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 effects of different culture conditions on PCLS from cirrhotic animals has not been well characterized and there is no consensus on optimal methods. 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-transferrin-selenium and dexamethasone, and when incubated on a rocking platform. Culturing with insulin-transferrin-selenium 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 of key genes by day 2. Interestingly, we also observed that cirrhotic PCLS maintain viability in culture longer than slices from healthy animals. 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.

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

Fractional 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 toward 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. Healthy and cirrhotic rats were treated with a fractional laser then euthanized from 0 h up to 14 days 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. Healthy rats showed little damage beyond the initial microinjury and healed completely by 7 days without scarring. In cirrhotic rats, hepatocytes surrounding microinjury sites died 4-6 h 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 4 h. Laser-treated regions had reduced oxygen saturation and broadly disrupted perfusion of nodule microvasculature, which matched the zones of cell death. Our results demonstrate that the 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.

When Wounds Are Good for You: The Regenerative Capacity of Fractional Resurfacing and Potential Utility in Chronic Wound Prevention.

Significance: Fractional resurfacing involves producing arrays of microinjuries on the skin, by thermal or mechanical means, to trigger tissue regeneration. Originally developed for cosmetic enhancement, fractional resurfacing induces a broad array of improvements in the structural and functional qualities of the treated skin and is especially effective at returning defective skin to a more normal state. In addition to fascinating questions about the nature of this remarkable regenerative capacity, there may be potential utility in ulcer prevention by halting or even reversing the progressive decline in overall skin quality that usually precedes chronic wound development. Recent Advances: Photoaging and scarring are the two skin defects most commonly treated by fractional resurfacing, and the treatment produces profound and long-lasting improvements in skin quality, both clinically and at the cellular/histologic level. Chronic wounds usually occur in skin that is compromised by various pathologic factors, and many of the defects found in this ulcer-prone skin are similar to those that have seen improvements after fractional resurfacing. Critical Issues: The mechanisms responsible for the regenerative capacity of fractional resurfacing are mostly unknown, as is how ulcer-prone skin, which is usually afflicted by stressors external to the skin tissue itself, would respond to fractional resurfacing. Future Directions: Better understanding of the cellular and molecular mechanisms underlying the unique healing response to fractional resurfacing could reveal fundamental information about adult tissue regeneration, lead to improvements in current applications, as well as new therapies in other pathologic conditions.