Biology subjects

Leaker, B. D.

Publications and source records attributed to Leaker, B. D..

3 recordsLinked to original sources

bioRxiv2024

Interstitial infusion of purified collagenase Clostridium histolyticum in the cirrhotic liver causes rapid reduction in fibrosis with minimal liver toxicity

Crude mixtures of matrix-degrading enzymes called collagenase Clostridium histolyticum (CCH) have been used to efficiently breakdown tissue for many years. Recently, direct injection of purified CCH has been successfully developed as a treatment for Dupuytrens contracture (DC), a fibrotic disorder of the hand. Given similar histologic and mechanical features between the fibrous bands in DC and cirrhosis, a similar approach may be feasible for the treatment of cirrhosis. Crude and purified CCH were first compared through composition and substrate specificity. The biodistribution of a macromolecule delivered via interstitial infusion in the liver was mapped and quantified with a fluorescent dextran tracer to design a protocol for efficient delivery throughout the liver with minimal off-target exposure. Safety and efficacy of interstitial CCH infusion in the liver was investigated in cirrhotic mice using serum markers of injury and histological analysis of fibrosis. Purified CCH showed high purity and efficient degradation of type I collagen. Tracer experiments showed that interstitial infusion in one lobe of the liver will reach the entire organ in the mouse. A significant amount of the tracer was also found to enter the bloodstream where it is cleared by the kidneys, but was not found to significantly infiltrate other organs. Mild elevation in liver enzymes AST and ALT were observed 1d after infusion of purified CCH, but this was not significantly different than infusion with saline. No elevation in creatinine was observed. Cirrhotic mice infused with purified CCH showed 38% reduction in collagen proportionate area compared to mice infused with saline. These results show interstitial delivery of purified CCH in the cirrhotic liver rapidly decreases collagen content with minimal liver toxicity. This strategy merits further study as a potential treatment for cirrhosis.

bioengineering↗

bioRxiv2023

Increased susceptibility to ischemia causes exacerbated response to microinjuries in the cirrhotic liver

BackgroundFractional laser ablation is a technique developed in dermatology to induce remodeling of skin scars by creating a dense pattern of microinjuries. Despite remarkable clinical results, this technique has yet to be tested for scars in other tissues. As a first step towards determining the suitability of this technique, we aimed to (1) characterize the response to microinjuries in the healthy and cirrhotic liver, and (2) determine the underlying cause for any differences in response. MethodsHealthy and cirrhotic rats were treated with a fractional laser then euthanized from 0hr up to 14d after treatment. Differential expression was assessed using RNAseq with a difference-in-differences model. Spatial maps of tissue oxygenation were acquired with hyperspectral imaging and disruptions in blood supply were assessed with tomato lectin perfusion. ResultsHealthy rats showed little damage beyond the initial microinjury and healed completely by 7d without scarring. In cirrhotic rats, hepatocytes surrounding microinjury sites died 4-6hr after ablation, resulting in enlarged and heterogeneous zones of cell death. Hepatocytes near blood vessels were spared, particularly near the highly vascularized septa. Gene sets related to ischemia and angiogenesis were enriched at 4hr. Laser-treated regions had reduced oxygen saturation and broadly disrupted perfusion of nodule microvasculature, which matched the zones of cell death. ConclusionsThe cirrhotic liver has an exacerbated response to microinjuries and increased susceptibility to ischemia from microvascular damage, likely related to the vascular derangements that occur during cirrhosis development. Modifications to the fractional laser tool, such as using a femtosecond laser or reducing the spot size, may be able to prevent large disruptions of perfusion and enable further development of a laser-induced microinjury treatment for cirrhosis.

bioengineering↗

bioRxiv2023

Analysis of culture and RNA isolation methods for precision-cut liver slices from cirrhotic rats

Precision-cut liver slices (PCLS) are increasingly used as a model to investigate anti-fibrotic therapies. However, many studies use PCLS from healthy animals treated with pro-fibrotic stimuli in culture, which reflects only the early stages of fibrosis. The performance of PCLS from cirrhotic animals has not been well characterized and there is no consensus on optimal culture conditions. In this study, we report a method for the collection and culture of cirrhotic PCLS and compare the effect of common culture conditions on viability, function, and gene expression. Additionally, we compared three methods of RNA isolation and identified a protocol with high yield and purity. We observed significantly increased albumin production when cultured with insulin and dexamethasone, and when incubated on a rocking platform. Culturing with insulin and dexamethasone maintained gene expression closer to the levels in fresh slices. However, despite stable viability and function up to 4 days, we found significant changes in expression by day 2. Due to the influence of matrix stiffness on fibrosis and hepatocellular function, it is important to evaluate prospective anti-fibrotic therapies in a platform that preserves tissue biomechanics. PCLS from cirrhotic animals represent a promising tool for the development of treatments for chronic liver disease.

bioengineering↗