Discrimination of lipid composition and cellular localization in human liver tissues by stimulated Raman scattering microscopy.

Significance: The molecular mechanisms driving the progression from nonalcoholic fatty liver (NAFL) to fibrosing steatohepatitis (NASH) are insufficiently understood. Techniques enabling the characterization of different lipid species with both chemical and spatial information can provide valuable insights into their contributions to the disease progression. Aim: We extend the utility of stimulated Raman scattering (SRS) microscopy to characterize and quantify lipid species in liver tissue sections from patients with NAFL and NASH. Approach: We applied a dual-band hyperspectral SRS microscopy system for imaging tissue sections in both the C-H stretching and fingerprint regions. The same sections were imaged with polarization microscopy for detecting birefringent liquid crystals in the tissues. Results: Our imaging and analysis pipeline provides accurate classification and quantification of free cholesterol, saturated cholesteryl esters (CEs), unsaturated CE, and triglycerides in liver tissue sections. The subcellular resolution enables investigations of the heterogeneous distribution of saturated CE, which has been under-examined in previous studies. We also discovered that the birefringent crystals, previously found to be associated with NASH development, are predominantly composed of saturated CE. Conclusions: Our method allows for a detailed characterization of lipid composition in human liver tissues and enables further investigation into the potential mechanism of NASH progression.

Genetic deletion or pharmacologic inhibition of the Nlrp3 inflammasome did not ameliorate experimental NASH.

It has been postulated that inflammasomes, in particular the NLRP3 (NLR family pyrin domain containing 3) inflammasome, mediate the necroinflammation and fibrosis that characterize nonalcoholic steatohepatitis (NASH) by engaging innate immune responses. We aimed to investigate the impact of genetic deletion or pharmacologic inhibition of the NLRP3 inflammasome on experimental steatohepatitis. Global Nlrp3 KO (expected to inhibit the NLRP3 inflammasome) or Casp1 KO (expected to inhibit all inflammasomes) mice were compared to wild type controls after 6 months on a high-fat, high-cholesterol (HFHC, 1% cholesterol) diet known to induce fibrosing steatohepatitis. Additionally, wildtype mice on a HFHC diet (0.75% or 0.5% cholesterol) for 6 months were either treated or not treated with an oral, pharmacologic inhibitor of Nlrp3 (MCC950) that was delivered in the drinking water (0.3 mg/ml). We found that genetic deletion or pharmacologic inhibition of the NLRP3 inflammasome did not ameliorate any of the histological components of fibrosing NASH in HFHC-fed mice. Collectively, these results do not support NLRP3 inhibition as a potential target for human NASH.

Role of Cholesterol-Associated Steatohepatitis in the Development of NASH.

The rising prevalence of nonalcoholic fatty liver disease (NAFLD) and NAFLD-related cirrhosis in the United States and globally highlights the need to better understand the mechanisms causing progression of hepatic steatosis to fibrosing steatohepatitis and cirrhosis in a small proportion of patients with NAFLD. Accumulating evidence suggests that lipotoxicity mediated by hepatic free cholesterol (FC) overload is a mechanistic driver for necroinflammation and fibrosis, characteristic of nonalcoholic steatohepatitis (NASH), in many animal models and also in some patients with NASH. Diet, lifestyle, obesity, key genetic polymorphisms, and hyperinsulinemia secondary to insulin resistance are pivotal drivers leading to aberrant cholesterol signaling, which leads to accumulation of FC within hepatocytes. FC overload in hepatocytes can lead to ER stress, mitochondrial dysfunction, development of toxic oxysterols, and cholesterol crystallization in lipid droplets, which in turn lead to hepatocyte apoptosis, necrosis, or pyroptosis. Activation of Kupffer cells and hepatic stellate cells by hepatocyte signaling and cholesterol loading contributes to this inflammation and leads to hepatic fibrosis. Cholesterol accumulation in hepatocytes can be readily prevented or reversed by statins. Observational studies suggest that use of statins in NASH not only decreases the substantially increased cardiovascular risk, but may ameliorate liver pathology. Conclusion: Hepatic FC loading may result in cholesterol-associated steatohepatitis and play an important role in the development and progression of NASH. Statins appear to provide significant benefit in preventing progression to NASH and NASH-cirrhosis. Randomized controlled trials are needed to demonstrate whether statins or statin/ezetimibe combination can effectively reverse steatohepatitis and liver fibrosis in patients with NASH.

Thyrotoxicosis and Choledocholithiasis Masquerading as Thyroid Storm.

A 26-year-old female, thirteen months postpartum, presented to the emergency department for four weeks of epigastric abdominal pain, pruritus, new onset jaundice, and 11.3 kgs (25 lbs) unintentional weight loss. On examination, she was afebrile, tachycardic, alert, and oriented and had jaundice with scleral icterus. Labs were significant for undetectable TSH, FT4 that was too high to measure, and elevated total bilirubin, direct bilirubin, alkaline phosphatase, and transaminases. Abdominal ultrasound revealed cholelithiasis without biliary ductal dilation. Treatment for presumed thyroid storm was initiated. Further work-up with magnetic resonance cholangiopancreatography (MRCP) revealed an obstructing cholelith within the distal common bile duct. With the presence of choledocholithiasis explaining the jaundice and abdominal pain, plus the absence of CNS alterations, the diagnosis of thyroid storm was revised to thyrotoxicosis complicated by choledocholithiasis. Endoscopic retrograde cholangiopancreatogram (ERCP) with sphincterotomy was performed to alleviate the biliary obstruction, with prompt symptomatic improvement. Thyroid storm is a rare manifestation of hyperthyroidism with a high rate of morbidity and mortality. The diagnosis of thyroid storm is based on clinical examination, and abnormal thyroid function tests do not correlate with disease severity. Knowledge of the many manifestations of thyroid storm will facilitate a quick and accurate diagnosis and treatment.