Electron microscopic observations in perfusion-fixed human non-alcoholic fatty liver disease biopsies

Abstract

Non-alcoholic fatty liver disease (NAFLD) is a widespread liver disease in Western society, but its multifactorial pathogenesis is not yet fully understood. Ultrastructural analysis of liver sinusoidal endothelial cells (LSECs) in animal models and in vitro studies shows defenestration early in the course of NAFLD, promoting steatosis. LSECs and fenestrae are important in the transport of lipids across the sinusoids. However, human ultrastructural data, especially on LSECs and fenestrae, are scarce. This study aimed to explore the ultrastructural changes in perfusion type fixed liver biopsies of NAFLD patients with and without nonalcoholic steatohepatitis (NASH), with a special focus on LSECs and their fenestration. Liver biopsies from patients with NAFLD were fixed using two perfusion techniques, jet and injection fixation, for needle and wedge biopsies, respectively. Ultrastructural changes were studied using transmission electron microscopy. NASH was diagnosed by bright-field microscopy using the SAF score (steatosis, activity, fibrosis). Thirty-seven patients were included, of which 12 (32.4%) had NASH. Significantly less defenestration was found in NASH compared to noNASH samples (p=0.002). Other features, i.e., giant mitochondria and fenestrae size did not differ between groups. Furthermore, we described new structures, i.e., single cell steatonecrosis and inflammatory fat follicles (IFF) that were observed in both groups. Concluding, defenestration was more common in noNASH compared to NASH in human liver samples. Defenestration was not related to the degree of steatosis or fibrosis. We speculate that defenestration can be a protective mechanism in simple steatosis which is lacking in NASH.

Overlooked subclinical portal hypertension in non-cirrhotic NAFLD: Is it real and how to measure it?

“…At physiological perfusion pressures, sinusoidal fenestrae act as a mechanical sieve to macromolecules and cellular projections that may come into contact with the microvilli of hepatocytes across the space of Disse. As recently reported, loss of fenestration in LSECs is more commonly seen in isolated steatosis compared to steatohepatitis [33]. These surprising observations suggest that in some situations loss of fenestration could be a protective mechanism intended to prevent exposure of hepatocytes to excess large molecules of lipids or albumin-bound small molecules such as toxic substances or microbial metabolites received through the portal circulation [33].…”

Fat causes necrosis and inflammation in parenchymal cells in human steatotic liver

“…Naturally, this indicates defenestration that was apparently present in these livers. Importantly, fenestrae were again present in NASH livers (Verhaegh et al 2021) (Fig. 6a, b).…”

“…Reported defenestration in steatotic livers results in the creation of capillaries with more or less continuous endothelium (DeLeve 2015;Horn et al 1987;Schaffner et al 1963a). Importantly, defenestration occurs in early phases in NAFLD patients (Verhaegh et al 2021). However, according to our present observations, fenestrae return in NASH (Fig.…”

“…They were collected mainly during bariatric surgery as part of a project approved by the Medical Ethics Committee Zuyd, registered at clinicaltrials.gov (NCT02717000). All participants gave written informed consent prior to their participation (Verhaegh et al 2021). The study was performed according to the Declaration of Helsinki (latest amendment of 2013, Fortaleza, Brazil).…”

Remodeling of Mitochondrial Plasticity: The Key Switch from NAFLD/NASH to HCC

“…However, NASH patients, much more than NAFLD ones, showed higher mitochondrial diameters, intra-mitochondria crystalline inclusions and granules in the matrix which correlated with both mitochondrial swelling and OXPHOS failure [85]. Recently, Verhaegh et al have observed ultrastructural changes by using transmission electron microscopy in 37 NAFLD patients, of whom 12 had NASH [88]. In this study, the presence of giant mitochondria showed no differences between patients with or without NASH, according to the results of Ahishali et al [87], supporting that the appearance of megamitochondria could represent a transitory phase between simple steatosis and NASH.…”