Investigations into the anti-collagenase efficacy of serum and plasma

摘要

Corneal ulcers are a commonly encountered problem in domesticated species and can cause significant pain, incur a high cost for the owner, and result in blindness or eventual enucleation. The progressive deepening of corneal ulcers is mediated by enzymatic destruction of stromal collagen (termed keratomalacia) when collagenases from endogenous and exogenous sources are active on the corneal surface. The inhibition of collagenases can therefore be crucial to mitigating the damage that breakdown of corneal stromal collagen causes. In ulcers where collagenase activity is suspected, the use of topical serum has been recommended, in both human and veterinary medicine, primarily due to the presence of an endogenous serum anti-collagenase protein molecule: alpha-2 macroglobulin. Alpha-2 macroglobulin is produced by the liver and is able to inactivate collagenase molecules from various endogenous and exogenous sources. Plasma has also been recommended for use, in various forms, for the treatment of various corneal conditions. Plasma, in addition to containing alpha-2 macroglobulin, also contains platelets and other growth factors that may be useful in promoting corneal healing. Despite widespread recommendations for the topical use of serum to treat keratomalacia, there is little data on its storage and usage in domestic species. Additionally, there is little information available comparing the anti-collagenase efficacy of serum to that of plasma. The goals of this research were to determine the effects of storage time and temperature on the anti-collagenase efficacy of serum in an in vitro corneal degradation model. This model uses a bacterial collagenase to cause collagen degradation, which is then measured by calculating percent corneal weight loss and quantifying the level of HP (a breakdown product of collagen) present; serum from different time and temperature storage is added to try to decrease weight loss and levels of HP. A reduction in percent corneal weight loss or HP would be indicative of a protective, anti-collagenase effect of the tested substance. An investigation into interspecies use of serum in the same model was also undertaken, to determine if any differences in anti-collagenase efficacy are seen with homologous serum compared to heterologous serum. In a second experiment, the anti-collagenase efficacy of fresh serum was compared to that of plasma for feline, canine and equine species. The hypothesis was that storage conditions would not affect the anti-collagenase efficacy of serum and that homologous and heterologous serum would have equal anti-collagenase efficacy. A final hypothesis was that fresh serum and plasma would have equal anti-collagenase efficacy. For the corneal degradation model, normal corneas from recently euthanized cats, dogs and horses were collected and stored at -80°C until use. For the first experiment serum was collected from healthy cats, dogs and horses and pooled by species, then stored for 30, 90 or 180 days at both -20°C and -80°C. Serum was pooled to control for any individual variation in serum composition. Sections of cornea were dried, weighed and incubated with clostridial collagenase and serum (homologous/heterologous) from each time/temperature point for the first experiment. Negative control samples were incubated only in saline with added calcium chloride while positive controls contained saline, calcium chloride and clostridial collagenase. Corneal damage was assessed by percent corneal weight loss and HP concentration of the incubation fluid compared to positive and negative control samples. A Shapiro-Wilk statistic was used to test for normality on percent corneal weight loss and HP. If the distribution was normal, mean and standard deviation (SD) were reported, if the distribution was not normal then median and range were reported. If the data was normal, then a general linear model was used to compare the means across groups, otherwise the Kruskal-Wallis test was used. If the Kruskal-Wallis test or the general linear model was significant, then pairwise comparisons with Bonferroni adjustment. A P value of <0.05 was considered significant. Statistical software was used for all analyses. The inclusion of serum resulted in significantly less percent corneal weight loss compared to positive controls (P<0.001). Storage time ( P=0.074) and temperature (P=0.526) did not affect percent corneal weight loss, while interspecies cornea/serum combinations (i.e. heterologous serum) (P=0.028) did affect percent corneal weight loss. The inclusion of feline or equine sera significantly reduced HP concentration (P<0.001) compared to positive controls. Significantly more HP was present in samples incubated with serum stored for 90 days compared to all other time points (P<0.001) while temperature did not significantly affect HP concentrations (P=0.132). For the second experiment, the ulcer degradation model used was the same as for the first experiment. However, in the second experiment, the sections of feline, canine and equine cornea were dried, weighed and incubated with clostridial collagenase and fresh serum or plasma. For the second experiment, blood was collected from healthy cats, dogs and horses; fresh serum or plasma was pooled by species and used in the model. Serum and plasma were pooled to control for any individual variation in composition. To analyze the data from the second experiment, percent corneal weight loss and HP levels were compared among groups for positive control, serum and plasma samples with Kruskal-Wallis tests. If the Kruskal-Wallis test was significant (i.e., P<0.05), then Dunn's tests were performed for pairwise comparisons. A Spearman correlation coefficient was used to assess correlation between percent corneal weight loss and HP concentration. In the second experiment, both serum and plasma were significantly effective at reducing percent corneal weight loss in this model compared to positive control samples. No significant difference was found between feline (P=0.579), canine ( P=0.249) or equine (P=0.406) corneas incubated with serum or plasma with regards to percent corneal weight loss. Canine serum and plasma significantly reduced HP levels while inclusion of feline and equine serum or plasma did not, compared to positive controls. Levels of HP were moderately correlated with percent corneal weight loss for feline sample ( P=0.002), weakly correlated for equine samples (P=0.096) but were not correlated with percent corneal weight loss for canine samples (P=0.842). These studies confirm that both serum and plasma exert an anti-collagenase effect. The results of these studies suggest that serum can be stored for up to 6 months, at either -20°C or -80C°C without loss of anti-collagenase efficacy. The use of heterologous serum may be beneficial but further research is needed before any clinical recommendations can be made. Additionally this research indicates that plasma may be an acceptable substitute for serum as a topical corneal anti-collagenase treatment.