Combination of Violet Light Irradiation and Collagenase Treatments in a Rabbit Model of Keratoconus

Purpose: We evaluated the use of collagenase treatment to generate a rabbit model of keratoconus and the impact of violet light (VL) irradiation on the disease model in six Japanese White rabbits. Methods: After epithelial debridement, the collagenase group was treated with a collagenase type II solution for 30 min; the control group was treated with a solution without collagenase. Three rabbits also underwent VL irradiation (375 nm, irradiance 310 μW/cm2) for 3 hours daily for 7 days after topical collagenase application. Slit-lamp microscopy results, steep keratometry (Ks), corneal astigmatism, central corneal thickness, and axial length were examined before and after the procedure. The corneas were obtained on day 7 for biomechanical evaluation. Results: A significant increase in Ks and corneal astigmatism was observed in the collagenase and VL irradiation groups compared with the control group at day 7. No significant difference was found in the change in corneal thickness between the groups. The elastic modulus at 10% strain, but not at 3% and 5% strain, was significantly lower in the collagenase group than in the control group. There was no significant difference in the elastic modulus at each level of strain between the collagenase and VL irradiation groups. The average axial length at day 7 was significantly longer in the collagenase group than in the control group. Collagenase treatment induced a keratoconic model by steepening the keratometric and astigmatic values. There was no significant difference in the observed elastic behaviour of normal and ectatic corneas under physiologically relevant stress levels. Conclusion: VL irradiation did not cause regression of corneal steepening in collagenase-induced model during short-term observation.

Clostridium Collagenase Impact on Zone of Stasis Stabilization and Transition to Healthy Tissue in Burns

Clostridium collagenase has provided superior clinical results in achieving digestion of immediate and accumulating devitalized collagen tissue. Recent studies suggest that debridement via Clostridium collagenase modulates a cellular response to foster an anti-inflammatory microenvironment milieu, allowing for a more coordinated healing response. In an effort to better understand its role in burn wounds, we evaluated Clostridium collagenase’s ability to effectively minimize burn progression using the classic burn comb model in pigs. Following burn injury, wounds were treated with Clostridium collagenase or control vehicle daily and biopsied at various time points. Biopsies were evaluated for factors associated with progressing necrosis as well as inflammatory response associated with treatment. Data presented herein showed that Clostridium collagenase treatment prevented destruction of dermal collagen. Additionally, treatment with collagenase reduced necrosis (HMGB1) and apoptosis (CC3a) early in burn injuries, allowing for increased infiltration of cells and protecting tissue from conversion. Furthermore, early epidermal separation and epidermal loss with a clearly defined basement membrane was observed in the treated wounds. We also show that collagenase treatment provided an early and improved inflammatory response followed by faster resolution in neutrophils. In assessing the inflammatory response, collagenase-treated wounds exhibited significantly greater neutrophil influx at day 1, with macrophage recruitment throughout days 2 and 4. In further evaluation, macrophage polarization to MHC II and vascular network maintenance were significantly increased in collagenase-treated wounds, indicative of a pro-resolving macrophage environment. Taken together, these data validate the impact of clostridial collagenases in the pathophysiology of burn wounds and that they complement patient outcomes in the clinical scenario.

Applicability of Artificial Vascularized Liver Tissue to Proteomic Analysis

Artificial vascularized tubular liver tissue has perfusable blood vessels that allow fluid access to the tissue interior, enabling the injection of drugs and collection of metabolites, which are valuable for drug discovery. It is amenable to standard evaluation methods, such as paraffin-embedded sectioning, qPCR, and RNA sequencing, which makes it easy to implement into existing research processes. However, the application of tissues vascularized by the self-assembly of cells, (including tubular liver tissue, has not yet been tested in comprehensive proteomic analysis relevant for drug discovery. Here, we established a method to efficiently separate cells from the tubular liver tissue by adding a pipetting step during collagenase treatment. By using this method, we succeeded in obtaining a sufficient number of cells for the proteomic analysis. In addition, to validate this approach, we compared the cells separated from the tissue with those grown in 2D culture, focusing on the proteins related to drug metabolism. We found that the levels of proteins involved in metabolic phases II and III were slightly higher in the tubular liver tissue than those in the 2D cell culture. Taken together, our suggested method demonstrates the applicability of tubular liver tissue to the proteomic analysis in drug assays.