Gel-to-gel phase transition may occur in mammalian cells: Mechanism of formation of "dark" (compacted) neurons

In the course of many diseases, individual non-apoptotic cells that are randomly distributed among undamaged cells in various mammalian tissues become shrunken and hyperbasophilic ("dark"). The light microscopic shrinkage is caused by a potentially reversible, dramatic compaction of all ultrastructural elements inside the affected cells, and escape of the excess water through apparently intact plasma membrane. In the case of neurons, the ultrastructural compaction rapidly involves the soma-dendrite domains in an all-or-nothing manner, and also mm-long axon segments. The present paper demonstrates that such ultrastructural compaction in neurons, which affects the whole soma-dendrite domain or long axon segments, can take place both immediately after an in vivo head injury and in rat brains perfusion-fixed for 30 min., and then chilled to just above the freezing point before the same kind of head injury was inflicted. This argues strongly against any enzyme-mediated compaction mechanism. On the analogy of gel-to-gel phase transitions in polymer chemistry, we hypothesize a pure physico-chemical compaction mechanism. Specifically, after initiation at a single site in each affected cell, the ultrastructural compaction is propelled throughout the whole cell on the domino principle by the free energy stored in the form of non-covalent interactions among the constituents of some cytoplasmic gel structure.     

Differential effects of inescapable footshocks and of stimuli previously paired with inescapable footshocks on dopamine turnover in cortical and limbic areas of the rat

The effect of electric footshocks and of exposure to environmental stimuli paired with electrical shocks upon the dopaminergic activity in various cortical and limbic areas of the rat were evaluated by measuring dihydroxyphenylacetic acid (DOPAC) levels in these areas. In animals exposed to a 20 min electric footshock session DOPAC concentrations were significantly increased in the antero-medial and sulcal frontal cortices, olfactory tubercle, nucleus accumbens and amygdaloid complex (by 66, 37, 28, 55 and 90% respectively). Re-exposure of rats to an environment where they had been shocked 24 h earlier induced an elevation of DOPAC content only in the anteromedial frontal cortex (by 47%). Plasma corticosterone levels were elevated in both situations. No change in serotonin or 5-hydroxyindolacetic acid content of these areas could be detected in either situation. The results show that electric footshocks and environmental stimuli associated to previous shocks both activate central dopaminergic systems, although the patterns of activation are different.     

Differences between left and right ventricular anatomy determine the types of reentrant circuits induced by an external electric shock. A rabbit heart simulation study

Despite the fact that elucidating the mechanisms of cardiac vulnerability to electric shocks is crucial to understanding why defibrillation shocks fail, important aspects of cardiac vulnerability remain unknown. This research utilizes a novel anatomically based bidomain finite-element model of the rabbit ventricles to investigate the effect of shock polarity reversal on the reentrant activity induced by an external defibrillation-strength shock in the paced ventricles. The specific goal of the study is to examine how differences between left and right ventricular chamber anatomy result in differences in the types of reentrant circuits established by the shock. Truncated exponential monophasic shocks of duration 8 ms were delivered via two external electrodes at various timings. Vulnerability grids were constructed for shocks of reversed polarity (referred to as RV- or LV- when either the RV or the LV electrode is a cathode). Our results demonstrate that reversing electrode polarity from RV- to LV- changes the dominant type of post-shock reentry: it is figure-of-eight for RV- and quatrefoil for LV- shocks. Differences in secondary types of post-shock arrhythmia also occur following shock polarity reversal. These effects of polarity reversal are primarily due to the fact that the LV wall is thicker than the RV, resulting in a post-shock excitable gap that is predominantly within the LV wall for RV- shocks and in the septum for LV- shocks.     

Gastric and cardiac organoprotection by lidocaine

The concept of cytoprotection has been applied to many tissues afforded protection by drugs or endogenous chemicals against organelle, cyto- or histopathologic damage. We review here the "organoprotection" by lidocaine in rats and dogs as appraised by in vitro, ex vivo, and in vivo experiments with the stomach and heart, and as revealed at organelle to organ functional levels. Gastric mucosal lesions induced by 80% ethanol with 100 mM HCl on the ex vivo rat stomach were significantly reduced by lidocaine (2.2-4.4 mg/kg bolus followed by 66-132 micrograms/kg/min i. v. infusion). In anesthetized dogs with gastric corporeal lesions induced by increased gastric intraluminal pressure (50 mm Hg, 2.5 hrs), lidocaine (2.2 mg/kg bolus plus 66 micrograms/kg/min infusion) significantly reduced lesion severity. In the isolated rat heart, reperfusion after a 60 min period of ischemia induced localized cardiac mitochondrial swelling and disruption in ventricular apices which was greatly reduced if hearts were pretreated (15 min perfusion with lidocaine). In intact rats subjected to hemorrhagic shock, lidocaine pretreatment also facilitated shock resuscitation and reduced ultrastructural damage. In these diverse experiments, lidocaine organoprotection was likely mediated in part through reduction of ischemia induced organelle membrane damage and through reduction of reperfusion-induced superoxide and other oxygen-derived free radical related damage.     

Studies on [3H]Diazepam and [3H]Ethyl-β-Carboline Carboxylate Binding to Rat Brain In Vivo. II. Effects of Electroconvulsive Shock

In vivo specific binding of [3H]diazepam was not altered by a single electroconvulsive shock given 5, 30, or 60 min, or 24 h previously, nor 24 h after the last of 10 daily shocks. Similarly, in vivo [3H]ethyl-beta-carboline carboxylate binding was not changed in the brains of animals that had been given a single electroconvulsive shock 30 min previously or a series of 10 daily shocks. Brain areas examined included cerebral cortex, hippocampus, cerebellum, and striatum. However, cortical binding of [3H]diazepam was increased by 32% in animals which were present in the same room while another was being injected and killed. This may represent a response to stress and/or anxiety.     

Progesterone prevents corticosterone mediated inhibition of estrous behaviour in rats

Stress induced by application of electric foot shocks (300 microA/shock, five shocks per episode, 4 episodes at 1800, 1830, 1900 and 1930 hrs on the proestrus day) to rats at the time of pre-ovulatory progesterone secretion, abolished lordosis and resulted in maximum rejection co-efficient, whereas treatment with a CRF receptor antagonist (alpha-helical CRF9-41) or metapirone, an inhibitor of corticosterone synthesis, prior to application of the electric foot shocks, resulted in normal lordosis and a significant reduction in rejection coefficient. Further, administration of a single dose of corticosterone (40 microg) at 1800 hrs of proestrus caused inhibition of lordosis and resulted in maximum rejection co-efficient. On the other hand, corticosterone + progesterone treatment at 1800 hrs of proestrus resulted in normal lordosis and a significant reduction in rejection coefficient. The facts that stress induced inhibition of lordosis is prevented by CRF receptor antagonist or metapirone and that corticosterone inhibits lordosis indicate that stress induced inhibition of lordosis is mediated by corticosterone. Further, normal display of lordosis by rats treated with corticosterone + progesterone in contrast to its absence in corticosterone alone treated rats suggests that impaired progesterone secretion due to action of corticosterone leads to inhibition of lordosis.     

The effect of low energy electric shock on cortisol, β-endorphin, heart rate and behaviour of cattle

Electrical stimuli are used increasingly to confine cattle, whether through conventional electric fencing or the development of ‘virtual’ fencing systems. Two experiments were conducted to assess behavioural, heart rate and stress hormone responses of cattle to electrical stimuli typically used in such confinement applications. In the first experiment, 30 steers (18-months old; n = 10 per treatment) were held in a handling crush for 15 min after receiving one of the following treatments: nothing (control); delivery of three shocks at 2 s intervals (600 V, 250 mW); and restraint in a head bail for 3 min. Plasma cortisol and β-endorphin concentrations were measured at 0, 5, 10, 15 min, 1, 2, 3, and 4 h. In a second experiment, heart rate and behaviour were measured in 17 heifers (18 months of age) subjected to one of the following treatments whilst held in a crush for 10 min: nothing (control; n = 5); delivery of three shocks at 2 s intervals (600 V, 250 mW; n = 6); and restraint in a head bail for 3 min (n = 6). Cortisol and β-endorphin concentrations did not differ between treatments (P > 0.05). Whilst animals were receiving the treatments, heart rate was lower when head restrained compared with shock or control treatments (P = 0.009) and did not differ between control and electric shock treatments (P = 0.35). Upon release from the crush, heart rate was higher in shock and head restrained treatments than the control treatment (P = 0.005). Animals receiving the electric shock treatment tossed their heads more frequently whilst in the crush than control animals (P = 0.012) but did not differ from the other treatments in the number of vocalisations, tail swishes, steps back and forward, head tilts and head turns. There was a significant effect of treatment on flight time (P = 0.005); animals receiving the electric shocks were faster to leave the crush than control animals (P = 0.005) and there was no difference between head restraint and shock treatment (P = 0.86). In 10 min following release from the crush, there was no treatment difference in the time to start feeding. This study suggests that the stress response of cattle to low energy electric shocks is minimal and is similar to that induced by restraint in a crush.     

Damage induction by direct electric current in tumoural target cells

Damage induction to tumour target cells (P815) by direct electric current (DC) was investigated. A 6 min treatment of P815 cells with DC generated decreased levels of cell viability and proliferation. The ultrastructural analysis of DC-treated cells revealed the presence of blebs, loss of cell surface filopodia, and ruptures in cell membrane. Mitochondrial alterations, swelling of cells, cytoplasmic matrix rarefaction, and cellular debri formation were also observed. The study shows that tumoural target cells can be damaged by direct electric current and this approach may provide means to understand the mechanism of tumour regression induced by electrochemical therapy.     

Atomic force and electron microscopy of high molecular weight circular DNA complexes with synthetic oligopeptide trivaline

Intramolecular compact structures formed by high molecular weight circular superhelical DNA molecules due to interaction with synthetic oligopeptide trivaline (1) were studied by atomic force and electron microscopy. Three DNA preparations were used: plasmids pTbol, pRX10 and cosmid 27,877, with sizes 6,120 bp, 10,500 bp and 44,890 bp respectively. Plasmid pTbo1 and pRX10 preparations along with monomers contained significant amount of dimers and trimers. Main structures in all preparations observed were compact particles, which coincide in their appearance and compaction coefficient (3,5-3,7) with triple rings described earlier. The size and structure characteristics of triple rings and other compact particles on atomic force images in general coincide with those obtained by EM (2). AFM (3) images allow to get additional information about the ultrastructural organization and arrangement of DNA fibers within the compact structures. Along with triple rings in pTbol and pRX10-TVP complexes significant amount of compact structures were observed having the shape of two or three compact rings attached to each other by a region of compact fibre. Basing on the data of contour length measurements and the shape of the particles it was concluded that these structures were formed due to compaction of dimeric and trimeric circular DNA molecules. Structures consisting of several attached to each other triple rings were not found for pTbol, pRX10 monomers or cosmid preparations--TVP complexes where only single triple rings were observed. The conclusion is made that initiation of compact fibre formation within the circular molecules depends on the primary structure and for dimeric or trimeric circular molecules two or three compaction initiation points are present, located in each monomer unit within one circular DNA molecule. The nucleotide sequence dependent compaction mechanism providing independent compaction of portions of one circular molecule can be of interest for understanding of DNA compaction processes in vivo.     

Effect of acute global ischemia on the upper limit of vulnerability: a simulation study

The goal of this modeling research is to provide mechanistic insight into the effect of altered membrane kinetics associated with 5-12 min of acute global ischemia on the upper limit of cardiac vulnerability (ULV) to electric shocks. We simulate electrical activity in a finite-element bidomain model of a 4-mm-thick slice through the canine ventricles that incorporates realistic geometry and fiber architecture. Global acute ischemia is represented by changes in membrane dynamics due to hyperkalemia, acidosis, and hypoxia. Two stages of acute ischemia are simulated corresponding to 5-7 min (stage 1) and 10-12 min (stage 2) after the onset of ischemia. Monophasic shocks are delivered in normoxia and ischemia over a range of coupling intervals, and their outcomes are examined to determine the highest shock strength that resulted in induction of reentrant arrhythmia. Our results demonstrate that acute ischemia stage 1 results in ULV reduction to 0.8A from its normoxic value of 1.4A. In contrast, no arrhythmia is induced regardless of shock strength in acute ischemia stage 2. An investigation of mechanisms underlying this behavior revealed that decreased postshock refractoriness resulting mainly from 1) ischemic electrophysiological substrate and 2) decrease in the extent of areas positively-polarized by the shock is responsible for the change in ULV during stage 1. In contrast, conduction failure is the main cause for the lack of vulnerability in acute ischemia stage 2. The insight provided by this study furthers our understanding of mechanisms by which acute ischemia-induced changes at the ionic level modulate cardiac vulnerability to electric shocks.     

Alterations in ultrastructural morphology of two-cell bovine embryos produced in vitro and in vivo following a physiologically relevant heat shock

Exposure of cultured preimplantation embryos to temperatures similar to those experienced by heat-stressed cows inhibits subsequent development. In this study, the effects of heat shock on the ultrastructure of two-cell bovine embryos were examined to determine mechanisms for inhibition of development. Two-cell embryos produced in vitro were harvested at approximately 28 h postinsemination and cultured for 6 h at one of three temperatures: 38.5 degrees C (cow body temperature), 41.0 degrees C (characteristic temperature for heat-stressed cows), or 43.0 degrees C (severe heat shock). Ultrastructural examinations revealed that both heat shocks resulted in the movement of organelles towards the center of the blastomere. In addition, heat shock increased the percentage of mitochondria exhibiting a swollen morphology. Distance between the membranes comprising the nuclear envelope was increased but only when embryos were treated at 43.0 degrees C. To determine whether ultrastructural responses to heat shock in culture were similar for embryos produced in vitro and in vivo, two-cell embryos were collected from superovulated Angus cows 48 h postinsemination and treated ex vivo for 6 h at 38.5 degrees C or 41.0 degrees C. Again, heat shock caused an increase in number of swollen mitochondria and movement of organelles away from the periphery of the blastomere. Exposure of two-cell bovine embryos to physiologically relevant elevated temperatures causes disruption in ultrastructural morphology that is inimical to development. The observation that overall morphology and response to heat was similar for embryos produced in vitro and in vivo implies that the former can be a good model for understanding embryonic responses to heat shock.     

Triploidy Induction in the Pacific White Shrimp Litopenaeus vannamei: An Assessment of Induction Agents and Parameters, Embryo Viability, and Early Larval Survival

In this study, we trialed 6-dimethylaminopurine (6-DMAP) chemical shocks to induce meiosis I or meiosis II Pacific White shrimp, Litopenaeus vannamei, triploids for the first time, and cold temperature shocks to induce meiosis II L. vannamei triploids as done previously. Inductions were performed on 37 spawnings in total with experiments being progressively designed in a factorial manner to allow optimization of induction parameters. Treatment with a 200-μm 6-DMAP final concentration at 1?min post-spawning detection for a 6 to 8?min duration resulted in the most consistent induction of chemically induced meiosis I triploids while treatment at 7?min 30?s post-spawning detection for a 10-min duration resulted in the most consistent induction of chemically induced meiosis II triploids. A cold temperature shock of 11.7°C to 13.25°C (final treatment temperature; spawning water temperature 28.5°C) applied at 8?min post-spawning detection for a 4 to 10?min duration resulted in the most consistent induction of cold-temperature-induced meiosis II triploids. 6-DMAP shocks resulted in meiosis I induction rates from 29% to 100% in unhatched embryos and 50% in nauplii, and meiosis II induction rates from 65% to 100% in unhatched embryos and 52% to 100% in nauplii. Cold shocks resulted in induction rates from 5% to 100% in unhatched embryos and nauplii. Confocal microscopy analysis of embryos revealed that there are major developmental abnormalities in a large proportion of later stage triploid L. vannamei embryos compared to their diploid sibling controls. Despite this, however, some triploid embryos did appear normal and both shock agents induced small numbers of viable triploid L. vannamei nauplii which were successfully reared to protozoeal stage 3 as confirmed by flow cytometry. Triploids beyond this life-history stage were not observed in the present study as confirmed by flow cytometry at mysis stages. This study adds to our knowledge base of triploid induction in L. vannamei and further highlights the inherent difficulties with triploid embryonic and larval viability in this species.     

Ultrastructural observation of human neutrophils during apoptotic cell death triggered by Entamoeba histolytica

Neutrophils are important effector cells against protozoan extracellular parasite Entamoeba histolytica, which causes amoebic colitis and liver abscess in human beings. Apoptotic cell death of neutrophils is an important event in the resolution of inflammation and parasite's survival in vivo. This study was undertaken to investigate the ultrastructural aspects of apoptotic cells during neutrophil death triggered by Entamoeba histolytica. Isolated human neutrophils from the peripheral blood were incubated with or without live trophozoites of E. histolytica and examined by transmission electron microscopy (TEM). Neutrophils incubated with E. histolytica were observed to show apoptotic characteristics, such as compaction of the nuclear chromatin and swelling of the nuclear envelop. In contrast, neutrophils incubated in the absence of the amoeba had many protrusions of irregular cell surfaces and heterogenous nuclear chromatin. Therefore, it is suggested that Entamoeba-induced neutrophil apoptosis contribute to prevent unwanted tissue inflammation and damage in the amoeba-invaded lesions in vivo.     

Adaptive MscS gating in the osmotic permeability response in E. coli: the question of time

Microorganisms adapt to osmotic downshifts by releasing small osmolytes through mechanosensitive (MS) channels. We want to understand how the small mechanosensitive channel's (MscS) activation and inactivation, both driven by membrane tension, optimize survival in varying hypoosmotic shock situations. By measuring light scattering with a stopped-flow device, we estimate bacterial swelling time as 30-50 ms. A partial solute equilibration follows within 150-200 ms, during which optical responses from cells with WT MscS deviate from those lacking MS channels. MscS opening rates estimated in patch clamp show the channels readily respond to tensions below the lytic limit with a time course faster than 20 ms and close promptly upon tension release. To address the role of the tension-insensitive inactivated state in vivo, we applied short, long, and two-step osmotic shock protocols to WT, noninactivating G113A, and fast-inactivating D62N mutants. WT and G113A showed a comparable survival in short 1 min 800 mOsm downshock experiments, but G113A was at a disadvantage under a long 60 min shock. Preshocking cells carrying WT MscS for 15 s to 15 min with a 200 mOsm downshift did not sensitize them to the final 500 mOsm drop in osmolarity of the second step. However, these two-step shocks induced death in D62N more than just a one-step 700 mOsm downshift. We conclude MscS is able to activate and exude osmolytes faster than lytic pressure builds inside the cell under abrupt shock. During prolonged shocks, gradual inactivation prevents continuous channel activity and assists recovery. Slow kinetics of inactivation in WT MscS ensures that mild shocks do not inactivate the entire population, leaving some protection should conditions worsen.     

Biochemical and ultrastructural changes in rat liver plasma membranes after temporary ischemia

In this study we have attempted to correlate reversible and irreversible cell damage induced by in vivo or in vitro ischemia with characteristics of the plasma membranes of liver parenchymal cells, as detected biochemically and ultrastructurally. The effects of in vivo or in vitro ischemia appeared to be similar. It was virtually impossible to isolate a substantial membrane fraction from ischemic livers, probably because of changes in the physical properties of the membranes by ischemia. The isolated membranes of ischemic liver cells show ultrastructural changes including the occurrence of many vesicular profiles and alterations in junctional complexes expressed by extended and smudged electron densities along the lateral surfaces. The microvilli of the bile canaliculi disappeared after only 15 min ischemia and cytoplasmic densities associated with junctional complexes also appeared extended and smudged. These changes correspond with the alterations observed in ischemic isolated membranes. After 30 min in vivo ischemia the activity of 5'-mononucleotidase used as a marker enzyme for plasma membranes, decreased by 75%, whereas the activity of thymidine 5'-phosphodiesterase was reduced only slightly. The changes in these enzyme activities were more prominent after in vitro ischemia than after in vivo. The morphological and biochemical changes observed in rat hepatocyte plasma membrane during the early stage of injury have no value in predicting the occurrence of necrosis in a later phase of the process since profound changes occur in plasma membrane properties after even short periods of ischemia (i.e. during the reversible stage).     

Experimental puffs in salivary gland chromosomes of Drosophila hydei

Summary In Drosophila hydei abnormal puffing activities could be induced by temperature shocks and treatments with 1.2% and 1.4% KCl solutions. After temperature shocks in vivo and in vitro, a number of puffs showed a similar change in activity.Other puffs were found to show a change in activity only after a distinct treatment.Some of the puffs, specific for temperature shocks, showed a change in activity only at a distinct stage of development.In discussing the results, particular attention is paid to puffs observed in common after all treatments.Dedicated to Prof. H. Bauer on the occasion of his sixtieth birthday.     

Protein synthesis patterns following stage-specific heat shock in early Drosophila embryos

Summary Very short heat shocks are administered to carefully staged early embryos of Drosophila melanogaster, and the effects on protein synthesis pattern investigated. A shock as short as 2 min will induce the heat shock response (reduction of normal protein synthesis, increased synthesis of the heat shock proteins) in syncytial blastoderm or later stages. Thus the initial events of the heat shock response must occur within 2 min, and not reverse upon rapid return to 22° C. A low level of synthesis of the 70 kDa heat shock protein is sometimes visible in unshocked animals, but may be induced by the labeling procedure. Survival following a short shock is not strictly correlated with a high level of heat shock response. Pre-blastoderm embryos do not produce significant heat shock protein, but survive a 2 min 43°C heat shock better than do heat shock response competent blastoderm embryos. The protein synthesis pattern prior to the blastoderm stage is very stable, possibly enhancing survival following a short shock. Shocks of 3 min or longer are more detrimental to pre-blastoderm embryos than to later stages, confirming the role of the heat shock response in survival following a longer shock. Stage-specific developmental defects (phenocopies) may be induced by heat shock at the blastoderm or later stages. Induction of these defects may require disruption of the normal protein synthesis pattern. Use of very short heat shocks to induce the heat shock response will be valuable in identifying the precise time at which a specific defect can be induced.     

Ultrastructure of Purkinje cells in the subendocardium and false tendons in early experimental myocardial infarction complicated by fibrillation in the dog

The effect of regional myocardial ischemia complicated by ventricular fibrillation (VF) on the ultrastructure of subendocardial (SE) and false tendon (FT) Purkinje cells (PC) was studied in anesthetized dogs. In all cases of early ischemia with spontaneous VF, many PC exhibited ultrastructural damage as early as 2 min after the onset of ischemia. The changes noted were: intercalated disk dissociation, sarcoplasmic reticulum vacuolization (SRV), supercontraction, mitochondrial swelling, and sarcolemmal defects (rigor cells). The appearance of at least some rigor PC seemed to precede spontaneous VF, since these cells were absent from the conduction systems in control hearts in which VF was induced by electric shock or reperfusion, from hearts from sham-operated dogs, or from hearts subjected to longer periods of uncomplicated myocardial infarction. These observations indicate that alterations in SE and FTPC may play a role in the pathogenesis of sudden death due to early myocardial ischemia. The mechanism of this rapid damage of PC remains obscure.     

Morphological effects of transcardially perfused SDS on the rat brain

Background information. For explanation of the formation of ‘dark’ neurons, an enigmatic phenomenon in neuropathology, we hypothesized recently that all spaces between the ultrastructural elements visible in the traditional transmission electron microscope are filled with a gel structure that stores free energy in the form of non‐covalent interactions, is continuous in the whole soma—dendrite domains of neurons, and is capable of whole‐cell phase transition. This hypothesis was deduced from the fact that ‘dark’ neurons can be formed, even under conditions extremely unfavourable for enzyme‐mediated biochemical processes, if initiated by a physical damage. In order to gain further information on this gel structure, we perfused transcardially rats for 5 min with physiological saline containing 1 mM SDS before the perfusion of a fixative for electron microscopy. Results. Dramatic compaction of visibly intact ultrastructural elements was caused in the whole soma—dendrite domains of thinly scattered neurons (‘dark’ neurons), whereas substantial cytoplasmic swelling and patchy ultrastructural disintegration occurred in numerous other neurons (‘light’ neurons). Similar morphological changes were observed in scattered astrocytes, oligodendrocytes, pericytes and endothelial cells. Conclusions. These observations: (i) support the existence of the above intracellular gel structure in neurons; (ii) allow the conclusion that this gel structure is present in the form of an ubiquitous trabecular network surrounded by a confluent system of fluid cytoplasm; (iii) draw attention to the possibility that the previous two statements also apply to other cell types of the brain tissue; and (iv) suggest that pressure‐induced direct channels exist between neurons and astrocytes.