Development of corneal ulcers is a frequent problem in domestic species. Progression of corneal ulcers via degradation of corneal stroma is mediated by collagenases that can come from infectious organisms, leukocytes, or corneal epithelial cells.1–3 Activities of such enzymes are increased in the tears of horses with ulcerative keratitis,4 dogs with Pseudomonas aeruginosa-associated keratitis,5 and humans with melting corneal ulcers6; a decrease in enzyme activities is evident as the ulcers resolve.4–6 Thus, inhibition of these destructive enzymes can be crucial to preventing further progression of corneal degradation.2 Control of these enzymes in vivo is mediated by endogenous tissue inhibitors of matrix metalloproteinases.7–9 In cases of keratomalacia, treatment with anticollagenase agents (tetracyclines, EDTA, N-acetylcysteine, or topically applied serum) can be used.2,10–12
Topical application of serum has been used in human and veterinary medicine for various ocular conditions. In human medicine, topical application of serum is used to treat dry eye, persistent epithelial defects, neurotrophic keratopathy, superior limbic kerato-conjunctivitis, and Mooren ulcers.13,14 Umbilical cord serum has also been investigated as a treatment for acute ocular chemical burns.15 In veterinary medicine, topical application of serum has been widely used as an anticollagenase for treating corneal stromal ulcers in an attempt to slow or arrest progression.2,3,16 The anticollagenase effect of topically applied serum is attributed to the presence of α2-macroglobulin, which is a serum protein that complexes with and inhibits corneal collagenase.17,18
Plasma has also been investigated in human medicine for potential benefits when used topically. Plasma has some possible advantages over serum, namely that plasma is commercially available and therefore perhaps easier to obtain. Plasma also contains clotting proteins and platelets, which provide sustained release of growth factors.19,20 Another potential benefit of plasma over serum is that it can be obtained quickly in a clinical setting because coagulation of the blood is not required. In 1 study,21 investigators compared serum and FFP in experiments involving human corneal epithelial cells to determine the relative effects on various aspects of corneal wound healing and found that serum was superior to FFP with regard to enhancing growth, differentiation, and migration of epithelial cells. Numerous other studies19,20,22–28 have been conducted to evaluate FFP, PRP, and platelet releasate (a product in which growth factors in platelets are released by thrombin stimulation and the remaining platelet membranes are removed by centrifugation) for treatment of ocular surface defects, ocular burns, nonhealing corneal ulcers, corneal perforations, and dry eye, and clinical improvement was detected with their use.
Despite investigation of the effects of plasma and its constituents on corneal conditions in humans, there has been little investigation of the anticollagenase efficacy of plasma in veterinary medicine. In 2 studies,29,30 investigators evaluated canine plasma in vitro in comparison to serum with regard to anticollagenase efficacy, but to the authors' knowledge, no studies have been conducted to evaluate the anticollagenase efficacy of feline or equine plasma in comparison to serum. Therefore, the purpose of the study reported here was to compare the anticollagenase efficacy of fresh feline, canine, and equine serum with that of plasma for preventing in vitro corneal degradation.
Materials and Methods
Corneal samples
Corneas were collected from recently euthanized dogs, cats, and horses at the Purdue University Veterinary Teaching Hospital or a local animal shelter. Animals were euthanized for reasons unrelated to this study and had no evidence of corneal disease as determined by examination with a direct light source. Each cornea was removed and sectioned into 4 pieces (feline and canine) or 6 pieces (equine) of approximately equal size. Each piece of cornea was labeled by species and individually stored at −80°C until use. Corneal sections were stored for 1 to 18 months before use. Storage times for corneal samples were evenly distributed among control and test groups.
Serum and plasma samples
Blood samples were obtained from healthy canine (n = 4), feline (4), and equine (4) donors. The dogs and cats were employee-owned animals, whereas the horses were teaching animals owned by Purdue University. Employees signed consent forms authorizing the use of their pets for blood collection. All procedures were approved by the Purdue University Animal Care and Use Committee.
Cats were anesthetized for blood collection, whereas dogs and horses were manually restrained. Blood was collected into serum separator tubes,a allowed to clot, and centrifuged to separate serum. The serum was then removed and pooled by species. Blood was collected into lithium heparin tubesb (EDTA was not used as an anticoagulant because EDTA has anticollagenase properties) and centrifuged to separate plasma. Plasma was then removed and pooled by species. Pooled serum and plasma were used within 24 hours after blood collection. Sterile technique was used throughout the experiment, but bacterial culture was not performed on the pooled serum or plasma.
In vitro corneal degradation
Individual corneal samples were placed in plastic weigh boats and dried in an oven at 40°C for 3 hours. After samples were dry, pretreatment corneal weight was recorded. Incubation fluid was prepared by adding collagenase derived from Clostridium histolyticumc (800 U/mL) to 5mM calcium chloride in saline (0.9% NaCl) solution. An aliquot (5 mL) of incubation fluid was added to a 10-mL tube that contained a corneal sample. Then, 500 μL of serum or 500 μL of plasma from the appropriate species was added to the tube so that corneal samples were incubated with homologous serum or homologous plasma. Negative control samples consisted of 5 mL of 5mM calcium chloride in saline solution. Positive control samples consisted of 5mM calcium chloride in saline solution with clostridial collagenase (800 U/mL) but without serum or plasma. For each species, 15 corneal samples were incubated with serum and 15 corneal samples were incubated with plasma. Positive and negative control samples for each species were in triplicate.
Tubes were incubated with agitation at 40°C for 4 hours. After incubation was complete, 1.8 mL of incubation fluid was collected from each tube and stored at −80°C for subsequent analysis of hydroxyproline concentrations. Posttreatment corneal samples were then collected by pouring the remaining solution through filter paperd and placing corneal tissue in its original plastic weigh boat. Posttreatment corneas were dried at 40°C for 3 hours and then weighed. Posttreatment percentage of corneal weight loss was recorded.
Hydroxyproline assay
Hydroxyproline is a breakdown product of collagen. In other similar studies,1,31 hydroxyproline concentrations have been used as a second method of evaluating corneal collagen degradation. The hydroxyproline concentration in incubation media was evaluated by use of a commercial hydroxyproline spectro-photometric assay kit.e Analyses were performed in accordance with kit instructions, with 2 modifications: samples were diluted 1:2 with ultrapure water prior to analysis to ensure measured hydroxyproline concentrations would fit on the standard curve, and samples were centrifuged for 10 minutes at 10,000 × g. A new standard curve was calculated for each plate, as per kit instructions.
Statistical analysis
Kruskal-Wallis tests were used to compare the percentage of corneal weight loss and hydroxyproline concentrations among serum, plasma, and positive control samples. When results for the Kruskal-Wallis test were significant, Dunn tests were performed for pairwise comparisons. Results for all statistical tests were considered significant at P < 0.05.
Correlation between the percentage of corneal weight loss and hydroxyproline concentration was assessed by use of the Spearman correlation coefficient. Only serum and plasma samples were analyzed for correlations; results for positive and negative control samples were not included in this assessment.
Results
In vitro corneal degradation
The method was effective for causing corneal weight loss by collagen degradation. Mean ± SD percentage corneal weight loss for feline, canine, and equine positive control samples was 85 ± 3%, 100 ± 4%, and 76 ± 13%, respectively, whereas the mean percentage corneal weight loss for feline, canine, and equine negative control samples was 4 ± 2%, 4 ± 4%, and 1 ± 2%, respectively. There was no recognizable cornea remaining in some positive control samples. Mean ± SD percentage corneal weight loss for feline, canine, and equine corneas incubated in serum was 62 ± 7%, 74 ± 15%, and 38 ± 12%, respectively, whereas the mean percentage corneal weight loss for feline, canine, and equine corneas incubated in plasma was 60 ± 10%, 60 ± 27%, and 41 ± 14%, respectively.
Serum and plasma were both effective at reducing the percentage of corneal weight loss, compared with results for the positive control samples. Inclusion of feline, canine, or equine serum significantly reduced corneal weight loss by 23% (P = 0.012), 26% (P = 0.018), and 38% (P = 0.005), respectively, compared with results for the respective positive control samples. Inclusion of feline, canine, or equine plasma significantly reduced corneal weight loss by 25% (P = 0.004), 40°% (P = 0.002), and 35% (P = 0.019), respectively, compared with results for the respective positive control samples. There was no significant difference in corneal weight loss between corneas incubated with serum or plasma for feline (P = 0.579), canine (P = 0.249), or equine (P = 0.406) samples (Figure 1).
Mean ± SD percentage of corneal weight loss for feline, canine, and equine corneal samples incubated with clostridial collagenase and homologous fresh serum (white bars) or plasma (diagonal-striped bars) or that served as positive control samples (gray bars) or negative control samples (black bars). There was no significant difference between incubation with serum or plasma for feline (P = 0.579), canine (P = 0.249), or equine (P = 0.406) corneas.
Citation: American Journal of Veterinary Research 77, 6; 10.2460/ajvr.77.6.627
Hydroxyproline assay
Concentrations of hydroxyproline were higher in incubation fluid from positive control samples, compared with concentrations in incubation fluid from negative control samples. Mean ± SD hydroxyproline concentration for feline, canine, and equine positive control samples was 0.04 ± 0.004 μg/mL, 0.04 ± 0.040 μg/mL, and 0.04 ± 0.020 μg/mL, respectively, whereas the mean hydroxyproline concentration for feline, canine, and equine negative control samples was 0.001 ± 0.001 μg/mL, 0.004 ± 0.007 μg/mL, and 0.007 ± 0.010 μg/mL, respectively. Mean ± SD hydroxyproline concentration for feline, canine, and equine corneas incubated in serum was 0.04 ± 0.01 μg/mL, 0.01 ± 0.01 μg/mL, and 0.06 ± 0.03 μg/mL, respectively, whereas the mean hydroxyproline concentration for feline, canine, and equine corneas incubated in plasma was 0.04 ± 0.01 μg/mL, 0.01 ± 0.01 μg/mL, and 0.06 ± 0.03 μg/mL, respectively.
The inclusion of canine serum (P = 0.028) or canine plasma (P = 0.009) significantly reduced the hydroxyproline concentration, compared with the concentration for the canine positive control samples (Figure 2). There was no significant (P = 0.497) difference in hydroxyproline concentration between corneas incubated with canine serum or canine plasma. There was no significant difference in hydroxyproline concentration between positive control samples and samples incubated with feline serum or plasma (P = 0.480) or equine serum or plasma (P = 0.846).
Mean ± SD hydroxyproline concentration of feline, canine, and equine corneal samples incubated with clostridial collagenase and homologous fresh serum or plasma or that served as positive or negative control samples. Incubation in canine serum (P = 0.028) or canine plasma (P = 0.009) significantly reduced the hydroxyproline concentration, compared with the concentration in canine positive control samples. There was no significant (P = 0.497) difference in hydroxyproline concentration between corneas incubated with canine serum or canine plasma. No significant difference was detected in the hydroxyproline concentration between positive control samples and samples incubated with feline serum or plasma (P = 0.480) or equine serum or plasma (P = 0.846). See Figure 1 for remainder of key.
Citation: American Journal of Veterinary Research 77, 6; 10.2460/ajvr.77.6.627
Correlation analysis
Spearman correlation coefficients to compare hydroxyproline concentration and percentage of corneal weight loss were determined. There was a moderate correlation (ρ = 0.54; P = 0.002) for feline samples and a weak correlation (ρ = 0.31; P = 0.096) for equine samples but no correlation (ρ = 0.04; P = 0.842) for canine samples (Figure 3).
Scatterplots of hydroxyproline concentration versus percentage of corneal weight loss for feline (A), canine (B), and equine (C) corneal samples in Figures 1 and 2. Each symbol represents results for 1 piece of cornea. Correlation analysis (Spearman correlation coefficient) revealed a moderate correlation (ρ = 0.54; P = 0.002) for feline samples, no correlation (ρ = 0.04; P = 0.842) for canine samples, and a weak correlation (ρ = 0.31; P = 0.096) for equine samples.
Citation: American Journal of Veterinary Research 77, 6; 10.2460/ajvr.77.6.627
Discussion
In the study reported here, plasma and serum were equally effective in all species tested for reducing corneal weight loss in vitro. This finding suggests that plasma or serum could be used topically on an eye with keratomalacia with equal benefit. Serum has been used extensively to treat many ocular diseases in humans and domestic species.2,3,14–16 The beneficial effects of topically applied serum can be attributed to constituent growth factors, vitamins, neuropeptides, and anticollagenases.13 A number of factors, including EGF, TGF-β, fibronectin, and substance P, promote migration and adhesion of corneal epithelial cells as well as corneal stromal and epithelial repair.13 Vitamin A can help to stop corneal squamous metaplasia, and α2-macroglobulin has anticollagenase effects. Fibronectin is a supportive factor in cellular migration.13 Serum also contains immunoglobulins, which can be bacteriostatic or bactericidal.13 Many of these factors (EGF, TGF-β1, vitamin A, and fibronectin) are present in higher concentrations in serum than in tears.13 Potential risks of topical use of serum include blood-borne disease transmission, deposition of immunoglobulins in the cornea, and contamination of serum with bacteria.13,14 Most of the benefits reported for humans in which topically applied serum has been used have been related to improvement in clinical signs, healing of persistent ulcers, and reduction in the recurrence of ulcers, rather than to anticollagenase activity.13,14 In veterinary medicine, topically applied serum has primarily been used for its anticollagenase effects, rather than for epithelial healing.2,3,16
Given that its composition is similar to that of serum, plasma has also been studied for ophthalmic use, although it also has been primarily evaluated for its effect on corneal epithelial growth and not for its anticollagenase effect. When activated, platelets in plasma release growth factors and other cytokines, which can contribute to the corneal healing process. Serum has been compared to FFP and to platelet releasate with regard to corneal epitheliotrophic capacities.21,26 Investigators of 1 study21 compared concentrations of EGF, PDGF, TGF-β, fibronectin, and vitamin A in serum and FFP; they also assessed the effects of serum and FFP on corneal epithelial cells in vitro. Serum contained significantly higher concentrations of all the factors, except for fibronectin, which was present at a concentration similar to that in FFP. Serum was significantly better at stimulating cell growth, migration, and differentiation, compared with results for FFP.21 In a 2006 study,26 investigators measured concentrations of EGF, PDGF, TGF-β, fibronectin, vitamin A, and vitamin E in serum, FFP, and platelet releasate. Serum contained significantly higher concentrations of fibronectin and vitamins A and E than did platelet releasate, whereas platelet releasate contained significantly higher concentrations of EGF, PDGF, and TGF-β than did serum. In that study,26 serum contained significantly more EGF, PDGF, and TGF-β than did FFP. In vitro corneal cell proliferation was best supported by platelet releasate, followed by serum and then by FFP, but cell differentiation and migration were best supported by serum.26 These results are similar to those of an earlier in vitro study25 in which serum was compared with platelet releasate, and it was found that platelet releasate resulted in significantly better cell growth, whereas cell differentiation and migration were slightly improved with serum. These findings are consistent with the information known about various growth factors regarding corneal epithelium (eg, the fact that PDGF stimulates cell proliferation, whereas fibronectin stimulates cell migration).25
Platelet-rich plasma has been used topically to treat acute corneal chemical burns, dry eye, and nonhealing corneal ulcers and has been used in conjunction with autologous fibrin membranes to treat corneal perforations.19,20,22,24 Platelet-rich plasma for use in eyes can be of 2 formulations (a drop or a clot), both of which have higher concentrations of growth factors than are found in serum.20 In 1 report,20 a PRP drop contained 800,000 platelets/μL, whereas a PRP clot contained 4,000,000 platelets/μL. Reference limits for the concentration of circulating platelets in humans is 150,000 to 450,000 platelets/μL.20 In 1 study,22 investigators found that topically applied PRP drops significantly improved the symptoms and clinical signs for humans with dry eye. In another study,20 it was also found that PRP, when used as a topically applied drop or as a PRP clot surgically placed under an amniotic membrane (for eyes with impending or current perforations), improved healing of corneal ulcers that had not responded to previous conventional treatment. In a recent report,24 details were provided on the use of a combination of an autologous PRP clot and an autologous fibrin membrane to surgically treat 11 patients with corneal perforations, with no reported complications and no evidence of relapse or perforation after 3 months; 7 of 11 eyes eventually received corneal transplants.
Plasma and its various iterations are supportive of corneal epithelial healing, but there has been a paucity of studies conducted on the anticollagenase efficacy of plasma. In 1 study,20 investigators used reduction of depth of an ulcer as a primary outcome measure but did not specifically evaluate anticollagenase activity. It was noted in 1 reportf that the concentration of α2-macroglobulin in human autologous platelet-integrated PRP was increased 5- to 10-fold, compared with the α2-macroglobulin concentration of blood. This suggests that the anticollagenase activity of PRP could be superior to that of serum, although studies to quantify the amount of α2-macroglobulin in serum of nonhuman species would be needed before direct comparisons could be made. The anticollagenase activity of fresh canine serum and plasma as well as frozen canine plasma was evaluated with a commercial gelatinase and collagenase kit.29 In that study,29 serum was superior to fresh canine plasma or frozen canine plasma with regard to decreasing the protease activity, although all 3 substances were effective. In another study,30 canine serum, FFP, and frozen-thawed cycled plasma and a hemodialysate (a commercial ultrafiltrate of calf blood that is free of protein) was evaluated by use of a gelatinase kit; results indicated that the hemodialysate only inhibited matrix metalloproteinase-9, whereas canine serum, FFP, and frozen-thawed plasma had comparable amounts of inhibition for both matrix metalloproteinase-2 and −9. These results are similar to results obtained in the present study, whereby serum and plasma did not differ with regard to anticollagenase efficacy.
In the study reported here, a commercially available clostridial collagenase was used to degrade corneal collagen. Although clostridial collagenase has been used in other in vitro studies,1,32 the resulting collagenolysis may not accurately approximate in vivo keratomalacia. This may be because there are other potential sources of collagenase in vivo, including exogenous infectious agents and endogenous cells (eg, leukocytes).1–3 Further in vivo studies of the anticollagenase effects of serum and plasma are therefore warranted.
Higher concentrations of hydroxyproline were detected in positive control samples than in negative control samples. Inclusion of canine serum or plasma resulted in significantly lower concentrations of hydroxyproline, whereas inclusion of feline and equine serum and plasma did not significantly reduce hydroxyproline concentrations. Overall, comparison of the hydroxyproline concentrations in incubation fluid for feline, canine, and equine corneas revealed no significant difference in the hydroxyproline concentration between serum and plasma.
The measurement of hydroxyproline concentrations has been used to quantify collagen degradation because most of the hydroxyproline found in the body is in collagen.1,31,33 Hydroxyproline is a breakdown product of collagen, and an increase in the amount of hydroxyproline is associated with an increase in collagen breakdown.1,31,33 In the present study, hydroxyproline concentrations were moderately correlated with the percentage of corneal weight loss for feline samples and weakly correlated for equine samples, but they were not correlated for canine samples. Investigators of 1 study1 found a strong positive correlation between equine corneal weight loss attributable to collagen degradation and hydroxyproline concentrations of the incubation fluid, whereas a previous study32 conducted by our laboratory group found only a weak correlation between the percentage of corneal weight loss and hydroxyproline concentrations. It is difficult to explain the disparity in these results; however, it may have been attributable to differences in the assay used. There is no obvious clinical explanation for the differences between feline, canine, and equine samples. Results for the study reported here, in addition to results for that previous study32 conducted by our research group, indicated that hydroxyproline concentrations may vary widely, which makes it difficult to interpret their relevance.
As previously discussed, plasma used in the present study was prepared without the use of EDTA to avoid confounding because EDTA has some anticollagenase efficacy,2,3,16,17 However, topically applied plasma produced by the use of EDTA may have anticollagenase effects greater than those for plasma produced with other anticoagulants, such as heparin. Therefore, the use of plasma produced with EDTA as an anticoagulant should be investigated further.
Results indicated that the anticollagenase efficacy of plasma and serum did not differ in the present in vitro study. This finding suggested that either of these 2 blood derivatives could be used for the treatment of keratomalacia. Additional studies are needed to determine whether the anticollagenase efficacy of plasma is comparable to that of serum in vivo and also to determine whether PRP or plasma releasate could be of value with regard to anticollagenase activity.
Acknowledgments
Supported by a grant from the Willis E. and Mary J. Armstrong Memorial Fund for Research in Anomalies of the Eye.
This manuscript represents a portion of a thesis submitted by Dr. Conway to the Department of Veterinary Clinical Sciences at Purdue University as partial fulfillment of the requirements for a Master of Science degree.
The authors thank Dr. Lynn Guptill and Anisa Dunham for technical assistance.
ABBREVIATIONS
| EGF | Epithelial growth factor |
| FFP | Fresh-frozen plasma |
| PDGF | Platelet-derived growth factor |
| PRP | Platelet-rich plasma |
| TGF | Transforming growth factor |
Footnotes
BD Vacutainer serum separator blood collection tube, Becton, Dickinson and Co, Franklin Lakes, NJ.
BD Vacutainer lithium heparin blood collection tube, Becton, Dickinson and Co, Franklin Lakes, NJ.
Collagenase from Clostridium histolyticum, type XI, Sigma-Aldrich Corp, St Louis, Mo.
Whatman filter paper, Sigma-Aldrich Corp, St Louis, Mo.
Sigma-Aldrich Corp, St Louis, Mo.
Browning SR, Carballo C, Golish SR, et al. Can cartilage degradation be prevented by platelet rich plasma (PRP) preparations on bovine cartilage explants? (abstr) Am J Phys Med Rehabil 2012:4:s193.
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