Morphological studies of fiber types of striated muscle fibers of the cremaster in the guinea pig

THe fine structure of the striated muscle fibers of the cremaster of the guinea pig was studied using the cholinesterase technique and light and electron microscopy. Under light microscopy, isolated single muscle fibers showed two types of nerve endings: the first one presented elliptic or oval areas having digit-like structures inside, some of the borders of which were heavily stained. These fibers had only one end-plate. The second type presented elongated clear areas with most of the density located on the borders. Several nerve endings were apparent in these fibers. By electron microscopy, the former had large and numerous sarcolemmal foldings and these characteristics were also observed in unstained fibers. In the latter, the foldings were scanty or absent. At the ultrastructural level, the fibers having only one end-plate presented a regular array of fibrils with an abundant sarcoplasmic reticulum ('Fibrillenstruktur' type) in contrast to the multi-innervated fiber with an irregular distribution pattern of fibrils and a scarce sarcoplasmic reticulum ('Felderstruktur' type). The striated muscle fiber layer of the cremaster probably contains both fast and slow fibers. The possible functional role for the slow striated muscle fibers is discussed.     

Progressive reorganization of the myenteric plexus during one year following reanastomosis of the ileum of the guinea pig

The enteric nervous system appears to play a pivotal role in the functional recovery of the gastrointestinal tract after partial resection and reanastomosis, but the structural changes following surgery are not fully understood. The present study was designed to clarify the processes of myenteric plexus regeneration up to one year after transection and reanastomosis of the ileum of the guinea pig. The following techniques were used: nicotinamide adenine dinucleotide (NADH) diaphorase histochemistry, immunostaining of neuron-specific enolase (NSE) in whole-mount preparations, and transmission electron microscopy. Two months after transection and reanastomosis, myenteric ganglion cells with NADH diaphorase reactions were scarce in the center of the lesion, and were less numerous in adjacent areas (3 mm in width) than in the control ileum. In the areas adjacent to the lesion, a few large extraganglionic neurons that did not completely compensate for the loss of ganglion neurons were observed. The remaining ileum showed no changes in NADH diaphorase staining pattern at this stage. Two to 12 months after transection and reanastomosis, ectopic large neurons gradually increased in number not only in the areas adjacent to the lesion but also in part of the remaining ileum, up to 10 cm from the lesion. Concomitantly, large ganglion neurons decreased in number in these areas. In other ileal regions (more than 10 cm distant from the site of transection), no obvious changes in NADH diaphorase staining were noted throughout the observation period. The outgrowth of NSE-containing nerve fibers from the severed stumps was seen two weeks after transection. Six weeks later, numerous bundles of fine nerve fibers with NSE were shown to interconnect the oral and anal cut ends of the myenteric plexus, but they exhibited no subsequent alterations. Transmission electron microscopy revealed that regenerating nerve fiber bundles appeared initially among irregularly arranged smooth muscle cells eight weeks after the operation, as expected from light-microscopic observations. These findings suggest that myenteric ganglion cell bodies, unlike myenteric nerve fibers, require a longer term of reconstruction than previously believed after transection and reanastomosis of the ileum of the guinea pig.     

A comparative study of the regeneration of the striated and smooth-muscle tissues of the esophagus]

By means of electron microscopy and observational histological techniques, using a similar experimental model, regeneration of the striated and smooth muscle tissues of the esophagus has been studied in rats. During early periods after lesion in both muscle tissues destructive-necrotic changes develop. Beginning from the 2nd-3d days regeneration processes are observed. The course and periodicity of the regenerative processes are specific for the types of the muscle tissues studied. Each of the muscle tissues of the esophagus has its own source of regeneration. For the smooth muscles those are myoblasts, that convert into smooth myocytes, for the striated ones--myosatellites, which after activation get out of the muscle fiber. During the restorative process of the muscular membrane no tissue interconnections are observed. This also proves certain specificity of the striated and smooth muscle tissues of the esophagus.     

Arrangement and structure of sinus hair muscles in the big-clawed shrew,Sorex unguiculatus

The arrangement and structure of sinus hair muscles in the snout of the shrew, Sorex unguiculatus, were studied by electron microscopy and serial section light microscopy. Both striated and smooth muscles are directly associated with sinus hair follicles. The striated muscle fibers originate from the base of a follicle and insert onto the superficial portion of adjoining caudally positioned follicles. Some fibers insert into the corium instead of inserting into a follicle. The fibers show a fine structure typical of red fibers. Smooth muscle cells form a network with elastic fibers beneath the corium. Some cells are directly attached to the capsule of the sinus, thus forming a type of M. arrector pili. Striated muscle fibers that appear to end in the corium are connected with the smooth muscle network through the elastic fibers which appear to function as the tendon of these two types of muscle cell.     

Rescue of enzyme deficiency in embryonic diaphragm in a mouse model of metabolic myopathy: Pompe disease

Several human genetic diseases that affect striated muscle have been modeled by creating knockout mouse strains. However, many of these are perinatal lethal mutations that result in death from respiratory distress within hours after birth. As the diaphragm muscle does not contract until birth, the sudden increase in diaphragm activity creates permanent injury to the muscle causing it to fail to meet respiratory demands. Therefore, the impact of these mutations remains hidden throughout embryonic development and early death prevents investigators from performing detailed studies of other striated muscle groups past the neonatal stage. Glycogen storage disease type II (GSDII), caused by a deficiency in acid alpha-glucosidase (GAA), leads to lysosomal accumulation of glycogen in all cell types and abnormal myofibrillogenesis in striated muscle. Contractile function of the diaphragm muscle is severely affected in both infantile-onset and late-onset individuals, with death often resulting from respiratory failure. The knockout mouse model of GSDII survives well into adulthood despite the gradual weakening of all striated muscle groups. Using this model, we investigated the delivery of recombinant adeno-associated virus (rAAV) vectors encoding the human GAA cDNA to the developing embryo. Results indicate specific high-level transduction of diaphragm tissue, leading to activity levels up to 10-fold higher than normal and restoration of normal contractile function. Up to an estimated 50 vector copies per diploid genome were quantified in treated diaphragms. Histological glycogen staining of treated diaphragms revealed prevention of lysosomal glycogen accumulation in almost all fibers when compared with untreated controls. This method could be employed with disease models where specific rescue of the diaphragm would allow for increased survival and thus further investigation into the impact of the gene deletion on other striated muscle groups.     

Histogenesis of human esophageal striated muscle tissue

Development of the esophageal striated muscle tissue has been studied in 60 human embryos and fetuses at the age of 6-40 weeks. Stages in differentiation of the muscle fiber have been demonstrated, process of myofibrillogenesis has been studied. In the process of differentiation of the esophageal striated muscle fiber under the basal membrane myosatellitocytes are laid. A conclusion is made about myotomic origin of the human esophageal striated muscle tissue. The developmental peculiarity of the human esophageal striated muscle is formation between muscle fibers of specialized connections. This is explained by conditions of the esophageal functioning, that realizes peristaltic movements.     

Smooth muscle cells in the rat testicular capsule: a developmental study.

This study of the postnatal development (from 1 to 60 days) of smooth muscle elements in the rat testicular capsule has demonstrated that while such elements are identifiable by light microscopy at 30 days, myocytes are present at birth as seen by electron microscopy. The differentiation of smooth muscle from birth to 30 days has been described, by which time it is of adult morphology and content. Perhaps significantly, it is at 30 days that the testis achieves a scrotal position, although sexual maturity does not occur until about 60 days. Presumably, at 30 days the testicular capsule of the rat is capable of the spontaneous contractions which are known to occur in the adult and which are assumed to aid the transport of non-motile spermatozoa from the testis to the spididymis. The presence of occasional striated muscle fibers in the rat testicular capsule as reported previously has not been confirmed by this investigation, although their possible origin is discussed.     

Alteration of the Systemic and Microcirculation by a Single Oral Dose of Flavan-3-Ols

Several systematic reviews have reported that flow mediated dilatation (FMD) was significantly increased in subjects after ingestion of chocolate that contains flavan-3-ols; however, the mechanisms responsible for this effect are not clear. In this study, we evaluated the effects of a single oral dose of flavan-3-ols on the systemic circulation and microcirculation in the cremaster muscle using intravital video microscopy in vivo. The cremaster muscle in rats was spread over a plastic chamber and a gastric tube was placed into the stomach. Blood flow in the cremasteric artery was determined using a laser Doppler flowmeter, while blood pressure and heart rate were measured by the tail-cuff method. Red blood cell velocity in arterioles and blood flow in the artery were significantly increased 5 min after the administration of 10 mg/kg flavan-3-ols compared with distilled water treatment. The number of capillaries recruited in the cremaster muscle was also significantly increased 15 min after treatment. Microscopic observation confirmed that increased shear stress on endothelial cells was maintained during the measurement period. The mean arterial blood pressure and heart rate were also significantly elevated soon after administration and returned to baseline before the end of the observation period. Plasma nitrate and nitrite levels, and NO phosphorylation of aortic tissue were significantly increased at 60 min after administration of flavan-3-ols. According to these results, a single oral dose of flavan-3-ols elevates blood pressure and flow transiently, and these effects induce NO production through increased shear stress on endothelial cells.     

Diaphragm muscle fiber function and structure in humans with hemidiaphragm paralysis

Recent studies proposed that mechanical inactivity of the human diaphragm during mechanical ventilation rapidly causes diaphragm atrophy and weakness. However, conclusive evidence for the notion that diaphragm weakness is a direct consequence of mechanical inactivity is lacking. To study the effect of hemidiaphragm paralysis on diaphragm muscle fiber function and structure in humans, biopsies were obtained from the paralyzed hemidiaphragm in eight patients with hemidiaphragm paralysis. All patients had unilateral paralysis of known duration, caused by en bloc resection of the phrenic nerve with a tumor. Furthermore, diaphragm biopsies were obtained from three control subjects. The contractile performance of demembranated muscle fibers was determined, as well as fiber ultrastructure and morphology. Finally, expression of E3 ligases and proteasome activity was determined to evaluate activation of the ubiquitin-proteasome pathway. The force-generating capacity, as well as myofibrillar ultrastructure, of diaphragm muscle fibers was preserved up to 8 wk of paralysis. The cross-sectional area of slow fibers was reduced after 2 wk of paralysis; that of fast fibers was preserved up to 8 wk. The expression of the E3 ligases MAFbx and MuRF-1 and proteasome activity was not significantly upregulated in diaphragm fibers following paralysis, not even after 72 and 88 wk of paralysis, at which time marked atrophy of slow and fast diaphragm fibers had occurred. Diaphragm muscle fiber atrophy and weakness following hemidiaphragm paralysis develops slowly and takes months to occur.     

Chronic denervation of rat hemidiaphragm: maintenance of fiber heterogeneity with associated increasing uniformity of myosin isoforms

During several months of denervation, rat mixed muscles lose slow myosin, though with variability among animals. Immunocytochemical studies showed that all the denervated fibers of the hemidiaphragm reacted with anti-fast myosin, while many reacted with anti-slow myosin as well. This has left open the question as to whether multiple forms of myosin co-exist within individual fibers or a unique, possibly embryonic, myosin is present, which shares epitopes with fast and slow myosins. Furthermore, one can ask if the reappearance of embryonic myosin in chronically denervated muscle is related both to its re-expression in the pre-existing fibers and to cell regeneration. To answer these questions we studied the myosin heavy chains from individual fibers of the denervated hemidiaphragm by SDS PAGE and morphologically searched for regenerative events in the long term denervated muscle. 3 mo after denervation the severely atrophic fibers of the hemidiaphragm showed either fast or a mixture of fast and slow myosin heavy chains. Structural analysis of proteins sequentially extracted from muscle cryostat sections showed that slow myosin was still present 16 mo after denervation, in spite of the loss of the selective distribution of fast and slow features. Therefore muscle fibers can express adult fast myosin not only when denervated during their differentiation but also after the slow program has been expressed for a long time. Light and electron microscopy showed that the long-term denervated muscle maintained a steady-state atrophy for the rat's life span. Some of the morphological features indicate that aneural regeneration events continuously occur and significantly contribute to the increasing uniformity of the myosin gene expression in long-term denervated diaphragm.     

Repair of spinal cord transection and its effects on muscle mass and myosin heavy chain isoform phenotype.

A number of significant advances have been developed for treating spinal cord injury during the past two decades. The combination of peripheral nerve grafts and acidic fibroblast growth factor (hereafter referred to as PNG) has been shown to partially restore hindlimb function. However, very little is known about the effects of such treatments in restoring normal muscle phenotype. The primary goal of the current study was to test the hypothesis that PNG would completely or partially restore 1) muscle mass and muscle fiber cross-sectional area and 2) the slow myosin heavy chain phenotype of the soleus muscle. To test this hypothesis, we assigned female Sprague-Dawley rats to three groups: 1) sham control, 2) spinal cord transection (Tx), and 3) spinal cord transection plus PNG (Tx+PNG). Six months following spinal cord transection, the open-field test was performed to assess locomotor function, and then the soleus muscles were harvested and analyzed. SDS-PAGE for single muscle fiber was used to evaluate the myosin heavy chain (MHC) isoform expression pattern following the injury and treatment. Immunohistochemistry was used to identify serotonin (5-HT) fibers in the spinal cord. Compared with the Tx group, the Tx+PNG group showed 1) significantly improved Basso, Beattie, and Bresnahan scores (hindlimb locomotion test), 2) less muscle atrophy, 3) a higher percentage of slow type I fibers, and 4) 5-HT fibers distal to the lesion site. We conclude that the combined treatment of PNG is partially effective in restoring the muscle mass and slow phenotype of the soleus muscle in a T-8 spinal cord-transected rat model.     

Histoenzymological and ultrastructural changes in lateral muscle fibers of Oreochromis niloticus (Teleostei: Cichlidae) after local injection of veratrine

The effects of veratrine have been investigated in mammalian, amphibian, and crustacean muscle, but not in fish. In this work, the action of veratrine was studied in the lateral muscle of the freshwater teleost Oreochromis niloticus after intramuscular injection. Histoenzymological typing and electron microscopy of muscle fibers before and 15, 30, and 60 min after veratrine injection (10 ng/kg fish) were used to indirectly assess the morphological changes and the oxidative and m-ATPase activities. In some cases, muscles were pretreated with tetrodotoxin to determine whether the ultrastructural changes were the result of Na(+) channel activation by veratrine. Veratrine altered the metabolism of fibers mainly after 30 min. Oxidative fibers showed decreased NADH-TR activity, whereas that of glycolytic and oxidative-glycolytic type fibers increased. There was no change in the m-ATPase activity of the three fiber types, except at 60 min postveratrine, when a novel fiber type, which showed no reversal after acidic and alkaline preincubations, appeared. Ultrastructural damage involved sarcomeres, myofibrils, and mitochondria, but the T-tubules remained intact. Pretreatment with tetrodotoxin (1 ng/ml) prevented the ultrastructural changes caused by veratrine. These results show that in fish skeletal muscle veratrine produces some effects that are not seen in mammalian muscle.     

Striated acto-myosin fibers can reorganize and register in response to elastic interactions with the matrix

The remarkable striation of muscle has fascinated many for centuries. In developing muscle cells, as well as in many adherent, nonmuscle cell types, striated, stress fiberlike structures with sarcomere-periodicity tend to register: Based on several studies, neighboring, parallel fibers at the basal membrane of cultured cells establish registry of their respective periodic sarcomeric architecture, but, to our knowledge, the mechanism has not yet been identified. Here, we propose for cells plated on an elastic substrate or adhered to a neighboring cell, that acto-myosin contractility in striated fibers close to the basal membrane induces substrate strain that gives rise to an elastic interaction between neighboring striated fibers, which in turn favors interfiber registry. Our physical theory predicts a dependence of interfiber registry on externally controllable elastic properties of the substrate. In developing muscle cells, registry of striated fibers (premyofibrils and nascent myofibrils) has been suggested as one major pathway of myofibrillogenesis, where it precedes the fusion of neighboring fibers. This suggests a mechanical basis for the optimal myofibrillogenesis on muscle-mimetic elastic substrates that was recently observed by several groups in cultures of mouse-, human-, and chick-derived muscle cells.     

A comparative analysis of the vasomotor responses and innervation of small arteries in rat locomotor and respiratory muscles

Characteristics of the small arteries (with a diameter of 200-250 μm) feeding the medial gastrocnemius muscle and diaphragm were studied. Recording of the mechanical activity of ring segments under isometric conditions demonstrated that, similar to other arteries feeding the muscles with a high content of slow fibers, the diaphragm arteries are highly sensitive to adrenoceptor agonists and acetylcholine. The differences in the endothelium-dependent relaxation in response to acetylcholine were retained in the presence of L-NAME and diclofenac. The diaphragm and gastrocnemius arteries similarly responded to serotonin. On the other hand, a high innervation density was characteristic of the diaphragm arteries unlike the arteries of other slow muscles. The density of adrenergic nerve plexus in the diaphragm arteries was considerably higher than in the gastrocnemius arteries. The results suggest that the characteristics of small diaphragm arteries are determined not only by the oxidative capacity of diaphragm muscle fibers, but also by the fact that this is a respiratory muscle.     

Atrophy, but not necrosis, in rabbit skeletal muscle denervated for periods up to one year

Our understanding of the effects of long-term denervation on skeletal muscle is heavily influenced by an extensive literature based on the rat. We have studied physiological and morphological changes in an alternative model, the rabbit. In adult rabbits, tibialis anterior muscles were denervated unilaterally by selective section of motor branches of the common peroneal nerve and examined after 10, 36, or 51 wk. Denervation reduced muscle mass and cross-sectional area by 50–60% and tetanic force by 75%, with no apparent reduction in specific force (force per cross-sectional area of muscle fibers). The loss of mass was associated with atrophy of fast fibers and an increase in fibrous and adipose connective tissue; the diameter of slow fibers was preserved. Within fibers, electron microscopy revealed signs of ultrastructural disorganization of sarcomeres and tubular systems. This, rather than the observed transformation of fiber type from IIx to IIa, was probably responsible for the slow contractile speed of the muscles. The muscle groups denervated for 10, 36, or 51 wk showed no significant differences. At no stage was there any evidence of necrosis or regeneration, and the total number of fibers remained constant. These changes are in marked contrast to the necrotic degeneration and progressive decline in mass and force that have previously been found in long-term denervated rat muscles. The rabbit may be a better choice for a model of the effects of denervation in humans, at least up to 1 yr after lesion. force; shortening velocity; electron microscopy; histochemistry     

Morphological and functional recovery from diaphragm injury: an in vivo rat diaphragm injury model.

Our objective was to develop an in vivo model to study the timing and mechanisms underlying diaphragm injury and repair. Diaphragm injury was induced in anesthetized rats by the application of a 100 mM caffeine solution for a 10-min period to the right abdominal diaphragm surface. Diaphragms were removed 1, 4, 6, 12, 24, 48, 72, and 96 h and 10 days after the injury, with contractile function being assessed in strips in vitro by force-frequency curves. The extent of caffeine-induced membrane injury was indicated by the percentage of fibers with a fluorescent cytoplasm revealed by inward leakage of the procion orange dye. One hour after caffeine exposure, 32.9 +/- 3.1 (SE) % of fibers showed membrane injury that resulted in 70% loss of muscle force. Within 72-96 h, the percentage of fluorescent cells decreased to control values. Muscle force, however, was still reduced by 30%. Complete muscle strength recovery was observed 10 days after the injury. Whereas diaphragmatic fiber repair occurred within 4 days after injury induction, force recovery took up to 10 days. We suggest that the caffeine-damaged rat diaphragm is a useful model to study the timing and mechanisms of muscle injury and repair.     

A comparative study of the membrane structure in different types of muscle fibers in the frog

The muscle membrane of slow and fast fibers in cruralis and iliofibularis muscles and of intermediate fibers in submaxillaris muscle of the frog is studied in freeze-fracture replicas. A comparison of membrane folds, number, size and distribution of caveolae and of intramembrane particles (IMP) is given. In slow muscle fibers, the membrane folds are systematically present at the level of the I zone with a transversal continuity, whereas in fast and intermediate types the membrane folds are small and are randomly distributed. In slow muscle the caveolae are more numerous at the I zone than in the part corresponding to the center of the sarcomere. In fast muscle, small groups of caveolae form linear patterns, and in intermediate fibers the distribution is random. The number of caveolae in slow muscle fibers is two times more than in fast and intermediate fibers. The mean area of caveolae opening is largest in fast and smallest in slow muscle fibers. The number of IMP is significantly different in the three types of fibers, being highest in slow and lowest in intermediate fibers. The different pattern of folds in slow fibers may correspond to the different contractile properties of this fiber type. The presence of double the number of caveolae in slow fibers correlated to the less elaborate T system in this fiber type shows the possibility that slow fibers may be the result of an arrest during development for the performance of a different function. The difference in IMP density in the three muscle fiber types may be interpreted as the difference in their electrical properties.     

Effects of inactivity on fiber size and myonuclear number in rat soleus muscle.

The effects of short-term (4 days) and long-term (60 days) neuromuscular inactivity on myonuclear number, size, and myosin heavy chain (MHC) composition of isolated rat soleus fibers were determined using confocal microscopy and gel electrophoresis. Inactivity was produced via spinal cord isolation (SI), i.e., complete spinal cord transections at a midthoracic and a high sacral level and bilateral deafferentation between the transection sites. Compared with control, there was an increase in the percentage of fibers containing the faster MHC isoforms after 60, but not 4, days of SI. The mean sizes of type I and type I+IIa fibers were 41 and 27% and 66 and 56% smaller after 4 and 60 days of SI, respectively. Thus atrophy occurred earlier than the shift in myosin heavy chain (MHC) profile. The number of myonuclei was approximately 30% higher in type I than type I+IIa fibers in control soleus, but after 60 days of SI these values were similar. The number of myonuclei per millimeter in type I fibers was significantly lower than control after 60 days of SI, whereas there was no change in type I+IIa fibers. Thus myonuclei were eliminated from fibers containing only type I MHC. Because the magnitude of the loss of myonuclei was less than the level of atrophy, the myonuclear domains of both type I and type I+IIa fibers were significantly lower than control. Thus chronic (60 days) inactivity results in smaller, faster fibers that contain a higher than normal amount of DNA per unit of cytoplasm. The absence of activation of muscle fibers that are normally the most active (pure type I fibers) resulted in most, but not all, fibers expressing some fast MHC isoforms. The results also indicate that a loss of myonuclei is not a prerequisite for sustained muscle fiber atrophy.     

Ultrastructure and mechanical activity expressed by striated muscle in culture

Newly devised assay procedures for quantitating the mechanical capabilities of striated muscle fibers grown in cell culture have permitted the correlation of cytological features with the ability to respond mechanically to electrical and chemical stimuli during development. By developmental timing and by physiological characteristics, three distinct mechanical activities can be distinguished: : TWITCH, contracture and wave propagation (escalation). Parallel electron microscopy studies suggest that contracture and escalation require significantly greater internal membrane development than twitch. The assay procedures have revealed that fibers developed in culture from genetically dystrophic chick muscle cells display a heightened electrical threshold for a twich response, but are otherwise similar to normal fibers. Cultured chick fibers, whether of leg or breast origin, exhibit similar ultrastructural and mechanical properties; yet these are different from those of in vivo adult muscle and may represent the avian striated muscle archetype expressed in the absence of innervation. Primary or cell line cultures of rat muscle produce far fewer mechanically active fibers than do avian cell cultures. The influence of culture conditions and cell source, whether avian or mammalian, on the extent of differentiation expressed in culture is so great that our understanding of studies on cultured muscle fibers would benefit from some characterization of both morphological and contractile properties of the fibers being used.     

A study of myonecrosis induced by the venom of the scorpion tityus serrulatus

The pathogenesis of skeletal muscle necrosis produced by Tityus Serrulatus venom was studied by means of light microscopy and electron microscopy. Wistar rats were inoculated subcutaneously, at some distance from the muscles under study, with a sublethal dose of scorpion venom. Samples were taken of the tibialis anterior muscles of both rear legs, 2, 7 and 24 hours postinoculation. Light microscopy analysis after 2 hours revealed certain changes identified as "delta lesions", and also the presence of hyperconcentrated muscle cells. Electron microscopy confirmed these lesions and also enabled us to identify a degree of discontinuity in the plasma membrane with a persistence of the basal membrane. Hyperconcentrated fibers could still be observed 7 hours postinoculation. Histochemical analysis revealed high levels of calcium within the fibers. 24 hours after inoculation with the venom, numerous phagocytic cells were found in the degenerated fibers. Muscle cells were also found to have undergone alterations indicative of an ischemic process. The most characteristic finding 7 days postinoculation was the appearance of regenerative fibers. After thirty days the muscles regained their normal appearance. It is suggested that Tityus Serrulatus venom induces myonecrosis by means of a twofold action: direct action, which gives rise in the first place to a rupture of the plasma membrane, permitting a massive entry of calcium this being a key factor in the process of cell lesion and an assumed indirect action due to ischemia.