Immunohistochemical Study of Dark and Progenitor Carotid Body Cells: Artefacts or Real Subtypes?

Postmortem changes occurring in human carotid body were simulated on the Wistar rat model. It was shown that light, dark, and pyknotic (progenitor) subtypes of human carotid body cells are an artifact and cannot be used in clinical practice to study the characteristics of various human diseases. The differences between the control group of healthy individuals and individuals with the various pathologies are most likely due to the different levels of premortal hypoxia that the tissue had been exposed to. Moreover, widespread antigens used in practice were divided into 2 groups by their tolerance to autolysis: stable and unstable ones. This can be useful for the development of immunohistochemical test algorithms for the diagnostics on autopsy material.

Increased Autolysis of µ-Calpain in Skeletal Muscles of Chronic Alcohol-Fed Rats.

BACKGROUND: Proteolysis can proceed via several distinct pathways such as the lysosomal, calcium-dependent, and ubiquitin-proteasome-dependent pathways. Calpains are the main proteases that cleave a large variety of proteins, including the giant sarcomeric proteins, titin and nebulin. Chronic ethanol feeding for 6 weeks did not affect the activities of µ-calpain and m-calpain in the m. gastrocnemius. In our research, changes in µ-calpain activity were studied in the m. gastrocnemius and m. soleus of chronically alcohol-fed rats after 6 months of alcohol intake. METHODS: SDS-PAGE analysis was applied to detect changes in titin and nebulin contents. Titin phosphorylation analysis was performed using the fluorescent dye Pro-Q Diamond. Western blotting was used to determine µ-calpain autolysis as well as µ-calpain and calpastatin contents. The titin and nebulin mRNA levels were assessed by real-time PCR. RESULTS: The amounts of the autolysed isoform (78 kDa) of full-length µ-calpain (80 kDa) increased in the m. gastrocnemius and m. soleus of alcohol-fed rats. The calpastatin content increased in m. gastrocnemius. Decreased intact titin-1 (T1) and increased T2-proteolytic fragment contents were found in the m. gastrocnemius and m. soleus of the alcohol-fed rats. The nebulin content decreased in the rat gastrocnemius muscle of the alcohol-fed group. The phosphorylation levels of T1 and T2 were increased in the m. gastrocnemius and m. soleus, and decreased titin and nebulin mRNA levels were observed in the m. gastrocnemius. The nebulin mRNA level was increased in the soleus muscle of the alcohol-fed rats. CONCLUSIONS: In summary, our data suggest that prolonged chronic alcohol consumption for 6 months resulted in increased autolysis of µ-calpain in rat skeletal muscles. These changes were accompanied by reduced titin and nebulin contents, titin hyperphosphorylation, and development of hindlimb muscle atrophy in the alcohol-fed rats.

[The specific features of corpse putrification under the influence of necrobiome enzymatic systems]. / Spetsifika putrifikatsii trupa pod deistviem fermentnykh sistem nekrobioma.

The objective of the present study was to characterize the specific features of corpse putrification under the influence of necrobiome enzymatic systems depending on the duration of the post-mortem period. We present the results of investigations into the enzymatic activity of the dominant species of microorganisms making up the post-mortem microbiome. The domestic pork carcasses weighing 50-70 kg were used as an experimental putrification model. The study revealed the characteristic features of protein decomposition under the influence of proteolytic enzymes of pseudomonads, bacilli, and clostridia, such as alteration in the amount of necrobionts producing proteases in the entire carcass and its fragments during biodegradation in the air over 30 and 136 days of the post-mortem period. A series of experiments designed to evaluate the effectiveness of protein hydrolysis by necrobionts have demonstrated the dependence of the rate of biodegradation on the environmental temperature, duration of the putrification pocess, and the species composition of the necrobiome.

[Dynamics of postmortem autolysis of cardiomyocytes].

Dynamics of postmortem autolysis of cardiocytes was evaluated using cells and tissues obtained from the patients who died from acute forms of ischemic heart disease, such as acute coronary insufficiency and acute myocardial infarction in the pre-necrotic phase. The studies were carried out at a temperature of 7, 20, and 37 degrees C. It was shown that autolysis of cardiac muscular fibers proceeds through three successive stages. A rise in temperature from 7 to 20 degrees C accelerated autolysis by one third while further elevation of the temperature up to 37 degrees C was associated with a 9-fold decrease in the duration of autolysis.

Autolysis in Crustacean Tissues after Death: A Case Study Using the Procambarus clarkii Hepatopancreas.

Autolysis is an internal phenomenon following the death of an organism that leads to the degradation of tissues. In order to explore the initial stages of autolysis and attempt to establish reference standards for tissue changes after death, we studied the rapidly autolyzing tissue of the crayfish hepatopancreas. Samples from the hepatopancreas of crayfish were examined 0, 5, 10, 30, 60, and 120 minutes after death. Histological and ultrapathological examinations and evaluations and apoptotic cell counts were conducted to determine the initiation time and degree of autolysis. The results showed that autolysis in the hepatopancreas of crayfish began within 5 minutes. Initially, autolysis manifested in the swelling of hepatic tubular cells and the widening of mesenchyme. Cells undergoing autolysis showed severe organelle necrolysis. Based on these observations, tissue samples should be collected and preserved within five minutes to avoid interfering with histopathological diagnoses.

Extensive autolytic fragmentation of membranous versus cytosolic calpain following myocardial ischemia-reperfusion.

We investigated calpain activation in the heart during ischemia-reperfusion (I-R) by immunologically mapping the fragmentation patterns of calpain and selected calpain substrates. Western blots showed the intact 78 kDa large subunit of membrane-associated calpain was autolytically fragmented to 56 and 43 kDa signature immunopeptides following I-R. Under these conditions, the 78 kDa calpain large subunit from crude cytosolic fractions was markedly less fragmented, with only weakly stained autolytic peptides detected at higher molecular weights (70 and 64 kDa). Western blots also showed corresponding calpain-like degradation products (150 and 145 kDa) of membrane-associated alpha-fodrin (240 kDa) following I-R, but in crude myofibrils alpha-fodrin degradation occurred in a manner uncharacteristic of calpain. For control hearts perfused in the absence of ischemia, autolytic fragmentation of calpain and calpain-like alpha-fodrin degradation were completely absent from most subcellular fractions. The exception was sarcolemma-enriched membranes, where significant calpain autolysis and calpain-like alpha-fodrin degradation were detected. In purified sarcoplasmic reticulum membranes, RyR2 and SERCA2 proteins were also highly degraded, but for RyR2 this did not occur in a manner characteristic of calpain. When I-R-treated hearts were perfused with peptidyl calpain inhibitors (ALLN or ALLM; 25 micromol/L), calpain autolysis and calpain-like degradation of alpha-fodrin were equally attenuated by each inhibitor. However, only ALLN protected against early loss of developed pressure in hearts following I-R, with no functionally protective effect of ALLM observed. Our studies suggest calpain is preferentially activated at membranes following I-R, possibly contributing to impaired ion channel function implicated by others in I-R injury.

Postmortem MRI changes of the brains of the rats of different ages.

Brain degenerative changes were studied in the rats of different ages after cervical dislocation by T(2) MRI and histology. Appearance of dark spots in the T(2) images increased with increased duration after death. Quantitative analysis of the density of these spots revealed that the neonatal (1 week) and the old (9 months) animals had accelerated degenerative changes when compared with the young adult (1 month). The degenerative changes correlated with the accumulation of vacuoles or spaces in the brain tissue histologically. This study pointed out not only brain degenerative changes after death were associated with age, it also revealed that the MRI T(2) evaluations could be used as a way in postmortem investigation.

Post Mortem Redistribution of Drugs: Current State of Knowledge.

BACKGROUND: Drug concentrations obtained from post mortem samples do not necessarily reflect the concentrations at the time of death, and variations of concentration may be observed between different sites and/or different sampling times. These phenomena, collectively termed post mortem redistribution, concern numerous molecules (medications, drugs of abuse, gases, etc.) and can complicate the interpretation of toxicological analyses. METHODS: Literature review. RESULTS: The mechanisms that cause these phenomena are complex and often intricate. Certain organs, which concentrate the molecules before death, may release them very early in the vascular sector. The gastrointestinal tract, liver, lungs and myocardium are mainly concerned. Cell autolysis also plays a part in drug release. Furthermore, micro-organisms (mainly bacteria and yeasts) which colonize the organism during putrefaction may cause neoformation and/or the degradation of certain molecules. Lastly, it appears that the physicochemical and pharmacokinetic profile of xenobiotics, notably their lipophilic nature, their ionization state and their volume of distribution may be factors likely to influence redistribution phenomena. Some recommendations concerning anatomic sampling sites, sampling methods and sample storage make it possible to limit these phenomena.

Changes in collagenous tissue microstructures and distributions of cathepsin L in body wall of autolytic sea cucumber (Stichopus japonicus).

The autolysis of sea cucumber (Stichopus japonicus) was induced by ultraviolet (UV) irradiation, and the changes of microstructures of collagenous tissues and distributions of cathepsin L were investigated using histological and histochemical techniques. Intact collagen fibers in fresh S. japonicus dermis were disaggregated into collagen fibrils after UV stimuli. Cathepsin L was identified inside the surface of vacuoles in the fresh S. japonicus dermis cells. After the UV stimuli, the membranes of vacuoles and cells were fused together, and cathepsin L was released from cells and diffused into tissues. The density of cathepsin L was positively correlated with the speed and degree of autolysis in different layers of body wall. Our results revealed that lysosomal cathepsin L was released from cells in response to UV stimuli, which contacts and degrades the extracellular substrates such as collagen fibers, and thus participates in the autolysis of S. japonicus.

Temporal Sequence of Autolysis in the Cerebellar Cortex of the Mouse.

This study examined the temporal sequence of post-mortem changes in the cerebellar cortical granular and Purkinje cell layers of mice kept at a constant ambient temperature for up to 4 weeks. Nuclei of granule cell microneurons became pyknotic early after death, increasing progressively until, by 7 days, widespread nuclear lysis resulted in marked cellular depletion of the granular layer. Purkinje cells were relatively unaltered until about 96 h post mortem, at which time there was shrinkage and multivacuolation of the amphophilic cytoplasm, nuclear hyperchromasia and, sometimes, a perinuclear clear space. By 7 days, Purkinje cells had hypereosinophilic cytoplasm and frequent nuclear pyknosis. By 2 weeks after death, Purkinje cells showed homogenization, the cytoplasm being uniformly eosinophilic, progressing to a 'ghost-like' appearance in which the cytoplasm had pale eosinophilic staining with indistinct cell boundaries, and nuclei often absent. The results of this study could assist in differentiating post-mortem autolysis from ante-mortem lesions in the cerebellar cortex and determining the post-mortem interval. Moreover, this information could be useful when interpreting brain lesions in valuable mice found dead unexpectedly during the course of biomedical experiments.

A comparison of rapid bovine spongiform encephalopathy testing methods on autolyzed bovine brain tissue.

In 1999, the European Union (EU) approved 3 rapid methods for the testing of bovine brain samples for the presence of bovine spongiform encephalopathy (BSE). The evaluation that led to the approval did not include an analysis of autolyzed material. Member states of the EU have active surveillance programs for BSE, which target fallen stock as well as other categories of cattle. Autolysis is a common feature of fallen stock samples because there can be a considerable delay between death and collection of samples. Therefore, it is important to know whether these tests perform optimally on autolyzed samples. The Veterinary Laboratories Agency (VLA) selected 250 positive fallen stock samples. These had been detected during routine testing using the Prionics-Check Western blot and confirmed as BSE cases by immunohistochemistry or electron microscopy. Samples were graded according to the degree of autolysis and then tested by the 3 methods: Prionics-Check Western blot, Platelia test, and Enfer test. All 3 methods correctly classified the samples as positive BSE cases, therefore alleviating doubt about their ability to do so. Subsequent EU validation exercises, such as those conducted in 2002--2003, have included the testing of autolyzed material. It is important that all new methods be evaluated on autolyzed tissue before approval for official use.

Diagnosis of Alzheimer's disease in an exhumed decomposed brain after twenty months of burial in a deep grave.

After 20 months of interment in a deep grave, the decomposed body of the 81-year old testator of a will was exhumed to sustain the burden of proof that he lacked testamentary capacity when the will was rewritten two days prior to his death. The brain was mushy and pulverized with complete disappearance of the brainstem, cerebellum and subcortical ganglia. Small foci of relatively intact dorsal frontal neocortex were identified. Sections from these foci were stained with hematoxylin and eosin, bielchowsky silver stain and immunostains for beta amyloid peptide (betaA4), tau and alpha-synuclein. Despite severe autolysis and decomposition, the bielchowsky stain and the betaA4 immunostains showed preserved frequent neuritic amyloid plaques with very few residual neurofibrillary tangles. Cerebral Amyloid Angiopathy was present. At the present time this case represents the first documented and reported case of direct tissue diagnosis of Alzheimer's Disease pathology in a decomposed brain following long term burial in a deep grave.

Analysis of rugae in burn victims and cadavers to simulate rugae identification in cases of incineration and decomposition.

The most challenging situations in Forensic Odonto-Stomatology are mass disasters, where the forensic dentist is usually confronted with charred human remains or heavily decomposed or fragmented bodies. This article determines the extent of preservation of palatal rugae for use as an alternative identification tool in such situations, using a study group comprising burn victims and cadavers simulating forensic cases of incineration and decomposition. The thermal effects and the decomposition changes on the palatal rugae of burn victims with panfacial third degree burns and human cadavers in storage were respectively assessed and graded on a new scale. Ninety three percent of burn victims and 77% of human cadavers had Grade 0 changes (normal). When changes were noted, they were less pronounced than the generalized body involvement of burns in burn victims and the generalized body decomposition of human cadavers.

Acinar autolysis and mucous extravasation in human sublingual glands: a microscopic postmortem study.

UNLABELLED: Although some morphological investigations on aged human sublingual glands (HSG) found eventual phenomena identified as autolysis and mucous extravasation, the exact meaning of these findings has not been elucidated. OBJECTIVE: The aim of this work is to investigate whether acinar autolysis and mucous extravasation are related to the aging process in human sublingual glands. We also speculate if autolytic changes may assist forensic pathologists in determining time of death. MATERIAL AND METHODS: 186 cadavers' glands were allocated to age groups: I (0-30 years); II (31-60), and III (61-90). Time and mode of death were also recorded. Acinar autolysis and mucous extravasation were classified as present or absent. Ultrastructural analysis was performed using transmission electron microscopy (TEM). Data were compared using Mann-Whitney U, Spearman's correlation coefficient, Kruskal-Wallis, and Dunn tests (p<0.05). RESULTS: There was correlation between age and acinar autolysis (r=0.38; p=0.0001). However, there was no correlation between autolysis and time of death. No differences were observed between genders. TEM showed mucous and serous cells presenting nuclear and membrane alterations and mucous cells were more susceptible to autolysis. CONCLUSION: Acinar autolysis occurred in all age groups and increased with age while mucous extravasation was rarely found. Both findings are independent. Autolysis degrees in HSG could not be used to determine time of death.

Myofibrillar degeneration and necrosis of the visceral smooth musculature: an ischemic visceral myopathy.

Pathologic studies of the visceral smooth musculature in humans are scant despite the relatively frequent occurrence of alterations in these muscles in autopsy material. We investigated the different types of lesions of this musculature observed in various conditions associated with ischemia--acute tubular necrosis, congenital heart disease (low output syndrome due to open heart surgery), and necrotizing enterocolitis in premature babies. Control cases included normal rat tissue undergoing autolysis and rigor mortis and bowel resected from patients with ulcerative colitis and Hirschsprung's disease. Four histologically distinct lesions were present on hematoxylin--eosin staining in the ischemic group: contraction bands, wavy fibers, thick waves, and coagulation necrosis. These lesions were absent in the control groups. We conclude that myofibrillar degeneration and necrosis of the visceral musculature are common in disorders associated with visceral ischemia. These changes are not artifacts produced by autolysis, rigor mortis, or technical handling, nor are they induced by nonischemic inflammatory conditions. Catecholamines may play a role in their genesis.

Structural basis of ventricular arrhythmias in human myocardial infarction: a hypothesis.

The present study was undertaken using light and electron microscopic techniques to determine whether Purkinje fibers survive in the subendocardial region of anteroseptal infarcts in humans. Tissue was obtained for this purpose from 11 patients with 12 documented infarctions at the time of autopsy; six patients died within 72 hours of the infarction and five had healed infarcts. Seven of the 11 patients had ventricular arrhythmias. Light microscopic study indicated that intact cells with a normal appearance remained on the subendocardial surface, although the underlying ventricular muscle either was necrotic or was replaced by fibrous tissue. Electron microscopy demonstrated that these intact surviving cells over the surface of the infarct had few randomly oriented myofibrils, abundant glycogen, and other characteristics of Purkinje fibers. These cells could be readily distinguished from normal or infarcted ventricular muscle cells. Purkinje fibers, the most peripheral part of the conduction system, survive in extensive anteroseptal infarcts and may be the site of origin of ventricular arrhythmias.

Respirator retina.

Retinas from 14 comatose patients, who had been sustained with a respirator for one or more days before death, had selective characteristic alterations (ie, autophagy, cell swelling, and coagulation necrosis) of inner nuclear layer in a patchy pattern in posterior fundus. Bipolar cells were most often affected, but amacrine and horizontal cells also were substantially damaged. Cells of Müller and vascular cells were largely spared. These lesions are ascribed to oligemia (ischemia) since the inner nuclear layer is a microscopic vascular watershed (boundary zone) between the choroidal and retinal circulations.

A simple method for the prevention of endometrial autolysis in hysterectomy specimens.

AIMS: Uteri are among the most common surgical pathology specimens. Assessment of the endometrium is often difficult because of pronounced tissue autolysis. This study describes a simple method to prevent endometrial autolysis and aid in interpretation of the endometrium. METHODS: Sixty uteri were injected with formalin using a needle and syringe directed alongside a probe, which was inserted through the external cervical os into the endometrial cavity. Injection was performed on the same day as removal of the uterus. As controls, 60 uteri that were not injected with formalin were examined. The degree of endometrial autolysis was assessed on a four point scale (0-3), with a score of 0 representing no or minimal autolysis and a score of 3 representing extensive autolysis, such that histological interpretation of the endometrium was impossible. RESULTS: In the injected group, the number of cases with scores of 0, 1, 2, and 3 was 42, 13, four, and one, respectively. The corresponding values for the control group were 17, 23, eight, and 12, respectively. This was highly significant (p < 0.001) CONCLUSIONS: There was significantly less endometrial autolysis in uteri injected with formalin. The use of this simple procedure should be encouraged in hysterectomy specimens.

Postmortem detection of inapparent myocardial infarction.

Two methods of detecting early inapparent myocardial infarcts have been studied and their value in diagnostic practice compared. The better method proved to be the determination of the potassium to sodium ratio (ionic ratio) which falls in infarcted tissue within minutes of the onset of anoxia. The second method was nitro blue tetrazolium staining of gross sections of myocardium which revealed any infarct older than three and a half hours. As staining is dependent upon enzyme activity, the latter method is disturbed by autolysis. It was shown, on the other hand, that the ionic ratio (K(+)/Na(+)) was not affected by autolysis and was therefore well suited to forensic practice. Sixteen non-infarcted control hearts, plus the nine from cases of sudden death due to causes other than myocardial infarction, all yielded high ionic ratios (K(+)/Na(+)), average 1.4, and stained normally with tetrazolium (the normal controls). Positive control was provided by 20 histologically proven infarcts of which the ionic ratios (K(+)/Na(+)) were all low (average 0.7). Histochemical staining with tetrazolium delineated infarcted areas in each case. In a series of 29 sudden deaths, a cause of death other than myocardial infarction was found at necropsy in nine, mentioned above as normal controls. The remaining 20 hearts were not infarcted histologically, but were shown to be infarcted by examination of the ionic ratios (K(+)/Na(+)). These ratios were low (average 0.8) including three borderline ratios. Confirmatory evidence of infarction included nitro blue tetrazolium staining which revealed infarcts in 10 of the 20 cases, and clinical and necropsy observations. The ionic ratio (K(+)/Na(+)) decreases as the age of the infarct increases for at least 24 hours. Thereafter as healing proceeds, the ratio gradually reverts to normal. Thus, previous infarction and replacement fibrosis do not significantly alter the ionic ratio (K(+)/Na(+)). Nor is it changed by left ventricular hypertrophy, the presence of congestive cardiac failure, or digitalis therapy. It is suggested that macroscopic tetrazolium staining is a useful screening test for early inapparent myocardial infarcts. In cases where no infarct is delineated with that method estimation of the ionic ratio (K(+)/Na(+)) should be carried out on myocardium removed from standard areas on the anterior and posterior left ventricular walls.

Paleohistopathology of bone: a new approach to the study of ancient diseases.

Light microscopy, particularly the use of polarized light, has such a high value for the differential diagnosis of dry bones that it can no longer be neglected. Alterations caused intra vitam by disease or other living conditions can clearly be differentiated by this technique from changes due to postmortem reactions (e.g., pseudopathology). As a reliable diagnosis is the basis not only of the study of case reports but also of the etiology and epidemiology of diseases in ancient populations, paleopathologists would be well-advised to employ histological analysis for their research, to avoid false diagnoses. The necessary basis for such research is the knowledge of the general histology, histogenesis, and growth as well as pathophysiology of bone. Some new techniques which facilitate the practical use of microscopic analysis, such as the preparation of thin-ground sections from undecalcified bone samples and nonrehydrated mummified soft tissues, are described. Selected examples of mechanisms of pathological bone changes, particularly the determination of vestiges of diseases in macerated bones by microscopy, are presented. Emphasis is placed on the differential diagnoses of proliferative reactions (e.g., periosteal processes of long bones and the skull). In this context, the importance of meningeal reactions on the endocranial lamina of the skull for morbidity and mortality in ancient populations is demonstrated. Furthermore, porotic hyperostosis of the skull vault and the orbital roof, i.e., the cribra cranii externa and cribra orbitalia, is discussed. Selected examples of the etiology and epidemiology of ancient diseases are presented (e.g., anemia, scurvy, rickets, and meningeal diseases), and ideas on living conditions and their implications for the origin and the spread of disease are given to establish a better understanding of deficiency and infectious diseases in the past.