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.

Precision-Cut Liver Slices as an ex vivo model to evaluate antifibrotic therapies for liver fibrosis and cirrhosis.

Background: Precision-Cut Liver Slices (PCLS) are an ex vivo culture model developed to study hepatic drug metabolism. One of the main benefits of this model is that it retains the structure and cellular composition of the native liver. PCLS also represents a potential model system to study liver fibrosis in a setting that more closely approximates in vivo pathology than in vitro methods. The aim of this study was to assess whether responses to antifibrotic interventions can be detected and quantified with PCLS. Methods: PCLS of 250 µm thickness were prepared from four different murine fibrotic liver models: choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD), thioacetamide (TAA), diethylnitrosamine (DEN), and carbon tetrachloride (CCl4). PCLS were treated with 5 µM Erlotinib for 72 hours. Histology and gene expression were then compared with in vivo murine experiments and TGF-ß1 activated hepatic stellate cells (HSCs). These types of PCLS characterization were also evaluated in PCLS from human cirrhotic liver. Results: PCLS viability in culture was stable for 72 hours. Treatment of erlotinib, an EGFR inhibitor significantly inhibited the expression of profibrogenic genes Il6, Col1a1 and Timp1 in PCLS from CDAHFD-induced cirrhotic mice, and Il6, Col1a1 and Tgfb1 in PCLS from TAA-induced cirrhotic rats. Erlotinib treatment of PCLS from DEN-induced cirrhotic rats inhibited the expression of Col1a1, Timp1, Tgfb1 and Il6, which was consistent with the impact of erlotinib on Col1a1 and Tgfb1 expression in in vivo DEN-induced cirrhosis. Erlotinib treatment of PCLS from CCl4-induced cirrhosis caused reduced expression of Timp1, Col1a1 and Tgfb1, which was consistent with the effect of erlotinib in in vivo CCl4-induced cirrhosis. In addition, in HSCs at PCLS from normal mice, TGF-ß1 treatment upregulated Acta2 (αSMA), while treatment with erlotinib inhibited the expression of Acta2. Similar expression results were observed in TGF-ß1 treated in vitro HSCs. Expression of MMPs and TIMPs, key regulators of fibrosis progression and regression, were also significantly altered under erlotinib treatment in PCLS. Expression changes under erlotinib treatment were also corroborated with PCLS from human cirrhosis samples. Conclusion: The responses to antifibrotic interventions can be detected and quantified with PCLS at the gene expression level. The antifibrotic effects of erlotinib are consistent between PCLS models of murine cirrhosis and those observed in vivo and in vitro. Similar effects were also reproduced in PCLS derived from patients with cirrhosis. PCLS is an excellent model to assess antifibrotic therapies that is aligned with the principles of Replacement, Reduction and Refinement (3Rs).

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.

Optogenetic Peripheral Nerve Immunogenicity.

Optogenetic technologies have been the subject of great excitement within the scientific community for their ability to demystify complex neurophysiological pathways in the central (CNS) and peripheral nervous systems (PNS). The excitement surrounding optogenetics has also extended to the clinic with a trial for ChR2 in the treatment of retinitis pigmentosa currently underway and additional trials anticipated for the near future. In this work, we identify the cause of loss-of-expression in response to transdermal illumination of an optogenetically active peroneal nerve following an anterior compartment (AC) injection of AAV6-hSyn-ChR2(H134R) with and without a fluorescent reporter. Using Sprague Dawley Rag2-/- rats and appropriate controls, we discover optogenetic loss-of-expression is chiefly elicited by ChR2-mediated immunogenicity in the spinal cord, resulting in both CNS motor neuron death and ipsilateral muscle atrophy in both low and high Adeno-Associated Virus (AAV) dosages. We further employ pharmacological immunosuppression using a slow-release tacrolimus pellet to demonstrate sustained transdermal optogenetic expression up to 12 weeks. These results suggest that all dosages of AAV-mediated optogenetic expression within the PNS may be unsafe. Clinical optogenetics for both PNS and CNS applications should take extreme caution when employing opsins to treat disease and may require concurrent immunosuppression. Future work in optogenetics should focus on designing opsins with lesser immunogenicity.