November 2012
Volume 53, Issue 12
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Cornea  |   November 2012
Short-Term Effects of Extremely Low Frequency Pulsed Electromagnetic Field on Corneas with Alkaline Burns in Rabbits
Author Affiliations & Notes
  • Mozhgan Rezaei Kanavi
    From the Ophthalmic Research Center, Shahid Beheshti University of Medical Sciences, and Central Eye Bank of Iran, Tehran, Iran; the
  • Farzin Sahebjam
    From the Ophthalmic Research Center, Shahid Beheshti University of Medical Sciences, and Central Eye Bank of Iran, Tehran, Iran; the
  • Faraj Tabeie
    Department of Nuclear Medicine and Medical Physics, Shahid Beheshti University of Medical Sciences, Tehran, Iran; and the
  • Paniz Davari
    From the Ophthalmic Research Center, Shahid Beheshti University of Medical Sciences, and Central Eye Bank of Iran, Tehran, Iran; the
  • Aminpasha Samadian
    From the Ophthalmic Research Center, Shahid Beheshti University of Medical Sciences, and Central Eye Bank of Iran, Tehran, Iran; the
  • Mehdi Yaseri
    Department of Epidemiology and Biostatistics, Tehran University of Medical Sciences, Tehran, Iran.
  • Corresponding author: Mozhgan Rezaei Kanavi, Ophthalmic Research Center, Shahid Beheshti University of Medical Sciences, No 23, Paydarfar-9th Boostan Street, Pasdaran Avenue, Tehran 1666663111, Iran; mrezaie47@yahoo.com
Investigative Ophthalmology & Visual Science November 2012, Vol.53, 7881-7888. doi:10.1167/iovs.12-10248
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      Mozhgan Rezaei Kanavi, Farzin Sahebjam, Faraj Tabeie, Paniz Davari, Aminpasha Samadian, Mehdi Yaseri; Short-Term Effects of Extremely Low Frequency Pulsed Electromagnetic Field on Corneas with Alkaline Burns in Rabbits. Invest. Ophthalmol. Vis. Sci. 2012;53(12):7881-7888. doi: 10.1167/iovs.12-10248.

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Abstract

Purpose.: To investigate the short-term effects of extremely low frequency pulsed electromagnetic fields (ELF-PEMF) on the healing of alkaline-burned corneas in rabbits.

Methods.: Fifty-six alkaline-burned corneas from 56 rabbits were categorized into four groups: ELF-PEMF therapy with 2 mTesla (mT) intensity (ELF 2) for 30 minutes twice daily, ELF-PEMF therapy with 5 mT intensity (ELF 5) for 30 minutes twice daily, medical therapy (MT), and controls. Clinical examination together with digital photography of the corneas was performed on days 0, 2, 7, and 14. After euthanizing the rabbits, affected eyes were evaluated by way of histopathology. Finally the clinical and the histopathologic results of the four groups were compared.

Results.: None of the cases developed limbal ischemia, symblepharon formation, Descemetocele, or corneal perforation. Although the area of corneal defect in the ELF groups on day 2 was significantly less than the defects in MT, it was not notably different from those on days 7 and 14. Rate of significant corneal neovascularization on days 7 and 14 was not statistically different between the groups. The keratocyte loss in MT was significantly higher than in the ELF groups. Mild stromal scar formation was observed more frequently in ELF-PEMF groups than the control.

Conclusions.: Short-term ELF-PEMF therapy is a safe, noninvasive, and markedly effective method in healing alkaline-burned corneas, and its therapeutic results are comparable with those of MT.

Introduction
Alkaline burns of the cornea induce persistent and long standing corneal ulcers that are difficult to resolve and result in decreased corneal clarity. 1 Treatment modalities in such cases have variable results; they include local or systemic medical therapies and surgical techniques such as amniotic membrane transplantation and corneal lamellar graft. 13 However, ocular surface failure may occur despite medical and surgical treatments, 4 which necessitates seeking new strategies with regard to the ideal treatment of corneal chemical burn. 
Pulsed electromagnetic fields exist whenever electricity flows and are safe on short term exposure. 5 They are currently used for the treatment of chronic wounds 6 and have been reported as a successful treatment for corneal wound healing. 710 In normal corneas, there is an endogenous electric field that is significantly increased after corneal wounding. 9 Enhancement of the endogenous electric field by an external electric field may induce cellular galvanotropism in form of cellular polarity, directional migration, and outgrowth. 8 Through interacting with cell membranes and modulating the rate of ion binding and/or transport, treatment with pulsed electromagnetic field may alter the biophysiological cascade of cellular response to injuries such as decrease in tissue edema, debridement of necrotic tissue, stimulation of receptor sites for growth factors, stimulation of the growth of fibroblasts and granulation tissue, induction of epithelial cell migration, stimulation of neurite growth, prevention of free-radical-mediated damage, and inhibition of bacteria. 1114  
To the best of our knowledge, there is no study concerning the effects of magnetic therapy on corneas with alkaline burns. Therefore, this study was conducted to specify the short-term effects of extremely low frequency pulsed electromagnetic fields (ELF-PEMF) on the healing of corneas with alkaline burns in rabbits. Given the effectiveness of the PEMF with field intensity of 10 mTesla (mT) on the reduction of the rate of complications in a rabbit hyphema model 15 and the effectiveness of the ELF-PEMF of 2 mT intensity on increased collagen synthesis in rat skin, 16 the field intensities of 2 and 5 mT were used in this experiment because of the superficial location of the cornea compared to the anterior chamber and the histologic similarities between the cornea and the skin tissue. 
Methods
Animals and Experimental Procedures
In this experimental study, 56 New Zealand albino female rabbits weighing approximately 2 kg were treated under the Guidelines for Animal Research at the Ophthalmic Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran, and the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. All the rabbits were initially examined for the presence of any ocular or systemic disease. On day 0 general anesthesia was achieved with intramuscular 10% ketamine HCl (Alfamine; Alfasan, Woerden, Holland), 30 mg/kg, and xylazine (Rompun; Bayer, Leverkusen, Germany), 3 mg/kg. 17 The right eye of each animal received a topical 0.5% tetracaine eye drops (trade name; Sina Darou Laboratories, Tehran, Iran), and the sharply demarcated central corneal burn was produced by the adherence for 30 seconds of a 6-mm-diameter round filter paper that had been immersed in 2.5 N sodium hydroxide (NaOH). The surface of the eye was then thoroughly irrigated with isotonic saline solution (0.9% NaCl) for 3 minutes to remove the residual alkaline substance and any coagulated debris material. 
The rabbits were randomly categorized into one of the four groups: (1) ELF 2 group consisted of 14 eyes from 14 rabbits treated with ELF-PEMF of 2 mT field intensity and frequency of 25 Hz for 30 minutes twice daily during 14 days, (2) ELF 5 group consisted of 14 eyes from 14 rabbits treated with ELF-PEMF of 5 mT intensity and frequency of 25 Hz for 30 minutes twice daily in 14 days, (3) medical therapy (MT) group 18,19 composed of 14 eyes from 14 rabbits treated with sodium citrate 10% eye drops (Aurocitrate; Aurolab, Madurai, India) every 6 hours, ascorbic acid 10% solution (prepared from ascorbic acid ampule 500 mg in 5 mL, [trade name; Aboorihan Company, city, Iran) four times a day, chloramphenicol eye drops 0.5% (trade name; Sina Darou Laboratories) four times daily, atropine 1% eye drops (trade name; Sina Darou Laboratories) once a day and betamethasone 0.1% eye drops (trade name; Sina Darou Laboratories) four times a day for 14 days, and (4) control group composed of 14 eyes from 14 rabbits did not receive any treatment. 
ELF-PEMF Treatment
A ring-shaped solenoid electromagnet (Helmholtz coil) of 5-cm-diameter, composed of 250 wire loops attached to a plastic ocular shield, was used to generate ELF-PEMF. There was a 2-cm circular opening in the center of the shield where the rabbit's eye could be seen throughout the magnetic therapy. After restraining the head of the animal, the shield including the coil was placed on the right eye, and an elastic-cotton strip was fixed behind the head to stabilize the shield (Fig. 1). The whole procedure was performed without any anesthesia. The field intensities of 2 mT and 5 mT were achieved by altering the pulse amplitude of the direct electrical generator unit. 
Figure 1. 
 
The upper image illustrates the restrained rabbit's head with the shield, including the coil, on the right eye. Rabbit's eye is visible through the 2-cm circular central opening in the shield. The lower image shows a wire connecting the coil to a direct electrical generator unit.
Figure 1. 
 
The upper image illustrates the restrained rabbit's head with the shield, including the coil, on the right eye. Rabbit's eye is visible through the 2-cm circular central opening in the shield. The lower image shows a wire connecting the coil to a direct electrical generator unit.
Clinical Observations and Tissue Analysis
On days 0, 2, 7, and 14, the eyes of each group were examined using an ophthalmic operating microscope (OMS-300; Topcon, Tokyo, Japan) for the presence of corneal perforation, limbal ischemia, symblepharon formation, and apparent corneal vascularization. To measure the corneal defect, following topical anesthesia with tetracaine 0.5% eye drops (Sina Darou Laboratories), a fluorescein dye strip (Negah Fluorescein Paper; Toosnegah Co., Mashhad, Iran) was placed in the inferior fornix. The measurement of the corneal epithelial defect was performed using methods described previously. 20 A photograph was taken using a 12.1 megapixel high resolution digital camera (IXUS 100 IS; Canon, Tokyo, Japan) at 10× optical zoom of the corneal epithelial defect with the adjacent metric ruler. The image was imported to image editing software (Adobe Photoshop CS5 12.0 for Windows; Adobe, San Jose, CA) where the image of the ruler was copied and rotated 90° perpendicular to the image of the original ruler. Lines were drawn from the hash marks of both rulers across the image, creating 1-mm-squared grids. The surface area of the fluorescein-stained epithelial defect was measured by summating the number of grids covered. In areas where the defect did not encompass a complete grid, particularly at the margin of the defect, the percentage of grid covered was determined and included in the final tally. Corneal neovascularization was considered significant if it was observed at the central and/or the para-central region. 
On day 14, after clinical examinations, the rabbits were euthanized with an overdose of intravenous sodium phenobarbital, and the involved eyes were enucleated, fixed in 10% formalin, and sent for histopathology. The corneo-scleral disc was excised and bisected through the affected zone. After tissue processing and embedding into paraffin blocks, 5-μm tissue sections were prepared and stained with hematoxylin and eosin, periodic acid-Schiff (PAS), and Gram-Twort staining methods. Histologically, the keratocytes and endothelial cells in the central and two paracentral regions of the involved cornea were manually counted on three consecutive tissue levels (150 μm apart). Counting of the keratocytes in the anterior, middle, and posterior stroma was performed under 100 microscope power-fields, and the calculated mean value was considered for final scoring. The endothelial cells were counted under 40 microscope power-fields, and the average was calculated. The degree of keratocyte loss was defined as severe (0–2 keratocytes per 100 power-field), moderate (3–6 keratocytes per 100 power-field), and mild (7–10 keratocytes per 100 power-field). A keratocyte density of more than 11 keratocytes per 100 power-field was considered within normal limits. The stromal inflammatory cell infiltration was regarded mild when only the anterior stroma was involved. In cases where inflammation extended to the mid- or posterior stromal layers, the inflammation was considered moderate or severe, respectively. The degree of endothelial cell loss was defined as severe (0–2 endothelial cells per 40 power-field), moderate (3–8 endothelial cells per 40 power-field), and mild (9–12 endothelial cells per 40 power-field). A total of 13 to 15 endothelial cells per 40 power-field was considered to be normal. Histopathologically, central and/or para-central corneal neovascularization was considered significant. The PAS and Gram-stained histologic sections were also examined for any possible bacterial or fungal infection. 
Statistical Methods
In reviewing the data, we used the frequency, percentage, mean ± SD, and median. Percentage of change from the baseline was calculated as [(value at each time − Baseline)/Baseline] × 100. Since the Kolmogorov-Smirnov test shows deviation from the normal, we evaluated the differences between the groups by Mann-Whitney, Kruskall-Wallis, χ2, and Fisher exact tests. If the difference between the groups was significant (P value < 0.05), we used the Tukey test as a multiple comparison correction method. (The Tukey test was downloaded from: http://gjyp.nl/marta/). This test evaluates the differences between two groups at a time. The Wilcoxon signed rank test was used to specify the changes in the defective area of each group during the 14-day period. Differences in corneal neovascularization between the two groups were evaluated by means of the Bonferroni method. All the statistical analyses were performed using analytics software (SPSS Version 17.0; SPSS, Inc., Chicago, IL), and a P value of less than 0.05 was considered statistically significant. 
Results
Clinical Examinations
General Ocular Examinations.
Fifty-six adult female rabbits, each weighing approximately 2 kg, were enrolled in the study. None of the eyes with alkaline burns revealed any limbal ischemia, symblepharon formation, Descemetocele, or corneal perforation. 
Corneal Defect Area.
For each group, the areas of the corneal defects on days 0, 2, 7, and 14 and their changes over the 14-day period are illustrated in Table 1 and Figure 2. Since the percentages of the changes were usually highly skewed, we used median and range to describe them. There was a significant difference in the area of the corneal defect between all groups (Fig. 3) on days 2, 7, and 14 (P = 0.003, P = 0.001, and P = 0.008, respectively). By using the statistical Tukey test as a multiple comparison correction method (Table 1), the defective area of the ELF 2 and ELF 5 groups on day 2 compared to day 0 was considerably less than that of MT (P < 0.01 and P < 0.05, correspondingly). The defective area on days 7 and 14 and the changes compared to day 0 (Table 1) in the ELF 2 and the ELF 5 groups were not significantly different from those of MT. Decrease in the defective area (Table 1) in the ELF 2 and ELF 5 groups on day 7 (P < 0.01 and P < 0.05, respectively) and in the ELF 5 on day 14 (P < 0.01) was notably more than those of the controls. Despite a marked decrease in the defective area of the study groups on days 2 and 7 compared to day 0, the area of corneal defect on day 14, when compared to day 7 in the ELF 2, ELF 5, and the MT groups (Table 1), showed considerable enlargement (P = 0.04, P = 0.02, and P = 0.02, respectively), and this was more conspicuous in the MT but was not statistically significant in the control group (P = 0.55). 
Figure 2. 
 
Graphs (A) and (B), respectively, illustrate mean areas of corneal defect and changes of the defective areas in the control, MT, ELF 2, and ELF 5 groups on days 0, 2, 7, and 14 and show the greater rate of improvement in the corneal defect and smaller size of the defective areas in the ELF and MT groups compared to the controls by time.
Figure 2. 
 
Graphs (A) and (B), respectively, illustrate mean areas of corneal defect and changes of the defective areas in the control, MT, ELF 2, and ELF 5 groups on days 0, 2, 7, and 14 and show the greater rate of improvement in the corneal defect and smaller size of the defective areas in the ELF and MT groups compared to the controls by time.
Figure 3. 
 
Corneal defect photographs of rabbit corneas with alkaline burns on days 0, 2, 7, and 14 in the control (A), MT (B), 2ELF 2 (C), and ELF 5 (D) groups.
Figure 3. 
 
Corneal defect photographs of rabbit corneas with alkaline burns on days 0, 2, 7, and 14 in the control (A), MT (B), 2ELF 2 (C), and ELF 5 (D) groups.
Table 1. 
 
Comparing the Mean Areas of the Corneal Defects on Days 0, 2, 7, and 14 and Their Changes over the 14-Day Period between the Control, MT, ELF 2, and ELF 5 Groups
Table 1. 
 
Comparing the Mean Areas of the Corneal Defects on Days 0, 2, 7, and 14 and Their Changes over the 14-Day Period between the Control, MT, ELF 2, and ELF 5 Groups
Time Control MT ELF P§ Multiple Comparison‖
ELF 2 ELF 5
Day 0* 39.57 ± 7.2 38.19 ± 10.65 37.53 ± 5.69 42.37 ± 12.64 0.83 -
Day 2* 13.61 ± 12.08 18.19 ± 13.82 4.65 ± 4.08 7.9 ± 9.68 0.003 (ELF 2 vs. MT, P < 0.01)
(ELF 5 vs. MT, P < 0.05)
Change 2–0* −25.95 ± 12.57 −20 ± 10.42 −32.87 ± 7.34 −34.46 ± 9.26 0.001 (ELF 2 vs. MT, P < 0.05)
(ELF 5 vs. MT, P < 0.01)
% of change† −73 (−100 to 16.05) −62 (−91 to −2) −90 (−100 to −60) −85 (−100 to −42)
Within P 0.001 0.001 0.001 0.001
Day 7* 15.24 ± 13.47 7.2 ± 6.37 4.54 ± 7.02 2.36 ± 3.64 0.001 (ELF 2 vs. Control, P < 0.01)
(ELF 5 vs. Control, P < 0.05)
Change 7–0* −24.33 ± 15.16 −30.99 ± 10.69 −32.99 ± 8.22 −40.01 ± 11.38 0.01 ELF 5 vs. MT, P < 0.01
% of change† −76 (−100 to 23) −86 (−100 to −44) −94 (−100 to −37) −99 (−100 to −70)
Within P 0.002 0.001 0.001 0.001
Day 14* 17.55 ± 12 12.73 ± 7.49 9.68 ± 8.06 6 ± 3.82 0.008 ELF 5 vs. Control, P < 0.01
Change 14–0* −21.19 ± 10.84 −27.22 ± 9.21 −27.85 ± 8.13 −36.37 ± 15.13 0.01 ELF 5 vs. Control, P < 0.01
% of change† 55 (−5 to 95) 66 (41 to 89) 83 (31 to 96) 88 (58 to 98)
Within P 0.002 0.001 0.001 0.001
Change 14–7* −1.72 ± 20.11 −5.51 ± 8.09 −5.14 ± 8.69 −3.64 ± 5.29 0.65 -
% of change† −14 (−275 to 92) −140 (−400 to 76) −20 (−1470 to 33) 1 (−1633 to 86)
Within P 0.55 0.02 0.03 0.02
After merging the results of the two ELF-PEMF groups into one (Table 1), the area of the corneal defect in the ELF group on day 2 compared to day 0 was notably less than that of MT (P < 0.01). Changes in the defective area (decrease) of the ELF group on days 7 and 14 were not significantly different from those of MT but were remarkable when compared to the controls (P < 0.01). 
Corneal Neovascularization.
The rate of significant corneal neovascularization on days 7 and 14 (Table 2) was not statistically different between the groups (P = 0.22 and P = 0.07, respectively). However, over the 2-week period, the progression rate of neovascularization in the control, ELF 2, and ELF 5 groups was statistically significant (P = 0.005, P = 0.049 and P = 0.049, respectively). 
Table 2. 
 
Comparing the Rate of Significant Corneal Neovascularization on Days 7 and 14 and Their Progression over the 2-Week Period between the Control, MT, ELF 2, and ELF 5 Groups
Table 2. 
 
Comparing the Rate of Significant Corneal Neovascularization on Days 7 and 14 and Their Progression over the 2-Week Period between the Control, MT, ELF 2, and ELF 5 Groups
Time Control MT ELF 2 ELF 5 P*
Day 0 0 (0.0%) 0 (0.0%) 0 (0.0%) 0 (0.0%) -
Day 2 0 (0.0%) 0 (0.0%) 0 (0.0%) 0 (0.0%) -
Day 7 3 (21.4%) 1 (7.1%) 0 (0.0%) 3 (21.4%) 0.22
Day 14 7 (50.0%) 1 (7.1%) 3 (21.4%) 3 (21.4%) 0.07
P for progression† 0.005 0.37 0.049 0.049
Histopathologic Examinations
On histopathologic examinations (Table 3, Fig. 4) there was no significant difference in the findings, such as loss of corneal epithelial integrity (P = 0.77), focal or severe endothelial cell loss together with retro-corneal membrane formation (P = 0.28), and significant intrastromal neovascularization (P = 0.16) between the study groups. The histopathologic results for corneal neovascularization supported the clinical findings. With regard to the variable degrees of intrastromal inflammatory cell infiltrate, although the difference between the groups was not statistically significant (P = 0.06), only one case in the MT group revealed a moderate to severe stromal inflammation. The loss of stromal keratocytes was significantly different between the study groups (P < 0.001), histologically it was notably more frequent in the MT than the ELF groups (P < 0.01). A significant difference was also observed between the groups in development of mild corneal stromal scarring (P = 0.03); a mild degree of the scarring was observed more frequently in the ELF groups than the control (P = 0.001 and P = 0.03, respectively), but it was not significantly different between the MT and the other study groups. None of the eyes in the current study developed any significant corneal thinning (P = 0.45), although a mild degree of corneal thinning was observed in two eyes: one in the ELF 2 and the other in the control group. None of the histologic specimens stained with either PAS or Gram-Twort methods disclosed any microorganisms. 
Figure 4. 
 
(A) Microphotograph of a rabbit's full thickness cornea with membrane formation (arrow) posterior to Descemet's membrane (DM), (B) foci of intrastromal neovascularization (arrows), (C) moderately intense intrastromal inflammatory cell infiltrate involving anterior and midstromal layers, and (D) severe keratocyte loss; only one keratocyte (arrow) is visible in the examined field. All microphotographs are stained with hematoxylin and eosin. Magnification: ×100 (A), ×400 (B), ×100 (C), and ×1000 (D).
Figure 4. 
 
(A) Microphotograph of a rabbit's full thickness cornea with membrane formation (arrow) posterior to Descemet's membrane (DM), (B) foci of intrastromal neovascularization (arrows), (C) moderately intense intrastromal inflammatory cell infiltrate involving anterior and midstromal layers, and (D) severe keratocyte loss; only one keratocyte (arrow) is visible in the examined field. All microphotographs are stained with hematoxylin and eosin. Magnification: ×100 (A), ×400 (B), ×100 (C), and ×1000 (D).
Table 3. 
 
Comparing the Histopathologic Results of the Corneal Tissues between the Control, MT, ELF 2, and ELF 5 Groups
Table 3. 
 
Comparing the Histopathologic Results of the Corneal Tissues between the Control, MT, ELF 2, and ELF 5 Groups
Histopathologic Features Control MT ELF 2 ELF 5 P Multiple Comparisons
Epithelial integrity
 No 10 (66.7%) 8 (57.1%) 7 (50.0%) 7 (50.0%) 0.77*
 Yes 5 (33.3%) 6 (42.9%) 7 (50.0%) 7 (50.0%)
Bowmans layer
 No 14 (93.3%) 14 (100.0%) 14 (100.0%) 14 (100.0%) >0.99†
 Yes 1 (6.7%) 0 (0.0%) 0 (0.0%) 0 (0.0%)
Mild scarring
 No 10 (66.7%) 6 (42.9%) 2 (14.3%) 4 (28.6%) 0.03* (C vs. ELF 2 P = 0.001), (C vs. ELF 5, P = 0.03)
 Yes 5 (33.3%) 8 (57.1%) 12 (85.7%) 10 (71.4%)
Neovascularization
 Nonsignificant 8 (57.1%) 13 (92.9%) 11 (78.6%) 9 (64.3%) 0.16†
 Significant 6 (42.9%) 1 (7.1%) 3 (21.4%) 5 (35.7%)
Thinning
 No 14 (93.3%) 14 (100.0%) 12 (85.7%) 14 (100.0%) 0.45†
 Yes 1 (6.7%) 0 (0.0%) 2 (14.3%) 0 (0.0%)
Keratocyte loss
 No 3 (21.4%) 1 (7.1%) 10 (71.4%) 9 (64.3%) 0.001‡ (MT vs. ELF 2, P = 0.01), (MT vs. ELF 5, P = 0.01)
 Mild 10 (71.4%) 4 (28.6%) 4 (28.6%) 4 (28.6%)
 Moderate 1 (7.1%) 8 (57.1%) 0 (0.0%) 0 (0.0%)
 Severe 0 (0.0%) 1 (7.1%) 0 (0.0%) 1 (7.1%)
Inflammation
 None to mild 8 (57.1%) 13 (92.9%) 7 (50%) 7 (50%) 0.06* -
 Moderate to severe 6 (42.9%) 1 (7.1%) 7 (50%) 7 (50%)
Endothelial cell loss
 No 2 (13.3%) 0 (0.0%) 0 (0.0%) 0 (0.0%) 0.28†
 Severe 13 (86.7%) 14 (100.0%) 14 (100.0%) 14 (100.0%)
Discussion
Magnetic therapy with an extremely low frequency pulsed electromagnetic field is a noninvasive, safe, and easy method for the treatment of a variety of diseases and for the healing of injuries from various etiologies. 6 It has been shown that the endogenous electric fields in a variety of human cells can be enhanced by exogenous electric fields, leading to an increased rate of wound healing through induction of cellular polarity and a cathodal-directed migration of the cells. 21 Increased expression of integrin β1 subunit by electric field stimulation may play a role in various signaling pathways such as cell adhesion, migration, differentiation, angiogenesis, and wound healing. 22,23 Our study showed that the magnetic treatment of corneal chemical burns significantly decreased the ulcerative area when compared to the controls, especially in the first week of therapy, and its effectiveness was comparable to that of medical therapy. The rate of improvement in the corneal defects of the ELF groups at the early stage of therapy (day 2) was more than the MT, although the defective area was not markedly different between the two groups by the end of the first week. However, the improvement of the corneal defects, whether with magnetic therapy or with medical treatment, was, to a certain degree, unstable with partial regression of the corneal defects noted in the second week of treatment. These changes in the area of corneal defect were the basis for monitoring the regression in this current study. This demonstrates that although treatment with the ELF-PEMF is rapidly and markedly effective in improving corneal defects induced by chemical burns, its effectiveness is, to some extent, inconsistent. This may be due to aggravation of the background of the chemical burn, incurring a dry eye due to a lack of enough moisture in the magnetic therapy media. Therefore, to prolong the therapeutic effect of ELF-PEMF, the addition of lubricants in such cases might be helpful; this will need further study. 
Pulsed electromagnetic fields were shown to induce vascular endothelial cell growth and angiogenesis through influencing the vascular endothelial growth factor (VEGF)-related pathways. 24,25 In another study, Yuan et al. 26 reported induced angiogenesis and the improvement of cardiac function in a surgically-induced infarcted myocardium, after implementing a pulsed magnetic field. In this current study, although in both clinical and histopathologic investigations, there was not a remarkable difference in the rate of stromal neovascularization between the groups; the progression rate of neovascularization over a 2-week period was statistically significant in the magnetic therapy groups and was comparable with the control; such a progression, however, was not observed in the MT group; this was probably due to use of corticosteroids. Although corneal neovascularization may be associated with inadvertent consequences, such as lipid keratopathy, it can also be considered as a part of the reparative process in a cornea with alkaline burns, and this may contribute to the accelerated rate of improvement in the corneal defect induced by an alkaline burn. The use of low dose of a topical corticosteroid or topical anti-VEGF compounds, such as Avastin or Fasudil, along with the magnetic therapy may be associated with a decreased rate of corneal neovascularization; this too will require further investigation. 
In the current study, the rate of mild stromal scarring in the MT group was not significantly different from the other groups; however, there was a higher rate of mild stromal scar formation in the magnetic therapy group compared to the control, which appears to be a part of the corneal repair process. This is probably due to the influence of the electromagnetic fields on keratocyte transformation into fibroblasts and collagen synthesis within the corneal stroma. A few studies have demonstrated the effects of electromagnetic fields on the differentiation of fibroblasts from normal or transformed skin and lung and their influence on the increase rate of collagen synthesis in rat skin. 16,27 Although even a mild degree of corneal scarring may influence vision, it may induce increased consistency of a chemically burned cornea and may play a role in protecting the cornea from further possible melting. Given the short term duration of our study, we could not demonstrate the occurrence of corneal melting in the study groups. 
Although there was not a statistically significant difference between the groups in terms of variable degrees of stromal inflammation, it seemed that the MT group was associated with a lower rate of moderate to severe inflammation. Use of corticosteroids in this group appears to be the key factor. There are a few reports on improvement of inflammatory edema by electromagnetic fields through increase in the tonicity of the vessels, 2831 but their effects on inflammatory cells were not clearly addressed. Given the results of our study, it does not appear that the treatment with ELF-PEMF has a marked influence in the reduction of inflammatory cell infiltrates. The use of a low dose of topical corticosteroids along with the magnetic therapy may be effective in reduction of corneal inflammation, which needs further investigation. 
Loss or fragmentation of stromal keratocytes is one of the primary changes in corneas with alkaline burns. 32,33 After a time, there is migration and repopulation of keratocytes from the intact and posterior parts of the stroma into the injured area. 34 The histopathologic results of our study indicated a lower rate of keratocyte loss in the ELF-PEMF groups compared to the MT; although when no treatment was applied, the loss of keratocytes was greater than in the MT group. This means that the electromagnetic fields may play an important role in the restoration of corneal integrity either through the inhibitory effects on keratocyte apoptosis or the stimulation of keratocyte repopulation at the site of corneal stromal injury. 
Severe and focal loss of the corneal endothelial cells and retro-corneal fibro-inflammatory membrane formation are the histopathologic features described in corneas with alkaline burns. 35 Such findings were observed in all the corneas of the current study; this demonstrated the ineffectiveness of magnetic and medical therapies on the occurrence of such events. 
None of our cases showed limbal ischemia, symblepharon formation, Descemetocele, or corneal perforation. Furthermore, on histopathologic examinations, there was no significant corneal thinning. The lack of such features being observed may be due to the short duration of our study, but by prolonging the length of the study, the chance of these complications occurring may increase. 
Our study demonstrated no superiority between ELF 2 and ELF 5 in terms of healing of corneal defects, induction of corneal neovascularization, corneal scarring, stromal inflammation, loss of keratocytes, and endothelial cell loss. However, it seems logical that ELF 2 may be a better option for therapeutic purposes because lower intensities of the electromagnetic fields are a safer option. Given the favorable effects of magnetic therapy on the improvement of alkaline corneal defects, the stimulation of keratocyte repopulation, and the partial restoration of corneal integrity, this therapeutic modality may be applicable and be an effective treatment of persistent corneal ulcers of various etiologies; this requires further investigation. 
In conclusion, short-term magnetic therapy using extremely low frequency pulsed electromagnetic fields is a noninvasive and safe treatment method that is considerably effective in improving the outcome of corneas with alkaline burns, and its therapeutic effects are quite comparable to those of medical treatment. Such healing effects were, to some extent, transient, and after a time, partial regression of the corneal defect and progression of corneal neovascularization were observed. Given the results of this study and lack of superiority between the ELF groups, implementation of ELF-PEMF of 2 mT field intensity seems to be a proper option for therapeutic purposes. In summary, although magnetic therapy is effective in the healing of corneal defects from alkaline burns, it is not sufficient. Consequently, in order to obtain more favorable results, adjunctive therapies such as lubricants and/or topical corticosteroids or anti-VEGF compounds may be needed. The other advantages of implementation of magnetic therapy for treatment of corneas with alkaline burns are the stimulation of keratocyte repopulation into the injured area together with restoration of the integrity of the corneal layers. Indeed, further investigation with a longer follow up is needed to make comprehensive comments on the results of this study. 
Acknowledgments
We thank Ali-Asghar Aghdaei and Gholam-Hossein Abbasi for providing instrumental support contributing to this work. 
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Footnotes
 Disclosure: M. Rezaei Kanavi, None; F. Sahebjam, None; F. Tabeie, None; P. Davari, None; A. Samadian, None; M. Yaseri, None
Figure 1. 
 
The upper image illustrates the restrained rabbit's head with the shield, including the coil, on the right eye. Rabbit's eye is visible through the 2-cm circular central opening in the shield. The lower image shows a wire connecting the coil to a direct electrical generator unit.
Figure 1. 
 
The upper image illustrates the restrained rabbit's head with the shield, including the coil, on the right eye. Rabbit's eye is visible through the 2-cm circular central opening in the shield. The lower image shows a wire connecting the coil to a direct electrical generator unit.
Figure 2. 
 
Graphs (A) and (B), respectively, illustrate mean areas of corneal defect and changes of the defective areas in the control, MT, ELF 2, and ELF 5 groups on days 0, 2, 7, and 14 and show the greater rate of improvement in the corneal defect and smaller size of the defective areas in the ELF and MT groups compared to the controls by time.
Figure 2. 
 
Graphs (A) and (B), respectively, illustrate mean areas of corneal defect and changes of the defective areas in the control, MT, ELF 2, and ELF 5 groups on days 0, 2, 7, and 14 and show the greater rate of improvement in the corneal defect and smaller size of the defective areas in the ELF and MT groups compared to the controls by time.
Figure 3. 
 
Corneal defect photographs of rabbit corneas with alkaline burns on days 0, 2, 7, and 14 in the control (A), MT (B), 2ELF 2 (C), and ELF 5 (D) groups.
Figure 3. 
 
Corneal defect photographs of rabbit corneas with alkaline burns on days 0, 2, 7, and 14 in the control (A), MT (B), 2ELF 2 (C), and ELF 5 (D) groups.
Figure 4. 
 
(A) Microphotograph of a rabbit's full thickness cornea with membrane formation (arrow) posterior to Descemet's membrane (DM), (B) foci of intrastromal neovascularization (arrows), (C) moderately intense intrastromal inflammatory cell infiltrate involving anterior and midstromal layers, and (D) severe keratocyte loss; only one keratocyte (arrow) is visible in the examined field. All microphotographs are stained with hematoxylin and eosin. Magnification: ×100 (A), ×400 (B), ×100 (C), and ×1000 (D).
Figure 4. 
 
(A) Microphotograph of a rabbit's full thickness cornea with membrane formation (arrow) posterior to Descemet's membrane (DM), (B) foci of intrastromal neovascularization (arrows), (C) moderately intense intrastromal inflammatory cell infiltrate involving anterior and midstromal layers, and (D) severe keratocyte loss; only one keratocyte (arrow) is visible in the examined field. All microphotographs are stained with hematoxylin and eosin. Magnification: ×100 (A), ×400 (B), ×100 (C), and ×1000 (D).
Table 1. 
 
Comparing the Mean Areas of the Corneal Defects on Days 0, 2, 7, and 14 and Their Changes over the 14-Day Period between the Control, MT, ELF 2, and ELF 5 Groups
Table 1. 
 
Comparing the Mean Areas of the Corneal Defects on Days 0, 2, 7, and 14 and Their Changes over the 14-Day Period between the Control, MT, ELF 2, and ELF 5 Groups
Time Control MT ELF P§ Multiple Comparison‖
ELF 2 ELF 5
Day 0* 39.57 ± 7.2 38.19 ± 10.65 37.53 ± 5.69 42.37 ± 12.64 0.83 -
Day 2* 13.61 ± 12.08 18.19 ± 13.82 4.65 ± 4.08 7.9 ± 9.68 0.003 (ELF 2 vs. MT, P < 0.01)
(ELF 5 vs. MT, P < 0.05)
Change 2–0* −25.95 ± 12.57 −20 ± 10.42 −32.87 ± 7.34 −34.46 ± 9.26 0.001 (ELF 2 vs. MT, P < 0.05)
(ELF 5 vs. MT, P < 0.01)
% of change† −73 (−100 to 16.05) −62 (−91 to −2) −90 (−100 to −60) −85 (−100 to −42)
Within P 0.001 0.001 0.001 0.001
Day 7* 15.24 ± 13.47 7.2 ± 6.37 4.54 ± 7.02 2.36 ± 3.64 0.001 (ELF 2 vs. Control, P < 0.01)
(ELF 5 vs. Control, P < 0.05)
Change 7–0* −24.33 ± 15.16 −30.99 ± 10.69 −32.99 ± 8.22 −40.01 ± 11.38 0.01 ELF 5 vs. MT, P < 0.01
% of change† −76 (−100 to 23) −86 (−100 to −44) −94 (−100 to −37) −99 (−100 to −70)
Within P 0.002 0.001 0.001 0.001
Day 14* 17.55 ± 12 12.73 ± 7.49 9.68 ± 8.06 6 ± 3.82 0.008 ELF 5 vs. Control, P < 0.01
Change 14–0* −21.19 ± 10.84 −27.22 ± 9.21 −27.85 ± 8.13 −36.37 ± 15.13 0.01 ELF 5 vs. Control, P < 0.01
% of change† 55 (−5 to 95) 66 (41 to 89) 83 (31 to 96) 88 (58 to 98)
Within P 0.002 0.001 0.001 0.001
Change 14–7* −1.72 ± 20.11 −5.51 ± 8.09 −5.14 ± 8.69 −3.64 ± 5.29 0.65 -
% of change† −14 (−275 to 92) −140 (−400 to 76) −20 (−1470 to 33) 1 (−1633 to 86)
Within P 0.55 0.02 0.03 0.02
Table 2. 
 
Comparing the Rate of Significant Corneal Neovascularization on Days 7 and 14 and Their Progression over the 2-Week Period between the Control, MT, ELF 2, and ELF 5 Groups
Table 2. 
 
Comparing the Rate of Significant Corneal Neovascularization on Days 7 and 14 and Their Progression over the 2-Week Period between the Control, MT, ELF 2, and ELF 5 Groups
Time Control MT ELF 2 ELF 5 P*
Day 0 0 (0.0%) 0 (0.0%) 0 (0.0%) 0 (0.0%) -
Day 2 0 (0.0%) 0 (0.0%) 0 (0.0%) 0 (0.0%) -
Day 7 3 (21.4%) 1 (7.1%) 0 (0.0%) 3 (21.4%) 0.22
Day 14 7 (50.0%) 1 (7.1%) 3 (21.4%) 3 (21.4%) 0.07
P for progression† 0.005 0.37 0.049 0.049
Table 3. 
 
Comparing the Histopathologic Results of the Corneal Tissues between the Control, MT, ELF 2, and ELF 5 Groups
Table 3. 
 
Comparing the Histopathologic Results of the Corneal Tissues between the Control, MT, ELF 2, and ELF 5 Groups
Histopathologic Features Control MT ELF 2 ELF 5 P Multiple Comparisons
Epithelial integrity
 No 10 (66.7%) 8 (57.1%) 7 (50.0%) 7 (50.0%) 0.77*
 Yes 5 (33.3%) 6 (42.9%) 7 (50.0%) 7 (50.0%)
Bowmans layer
 No 14 (93.3%) 14 (100.0%) 14 (100.0%) 14 (100.0%) >0.99†
 Yes 1 (6.7%) 0 (0.0%) 0 (0.0%) 0 (0.0%)
Mild scarring
 No 10 (66.7%) 6 (42.9%) 2 (14.3%) 4 (28.6%) 0.03* (C vs. ELF 2 P = 0.001), (C vs. ELF 5, P = 0.03)
 Yes 5 (33.3%) 8 (57.1%) 12 (85.7%) 10 (71.4%)
Neovascularization
 Nonsignificant 8 (57.1%) 13 (92.9%) 11 (78.6%) 9 (64.3%) 0.16†
 Significant 6 (42.9%) 1 (7.1%) 3 (21.4%) 5 (35.7%)
Thinning
 No 14 (93.3%) 14 (100.0%) 12 (85.7%) 14 (100.0%) 0.45†
 Yes 1 (6.7%) 0 (0.0%) 2 (14.3%) 0 (0.0%)
Keratocyte loss
 No 3 (21.4%) 1 (7.1%) 10 (71.4%) 9 (64.3%) 0.001‡ (MT vs. ELF 2, P = 0.01), (MT vs. ELF 5, P = 0.01)
 Mild 10 (71.4%) 4 (28.6%) 4 (28.6%) 4 (28.6%)
 Moderate 1 (7.1%) 8 (57.1%) 0 (0.0%) 0 (0.0%)
 Severe 0 (0.0%) 1 (7.1%) 0 (0.0%) 1 (7.1%)
Inflammation
 None to mild 8 (57.1%) 13 (92.9%) 7 (50%) 7 (50%) 0.06* -
 Moderate to severe 6 (42.9%) 1 (7.1%) 7 (50%) 7 (50%)
Endothelial cell loss
 No 2 (13.3%) 0 (0.0%) 0 (0.0%) 0 (0.0%) 0.28†
 Severe 13 (86.7%) 14 (100.0%) 14 (100.0%) 14 (100.0%)
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