent epithelial defects, superior limbic keratoconjunctivitis, and recurrent corneal erosion We recently found that umbilical cord serum contains many growth factors and essential tear components, including EGF, vitamin A, and TGF-, and that umbilical cord serum eyedrops are safe and effective for the treatment of severe dry eye syndrome. 21 If umbilical cord serum contains many neurotrophic factors, it can also be useful for the treatment of patients with neurotrophic keratitis. In the present study, we investigated the efficacy of umbilical cord serum eyedrops for the treatment of patients with neurotrophic keratitis who were unresponsive to conventional treatment. We also analyzed and compared the levels of substance P, IGF-1, and NGF in umbilical cord serum with those in tears and normal peripheral blood serum of a volunteer control. Materials and Methods We prospectively analyzed the effect of umbilical cord serum eyedrops in patients with neurotrophic keratitis. Informed consent was obtained from each subject enrolled in this study. Institutional review board/ethics committee approval for the collection and use of umbilical cord serum was obtained from the Chonnam National University Medical School s institutional review board, and the study protocol followed the guidelines of the Declaration of Helsinki. Patient Selection Patients with neurotrophic keratitis who had partial or complete corneal anesthesia, who did not improve at all with conventional treatments with both artificial tears and therapeutic soft contact lenses for 2 months, and who did not undergo any surgical procedures were included. The diagnosis was based on keratopathy with low corneal sensitivity as measured by a Cochet Bonnet esthesiometer (Luneau, Chartres, France). Individuals who had an active corneal infection, other ocular disease, or ocular surgery within the previous 3 months were excluded from this study. Pregnant or lactating women were also excluded. Twenty-eight patients (28 eyes) were recruited for this study. The eyes were classified into 3 grades according to the clinical features as follows. 2 Briefly, grade 1 was characterized by punctate keratopathy, superficial vascularization, and stromal scarring; grade 2, by an epithelial defect, surrounding rim of loose epithelium, and stromal edema; and grade 3, by a corneal ulcer and stromal melting. Umbilical Cord Serum Eyedrop Preparation and Its Application Umbilical cord blood was obtained from mothers with vaginal or cesarean section delivery after obtaining informed consent. Laboratory data for hepatitis B and C viruses and human immunodeficiency virus (HIV) were examined twice at 8 and 38 gestational weeks. The umbilical cord serum eyedrops were prepared using a previously reported method with modification. 21 Briefly, a volume of 200 to 250 l of the umbilical cord blood was collected from the umbilical vein after fetal delivery. Umbilical cord blood was clotted for 2 hours at room temperature. After centrifugation at 3000 g for 15 minutes, the serum was isolated carefully under sterile conditions in a laminar air flow hood. Then, it was diluted to a 20% concentration with balanced salt solution (BSS; Alcon, Fort Worth, TX). The diluted serum was aliquoted into sterile 5- l bottles with ultraviolet light protection. Patients were instructed to keep an opened bottle in a refrigerator at 4 C and to store unopened eyedrop preparation bottles in a freezer at 20 C. Each opened bottle was discontinued after 1 week of use. The duration of maximum storage was 3 months. Before starting treatment with umbilical cord serum, the patients were treated with preservative-free artificial tears (Tears Naturale Free, Alcon) for 2 weeks as a washout period. Umbilical cord serum eyedrops were applied 6 to 10 times a day as required in addition to the preservative-free artificial tears. Levofloxacin eyedrops (Cravit, Santen, Osaka, Japan) were instilled 2 times a day simultaneously with the serum at 5-minute intervals. After resolution of the corneal lesions, umbilical cord serum eyedrops were instilled continuously 3 to 4 times a day. Evaluation Ophthalmic examinations including best-corrected visual acuity (VA) measurement, a corneal sensitivity test, corneal fluorescein staining, and anterior segment photography were performed before and after treatment with umbilical cord serum eyedrops. A baseline study was conducted after the washout period. All examinations were performed by the same investigators. Corneal sensitivity was measured using a Cochet Bonnet esthesiometer. The tip of the fully extended nylon filament was applied perpendicular to the surface of the central cornea and advanced steadily. When the subject felt its presence or had a blink reflex, the length of the filament was recorded in millimeters. A measurement of 45 mm was considered low corneal sensitivity. Corneal vital staining was performed using a 2 l volume of 1% fluorescein dye. Corneal photography was done after the administration of fluorescein dye at a magnification of 10 using a slit-lamp microscope attached to a camera (F-801s, Nikon, Miyagi, Japan). After measuring the longest linear diameter and longest vertical diameter of the epithelial defect area, the area of the equivalent rectangle was calculated by multiplying the 2 measured dimensions. 22 Measurement of Neurotrophic Factors in Umbilical Cord Serum, Peripheral Blood Serum, and Tears The concentrations of substance P, IGF-1, and NGF in umbilical cord serum from other pregnant donors (n 10) and those in peripheral blood serum and tears from healthy volunteers (n 10) were measured. Basal tear samples were collected atraumatically from the inferior tear meniscus of both eyes using glass capillary tubes or micropipettes. Care was taken to avoid touching the corneal and conjunctival surfaces. Tear samples were diluted in phosphate-buffered saline. A tear volume of 50 l was used to measure substance P, IGF-1, and NGF. Serum and tear samples were placed in microtubes and stored at 70 C until further examination. Substance P, IGF-1, and NGF concentrations were measured using a commercial enzyme-linked immunosorbent assay kit (Quantikine, R&D Systems, Minneapolis, MN) according to the manufacturer s instructions as described elsewhere

3 Yoon et al Umbilical Cord Serum Eyedrops Figure 1. Slit-lamp photographs in patients with neurotrophic keratitis: grade 2 neutrophic keratitis before (A) and after (B) treatment with umbilical cord serum eyedrops and grade 3 neutrophic keratitis before (C) and after (D) treatment with umbilical cord serum eyedrops. The corneal epithelial defect healed, with a significant decrease in corneal haze. Statistical Analysis The Wilcoxon signed rank test was used to determine the significance of VA and corneal sensitivity changes after umbilical cord serum therapy. The Mann Whitney U test was used to compare substance P, IGF-1, and NGF concentrations between the study groups. P 0.05 was considered statistically significant. Results The characteristics of patients with neurotrophic keratitis, treated with umbilical cord serum eyedrops, are presented in Table 1 (available at Among 28 patients (28 eyes) with neurotrophic keratitis, 19 were male and 9 were female. Mean age was years (range, 18 81). Causes of neurotrophic keratitis were herpetic infection in 8 patients (5 herpes zoster ophthalmicus and 3 herpes simplex keratitis), diabetes in 7, stroke in 5, trigeminal neuralgia in 3, leprosy in 2, a postneurosurgical procedure in 1, postirradiation in 1, and postkeratoplasty in 1. According to the stage, 2 eyes (7.1%) had stage 1 disease, 22 eyes (78.6%) had stage 2, and 4 (14.3%) had stage 3. The mean duration of neurotrophic keratitis before treatment was months (range, 2 6), and mean epithelial defect area was mm 2 (range, ). The results after treatment with umbilical cord serum eyedrops in neurotrophic keratitis patients are shown in Table 2 (available at The mean follow-up duration after treatment with umbilical cord serum was months (range, 2 18). The epithelial defect healed completely in all eyes (100.0%), and mean healing time was weeks (range, 2 20) (Fig 1). The epithelial defect healed within 2 weeks in 8 eyes (28.6%), between 2 and 4 weeks in 14 eyes (50.0%), and after 4 weeks in 6 eyes (21.4%). Of 2 eyes (7.1%) that healed after 8 weeks of treatment, one had grade 3 disease secondary to herpetic infection, and the other had grade 2 disease as a result of penetrating keratoplasty. There were no recurrences during the follow-up period. The mean pretreatment VA (logarithm of the minimum angle of resolution) was , and mean posttreatment VA was (P 0.01). Visual acuity improved by more than 1 line in 20 eyes (71.4%) and more than 2 lines in 17 eyes (60.7%). Visual acuity did not change in 7 eyes (25.0%) and was aggravated in 1 eye (3.6%). The mean pretreatment corneal sensitivity was mm, and mean posttreatment corneal sensitivity was mm (P 0.01). Thirteen eyes (46.4%) had improvement in corneal sensitivity, and 15 eyes (53.6%) did not have any improvement in corneal sensitivity. The corneal sensitivity was improved in 3 of 3 patients (100%) with herpes simplex keratitis, 5 of 7 patients (71.4%) with diabetes, 2 of 3 patients (66.7%) with trigeminal neuralgia, 2 of 5 patients (40.0%) with stroke, and 1 of 1639

4 Ophthalmology Volume 114, Number 9, September 2007 Figure 2. Concentration of substance P in umbilical cord serum, normal peripheral blood serum, and tears. Figure 4. Concentration of nerve growth factor (NGF) in umbilical cord serum, normal peripheral blood serum, and tears. 5 patients (20.0%) with herpes zoster infection. Patients who had leprosy and who underwent a neurosurgical procedure, irradiation, or keratoplasty did not show improvement in corneal sensitivity. No significant complications associated with the use of umbilical cord serum were observed. Mean concentrations of substance P in umbilical cord serum, peripheral blood serum, and tears were pg/ml, pg/ml, and pg/ml, respectively (Fig 2). Mean concentrations of IGF-1 in umbilical cord serum, peripheral blood serum, and tears were ng/ml, ng/ ml, and ng/ml, respectively (Fig 3). Mean concentrations of NGF in umbilical cord serum, peripheral blood serum, and tears were pg/ml, pg/ml, and Figure 3. Concentration of insulinlike growth factor 1 (IGF-1) in umbilical cord serum, normal peripheral blood serum, and tears. pg/ml, respectively (Fig 4). Substance P, IGF-1, and NGF levels of umbilical cord serum were significantly higher than those of tears. Umbilical cord serum contained higher NGF and lower IGF-1 levels than peripheral blood serum. Discussion Corneal sensory nerves play a significant role in maintaining the anatomic integrity and function of the corneal epithelium. Impairment of corneal sensation causes disruption and exfoliation of the corneal epithelium. 2 Such trophic disturbances and depletion of neurotrophic substances such as substance P and acetylcholine are important risk factors for neurotrophic keratitis. 3 The application of neuropeptides and growth factors such as substance P, IGF-1, and NGF to promote epithelial wound healing can be helpful in the treatment of neurotrophic keratitis. Substance P and IGF-1 synergistically promote corneal epithelial migration in a dose-dependent fashion in vitro and corneal wound closure in vivo, although neither substance P nor IGF-1 alone stimulates epithelial migration. 26,27 The combination of substance P and IGF-1 stimulates the expression of integrin 5 and 1, which are essential for the attachment of epithelial cells to the extracellular matrix proteins. Combined treatment with substance P and IGF-1 has been shown to improve corneal epithelial barrier function and stimulated corneal epithelial wound healing in an animal model of neurotrophic keratitis. 28,29 According to case reports, treatment with combined substance P and IGF-1 eyedrops has produced a complete recovery in patients with neurotrophic keratitis. 7,8 Nerve growth factor is a well-characterized neurotrophin that is essential for the survival and growth of sensory and sympathetic neurons, induces neurite sprouting by neural cells, and restores the function of injured neurons. 30 Specific 1640

5 Yoon et al Umbilical Cord Serum Eyedrops NGF receptors have been identified on both normal and abnormal cornea and conjunctiva, and NGF has been shown to promote corneal epithelial healing. 31,32 In clinical studies, topical treatment with murine NGF eyedrops improved corneal sensitivity and visual function and restored corneal integrity in moderate and severe neurotrophic keratitis However, ocular side effects such as conjunctival hyperemia, photophobia, and ocular pain were observed during the early period of NGF treatment. 10,11 Autologous serum contains many growth factors and tear components, which are EGF, vitamin A, TGF-, acidic and basic fibroblast growth factors, platelet-derived growth factor, hepatocyte growth factor, fibronectin, and serum antiproteases such as 2 macroglobulin, and can facilitate the proliferation, migration, and differentiation of the ocular surface epithelium. Autologous serum also harbors neurotrophic substances such as substance P, IGF-1, and NGF and can be useful for the restoration of ocular surface integrity in patients with neurotrophic keratitis. Poon et al 13 used autologous serum eyedrops in 4 patients with neurotrophic keratitis and achieved a successful result in 2. Young et al 15 reported that a corneal epithelial defect healed within 1 month in 2 of 3 patients treated with autologous serum eyedrops. Matsumoto et al 14 also used autologous serum in 11 patients with neurotrophic keratitis and reported that the epithelial disorders healed completely in all eyes within an average of 17.1 days after treatment. Both VA and corneal sensitivity improved significantly after treatment. Umbilical cord serum also contains essential tear component, growth factors, and neurotrophic factors and can be used in many ocular surface diseases, including dry eye syndrome, persistent epithelial defect, and neurotrophic keratitis. We previously reported that umbilical cord serum contains EGF, vitamin A, and TGF-. 21 We found that EGF and TGF- concentrations were much higher in umbilical cord serum than in peripheral blood serum and tears. Although vitamin A concentration in umbilical cord serum was lower than that in peripheral blood serum, it was much higher than in tears. In the present study, we analyzed and compared the concentrations of substance P, IGF-1, and NGF in umbilical cord serum with those in normal peripheral blood serum and tears. Substance P, IGF-1, and NGF concentrations in umbilical cord serum were much higher than those in tears. Umbilical cord serum contained higher NGF and lower IGF-1 levels compared with levels in peripheral blood serum. Vajpayee et al 33 used umbilical cord serum eyedrops in 8 patients with neurotrophic keratitis and reported that complete epithelialization was achieved in 5 cases. They found that umbilical cord serum led to faster healing of persistent epithelial defects refractory to medical treatment compared with autologous serum. In a previous preliminary study in 8 patients with neurotrophic keratitis, we reported that the epithelial defect healed within 2 weeks in 62.5% and between 2 and 4 weeks in 25.0% and did not heal within 4 weeks in 12.5% after umbilical cord serum therapy. 34 In the present study, we demonstrated that 20% of umbilical cord serum eyedrops appeared to be effective for the treatment of neurotrophic keratitis that was unresponsive to conventional treatment. The epithelial defect healed completely in all eyes, with a mean healing time of weeks. The epithelial defect healed within 2 weeks in 28.6% and within 4 weeks in 78.6%. Visual acuity and corneal sensitivity were significantly improved after treatment. Corneal sensitivity was improved in all herpes simplex keratitis patients, 71.4% of diabetes patients, 66.7% of patients with trigeminal neuralgia, 40.0% of stroke patients, and 20.0% of herpes zoster patients. Patients who had leprosy and who underwent a neurosurgical procedure, irradation, or keratoplasty did not show improvement in corneal sensitivity. The corneal epithelial disease resolved even in patients who had very low corneal sensitivity after treatment. This finding may be due to the continuous supply of neurotrophic and growth factors with the umbilical cord serum eyedrops. No recurrences were detected during the mean follow-up duration of 7.9 months, and no significant complications associated with the use of umbilical cord serum were observed. From patients and physicians perspectives, umbilical cord serum therapy has several advantages compared with autologous serum therapy. 21,34 Because a large amount of serum can be obtained from the umbilical vein at one time, many patients can benefit from this sampling without waiting for additional preparations. In addition, umbilical cord serum therapy is also feasible in patients with a poor general condition or blood dyscrasia. However, umbilical cord serum therapy may also have several disadvantages. Despite 2 laboratory examinations in pregnant donors, the possibility of transmission of bloodborne infectious disease or bloodborne diseases that at present cannot be detected cannot be absolutely ruled out. Additional HIV testing, with a shortened window, with the p24 antigen detection method as well as routine laboratory examination for the virus should be performed on all pregnant donors. 35,36 Additional possible problems include freezer storage, risk of allergies, and the potential risk of bacterial contamination. As the preparation of umbilical cord serum eyedrops is time consuming and labor intensive, the costs should be considered. Though our study was done in Korea, complex political, legal, and regulatory issues also should be considered in advance of the serum use in other countries. During preparation of the serum, one should keep in mind that clotting time, centrifugation, and diluents have an effect on the composition and epitheliotrophic effects of serum. A long clotting time, more than 2 hours; extensive and high-speed centrifugation; and dilution with BSS can improve the ability of serum eyedrops to support proliferation, migration, and differentiation of the ocular surface epithelium. 37,38 The main limitations of our study include lack of controls or masking and a sample size not large enough to detect infrequent but potentially severe unanticipated adverse events. Further studies need a larger sample size and a more vigorous study design to compare umbilical cord serum and autologous serum eyedrops for safety and efficacy in the treatment of neurotrophic keratitis. In conclusion, umbilical cord serum contains many neurotrophic factors such as substance P, IGF-1, and NGF, and umbilical cord serum eyedrops appear to be effective for the treatment of neurotrophic keratitis. 1641

6 Ophthalmology Volume 114, Number 9, September 2007 References 1. Heigle TJ, Pflugfelder SC. Aqueous tear production in patients with neurotrophic keratitis. Cornea 1996;15: Bonini S, Rama P, Olzi D, Lambiase A. Neurotrophic keratitis. Eye 2003;17: Lambiase A, Rama P, Aloe L, Bonini S. Management of neurotrophic keratopathy. Curr Opin Ophthalmol 1999;10: Lugo M, Arentsen JJ. Treatment of neurotrophic ulcers with conjuntival flaps. Am J Ophthalmol 1987;103: Khokhar S, Natung T, Sony P, et al. Amniotic membrane transplantation in refractory neurotrophic corneal ulcers: a randomized, controlled clinical trial. Cornea 2005;24: Chen HJ, Pires RT, Tseng SC. Amniotic membrane transplantation for severe neurotrophic corneal ulcers. Br J Ophthalmol 2000;84: Brown SM, Lamberts DW, Reid TW, et al. Neurotrophic and anhidrotic keratopathy treated with substance P and insulinlike growth factor 1 [letter]. Arch Ophthalmol 1997;115: Chikama T, Fukuda K, Morishige N, Nishida T. Treatment of neurotrophic keratopathy with substance-p-derived peptide (FGLM) and insulin-like growth factor I [letter]. Lancet 1998; 351: Tan MH, Bryars J, Moore J. Use of nerve growth factor to treat congenital neurotrophic corneal ulceration. Cornea 2006; 25: Lambiase A, Rama P, Bonini S, et al. Topical treatment with nerve growth factor for corneal neurotrophic ulcers. N Engl J Med 1998;338: Bonini S, Lambiase A, Rama P, et al. Topical treatment with nerve growth factor for neurotrophic keratitis. Ophthalmology 2000;107: , discussion Brown SM, Khanani AM, Stetson CL, Reid TW. Vesicular eruption in a child with trigeminal nerve palsy during topical therapy with substance P and insulinlike growth factor I for neurotrophic keratitis. Arch Ophthalmol 2004;122: Poon AC, Geerling G, Dart JK, et al. Autologous serum eyedrops for dry eyes and epithelial defects: clinical and in vitro toxicity studies. Br J Ophthalmol 2001;85: Matsumoto Y, Dogru M, Goto E, et al. Autologous serum application in the treatment of neurotrophic keratopathy. Ophthalmology 2004;111: Young AL, Cheng AC, Ng HK, et al. The use of autologous serum tears in persistent epithelial defect. Eye 2004;18: Tsubota K, Goto E, Shimmura S, Shimazaki J. Treatment of persistent epithelial defect by autologous serum application. Ophthalmology 1999;106: Tsubota K, Goto E, Fujita H, et al. Treatment of dry eye by autologous serum application in Sjogren s syndrome. Br J Ophthalmol 1999;83: Kojima T, Ishida R, Dogru M, et al. The effect of autologous serum eyedrops in the treatment of severe dry eye disease: a prospective randomized case-control study. Am J Ophthalmol 2005;139: Goto E, Shimmura S, Shimazaki J, Tsubota K. Treatment of superior limbic keratoconjunctivitis by application of autologous serum. Cornea 2001;20: del Castillo JM, de la Casa JM, Sardina RC, et al. Treatment of recurrent corneal erosions using autologous serum. Cornea 2002;21: Yoon KC, Im SK, Park YG, et al. Application of umbilical cord serum eyedrops for the treatment of dry eye syndrome. Cornea 2006;25: Mukerji N, Vajpayee RB, Sharma N. Technique of area measurement of epithelial defects. Cornea 2003;22: Varnell RJ, Freeman JY, Maitchouk D, et al. Detection of substance P in human tears by laser desorption mass spectrometry and immunoassay. Curr Eye Res 1997;16: Singer CF, Mogg M, Koestler W, et al. Insulin-like growth factor (IGF)-I and IGF-II serum concentrations in patients with benign and malignant breast lesions: free IGF-II is correlated with breast cancer size. Clin Cancer Res 2004;10: Lee HK, Lee KS, Kim HC, et al. Nerve growth factor concentration and implications in photorefractive keratectomy vs laser in situ keratomileusis. Am J Ophthalmol 2005;139: Nishida T, Nakamura M, Ofuji K, et al. Synergistic effects of substance P with insulin-like growth factor-1 on epithelial migration of the cornea. J Cell Physiol 1996;169: Nakamura M, Ofuji K, Chikama T, Nishida T. 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7 Patient No. Table 1. Characteristics of Patients with Neurotrophic Keratitis Who Were Treated with Umbilical Cord Serum Eyedrops Age (yrs) Gender Cause Yoon et al Umbilical Cord Serum Eyedrops Grade of Neurotrophic Keratopathy Duration (mos) Defect Area (mm 2 ) Pretreatment Visual Acuity Pretreatment Corneal Sensitivity Test (mm) 1 35 M Diabetes 1 3 NA F Stroke 1 3 NA M Herpes zoster HM M Herpes simplex F Herpes simplex M Herpes zoster HM M Herpes simplex F Herpes zoster F Herpes zoster M Diabetes M Diabetes M Diabetes HM M Diabetes CF, 30 cm M Diabetes F Diabetes HM F Stroke F Stroke M Stroke M Stroke M Keratoplasty HM M Neurosurgery M Trigeminal neuralgia M Leprosy F Leprosy M Herpes zoster HM M Postirradiation HM M Trigeminal neuralgia F Trigeminal neuralgia CF counting fingers; F female; HM hand movements; M male; NA not applicable e1

8 Table 2. Treatment Results in Patients with Neurotrophic Keratitis Who Were Treated with Umbilical Cord Serum Eyedrops Patient No. Follow-up Duration (mos) Posttreatment Visual Acuity Posttreatment Corneal Sensitivity Test (mm) Epithelial Healing Time (wks) HM HM CF, 50 cm HM CF, 20 cm NLP CF, 20 cm Ophthalmology Volume 114, Number 9, September 2007 CF counting fingers; HM hand movements; NLP no light perception e2