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Podocyte injury is an important factor in the pathogenesis of diabetic nephropathy. Podocytes are characterized by large numbers of mitochondria. However, mitochondrial dysfunction as it relates to kidney pathology remains poorly understood. The present study found that podocyte mitochondria in different animal models of diabetes mellitus became elongated with the development of albuminuria, suggesting a change in mitochondrial dynamics. We then treated cells with a combination of glucose, fatty acids, and angiotensin II (GFA) to mimic the diabetic milieu. Cultured podocytes exposed to GFA showed megamitochondria formation and decreased autophagosome degradation. We also found that GFA treatment decreased the binding of the autophagosome to the lysosome. Our results suggest that megamitochondria are common in podocytes during diabetic nephropathy and that insufficient autophagy flux may underlie this effect. These findings have expanded our understanding of the pathogenesis of diabetic nephropathy and identified a potential pharmacological target for treatment.

Giant mitochondria are peculiarly shaped, extremely large mitochondria in hepatic parenchymal cells, the internal structure of which is characterised by atypically arranged cristae, enlarged matrix granules and crystalline inclusions. The presence of giant mitochondria in human tissue biopsies is often linked with cellular adversity, caused by toxins such as alcohol, xenobiotics, anti-cancer drugs, free-radicals, nutritional deficiencies or as a consequence of high fat Western diets. To date, non-alcoholic fatty liver disease is the most prevalent liver disease in lipid dysmetabolism, in which mitochondrial dysfunction plays a crucial role. It is not well understood whether the morphologic characteristics of giant mitochondria are an adaption or caused by such dysfunction. In the present study, we employ a complementary multimodal imaging approach involving array tomography and transmission electron tomography in order to comparatively analyse the structure and morphometric parameters of thousands of normal- and giant mitochondria in four patients diagnosed with non-alcoholic fatty liver disease. In so doing, we reveal functional alterations associated with mitochondrial gigantism and propose a mechanism for their formation based on our ultrastructural findings.

Acute alcoholic microvesicular steatosis (MIC) may complicate heavy alcohol intake and present as alcoholic hepatitis (AH) syndrome. However, detailed clinical, biological, and histologic data associated with MIC are scarce. We compared the clinical presentation, histologic features, and hepatic transcriptomic of patients presenting with AH due to either MIC or severe alcoholic steatohepatitis (ASH). In this case‐control study, patients who drank heavily (>100 g/day) with the AH syndrome were included either in the MIC group (>50% severe microvesicular steatosis, no inflammation) or in the severe ASH group (polynuclear neutrophil infiltration, macrosteatosis, ballooned hepatocytes). All patients received standard supportive care plus steroids for those with severe ASH and were followed up for 3 months. Whole‐liver transcriptome profiling was performed on liver snap‐frozen biopsies. Compared to ASH (n = 24, mean age 49.3 years), patients in the MIC group (n = 12, mean age 49.1 years) had a higher reported alcohol intake (P

Considering the increase in cases of non-alcoholic steatohepatitis (NASH), the use of appropriate animal model of NASH is essential to understand the underlying pathogenesis mechanism. To date, several mice models have been used; however, significant differences in the etiologies and food administered affected the results, with inconsistent conclusions. Therefore, it is necessary to understand these models and their differences to be able to choose appropriate models. Inspired by the fact that mitochondrial (mt)DNA content is changed in non-alcoholic fatty liver disease in humans, we investigated the mtDNA copy number in the NASH mice models induced by high-fat diet (HFD) and methionine/choline-deficient diet (MCD) to understand the differences between these models. Megamitochondria were observed in both MCD and HFD groups. However, the MCD group showed significant decrease in liver mtDNA content compared with that in the HFD group. These changes were associated with significant upregulation of mitochondrial biogenesis- and degradation-related genes in MCD model than in HFD model. Thus, stability of mtDNA is associated with the differences between MCD and HFD-induced NASH models often used in studies; these findings could help in choosing appropriate models for studies on NASH.

In histological studies on liver stiffness (LS), fibrosis shows the closest association with correlation coefficients ranging from 0.7 to 0.8. These fibrosis features include width and number of septa. Other extracellular deposits of elevated LS include amyloid. Fibrosis features are followed by signs of ballooning, accumulation of Mallory’s hyaline in the now called Mallory–Denk bodies that are largely stuffed with CK18 cytokeratin filaments. Features of ballooning are followed by signs of inflammation such as infiltrates of neutrophils or other inflammatory cells. In the context of inflammation, it is highly conceivable that both recruitment of more cells and increased perfusion (pressure) will cause LS elevation. Inflammation is followed by other histological features of cell death that are significantly associated with LS: microgranulomas, acidophil bodies, megamitochondria, glycogenated nuclei, and large lipo-granulomas. Notably, steatosis in form of large and small lipid droplets is not associated with LS. Some controversies may result from the fact that in many biopsied cohorts, steatosis often coexists with liver damage. When ruling out these confounders, fat can even have a negative impact on stiffness. Since steatosis is low in normal, healthy livers but also end-stage cirrhotic livers, it remains to be studied whether viscoelastic properties of fat have a biomechanical beneficial effect on disease progression.

Tree shrews are small mammals with excellent vision and are closely related to primates. They have been used extensively as a model for studying refractive development, myopia, and central visual processing and are becoming an important model for vision research. Their cone dominant retina (∼95% cones) provides a potential avenue to create new damage/disease models of human macular pathology and to monitor progression or treatment response. To continue the development of the tree shrew as an animal model, we provide here the first measurements of higher order aberrations along with adaptive optics scanning light ophthalmoscopy (AOSLO) images of the photoreceptor mosaic in the tree shrew retina. To compare intra-animal in vivo and ex vivo cone density measurements, the AOSLO images were matched to whole-mount immunofluorescence microscopy. Analysis of the tree shrew wavefront indicated that the optics are well-matched to the sampling of the cone mosaic and is consistent with the suggestion that juvenile tree shrews are nearly emmetropic (slightly hyperopic). Compared with in vivo measurements, consistently higher cone density was measured ex vivo, likely due to tissue shrinkage during histological processing. Tree shrews also possess massive mitochondria ("megamitochondria") in their cone inner segments, providing a natural model to assess how mitochondrial size affects in vivo retinal imagery. Intra-animal in vivo and ex vivo axial distance measurements were made in the outer retina with optical coherence tomography (OCT) and transmission electron microscopy (TEM), respectively, to determine the origin of sub-cellular cone reflectivity seen on OCT. These results demonstrate that these megamitochondria create an additional hyper-reflective outer retinal reflective band in OCT images. The ability to use noninvasive retinal imaging in tree shrews supports development of this species as a model of cone disorders.

Although chronic hepatitis C virus (HCV) infection affect 185 million people world-wide, pathomechanism of liver damage is still unclear. Electron microscopy can reveal liver injury in very early stage and help understanding the mechanisms that is crucial in the pathogenesis of chronic hepatitis C. We present the morphological changes in the liver of HCV infected 24-year-old female patient, using light and transmission electron microscopy. Examination by TEM revealed wide range of specific subcellular abnormalities in hepatocellular ultrastructure. The most common observed changes were ring-shaped nuclei with intranuclear inclusion, megamitochondria, and “membranous web” structures – the hallmark of RNA-viruses infection.

The renal biopsy is a dynamic way of looking at renal disease, and tubular elements are an important part of this analysis. The mitochondria in 20 renal biopsies were examined by immunohistochemical (electron transport chain enzyme: cytochrome C oxidase IV [COX IV]) and enzyme histochemical methods (COX), both by light and electron microscopy. The distal convoluted tubules and thick ascending limbs showed the greatest intensity in the COX immunostains and enzyme activity in controls. The degree of mitochondrial COX protein and enzyme activity diminished as the tubules became atrophic. With proximal hypertrophic changes, there was great variation in both COX activity and protein expression. In contrast, in three cases of systemic lupus erythematosus, biopsied for high-grade proteinuria, the activity was consistently upregulated, whereas protein expression remained normal. These unexpected findings of heterogeneous upregulation in hypertrophy and the dyssynchrony of protein expression and activity may indicate mitochondrial dysregulation. Functional electron microscopy showed COX activity delineated by the intense mitochondrial staining in normal or hypertrophic proximal tubules. With atrophic changes, residual small mitochondria with diminished activity could be seen. With mitochondrial size abnormalities (enlargement and irregularity, adefovir toxicity), activity persisted. In the renal biopsy, mitochondrial analysis is feasible utilizing immunohistochemical and enzyme histochemical techniques.

Background Alcohol-attributable burden on global health is increasing, and the relationship between population alcohol consumption and liver-related deaths is strong. Longstanding scientific and clinical work has led to a relatively thorough, if not complete, understanding of the effects of alcohol consumption on the liver. Clinical and histological features of alcoholic liver disease (ALD) are used to assist clinicians in diagnosing and determining the severity of disease in patients with ALD. In this study, we provide salient points on their clinical and histological features. In addition, the benefits of liver biopsy and important differential diagnoses, including features distinguishing these entities. Aims This study intends to describe in detail the clinical and histological features of ALD. Methods Liver biopsy was required for the diagnosis of histologic ALD. A total of 83 patients with ALD were included in the study. Evaluate the stage of liver fibrosis according to Metavir classification (F0,F1,F2,F3,F4). Clinical and laboratory parameters were recorded. Results The average age was 51.41±9.69 years, the age group of 40–49 accounted for 43.4%. 83 patients included in the study were males. Clinical symptoms were hepatomegaly (62.7%), jaundice (67.5%). Histological results were mostly fatty liver (92.8%), significant fibrosis (F≥F2) accounted for 63.9%, foamy degeneration accounted for 84.3%, megamitochondria accounted for 63.4% and Mallory bodies accounted for 60.2%. Conclusions Patients in working age were often common. Common clinical symptoms were hepatomegaly and jaundice. The patients had salient points on histological features of ALD such as foamy degeneration, megamitochondria, Mallory body, fatty liver, significant fibrosis liver, widespread neutrophilic infiltration. Liver biopsy played an important role in the real-time clinical assessment of patients. Tissue histology not only yields diagnostic information but also important information about the patient‘s overall disease progression.

A comparative electron-microscopic study of ultrastructure of mitochondria in skeletal muscles of the 3- and 24-month-old Wistar and OXYS rats revealed age-dependent changes in both general organization of the mitochondrial reticulum and ultrastructure of mitochondria. The most pronounced ultrastructure changes were detected in the OXYS rats suffering from permanent oxidative stress. In the OXYS rats, significant changes in mitochondrial ultrastructure were detected already at the age of 3 months. Among them, there were the appearance of megamitochondria and reduction of cristae resulting in formation of cristae-free regions inside mitochondria. In the 24-month-old OXYS rats, mitochondrial reticulum was completely destroyed. In the isotropic region of muscle fiber, only small solitary mitochondria were present. There appeared regions of lysed myofibrils as well as vast regions filled with autophagosomes. A mitochondrial antioxidant SkQ1 (given to rats with food daily in the dose of 250 nmol/kg of body weight for 5 months beginning from the age of 19 months) prevented development of age-dependent destructive changes in both the Wistar and OXYS rats.

A 14-year-old boy with mitochondrial neurogastrointestinal encephalopathy (MNGIE) disease had a lifelong history of failure to thrive and gastrointestinal symptoms including vomiting, pain, and diarrhea, leading to progressive cachexia. At the age of 9 years, after an extensive workup, the diagnosis of Crohn disease was strongly suspected, and he underwent colonoscopy with multiple biopsies. At 11 years of age, vision change and poor balance lead to a diagnosis of leukodystrophy by magnetic resonance imaging. Investigations for metachromatic leukodystrophy, adrenal leukodystrophy, and globoid cell leukodystrophy were all negative. A diagnosis of MNGIE disease was suspected when he continued deteriorating with gastrointestinal symptoms, multiple neurologic deficits, and encephalopathy. Markedly diminished thymidine phosphorylase activity and increased thymidine plasma levels confirmed the diagnosis of MNGIE. At autopsy, megamitochondria were observed by light microscopy in submucosal and myenteric ganglion cells and in smooth muscle cells of muscularis mucosae and muscularis propria, along the entire gastrointestinal tract from the esophagus to the rectum. Megamitochondria in ganglion cells were also observed in a retrospective review of the endoscopic intestinal biopsies taken at age 9 and 13 years and in the appendectomy specimen obtained 1 month before his demise. This study corroborates the presence of megamitochondria in gastrointestinal ganglion cells in MNGIE disease, better illustrates their detailed morphology, and describes for the first time similar structures in the cytoplasm of gastrointestinal smooth muscle cells. Pathologists should be able to recognize these structures by light microscopy and be aware of their association with primary mitochondriopathies.

The effects of water-dispersed Ag nanoparticles on the small intestinal mucosa, liver, and spleen of rats were studied by transmission electron microscopy. Acute experiments demonstrated penetration of Ag nanoparticles injected into the isolated intestinal loop into the intestinal mucosa, liver, and splenic tissues. Ultrastructural changes (lobed nucleus, megamitochondria) were found in the studied organs. These data indicated that injection of water-dispersed Ag nanoparticles into the gastrointestinal tract was followed by their penetration through the epithelium of the small intestinal mucosa into other organs, e.g. into the liver and spleen. This fact is essential for evaluation of potential risks of the nanoparticle effects on human health and environment.

Familial dilated cardiomyopathy is a primary myocardial disease that can result in the development of congestive heart failure and sudden cardiac death. Spontaneous animal models of familial dilated cardiomyopathy exist and the Doberman pinscher dog is one of the most commonly reported canine breeds. The objective of this study was to evaluate familial dilated cardiomyopathy in the Doberman pinscher dog using a genome-wide association study for a genetic alteration(s) associated with the development of this disease in this canine model. Genome-wide association analysis identified an area of statistical significance on canine chromosome 14 (p(raw) = 9.999e-05 corrected for genome-wide significance), fine-mapping of additional SNPs flanking this region localized a signal to 23,774,190-23,781,919 (p = 0.001) and DNA sequencing identified a 16-base pair deletion in the 5' donor splice site of intron 10 of the pyruvate dehydrogenase kinase 4 gene in affected dogs (p < 0.0001). Electron microscopy of myocardium from affected dogs demonstrated disorganization of the Z line, mild to moderate T tubule and sarcoplasmic reticulum dilation, marked pleomorphic mitochondrial alterations with megamitochondria, scattered mitochondria with whorling and vacuolization and mild aggregates of lipofuscin granules. In conclusion, we report the identification of a splice site deletion in the PDK4 gene that is associated with the development of familial dilated cardiomyopathy in the Doberman pinscher dog.

Background and Aims: Non-alcoholic fatty liver disease has long been accepted as benign; however, recent evidence suggests that the disease may progress to cirrhosis and hepatocellular carcinoma, although the natural course of the disease is still unclear. This study was designed to comparatively evaluate electron microscopic features of non-alcoholic fatty liver (NAFL) and non-alcoholic steatohepatitis (NASH). Methods: Quantitative and semi-quantitative ultrastructural evaluations were performed on liver biopsies from 23 patients, 10 with NAFL and 13 with NASH. Results: No statistically significant difference was noted between NAFL and NASH patients in ultrastructural features of hepatocytes including megamitochondria, intramitochondrial crystalline inclusions, mitochondrial matrix granules, foamy cytoplasmic appearance, electron-lucent and glycogen-containing nuclear regions, lipofuscin granules, or an increased frequency of vesicles containing electron-dense material in peribiliary Golgi zone; however, the mitochondrial diameter was significantly higher in the NASH patients. Intercellular distance and microvilli between hepatocytes, collagen and electron-dense material accumulation in the space of Disse, electron-dense material accumulation and microvillus density in bile canaliculi did not differ significantly between the groups. Conclusions: Our data show that, although NAFL and NASH can be distinguished by their distinct light microscopic features, ultrastructural characteristics are similar, which suggests that NAFL may also have the potential to progress to fibrosis and cirrhosis like NASH.

To show what damage occurs in the hepatocytes of psoriatics receiving Methotrexate (MTX) therapy liver biopsies from 24 psoriatics with severe psoriasis before and after MTX therapy were studied blind by light and electron microscopy. We also aimed to determine the severity of lesions and find possible correlations, and to seek a relationship of these observations with light microscopical and clinical findings. The present study has shown that MTX probably caused damage to the hepatocytes reflected in the membrane whorls (p less than 0.05) and the accumulation of lipid droplets (p less than 0.05). There was an increase (p less than 0.05) in autophagic vacuoles, which mostly contained glycogen and cell sap with residual bodies. These residual bodies were then found in an increased number in the nearby Kupffer cells. Crystals were found in megamitochondria in most patients before and after MTX therapy. Some of these crystals were found free in the cytoplasm. Mitochondria containing crystals were shown in autophagic vacoules, representing possible pathways of their breakdown. Bile canaliculi commonly contained debris but only one patient had evidence of cholestasis. There was no significant change in nuclei, Golgi apparatus, or the endoplasmic reticulum and no statistically significant correlation between the shown changes and the total dose of MTX given.

Obesity and alcohol consumption are risk factors for hepatic steatosis, and both commonly coexist. Our objective was to evaluate the effect of ethanol and acetaldehyde on primary hepatocytes obtained from mice fed for two days with a high cholesterol (HC) diet. HC hepatocytes increased lipid and cholesterol content. HC diet sensitized hepatocytes to the toxic effect of ethanol and acetaldehyde. Cyp2E1 content increased with HC diet, as well as in those treated with ethanol or acetaldehyde, while the activity of this enzyme determined in microsomes increased in the HC and in all ethanol treated hepatocytes, HC and CW. Oxidized proteins were increased in the HC cultures treated or not with the toxins. Transmission electron microscopy showed endoplasmic reticulum (ER) stress and megamitochondria in hepatocytes treated with ethanol as in HC and the ethanol HC treated hepatocytes. ER stress determined by PERK content was increased in ethanol treated hepatocytes from HC mice and CW. Nuclear translocation of ATF6 was observed in HC hepatocytes treated with ethanol, results that indicate that lipids overload and ethanol treatment favor ER stress. Oxidative stress, ER stress, and mitochondrial damage underlie potential mechanisms for increased damage in steatotic hepatocyte treated with ethanol.

Aim: To document the histological appearances of liver biopsies in autochthonous hepatitis E virus (HEV) infection. Methods and results: Four patients were serologically positive for HEV; three had no traditional risk factors, the fourth had recently returned from China. All four consumed meat products. Liver histology of the three autochthonous (locally acquired) cases showed portal tracts expanded by a severe mixed polymorph and lymphocytic inflammatory infiltrate, with a geographical distribution of polymorphs at the interface and lymphocytes centrally. Moderate to severe interface hepatitis and cholangiolitis were present. There was a striking acinar mixed inflammatory infiltrate made up of polymorphs, lymphocytes and macrophages; frequent apoptotic hepatocytes, focal necrosis, cholestatic rosettes and zone 3 canalicular and cytoplasmic bilirubinostasis were noted. Significant steatosis, megamitochondria and Mallory bodies were not present. There was no evidence of iron, copper or α1-antitrypsin accumulation. By contrast, the histology of the imported case of HEV infection showed less intense portal and acinar inflammation, no cholangiolitis and no geographical distribution of the portal inflammatory infiltrate. Conclusion: The histological appearances of autochthonous HEV infection are sufficiently distinctive to consider the diagnosis in an acute setting and possibly to differentiate it from the endemic form of the disease.

We examined the histological findings of non-alcoholic steatohepatitis (NASH) and ALD to study resemblance and difference of these two diseases. At a glance of H&E staining, they looked same, however, careful examination on silver impregnation histology, the fibrosis of NASH showed lattice fibrosis on the other hand ALD showed solid fibrosis. The appearance of fibrosis was different due to qualitative difference. More examination on the change of hepatocytes, there were some difference in incidence of the findings such as megamitochondria, bile stasis, hemosiderin deposition, vacuolic nuclei, and lipogranuroma. So we are able to differentiate NASH and ALD histologically.

Mitochondria occasionally increase in size in response to metabolic injury. Numerous studies have reported giant mitochondria in patients with various diseases and animals with metabolic injuries, but there are few reports on giant mitochondria in normal cells under physiological conditions. Here, we report giant mitochondria in normal gastric parietal cells. Stomachs of guinea pigs fed freely, fasted or fasted and then injected with histamine were processed for electron microscopy. Giant mitochondria >2 microm in the diameter of their major axis were observed in resting-type parietal cells in the gastric glands of animals fasted for 60-72 h, whereas acid-secreting-type parietal cells found in those fed ad libitum did not contain giant mitochondria. Giant mitochondria showed unusual structures, especially in their cristae: they contained closely packed, tubular and concentric cristae as well as amorphous and pleomorphic inclusion bodies in their matrix. We observed giant mitochondria consisting of several segments, suggesting the fusion of several normal-sized mitochondria. Histamine injection decreased in a frequency of giant mitochondria in accordance with a decrease in a frequency of resting-type parietal cells. This is the first report of giant mitochondria in gastric parietal cells under physiological or near physiological conditions. Gastric parietal cells might be a good model for examining mitochondrial fusion and fission in a physiological state accompanied by the morphological change of the cells in the membrane system from an acid-secreting to resting type.

In patients with severe alcoholic liver disease (i.e., cirrhosis), a deficiency of S-adenosylmethionine (SAMe) develops as a result of decreased SAMe synthetase activity. Whether a sizeable SAMe depletion occurs already at earlier stages of alcoholic liver disease has been the subject of debate. To address this issue, rats were fed alcohol (or isocaloric carbohydrate) in Lieber-DeCarli liquid diets containing adequate amounts of protein, vitamins, and lipotropic factors, including methionine. Alcohol feeding resulted in hepatic steatosis (without fibrosis) and unchanged SAMe synthetase activity, yet SAMe concentration was already greatly decreased. This most likely resulted from oxidative stress associated with the metabolism of alcohol and the induction of cytochrome P4502E1 (CYP2E1), which generates free radicals. Indeed, the decrease in hepatic SAMe correlated with parameters of oxidative stress, such as increased 4-hydroxynonenal (measured by gas chromatography-mass spectrometry) and diminished glutathione (GSH). Decreased GSH, occurring as a result of excessive GSH consumption caused by the oxidative stress, probably generated by enhanced utilization of SAMe, a precursor of GSH, thereby explaining the depletion of SAMe. In view of the known differences between rodents and primates in the metabolism of lipotropes, my colleagues and I have also studied the interaction between alcohol and SAMe in baboons and found again that, at early stages preceding the development of cirrhosis, there was already a significant lowering of hepatic SAMe concentration, associated with a striking oxidative stress documented by decreased levels and accelerated turnover of GSH. This was associated with increased lipid peroxidation and damage to cellular membranes, including those of the mitochondria, assessed by electron microscopy. Oral administration of SAMe resulted in its hepatic repletion with a corresponding attenuation of the ethanol-induced oxidative stress and liver injury, with significantly less GSH depletion, less increases in plasma aspartate aminotransferase (AST) levels, less leakage of mitochondrial glutamic dehydrogenase into the plasma, and fewer megamitochondria. In conclusion, (1) both in rodents and in non-human primates, significant SAMe depletion occurs already at early stages of alcoholic liver disease, despite the consumption of adequate diets; (2) the decreased hepatic SAMe concentration and the associated liver lesions, including mitochondrial injury, can be corrected with SAMe supplementation; and (3) accordingly, therapeutic administration of SAMe should be the subject of a comprehensive clinical trial to assess its capacity to attenuate early stages of alcoholic liver injury in human beings.

To summarize the pathological features of nonalcoholic liver disease (NAFLD) in China based on a histological scoring system for NAFLD designed by the Pathology Committee of NASH Clinical Research Network (NASH-CRN), the specimens of liver needle biopsies from 130 patients with NAFLD were histopathologically analyzed by haematoxylin eosin, reticular fiber and Masson trichrome stain. Immunohistochemistry staining was used to exclude non-NAFLD cases combined with clinical data. Hepatic steatosis, lobular inflammation, hepatocytic ballooning and fibrosis were presented widespread in NAFLD liver tissues. Furthermore, macrovesicular steatosis predominantly located in acinar zone 3 was the main histologic feature of NAFLD and lobular inflammation was usually presented mildly. Hepatocyte ballooning was observed in 94.6% of all 130 cases. Mild perisinusoidal fibrosis and periportal fibrosis were often observed in stage 1 cases. According to the statistic analysis, hepatic steatosis was positively correlated with lobular inflammation, hepatocytic ballooning and fibrosis (r = 0.587, 0.488, 0.374, respectively, all P value < 0.01). The number of microgranulomas, lipogranulomas and apoptotic bodies increased following severity of steatosis, lobular inflammation and fibrosis. Meanwhile, the number of megamitochondria and glycogen nuclei was paralleveled to the degree of hepatocytic ballooning (P value all < 0.01). We suggest that the role of portal inflammation should be emphasized besides hepatic steatosis, lobular inflammation, hepatocyte ballooning and fibrosis in diagnosis and evaluation of NAFLD. It needs to be further verified whether microgranulomas, lipogranulomas and apoptosis bodies could be used as histopathological markers of development of NAFLD. The number of megamitochondria is more frequently be found in NAFLD, while in alcoholic liver diseases was Mallory bodies.

We assessed mitochondrial morphology by electron microscopy and the prevalence of a mitochondrial gene deletion in patients with non-alcoholic steatohepatitis (NASH), alcohol-related liver disease and non-fatty liver diseases. Respiratory chain function using a cytoplasmic hybrid (cybrid) assay was further studied in NASH patients and healthy controls.Electron microscopy was performed in 26 specimens. Fifteen patients were studied by polymerase chain reaction to detect a 520-bp deletion product of the mitochondrial genome (dmtDNA). Cybrids were created by fusion of platelets with anaerobic neuroblastoma cells in six NASH patients and 12 controls.Eight of ten NASH, one of seven alcoholics and two of nine other patients had linear crystalline inclusions in megamitochondria (p0.05). Three of five patients with alcohol-related liver disease had dmtDNA compared to one of five NASH patients and one of five non-steatohepatitis controls. Cybrid respiratory chain function in platelets was not different from that of controls.Respiratory chain dysfunction, if present in NASH, is not expressed in platelet-derived mitochondria. In contrast to alcohol-related liver disease with active drinking, NASH patients do not commonly express the 5-kb mitochondrial DNA gene deletion in liver tissue. As previously described in early alcohol-related liver disease, crystalline inclusions of unknown composition are seen in hepatic mitochondria in NASH. Their presence suggests either an adaptive process or mitochondrial injury.

A 14-year-old girl with the mitochondrial neurogastrointestinal encephalopathy syndrome had an 8-year history of intestinal pseudoobstruction with abdominal pain, persistent vomiting, gastric and duodenal dilatation, and duodenal diverticulosis. The child appeared chronically malnourished and had severe growth failure. Multisystem involvement was evident with the presence of ptosis, external ophthalmoplegia, muscle wasting, peripheral neuropathy, and diffuse white matter disease seen on magnetic resonance imaging. Lactic acidosis and increased cerebrospinal fluid protein were observed. Mitochondrial enzyme analysis of fresh-frozen skeletal muscle revealed a respiratory chain defect. Molecular genetic studies showed multiple mitochondrial DNA deletions. Pathologic findings in the intestine included atrophy of the external layer of the muscularis propria and an increased number of abnormal-appearing mitochondria in ganglion and smooth-muscle cells. Microvesicular steatosis was observed in liver, skeletal, and gastrointestinal smooth muscle, and Schwann cells of peripheral nerve. Brightly eosinophilic inclusions in the cytoplasm of gastrointestinal ganglion cells were visible by light microscopy, which were confirmed to be megamitochondria by ultrastructural studies. This is the first report of abnormal mitochondria observed in intestinal ganglion and smooth-muscle cells in this syndrome.

Background As part of a continuing investigation of the comparative ultrastructure of chiropteran salivary glands, we examined the submandibular glands of eight species of neotropical fruit bats in the genus Artibeus. We previously described secretory granules of unusual substructure in the seromucous demilunar cells of this organ in some species in this genus. In the present study, we turned our attention to the serous acinar cells in the same glands. Methods Specimens of seven species of Artibeus were collected in neotropical localities. Salivary glands were extirpated in the field and thin slices were fixed by immersion in triple aldehyde-DMSO or in modified half-strength Karnovsky's fixative. Tissues were further processed for electron microscopy by conventional means. Results In contrast to seromucous cells, which exhibit species-specific diversification in bats of this genus, the secretory apparatus and secretory granules in the serous acinar cells are highly conserved across all seven species. The single exception involves the mitochondria in one species. In this instance, some of the serous cell mitochondria in Artibeus obscurus are modified into megamitochondria. Such organelles usually have short, peripheral cristae; a laminar inclusion is present in the matrix compartment of every outsized organelle. Inclusions of this nature never are present in normal-size mitochondria in the serous cells. None of the megamitochondria were observed in the process of degeneration. Conclusions The giant mitochondria in A. obscurus have a matrical structure that is radically different from that of the only other megamitochondria reported to occur in bat salivary glands. The factors that lead to variation in megamitochondrial substructure in different species, as well as the functional capacities of such giant organelles, are unknown. Anat. Rec. 248:13–17, 1997. © 1997 Wiley-Liss, Inc.

An adult Bengal cat ( Felis catus × Prionailurus bengalensis) with a prolonged history of partial anorexia, regurgitation, and weight loss and a clinical, radiographic, and ultrasonographic diagnosis of persistent megaesophagus and gastrointestinal ileus was submitted for necropsy. The intestinal tract was diffusely distended by gas and fluid with appreciable loss of muscle tone and an absence of luminal obstruction, consistent with the clinical history of chronic intestinal pseudo-obstruction. Histologically, the autonomic nervous system was intact, but the smooth muscle within the gastrointestinal wall exhibited a marked basophilia that was most pronounced in the jejunum. Immunohistochemistry for neurofilament, synaptophysin, CD117, and desmin demonstrated that the number of myenteric ganglia, number of interstitial cells, and leiomyocyte desmin content were similar when compared with the unaffected age- and species-matched control. Immunohistochemistry for smooth muscle α-actin demonstrated a striking loss of immunoreactivity, predominantly in the circular layer of the jejunum, that corresponded with the tinctorial change in leiomyocytes. Transmission electron microscopy revealed loss of myofibrils, loss of organelle polarity, and significantly larger central mitochondria (megamitochondria) in affected leiomyocytes, as well as nonspecific degenerative changes. Although the presence of a primary leiomyopathy and a causal relationship could not be confirmed in this case, leiomyopathies are considered a cause of chronic intestinal pseudo-obstruction in human medicine, and loss of smooth muscle α-actin immunoreactivity is one recognized marker for intestinal dysmotility.

Elongated, enlarged mitochondria with crystalloid matrix arrays were discovered in periportal hepatocytes in 11 of 12 children (age 6 to 15 years) with portal hypertension, minimal alterations on light microscopy, and cavernous transformation of the portal vein. Eleven of the children were clinically well before onset of symptoms, one was anemic with megaloblastic bone marrow, and a second had undergone renal transplantation. Minimal findings by light microscopy included slight portal fibrosis (six cases), pericentral venular fibrosis (one case), mild, patchy sinusoidal sclerosis (one case), central venular and sinusoidal dilatation (two cases), and mild hepatocellular lipid accumulation (one case). Four were judged normal by routine histologic examination. Subtle depletion of periportal hepatocellular glycogen was present in six. In 10, subtle striation or granularity of periportal hepatocyte cytoplasm was visible with high-magnification light microscopy. Although similar mitochondria are seen sporadically in hepatocytes in diverse settings, enlarged mitochondria with crystalloid matrix inclusions have not been previously reported as a uniform feature in children with portal hypertension due to cavernous transformation of the portal vein and minimal other hepatic alteration. It is postulated that the mitochondria are adapting in response to an abnormal metabolic milieu created by hemodynamic alterations.

The effects of coenzyme Q10 (CoQ10) on the hydrazine-induced changes in the structure of mitochondria and those in antioxidant systems of the liver were investigated using rats as experimental animals. Animals were placed on a powdered diet containing 1.0% hydrazine for 7-8 days in the presence or absence of the combined treatment with CoQ10. Results obtained were as follows: (a) treatment of animals with CoQ10 prevented the hydrazine-induced formation of megamitochondria in the liver; (b) changes observed in the liver of the hydrazine-treated animals in comparison to the control were increases in the contents of α-tocopherol and CoQ analogs, increases in the levels of lipid peroxidation, decreases in the level of reduced glutathione with increases in that of oxidized glutathione, and increases in the ratio of unsaturated to saturated fatty acids in phospholipid domains of mitochondrial membranes; and (c) administration of CoQ10 to hydrazine-treated animals suppressed enhanced lipid peroxidation and improved lowered adenosine diphosphate/O ratios of mitochondria. The present data suggest that CoQ10 suppresses the hydrazine-induced formation of megamitochondria by scavenging free radicals generated from hydrazine and its metabolites.

Isolated Methylmalonic Acidemia (MMA) comprises a relatively common and heterogeneous group of inborn errors of metabolism. Most affected individuals display severe multisystemic disease with episodes of metabolic instability, chronic renal disease, and neurological complications. The most severe clinical presentations are typically associated with complete loss of methylmalonyl-CoA mutase (MUT) enzymatic activity. MUT converts methylmalonyl-CoA to succinyl-CoA within mitochondria but defects in the transport or synthesis of its cobalamin co-factor also variably impair MUT activity. Previous mouse models of Mut MMA generated by our group have been critical to developing gene therapy for MMA but have either displayed neonatal lethality or required concomitant transgenesis for viability. Thus, the need for ameliorated models to further investigate disease-associated pathophysiology and facilitate gene therapy studies exists. We have therefore constructed a mouse model of an attenuated form of isolated MMA, the cblA subtype, by using homologous recombination to create a deletion allele of Mmaa, the enzyme that performs the gated transfer of adenosylcobalamin to Mut and protects Mut from oxidative inactivation. Patients with the cblA subtype of MMA can have variable presentations, spanning the full spectrum of MMA associated symptoms and pathology, yet always harbor an element of clinical and biochemical responsiveness to injectable B12.Mmaa-/- mice were born in mendelian proportions, exhibited decreased survival after weaning (P

Intracytoplasmic inclusion bodies with an unusual immunohistochemical profile, histologically similar to alpha 1 antitrypsin globules and megamitochondria, were seen within hepatocytes in a liver allograft biopsy. Electron microscopy revealed that these structures were erythrocytes in various stages of degradation. Their appearance following an episode of Pneumocystis carinii pneumonia suggests a possible relationship between bouts of hypoxia and erythrocyte entry into hepatocytes.

Studies assessing mitochondrial function and structure in livers from humans or experimental animals with chronic liver disease, including liver cirrhosis, revealed a variety of alterations in comparison with normal subjects or control animals. Depending on the etiology of chronic liver disease, the function of the electron transport chain and/or ATP synthesis was found to be impaired, leading to decreased oxidative metabolism of various substrates and to impaired recovery of the hepatic energy state after a metabolic insult. Changes in mitochondrial structure include megamitochondria with reduced cristae, dilatation of mitochondrial cristae and crystalloid inclusions in the mitochondrial matrix. The most important strategies to maintain an adequate mitochondrial function per liver are mitochondrial proliferation and increases in the activity of critical enzymes or in the content of cofactors per mitochondrion. Possibilities to assess hepatic mitochondrial function and to treat mitochondrial dysfunction in patients with chronic liver disease are discussed.

Methylmalonic acidemia is an autosomal recessive inborn error of metabolism caused by defective activity of methylmalonyl-CoA mutase (MUT) that exhibits multiorgan system pathology. To examine whether mitochondrial dysfunction is a feature of this organic acidemia, a background-modified Mut-knockout mouse model was constructed and used to examine mitochondrial ultrastructure and respiratory chain function in the tissues that manifest pathology in humans. In parallel, the liver from a patient with mut methylmalonic acidemia was studied in a similar fashion. Megamitochondria formed early in life in the hepatocytes of the Mut−/− animals and progressively enlarged. Liver extracts prepared from the mutants at multiple time points displayed respiratory chain dysfunction, with diminished cytochrome c oxidase activity and reduced intracellular glutathione compared to control littermates. Over time, the exocrine pancreas and proximal tubules of the kidney also exhibited megamitochondria, and older mutant mice eventually developed tubulointerstitial renal disease. The patient liver displayed similar morphological and enzymatic findings as observed in the murine tissues. These murine and human studies establish that megamitochondria formation with respiratory chain dysfunction occur in a tissue-specific fashion in methylmalonic acidemia and suggest treatment approaches based on improving mitochondrial function and ameliorating the effects of oxidative stress.—Chandler, R. J., Zerfas, P. M., Shanske, S., Sloan, J., Hoffmann, V., DiMauro, S., Venditti, C. P. Mitochondrial dysfunction in mut methylmalonic acidemia.

Numerous mitochondria ranging from slightly larger than normal to several micrometers in diameter (giant) were found in about one-half the serous secretory cells in the surface epithelium of the normal gerbil trachea and proximal bronchi. Tracheal serous cells of mice also were found to contain numerous giant mitochondria. Clara cells of gerbil bronchioles contained abundant giant mitochondria in addition to normal tubular mitochondria and the second population of enlarged spherical mitochondria that have been described in Clara cells of several genera. In contrast, mouse Clara cells revealed the normal tubular and the enlarged spherical mitochondria but no giant mitochondria. A survey of a number of cell types in gerbils failed to disclose hypertrophied mitochondria outside tracheobronchial surface epithelium and bronchioles. The mitochondrial enlargement resulted from an increase of matrix but not cristae. The expansion of matrix displaced the relatively sparse cristae into small collections compressed against the outer membrane. The prevalence of giant mitochondria and of granular endoplasmic reticulum is similar among cells, and these two organelles are codistributed within cells. The megamitochondria and granular reticulum occupy a central stratum, whereas normal mitochondria occur in the apical and basal regions. The giant mitochondria are considered related to a normal biologic activity that is characteristic of respiratory tract epithelium of mice and gerbils selectively and is more prominent in secretory cells than in ciliated cells.

To clarify the possible role of oxidative stress in hepatocytes in nonalcoholic fatty liver disease, the hepatic expression of 8-hydroxydeoxyguanosine (8-OHdG), a good marker of oxidative DNA damage, was immunohistochemically investigated in nonalcoholic steatohepatitis (NASH) and steatosis. In double immunostaining, the cytoplasmic fine granular 8-OHdG expression was considered to reflect 8-OHdG-positive mitochondrial DNA affecting oxidation stress. In steatosis, 4 of 8 cases showed cytoplasmic 8-OHdG, 1 case showed nuclear 8-OHdG and 1 case showed both cytoplasmic and nuclear 8-OHdG. In contrast, 8-OHdG expression was more frequently detected in NASH (12 of 13 cases, 92%). Immunoreactivity for 8-OHdG was observed only in the cytoplasm with a fine granular pattern (1 of 13 cases, 8%), only in the nucleus (6 of 13 cases, 46%), and in both the cytoplasm and the nucleus (5 of 13 cases, 38%). Megamitochondria also exhibited 8-OHdG intensely. We indicate that 8-OHdG expression in the cytoplasm with a fine granular pattern reflects oxidative damage to the mitochondrial DNA of hepatocytes in both NASH and steatosis. We propose herein that the evaluation of cytoplasmic 8-OHdG may be a sensitive diagnostic marker of early nonalcoholic fatty liver disease events.

Systemic mitochondriopathies as chronic progressive external ophthalmoplegia (CPEO) are frequently associated with ptosis. We investigated whether mitochondrial abnormalities in the levator muscle are also found in patients with isolated congenital or acquired ptosis showing no other signs of mitochondrial cytopathy. Biopsies of levator muscle were taken during surgery from 24 patients with isolated congenital (group 1) or early-onset acquired ptosis (group 2). All patients were given a thorough clinical examination before and after surgery. Ultrathin muscle sections were examined by transmission electron microscopy. The findings were compared with biopsies from five patients with CPEO (positive control) and two patients with traumatic ptosis or pseudoptosis (negative control). The mean levator function equalled 7.3 mm (range 4–10 mm) in group 1 and 12.8 mm (range 9–15 mm) in group 2. Eight out of 11 patients in group 1 and eight out of 13 patients in group 2 were found to have mitochondrial alterations such as megamitochondria, mitochondrial matrix alterations and abnormal cristae, similar to CPEO. Within group 1 and 2, no significant clinical differences were found between patients with and without mitochondrial abnormalities. Mitochondrial alterations were found in a surprisingly large proportion of levator biopsies from patients with isolated congenital or early-onset acquired ptosis. There was no statistically significant correlation between mitochondrial alterations and levator function. Our findings suggest that the ultrastructural assessment of mitochondria in the eyelid muscle is a valuable tool, and may guide further biochemical and mutation screening tests that will help to understand the etiopathology of this disease.

It has been shown that portal hypertension in the rat causes microvesicular hepatocytic fatty infiltration. Formation of megamitochondria (MG) is one of the most prominent alterations in steatosis. Because nitric oxide (NO), tumor necrosis factor-alpha (TNFalpha), and interleukin-1beta (IL-1beta) impair mitochondrial function, these mediators have been studied in prehepatic portal hypertensive rats to verify their coexistence with MG and therefore with steatosis. Male Wistar rats were divided into two groups: a control group (n = 7) and a group with partial portal vein hgation (n = 19) at 6 weeks of evolution. TNFalpha and IL-1beta were quantified in liver by enzyme-linked immunosorbent assay, and NO was measured in the portal vein, suprahepatic inferior vena cava, and infrahepatic inferior vena cava by the Griess reaction. In portal hypertensive rats, the serum concentration of NO of hepatic origin increases (132.10 +/- 34.72 vs. 52.44 +/- 11.32 nmol/ml; p0.001), as do TNFalpha (2.02 +/- 0.20 vs. 1.12 +/- 0.43 micromol/mg protein) and IL-1beta (18.95 +/- 2.59 vs. 5.48 +/- 1.70 micromol/mg protein) (p = 0.005) in the liver. The most frequent hepatic histologic findings are the presence of MG (p0.001), steatosis, and hyperplasia. An increase in hepatic release of NO, TNFalpha and IL-1beta with MG formation is produced in rats with portal hypertension. Therefore these proinflammatory mediators and this morphologic mitochondrial alteration could both be involved in the etiopathogenesis of steatosis.

This study was conducted with 3 objectives in mind: first, to identify the toxic fraction (aqueous or organic) in leaves and flowers; second, to identify diagnostic marker(s) of toxicosis in cats; and, third, to evaluate the morphologic effects of intoxication. The study was conducted in 2 phases. Phase 1 was to identify which extract, organic or aqueous, was nephrotoxic and also to determine the appropriate dose for use in the phase 2 studies. Results indicated that only the aqueous extracts of leaves and flowers were nephrotoxic and pancreotoxic. To identify the proximate toxic compound, cats in the phase 2 study were orally exposed to subfractions of the aqueous flower extract, 1 subfraction per cat. Results confirmed vomiting, depression, polyuria, polydipsia, azotemia, glucosuria, proteinuria, and isosthenuria as toxic effects of the Easter lily plant. Another significant finding in serum was elevated creatinine kinase. Significant histologic kidney changes included acute necrosis of proximal convoluted tubules and degeneration of pancreatic acinar cells. Renal ultrastructural changes included swollen mitochondria, megamitochondria, edema, and lipidosis. Subfraction IIa3 of the aqueous floral extract contained most of the toxic compound(s). These studies reproduced the clinical disease, identified the most toxic fraction of the Easter lily, and helped characterize the clinical pathology, histopathology, and ultrastructural pathology associated with the disease.

Cytochemically evidenced cytochrome oxidase activity was morphometrically measured in the cerebellar cortex of adult and old rats. The ratio (R) between the area of the precipitate due to the cytochemical reaction and the overall area of each mitochondrion was calculated. While in adult rats an inverse correlation between mitochondrial size and R values (r = -.905) was envisaged, in old animals increasing values of R were paired by increases in mitochondrial area (r =.561). Paired-quartile comparisons of the R values from adult and old animals documented a marked age-related impairment of the mitochondrial metabolic competence in small (I quartile: -31.6%) and medium-sized (II quartile: -26.4; III quartile: -16.4) mitochondria, while large organelles showed the lowest age-related decrease (IV quartile: -3.0%). The present findings support that a marked dysfunction of small and medium-sized mitochondria contributes to the significant decay of energy metabolism currently reported in physiological aging.

Megamitochondria with crystalline inclusions (MMC) have been previously described in nonalcoholic fatty liver; however, their distribution within hepatic zones is unknown. We sought to determine this distribution from the core liver biopsy specimens of 31 patients: 8 males and 23 females, age range 21 to 72 years. Twenty-nine showed evidence of nonalcoholic steatohepatitis (NASH) on biopsy with steatosis, inflammation, varying degree of fibrosis, ballooned hepatocytes, and Mallory hyaline, and two patients had cryptogenic cirrhosis thought to represent “burned out” NASH. Identified by transmission electron microscopy, the abundance of MMC was compared between low-stage (fibrosis stages 1 and 2) and high-stage (fibrosis stages 3 and 4) groups and between zones with or without difference in fibrosis stage. Regardless of stage, the MMC were distributed equally in all zones and were abundant similarly in low- and high-stage groups. This abundance did not correlate with the degree of oxidative stress (4-hydroxynonenal staining) or with the abundance of ballooned hepatocytes. Consistent with age as a risk factor for more severe disease, the median age for the low-stage group was significantly lower than that of the high-stage group (P = .003). In conclusion, in NASH, the MMC seem to be distributed randomly among zones and without variation in abundance, regardless of the fibrosis stage. The exact function of these structures remains to be defined. In this study, their presence did not seem to correlate with the light microscopic injury pattern represented by ballooned hepatocytes or degree of oxidative stress defined by immunostaining for 4-hydroxynonenal. (HEPATOLOGY 2004;39:1423–1429.)

The constellation of histopathologic lesions that characterize alcoholic and nonalcoholic steatohepatitis has been well described and has served as the basis for clinical diagnosis, natural history studies, and experimental models for analyses of etiopathogenesis. The lesions common to both entities include, to varying degrees, steatosis, liver cell ballooning, lobular inflammation with a notable component of polymorphonuclear leukocytes, and a characteristic form of fibrosis that is initially located in the perisinusoidal regions of acinar zone 3. Cirrhosis with or without steatosis or steatohepatitis may occur in both entities. Mallory's hyaline is common but not necessary; megamitochondria and varying amounts of iron may be observed in either process. Hepatocellular carcinoma is a recognized complication of both processes, albeit with greater frequency in the former. Alcoholic hepatitis may present with more severe clinical and histologic manifestations than the nonalcoholic counterpart, including significant morbidity and mortality. The perivenular lesions collectively referred to as sclerosing hyaline necrosis are markers of severity, and are not common in nonalcoholics. In many instances, however, the microscopic lesions of these two processes are similar, likely as a reflection of common pathogenetic pathways, and the distinction between the two is ultimately clinically derived.

Our recent finding that free radical-induced formation of megamitochondria (MG) is followed by apoptosis has prompted us to investigate the correlation between the MG formation and the swelling of mitochondria which is considered to play a key role in early stages of apoptotic processes of the cell. Mitochondria of rat hepatocytes or RL-34 cells and those isolated from rat livers became enlarged up to three times in their diameters when they were exposed to a hypotonic medium. MG induced in the liver of rats placed on a 1% hydrazine-diet for 4-5 days or those induced in the liver of mice placed on a 2% chloramphenicol (CP)-diet for 9-10 days were endowed with a dense matrix whereas those fed with the toxic diets for longer periods of time became enlarged further and their matrix became extremely pale indicating that MG in the latter animals became swollen secondarily. The membrane potential, the content of cytochrome c and the rate of the generation of reactive oxygen species (ROS) of MG in the former animals were almost unchanged compared to those of mitochondria in control animals whereas those of MG in the latter animals became distinctly decreased. These results may suggest that free radical-induced MG possibly cause apoptosis via their secondary swelling.

AIM: To investigate the ultrastructure of abnormal hepatocyte mitochondria, including their cellular and hepatic zonal distribution, in bioptates in pediatric non-alcoholic steatohepatitis (NASH). METHODS: Ultrastructural investigations were conducted on biopsy liver specimens obtained from 10 children (6 boys and 4 girls) aged 2-14 years with previously clinicopathologically diagnosed NASH. The disease was diagnosed if liver biopsy revealed steatosis, inflammation, ballooned hepatocytes, Mallory hyaline, or focal necrosis, varying degrees of fibrosis in the absence of clinical, serological, or histological findings of infectious liver diseases, autoimmune hepatitis, metabolic liver diseases, or celiac disease. For ultrastructural analysis, fresh small liver blocks (1 mm3 volume) were fixed in a solution containing 2% paraformaldehyde and 2.5% glutaraldehyde in 0.1 mol/L cacodylate buffer. The specimens were postfixed in osmium tetroxide, subsequently dehydrated through a graded series of ethanols and propylene oxide, and embedded in Epon 812. The material was sectioned on a Reichert ultramicrotome to obtain semithin sections, which were stained with methylene blue in sodium borate. Ultrathin sections were contrasted with uranyl acetate and lead citrate, and examined using an Opton EM 900 transmission electron microscope. RESULTS: Ultrastructural analysis of bioptates obtained from children with non-alcoholic steatohepatitis revealed characteristic repetitive mitochondrial abnormalities within hepatocytes; mainly mitochondrial polymorphisms such as megamitochondria, loss of mitochondrial cristae, and the presence of linear crystalline inclusions within the mitochondrial matrix of an increased electron density. The crystalline inclusions were particularly evident within megamitochondria (MMC), which seemed to be distributed randomly both within the hepatic parenchymal cell and the zones of hepatic lobule, without special variations in abundance. The inclusions appeared as bundles viewed longitudinally, or as an evenly spaced matrix in cross section, and frequently caused mitochondrial deformation. The average diameter of these linear structures was 10 nm and the average space between them 20 nm. Sometimes enlarged intramitochondrial granules were seen in their vicinity. Foamy cytoplasm of hepatocytes was found, resulting from the proliferation of smooth endoplasmic reticulum and glycogen accumulation. The perivascular space of Disse was frequently dilated, and contained transitional hepatic stellate cells, as well as mature and/or newly forming collagen fiber bundles. CONCLUSION: Marked ultrastructural abnormalities observed in hepatocyte mitochondria, especially their polymorphism in the form of MMC and loss of mitochondrial cristae, accompanied by foamy cytoplasm, clearly indicate a major role of these organelles in the morphogenesis of pediatric NASH. Our findings seem to prove the high effectiveness of electron microscopy in the diagnosis of the disease.

The effects of prolonged administration (3 months) of a 3-thia fatty acid analogue and omega-3-fatty acids on cardiac fatty acid oxidation and the volume fraction of lipid droplets and mitochondria in cardiomyocytes were investigated. Doses were 1 g/day/kg body weight, except 150 mg/day/kg body weight for tetradecylthioacetic acid (a 3-thia fatty acid). One group served as control and did not receive any treatment. The volume fraction of lipid droplets in cardiomyocytes was significantly lower in the tetradecylthioacetic acid group compared to the other groups. Mitochondrial beta-oxidation was 60% greater and fatty acyl-CoA oxidase activity was increased by 430% in the tetradecylthioacetic acid group compared to control. This was accompanied by a greater volume fraction of mitochondria in cardiomyocytes (0.514 +/- 0.032% in tetradecylthioacetic acid v 0.318 +/- 0.007% in control) which was due to an increased size of mitochondria. The volume fraction of mitochondria was also greater in eicosapentaenoic acid (EPA) treated rats compared to control, but the enzymic activities were unaffected. Docosahexaenoic acid (DHA) treatment resulted in a greater volume fraction of lipid droplets in the cardiomyocytes, but the volume fraction of mitochondria and enzyme activities were unaltered. These results indicate that EPA and DHA have different effects on the modulation of mitochondrial biogenesis. Tetradecylthioacetic acid treatment results in megamitochondria formation and increased peroxisomal and mitochondrial beta-oxidation with a concomitant reduction of lipid droplets in the cardiomyocytes.

The effects of alpha-tocopherol on the hydrazine-induced changes in the structure of mitochondria and those in the enzymatic and nonenzymatic antioxidant systems of the liver were investigated using rats as experimental animals. Animals were divided into four groups: animals of the first group were placed on a powdered diet containing 1.0% hydrazine for 7 days; those of the second and third groups received a control diet and alpha-tocopherol (ip, daily, 700 mg/kg body wt). On the fourth day and thereafter for up to 7 days, the control diet was replaced by a 1.0% hydrazine diet for the animals of the second group; those of the fourth group served as the control. After 10 days (in the case of animals of the first group, 7 days), animals were sacrificed. Results obtained were as follows: (1) Treatment of animals with alpha-tocopherol partly prevented the hydrazine-induced formation of megamitochondria in the liver. (2) Treatment of animals with hydrazine induced remarkable increases in the contents of alpha-tocopherol in mitochondria (4.8 times), microsome (1.4 times), and homogenate (2.9 times) of the liver compared with those of the control. Treatment of animals with alpha-tocopherol did not raise the concentration of alpha-tocopherol in mitochondria of the liver. The highest concentration of alpha-tocopherol in mitochondria of the liver was obtained in animals given hydrazine plus alpha-tocopherol (7.2 times higher than the control). (3) The amount of lipid-soluble fluorophores as an indicator of nonenzymatic oxidative stress was remarkably increased in mitochondria, microsome, and homogenate of the liver of hydrazine-treated animals. (4) Among enzymes protecting the cell from the oxidative stress activities of superoxide dismutase and glutathione peroxidase were almost the same among four groups of animals, while the activity of catalase was decreased distinctly in hydrazine-treated animals. Administration of alpha-tocopherol to these animals did not improve its activity. The present study has clearly demonstrated that alpha-tocopherol, a typical scavenger for free radicals, prevents the hydrazine-induced formation of megamitochondria in the liver. However, we failed to correlate free radicals to biochemical and physicochemical changes of mitochondrial membranes induced by hydrazine. Previously, we have demonstrated that a ratio of unsaturated to saturated fatty acids in phospholipid domains of mitochondrial membranes increases with hydrazine treatment. Since increases in the ratio specified above are a key event in the membrane fusion process we are now studying how the ratio is modified by hydrazine focussing on desaturase activity in the liver, and results will be reported soon.

Late onset of symptoms in a 12 1/2-year-old male with ornithine transcarbamylase (OTC) deficiency were associated with unusual histological features in the liver. The patient presented with an acute onset of hyperammonemia and altered mental status after a 2-day prodrome of vomiting and lethargy. Physical examination showed a combative and disoriented male with icteric sclerae but with no fever or hepatomegaly. The plasma ammonia level was 282 microM. Enzyme assays of liver tissue obtained by percutaneous needle biopsy showed OTC activity of approximately 3% of normal; carbamyl phosphate synthetase was normal. Histopathological findings included severe microvesicular centrilobular steatosis. Hepatic architecture and reticulin framework were well preserved. Many hepatocyte nuclei were filled with glycogen. Electron microscopy showed mitochondria that were rounded and expanded with cristae at the edge of the mitochondrial membrane. In contrast to other reports, only slight variations in size and shape were seen. Megamitochondria and intramatrical paracrystalline inclusions were not identified. The cytoplasm contained scattered fat globules, peroxisomes, and dilated smooth endoplasmic reticulum. The prominent mitochondrial abnormalities commonly found in OTC deficiency were notably absent.

Liver biopsies were performed on 18 haemodialysis patients with clinically as well as clinico-chemically altered liver values and partially positive HBV marker serology, with the view to comparing these values with ultrastructural findings in correlation with histological and immunohistological results. The following characteristic combination of findings was recorded at ultrastructural level: hypertrophy and hyperplasia of agranular endoplasmic reticulum of hepatocytes, haemosiderin deposits in Kupffer cells and hepatocytes, invasion of lymphocytes into Disse and intercellular spaces; megamitochondria in hepatocytes and perisinusoidal fibrosis established in some cases. The same findings proved to be recordable in histological diagnosis of hepatitis or toxico-metabolic liver damage. The study shows that inflammatory lesions are not all the only causes for haemodialysis-related changes in clinico-chemical values, so that liver biopsies together with ultrastructural investigations are indicated for differential diagnosis.

Ultrastructural studies were conducted on the myocardium of rats fed corn oil, Tower RSO (0·88 per cent EA), 1788 RSO (3·6 per cent EA) and Target RSO (38·9 per cent EA) supplemented diets for 18 weeks. Cardiac myocytes of Tower RSO-fed rats showed some loosening of myofibrils and a slight increase in the number of mitochondria, few of which had lost their cristae. Large intravascular lipid droplets were observed in the myocardium of rats fed the 1788 RSO diet, as well as some small lipid droplets which were seen in close association with mitochondria. There was an apparent increase in the number of mitochondria of both normal and giant size. Many of the mitochondria exhibited distortion of shape and degeneration of cristae. The matrix of megamitochondria contained vesicles and electron- dense floccular inclusions and at times electron-lucent lipid-like material. The degenerative changes of mitochondria were most pronounced in the Target RSO group, where some megamitochondria showed a complete loss of cristae and a replacement of matrix with lipid-like material. These observations suggest that both intravascular lipid globules and the mitochondrial alterations are possible contributory factors involved in the development of cardiac lesions in RSO-fed rats.

The intestinal absorption of labeled folic acid ([3H]pteroylmonoglutamate) was determined from urinary and fecal recoveries of tritium in pairs of monkeys fed control liquid diets or diets containing 50% of energy as ethanol for a 24-mo period. Weight gain, fecal fat excretion, nitrogen balance, d-xylose absorption, serum folate levels, jejunal histology, and intestinal enzyme activities were similar in each group. Liver biopsies obtained after 12 and 24 mo of feeding demonstrated steatosis and megamitochondria in the ethanol-fed group, with decreased hepatic levels of folate at 24 mo. Intestinal malabsorption of labeled folic acid in the ethanol-fed monkeys was indicated by decreased urinary recovery of tritium but increased fecal recovery of tritium after intragastric administration of [3H]pteroylmonoglutamate. These studies suggest that folic acid malabsorption follows the chronic administration of ethanol together with a nutritious diet.

Summary A total ischaemia of one area of the myocardium was produced by ligature of a coronary arteryin dogs. After 30 min, 1 hr, 2 hrs, and 4 hrs the ligature was loosened and the artery was for 1–2 hrs reperfused up to 4–7 days. At the end of the ischaemia and after reperfusion the ultrastructure of the myocardium was qualitatively and morphometrically investigated. The findings concerned: loss of glycogen (partly glycogen accumulation), mitochondrial swellings and fusions and development of megamitochondria, loss of cristae, rupture of the mitochondrial outer membranes, relaxation or supercontraction of the myofibrils, vesicular and vacuolar changes of the sarcoplasmic reticulum, oedema of the sarcoplasm, rupture of the sarcolemma. No significant differences of the quantitative parameters could be observed during the ischaemia from 30 min to 4 hrs: myofibrils 53.65–59.74%, mitochondria 26.51–33.59%, sarcoplasm 8.22–11.86%, sarcoplasmic reticulum 1.41–2.63%, lipofuscin 0.05–0.87%, lipids 0.01–0.10%. In comparing the data of the single animals we could determine significant differences between some parameters at the ends of the ischaemic phase and of the reperfusion phase, but as far as the findings are concerned both increases and decreases could be observed. The investigations show that the limit of the reversibility of damages in myocardial cells is about 45 min; in special cases a longer survival time may be taken into account. Reperfusion has different, though not always positive, effects.

Processes of the formation of cuprizone-induced megamitochondria in mouse liver have been studied in detail by electron microscopy. The earliest change observed was the presence of large intramitochondrial granules. The next stage was the formation of myelin figures by which mitochondria were apparently connected. The third stage was characterized by megamitochondria connected with each other by their outer membranes. Continuity of mitochondria were further examined by serial sections, and megamitochondria were proved to be connected to each other far more frequentlythan expected on one plane of section. A model for the mechanism of megamitochondrial formation is proposed based on electron microscopic evidences, involving the fusion of mitochondrial membranes. Possibility is also discussed that cuprizone-induced megamitochondria may fuse to one single branching mitochondrion.

A light and electron microscopic study was undertaken on a surgical specimen of a hepatic fibrolamellar carcinoma with special emphasis on the mitochondrial changes in nontumor hepatocytes. The tumor, both light and electron microscopically, showed typical features for this special variant neoplasm. The nontumor liver maintained normal architecture with marked lymphoid hyperplasia and fibrosis in the portal regions in the vicinity of the tumor. No megamitochondria were appreciated under the light microscopic examination. The electron microscopic observation, however, revealed prominent morphological alterations of individual mitochondrion. The dividing mitochondria were frequently found. Pseudoinclusions or invaginations of the membrane were common. Many mitochondria were enlarged with various shapes. The cristae were disordered and crystalloid material was easily observed. The possible significance of these mitochondrial abnormalities in nontumor hepatocytes was discussed.