Contrast-Enhanced Tissue Processing of Fibrillin-Rich Elastic Fibres for 3D Visualization by Volume Scanning Electron Microscopy

Elastic fibres constitute an important component of the extracellular matrix and currently are the subject of intensive study in order to elucidate their assembly, function and involvement in cell–matrix interactions and disease. However, few studies to date have investigated the 3D architecture of the elastic fibre system in bulk tissue. We describe a protocol for preparation of tissue samples, including primary fixation and backscatter electron contrast-enhancement steps, through dehydration into stable resin-embedded blocks for volume electron microscopy. The use of low molecular weight tannic acid and alcoholic lead staining are critical stages in this procedure. Block preparation by ultramicrotomy and evaporative metal coating prior to microscopical examination are also described. We present images acquired from serial block face scanning electron microscopy of cornea and aorta showing target structures clearly differentiated from cells and other matrix components. The processing method imparts high contrast to fibrillin-containing elastic fibres, thus facilitating their segmentation and rendering into 3D reconstructions by image analysis software from large serial image datasets.

Collagen and elastic fibres in acute and chronic liver injury

The histological distinction between acute and chronic liver injury is a challenging aspect of liver histopathology. It is traditionally based on the interpretation of morphological changes to the extracellular matrix (ECM) at sites of hepatocyte loss using histochemical stains. Our aim was to investigate whether immunohistochemistry and multiplexing for collagen type (I & III) and elastic fibres and a modified Victoria blue method could be helpful. We studied 43 livers removed at transplantation for acute liver failure (ALF, 20 cases) or cirrhosis (23) plus 8 normal controls. In ALF the periportal ECM was normal in 2 cases, contained mainly collagen I associated with a ductular reaction in 6 cases, and delicate elastic strands in 11 cases. Periportal deposition of mainly collagen I and mature elastic fibres was observed in cirrhosis. In ALF the perisinusoidal ECM was intact in 4 cases, collapsed or condensed but of normal composition (predominantly collagen III) in 2 cases, and collapsed and condensed containing mostly collagen I in 17 cases (7 including delicate immature elastic strands). In contrast, bridging fibrous septa of cirrhosis contained abundant collagen 1 and bundles of mature elastin. We propose the use of a scale and the use of immunohistochemistry and multiplexing in additional to histochemical stains to characterise the ECM changes in acute and chronic liver injury.

Changes in the expression levels of elastic fibres in yak lungs at different growth stages

Background Yaks have a strong adaptability to the plateau environment, which can be attributed to the effective oxygen utilization rate of their lung tissue. Elastic fibre confers an important adaptive structure to the alveolar tissues in yaks. However, little research has been focused on the structural development of lung tissues and the expression levels of elastic fibres in yaks after birth. Therefore, this study aimed to investigate the morphological changes of elastic fibers and expression profiles of fibre-formation genes in yak lungs at different growth stages and the relationship between these changes and plateau adaptation. Results Histological staining was employed to observe the morphological changes in the lung tissue structure of yaks at four different ages: 1 day old, 30 days old, 180 days old and adult. There was no significant difference in the area of a single alveolus between the 1-day-old and 30-day-old groups (P-value > 0.05). However, the single alveolar area was gradually increased with an increase in age (P-value < 0.05). Elastic fibre staining revealed that the amount of elastic fibres in alveolar tissue was increased significantly from the ages of 30 days to 180 days (P-value < 0.05) and stabilized during the adult stage. Transcriptome analysis indicated that the highest levels of differentially expressed genes were found between 30 days of age and 180 days of age. KEGG analysis showed that PI3K-Akt signalling pathway and MAPK pathway, which are involved in fibre formation, accounted for the largest proportion of differentially expressed genes between 30 days of age and 180 days of age. The expression levels of 36 genes related to elastic fibre formation and collagen fibre formation were also analysed, and most of these genes were highly expressed in 30-day-old and 180-day-old yaks. Conclusions The content of elastic fibres in the alveolar tissue of yaks increases significantly after birth, but this change occurs only from 30 days of age to 180 days of age. Our study indicates that elastic fibres can improve the efficiency of oxygen utilization in yaks under harsh environmental conditions.

Collagen and Elastic Fibres in Acute and Chronic Liver Injury.

The histological distinction between acute and chronic liver injury is a challenging aspect of liver histopathology. It is traditionally based on the interpretation of morphological changes to the extracellular matrix (ECM) at sites of hepatocyte loss using histochemical stains. Our aim was to investigate whether immunohistochemistry and multiplexing for collagen type (I & III) and elastic fibres and a modified Victoria blue method could be helpful. We studied 43 livers removed at transplantation for acute liver failure (ALF, 20 cases) or cirrhosis (23) plus 8 normal controls. In ALF the periportal ECM was normal in 2 cases, contained collagen I associated with a ductular reaction in 6 cases, and delicate elastic strands in 11 cases. Periportal deposition of collagen I and mature elastic fibres was observed in cirrhosis. In ALF the perisinusoidal ECM was intact in 4 cases, collapsed or condensed but of normal composition (collagen III) in 2 cases, and collapsed and condensed containing collagen I in 17 cases (7 including delicate immature elastic strands). In contrast, bridging fibrous septa of cirrhosis contained abundant collagen 1 and bundles of mature elastin. We propose the use of a scale and the use of immunohistochemistry and multiplexing in additional to histochemical stains to characterise the ECM changes in acute and chronic liver injury.

Changes in Elastic Fibres in Yak Lungs at Different Developmental Stages

BackgroundYaks have a strong adaptability to the plateau environment, which is closely associated with the effective oxygen utilization rate of their lung tissue. The elastic fibre is an important adaptive structure of alveolar tissue. However, there are few studies on the development of the structure of lung tissue and the changes in elastic fibres of yak after birth. The purpose of this study was to investigate the changes of elastic fibers in the lungs of yaks after birth and the relationship between these changes and adaptation to hypoxic environment.ResultsIn this experiment, a histological method was employed to observe the changes in the lung tissue structure of yaks at four ages: 1 day old, 30 days old, 180 days old and adult. There was no significant difference in the area of a single alveolus between the 1-day-old and 30-day-old groups (P > 0.05). In yaks aged over 30 days, the single alveolar area gradually increased with age (P < 0.05). The observation of elastic fibres showed that elastic fibres in alveolar tissue increased significantly from the ages of 30 days to 180 days (P < 0.05) and stabilized after 180 days of age. Transcriptome analysis determined the highest levels of differentially expressed genes between 30 days of age and 180 days of age. KEGG analysis showed that the PI3K-Akt signalling pathway and MAPK pathway, which are involved in fibre formation, accounted for the largest proportion of differentially expressed genes between 30 days of age and 180 days of age. The expression levels of 36 genes related to fibre formation were analysed, and several genes related to elastic fibre formation and collagen fibre formation were determined to be highly expressed at the age of 30 days.ConclusionsThe content of elastic fibres in the alveolar tissue of yaks increases significantly after birth, but this change occurs only from 30 days of age to 180 days of age to make better use of oxygen in the environment.

Mendelian randomization and experimental IUGR reveal the adverse effect of low birth weight on lung structure and function

Intrauterine growth restriction (IUGR) and low birth weigth (LBW) are risk factors for neonatal chronic lung disease. However, maternal and fetal genetic factors and the molecular mechanisms remain unclear. We investigated the relationship between LBW and lung function with Mendelian randomisation analyses and studied angiogenesis in a low protein diet rat model of IUGR. Our data indicate a possible association between LBW and reduced FEV1 (p = 5.69E−18, MR-PRESSO) and FVC (6.02E-22, MR-PRESSO). Complimentary, we demonstrated two-phased perinatal programming after IUGR. The intrauterine phase (embryonic day 21) is earmarked by a reduction of endothelial cell markers (e.g. CD31) as well as mRNA expression of angiogenic factors (e.g., Vegfa, Flt1, Klf4). Protein analysis identified an activation of anti-angiogenic mTOR effectors. In the postnatal phase, lung capillaries (< 20 µm) were significantly reduced, expression of CD31 and VE-Cadherin were unaffected, whereas SMAD1/5/8 signaling and Klf4 protein were increased (p < 0.01). Moreover, elevated proteolytic activity of MMP2 and MMP9 was linked to a 50% reduction of lung elastic fibres. In conclusion, we show a possible link of LBW in humans and reduced lung function in adulthood. Experimental IUGR identifies an intrauterine phase with inhibition of angiogenic signaling, and a postnatal phase with proteolytic activity and reduced elastic fibres.

Elastic fibres in alcoholic liver disease

The literature on the contribution of elastic fibre deposition to alcohol-related liver disease (ARLD) is limited. We studied: (1) 180 liver biopsies from ARLD patients; (2) 20 ARLD explant livers; (3) 213 liver biopsies with non-ARLD injury. Elastic fibres were assessed in terms of their distribution around hepatocytes [pericellular elastosis (PCE)] and within bridging fibrous septa (septal elastosis) and scored using a semiquantitative system. We also investigated the composition of the elastic fibres (oxytalan, elaunin and mature elastic fibres) in 20 cases. PCE was associated with steatohepatitis in ARLD patients and with ARLD when compared to non-ARLD cases (p < 0.001). Oxytalan fibres were identified in PCE in ARLD biopsies and broken dense perisinusoidal mature elastic fibres in explanted livers. Septal elastosis increased from intermediate to advanced fibrosis stage. Early septal elastosis contained oxytalan fibres, whereas septal elastosis at more advanced stages contained mainly mature elastic fibres. PCE is a typical feature of steatohepatitis in ARLD and includes oxytalan fibres. Septal elastosis is a gradual process with a transition from oxytalan to mature elastic fibres usually present in explanted livers. There may be different dynamics in the assembly and reabsorption of pericellular and septal elastic fibres, and a potential role for stratification of patients with advanced stage ARLD.