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Keywords:

  • cornea;
  • metalloproteinases;
  • rat;
  • ulcerative keratitis

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgments
  9. References

Objective

To evaluate the effects of agents on corneal re-epithelization and metalloproteinase-2 and metalloproteinase-9 (MMP-2 and MMP-9) activities in corneas of rats submitted to ulceration.

Animals Studied

Ninety eight healthy rats.

Procedures

Corneal ulcers were created using 1N NaOH in their left eye. Eyes were treated every 6 h with 1% ethylenediaminetetraacetic acid (EDTA), 3% chondroitin sulfate (CS), 10% N-acetylcysteine NAc and saline (S) at 6-h intervals. Corneas were stained with fluorescein and photographed at the same time points. Following 20 h and 40–42 h of corneal injury, corneas were processed for scanning electron microscopy (SEM) to quantify microvilli density, and MMPs activities were analyzed using zymography.

Results

The percentage of wound area and the time in hours for corneal re-epithelization did not differ significantly among treatment groups (P > 0.05). In first and the second moments, latent MMP-2 was significantly elevated in the eyes treated with NAC and CS (P < 0.001). Active MMP-2 did not change significantly among treatment groups in the first moment (P > 0.05); significantly higher activity was observed in the second moment in the eyes treated with CS (P <0.001). In the second moment, latent MMP-9 decreased significantly in eyes treated with EDTA and S (P < 0.01). Microvilli corneal density did not change significantly between healthy subjects and treatment groups (P > 0.05).

Conclusion

Any of the studied substances did not accelerate corneal re-epithelization and did not add protection to the corneal microvilli. Significant higher levels of active form of MMP-2 in 3% chondroitin sulfate-treated group may indicate that the agent acts as substrate for such enzyme. At the end of the experiment, 1% EDTA was the most efficient agent to inhibit significantly the latent form of MMP-9. However, any of the substances add benefit over saline on reducing the proteolytic activity in the cornea of rats after alkali injury.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgments
  9. References

Following any corneal insult, numerous cellular interactions and alterations occur. Those are mediated by leukocytes, fibroblasts, endothelial cells as well as by combined actions of various substances such as proteinases, growth factors, and cytokines, aiming at corneal regeneration.[1]

Proteinases can be classified as matrix metalloproteinases (MMPs), serine proteinases, aspartic proteinases, and cysteine proteinases.[2, 3] MMPs constitute an enzymatic family responsible for the remodeling and degradation of extracellular matrix and basal membrane components.[4, 5] MMP-2 and MMP-9 are the enzymes of major importance in the remodeling and degradation of the corneal stromal collagen in rats,[6] humans,[7, 8] mice,[9] rabbits,[10] dogs,[11, 12] and horses.[13, 14]

Matrix metalloproteinase-2 is mainly produced by stromal keratocytes and epithelial cells and is found in its inactive form on the intact corneal epithelium and stroma. It performs a surveillance function, becoming locally activated to degrade collagen molecules that occasionally become damaged as a result of normal aging.[6, 14-16] Matrix metalloproteinase-9 is produced by corneal epithelial cells and keratinocytes, as well as by inflammatory cells following corneal wounding.[1, 6, 14, 16-18] This MMP plays an important role as it is able to destroy the adhesive structure, such as collagens, laminin, and proteoglycans of the epithelial basement membrane.[7, 16]

The activity of MMPs is tightly coordinated by tissue inhibitors of matrix metalloproteinases (TIMPs); an imbalance between MMPs and TIMPs in favor of proteinases may cause excessive degradation of normal healthy tissue impairing wound healing and leading to corneal melting.[19-22] Thus, the agents capable of inhibiting MMPs, such as N-acetylcysteine, ethylenediaminetetraacetic acid (EDTA), tetracyclines, and blood serum are recommended for topical use in cases of ulcerative keratitis.[2, 3, 21, 22]

EDTA, tetracyclines, and N-acetylcysteine chelate calcium and zinc, essential minerals for the proteinases activity.[22-24] By calcium chelation, EDTA interferes with the stability of MMPs and decreases the stimulation of the migration of polymorphonuclear cells to the corneal wound.[22, 24]

N-acetylcysteine is an MMP inhibitor commonly used in humans as well as veterinary ophthalmology.[23-26] In one study performed with tears of horses with ulcerative keratitis, 10% N-acetylcysteine decreased proteolytic activity by 98.9%.[26] Results of studies performed in living animals with this agent are based only on direct observation of corneal re-epithelization and are described as being beneficial or detrimental to the healing process in accordance with the concentration used.[27, 28]

Glycosaminoglycans such as sodium hyaluronate and chondroitin sulfate have been used for medical treatment of ulcerative keratitis.[29-33] Their antiproteolytic properties are observed mainly in cartilage and synovial fluid in humans, and it is reported that the chondroitin sulfate is able to inhibit proteolytic enzymes, such as collagenases and elastases, as well as to protect cellular membranes from the action of free radicals.[34]

To date, only one in vitro experiment tested the effects of commonly used substances in veterinary ophthalmology able to decrease the MMPs activity.[26] In another study, several agents were evaluated to determine the gelatinase activity present in the tear film of normal dogs.[32] To the author's knowledge, there are no studies performed in living animals with ulcerative keratitis, in which the effects of commonly used substances to inhibit MMPs were evaluated. Therefore, this study aimed to compare the effects of 1% EDTA, 3% chondroitin sulfate, and 10% N-acetylcysteine in alkali-burned rat corneas and to evaluate the effects of this treatment on the re-epithelization time, the expression of MMP-2 and MMP-9, and the density of corneal microvilli.

Material and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgments
  9. References

Animals

Ninety-eight Wistar healthy female rats (Rattus norvergicus albinus) weighing between 250 and 300 grams were used. They were individually housed in a ventilated environment and fed dry pellets twice a day. Water was available ad libitum. Only rats with no ocular surface abnormalities were included in the study based on slit-lamp biomicroscopy and fluorescein staining.

All procedures were approved by the Ethics Committee on Animal Use of the School of Agricultural and Veterinary Sciences of Sao Paulo State University, Jaboticabal, Sao Paulo, Brazil (protocol number 28628-08).

Alkali-induced corneal ulceration

This model of corneal ulceration was chosen because the alkaline agent is able to saponify plasmatic membranes, quickly penetrating the corneal layers and promoting degradation of the collagen causing stromal compaction.[35]

After induction of anesthesia with an intraperitoneal injection of ketamine chloride (50 mg/kg) combined with xylazine chloride (5 mg/kg), one drop of proxymetacaine (Anestalcon®, Alcon) was instilled to the left eye. Five minutes later, the ocular surface was rinsed with povidone-iodine solution diluted in saline (1:50). Alkali injuries were induced by a 60-s exposure of the central cornea to a 2-mm-diameter disk of filter paper soaked in 1N NaOH; afterward, the ocular surface was rinsed with sterile saline for 1 min. The contralateral eye was not included in the study.

Study design

A total of 96 rats were divided into four treatment groups composed of 12 individuals on each. The left eye was treated with 30 μL of 1% EDTA (pH 6.5), 3% chondroitin sulfate (pH 7.4), 10% N-acetylcysteine (pH 7.0), or sterile saline. All tested drugs were commercially manufactured (Ophthalmos®, São Paulo, Brazil) in sterile water free of preservatives. Immediately after corneal injury and every 6 h, wound healing was evaluated until re-epithelization has been completed. After fluorescein staining, corneas were photographed at a fixed distance of 3 cm, and the dimensions of the wound areas were measured using image analysis software (ImageJ, http://www.rsbweb.nih.gov/ij/). The percentage of wound area photographed was averaged and calculated as described previously.[40]

For laboratory purposes, four uninjured and untreated corneas of two rats were used as negative controls. Twelve animals of each group were euthanized 20 h (first moment) after corneal injury, while the other twelve were euthanized after completion of corneal re-epithelization, which occurred 40–42 h following corneal injury (second moment).

To achieve the minimum amount necessary to perform the protein analysis for zymography, the samples were grouped in pools composed of an entire cornea from one animal and half of a cornea from another animal of the same group. In both moments, pooled samples (6/treatment group) were frozen at −70 °Cand submitted to gelatin zymography for quantification of MMP-2 and MMP-9. The other samples (6 half corneas of each group collected in the second moment) were fixed in glutaraldehyde for further scanning electron microscopy analysis.

Zymography

To determine the MMPs activities, the corneas were macerated and homogenized at 20.000 rpm for 1 min (Polytron® PT 2100- Kinematica AG) in a solution containing 50 mm Tris–HCl (pH 7.4), 0.2 m NaCl, 0.1% Triton, 10 mm CaCl2, and 1% protease inhibitor cocktail (Sigma Chemical Co., Saint Louis MO, USA) and incubated for 2 h at 4 °C. The homogenate was centrifuged at 4000 g for 10 at 4 °C, and the supernatant was collected and separated in 4 aliquots and stored at −80 °C. Each one of the aliquots was thawed just once. The Bradford method was used for total protein quantification using serum albumin as a protein standard. Zymography assays were performed on 10% polyacrylamide electrophoresis gels containing 10% SDS and 0.1% gelatin, and 25 μg of protein was loaded into each well, to indentify MMP-2 and MMP-9. Standard nonreduced molecular weight markers (Amersham Biosciences) were also loaded into the gel, which was subsequently electrophoresed at 125 V for 2 h. Following electrophoresis, the gels were washed twice in a 2.5% Triton X-100 solution to remove SDS and incubated at 37 °C for 20–24 h in incubation buffer (50 mm Tris–HCl pH 7.4, 0.1 m NaCl, and 0.03% NaNO3). The gels were then stained with 2.5% Coomassie Brilliant Blue R-250 (Sigma) for 1 h, destained in acetic acid/methanol/water (1:3:6, v:v:v), and inspected for bands where gelatinolytic activity was observable.

Gels were photographed, and isoforms of MMP-2 and MMP-9 were identified based on their expected molecular weights and compared with gels in which human standards of latent and active forms of MMP-2 and MMP-9 were run as positive controls (MMP-2/MMP-9 control, Bio-Rad).

Quantitative assessment of band intensity was carried out by densitometry (ImageScanner III, General Electric) using Scion Image software (Scion Corporation, Frederick, MD, USA). Each sample was analyzed individually, and the experiments were repeated three times, with two gels used for MMP activity quantification, while in the third, EDTA (20 mm) was added to demonstrate a loss of proteolytic activity as a negative control.

Scanning electron microscopy

After 48 h of fixation in 3% glutaraldehyde, corneal samples collected at the second moment were washed in phosphate buffer, fixed in 1% osmium tetra oxide, dehydrated in graded concentrations of ethanol, critical point–dried at 37 ºC, and coated with gold–palladium to improve conductivity. Images were evaluated on a scanning electron microscope under 5000× magnification. Resulting images were printed on thermal paper by video printer (Mitsubishi® P90W video printer). Five images were randomly captured from the central cornea of each sample to quantify the microvilli density.

Images were binarized, and dark spots representing microvilli projections were quantified as described previously[36] using image analysis software (ImageJ, http://www.rsbweb.nih.gov/ij/). Values obtained were converted into μm2.

Statistical analysis

Statistical analysis was performed using Prism 4.0® (GraphPad Software inc – San Diego, CA, USA).

Repeated-measures anova followed by Tukey's HSD test was used to check for differences in the percentage of wound area. The corneal wound healing rate between groups and arbitrary densitometry units corresponding to the latent and active forms of MMPs obtained by zymography, as well as the area of microvilli projections in corneal samples, were evaluated by one-way anova with Tukey's HSD test. In each test, P < 0.05 was considered significant. All results are expressed as mean ± SD. Area under the curve was calculated after analysis of the percentage of wound area.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgments
  9. References

Corneal re-epithelization

Six hours after corneal injury, the diameter of the ulcerated area increased when compared to the initial size of the wound in the groups treated with 1% EDTA, 3% chondroitin sulfate, and 10% N-acetylcysteine (Fig. 1); however, only in individuals treated with 1% EDTA, statistical significance was observed (P < 0.05).

image

Figure 1. Mean ± SEM* of relative wound area size of corneal epithelium, after fluorescein instillation in treatment groups, from initial injury to complete re-epithelialization. *Differ significantly from time point zero by Tukey's test (P < 0.05).

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The percentage of wound area did not differ significantly among treatment groups at any time point (P > 0.05; Fig. 1). Nevertheless, a larger area under the curve was observed in individuals treated with 1% EDTA (2131), followed by 10% N-acetylcysteine (1873), and 3% chondroitin sulfate (1322). Average time in hours for corneal re-epithelization did not differ significantly among treatment groups (P = 0.97) (Table 1).

Table 1. Time of corneal re-epithelization following alkali-induced corneal ulceration among treatment groups
 10% N-acetylcysteine3% Chondroitin sulfate1% EDTASaline
  1. anova (P = 0.97).

Time (h)38.33 ± 7.0937.67 ± 7.0937.00 ± 6.6637.00 ± 3.95
Minimum24242430
Maximum42424240

Zymography

Latent form of MMP-2

In the first moment after corneal injury (20 h), the levels of latent MMP-2 decreased significantly following treatment with saline and 1% EDTA, when compared with the healthy corneas (P < 0.001; Fig. 2). In the second moment following corneal injury (40–42 h), significantly increased values of latent MMP-2 were observed in groups treated with N-acetylcysteine and 3% chondroitin sulfate (P < 0.05; Fig. 2); significantly decreased values were seen in saline-treated rats, when compared with the healthy corneas (P < 0.05; Fig. 2).

image

Figure 2. Mean ± SEM* densitometry readings of latent MMP-2 from zymogram gels loaded with corneal extracts of healthy animals (n = 2), and animals of treatment groups (n = 6). *Tukey test.

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Comparisons made among treatment groups in both moments showed significantly higher values of latent MMP-2 in individuals treated with 3% chondroitin sulfate and 10% N-acetylcysteine (P < 0.001; Fig. 2). Levels of this enzyme were significantly lower in rats treated with saline, when compared to treatment with 1% EDTA (P < 0.001; Fig. 2).

Active form of MMP-2

In the first moment after corneal injury (20 h), the levels of active MMP-2 increased significantly following treatment with 3% chondroitin sulfate, 10% N-acetylcysteine, and saline, when compared with the healthy corneas (P < 0.05; Fig. 3). In the second moment following corneal injury (40–42 h), significantly increased values of active MMP-2 were observed in groups treated with 1% EDTA, 10% N-acetylcysteine, and 3% chondroitin sulfate, when compared with the healthy corneas (P <0.05; Fig. 3). The levels of active MMP-2 did not change significantly among treatment groups in the first moment of evaluation (P > 0.05; Fig. 3). Comparisons made among treatment groups in the second moment showed significantly higher values of this MMP in individuals treated with 3% chondroitin sulfate (P < 0.001; Fig. 3).

image

Figure 3. Mean ± SEM* densitometry readings of active MMP-2 from zymogram gels loaded with corneal extracts of healthy animals (n = 2), and animals of treatment groups (n = 6). *Tukey test.

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Latent form of MMP-9

Latent MMP-9 was only expressed in rats that underwent corneal injury, and an increase in the levels of this enzyme was observed in the first moment when compared with unwounded controls. However, the levels of latent MMP-9 did not change significantly among treatment groups in the first moment of evaluation (P > 0.05; Fig. 4). Although not significant, when compared with the first moment, values of latent MMP-9 decreased in the second moment in all treatment groups. Comparisons among treatment groups, in the second moment, showed that significantly higher values of this enzyme were observed in individuals treated with 10% N-acetylcysteine, when compared with treatment with saline, 1% EDTA (P < 0.001; Fig. 4), and 3% chondroitin sulfate (P < 0.05; Fig. 4).

image

Figure 4. Mean ± SEM* densitometry readings of latent MMP-9 from zymogram gels loaded with corneal extracts of healthy animals (n = 2), and animals of treatment-groups (n = 6). *Tukey's test.

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A representative gelatin zymogram gel showing latent and active forms of MMPs in corneal extracts of healthy rats and among treatment groups is showed in Figure 5.

image

Figure 5. Polyacrylamide gel enriched with gelatin showing bands corresponding to active and latent metalloproteinases, in the first (1 m) and second moments (2 m) after alkali injury in corneas of rats treated with 3% chondroitin sulfate (CS), 1% EDTA (EDTA), 10% N-acetylcysteine (NAc), or sterile saline (Sal). Corneas of healthy rats were used as negative controls. Human standards corresponding to MMP complex (130 kDa), latent MMP-9 (92 kDa), active MMP-9 (82 kDa), latent MMP-2 (72 kDa), and active MMP-2 (62 kDa) were used as positive controls.

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Scanning electron microscopy

Average microvilli density was 1.24 ± 0.46 (saline), 1.38 ± 0.18 (10% N-acetylcysteine), 0.91 ± 0.32 (3% chondroitin sulfate), 0.96 ± 0.35 (1% EDTA), and 1.00 ±0.25 μ2 (healthy). Microvilli corneal density did not change significantly between healthy subjects and treatment groups (P > 0.05; Fig. 6).

image

Figure 6. Representative images of scanning electron microscopy of the corneal epithelium surface of healthy rats (a), saline (b), N-acetylcysteine (c), chondroitin sulfate (d), N-acetylcysteine (e), and EDTAtreated groups (f).

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgments
  9. References

The benefits of EDTA in inhibiting MMPs have been previously reported.[3, 22, 26, 32] However, this substance commonly used as a preservative and corneal penetrating enhancer in ophthalmic solutions induces oxidative DNA damage in the corneal cells at high concentrations.[37, 38] The high concentration used herein (1%) may have caused the significant larger ulcerated area observed at the 6-h time point following corneal injury, in association with a higher area under the curve in the EDTA treatment group. Although concentrations at 0.05–0.2% have been recommended in the veterinary literature,[3] 1% EDTA was chosen to be studied based on reports of its potent antiproteinase action in dogs.[32]

Topical application of 10% N-acetylcysteine every 1–4 h has been recommended to treat corneal ulcers of dogs and horses.[3] In this study, 10% N-acetylcysteine was not able to accelerate corneal re-epithelization and enhanced the diameter of the ulcerated area 6 h after corneal wounding. At concentrations of 10 and 20%, N-acetylcysteine did not accelerate corneal re-epithelization in rats[23] and in dogs.[28] However, ulcerated corneas of dogs[28] and rabbits[39] treated with 3% N-acetylcysteine re-epithelized faster than controls. Thermes et al.[40] reported that at concentration of 20%, N-acetylcysteine promoted focal necrosis of corneal and conjunctival epithelium cells of rabbits. Ramaesh et al.[41] demonstrated in vitro that human corneal cell migration is inhibited by N-acetylcysteine and that such inhibition is concentration-dependent. It has been postulated that lysis of disulfide linkages between mucopolysaccharides and tissue proteins by acetylcysteine is responsible for the apparent tissue destruction.[42] A possible alternative explanation is that the hyperosmotic solution draws fluid into the corneal stroma causing mechanical damage.[43] The concentration of 10% used herein may have impaired corneal migration at early stages of healing.

Similarly to the other tested substances, 3% chondroitin sulfate did not show any benefit on corneal re-epithelization. Similar results were reported in corneal cultures of rabbits,[44] as well as in dogs with corneal ulcers treated with commercially ophthalmic solutions of chondroitin sulfate associated with antibiotics.[32] One experiment demonstrated that polysulfated glycosaminoglycan causes a concentration-dependent effect on morphologic characteristics and migration of canine corneal epithelial cells in tissue culture.[45]

In this experiment, latent and active forms of MMP-2 were found in healthy and ulcerated rat corneas, while MMP-9 was only found in ulcerated corneas in its inactive form. In rabbits, the latent form of MMP-9 was only activated 3 days following keratectomy.[10] Shi et al.[46] have demonstrated by immunohistochemistry that MMP-9 can be found in the corneal epithelium and stromal cells of rats 24 h following alkali injury and that such expression increases seven days later in parallel with corneal neovascularization. However, once antibodies used in that study for MMP-2 and MMP-9 were able to recognize both latent and active forms of such enzymes,[46] it is not possible to make comparisons with our findings in regard of activation of MMP-9.

In vitro studies reported the efficacy of 10% N-acetylcysteine on reducing the proteolytic activity by up to 98% in equine tears affected by keratomalacia.[26] However, 10% N-acetylcysteine did not reduce enzyme activity in the tears of healthy dogs after 48 h of treatment.[32] In our study, when compared with saline group, the levels of the latent form of MMP-2, and MMP-9 were significantly increased in corneas treated with 10% N-acetylcysteine by the end of the experiment. The aforementioned information in regard to the corneal toxicity of the 10% N-acetylcysteine may explain the elevated levels of MMPs found in our study.[41-43]

Forty-two hours after corneal injury, significantly higher levels of the active form of MMP-2 was only observed in 3% chondroitin sulfate group. Based on the results of the present research, it can be postulated that the solution containing chondroitin sulfate might have acted as a substrate to the MMP-2, elevating the quantitative of this enzyme at the end of the experiment. Actually, it has been demonstrated in melanoma cell culture that the latent form of MMP-2 directly binds to chondroitin sulfate through the C-domain, facilitating the generation of the active form of MMP-2.[47] However, it has been demonstrated that MMP-2 participation in stromal remodeling may not depend on coexisting epithelial repair.[10]

In vitro, EDTA was able to reduce the gelatinolytic activity in the tears of horses with keratomalacia by up to 99.4%[26] and by up to 68% in the tears of healthy dogs following 48 h of treatment.[32] After re-epithelization has been completed, 1% EDTA was able to significantly reduce latent forms of MMP-2 and MMP-9, when compared with the eyes that receive chondroitin and N-acetylcysteine. Nevertheless, such reduction was not significant, when compared with the saline group.

The evaluation of corneal microvilli density by scanning electron microscopy has been shown to be a value method in assessing the corneal surface integrity after alkali injury[48] and experimentally induced dry eye in rabbits.[49]

Our experiment failed to show benefits of any tested agent in decreasing the levels of MMP-2 and MMP-9 after corneal alkali injury in rats. Despite the possible toxic corneal effects of EDTA[41-43] and N-acetylcysteine[37, 38] have been previously discussed herein, they may have acted only in acute phases of the corneal healing, once the corneal epithelium surface tended to appear restored at the end of the study.

Conclusion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgments
  9. References

Any of the studied substances did not accelerate corneal re-epithelization and did not add protection to the corneal microvilli. Significant higher levels of active form of MMP-2 in 3% chondroitin sulfate-treated group may indicate that the agent acts as substrate for such enzyme. At the end of the experiment, 1% EDTA was the most efficient agent to inhibit significantly the latent form of MMP-9. However, any of the substances add benefit over saline on reducing the proteolytic activity in the cornea of rats after alkali injury.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgments
  9. References

The authors would like to thank FAPESP for founding this experiment (process 2009/50080-5), CNPq providing the scholarship, UNESP vivarium (Botucatu – SP – Brazil), USP vivarium (Ribeirão Preto – SP – Brazil), and the Microbiology and Immunology Laboratory of the University of Campinas, SP, Brazil, for the technical support during the zymography.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgments
  9. References
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