Early Stages Of Necrosis Research Articles

Enterotoxic effects of Aeromonas hydrophila infection in the catfish, Clarias gariepinus: Biochemical, histological and proteome analyses

Aeromonas hydrophila is considered as a potential risk to fish populations in the aquaculture industry and could also pose a serious threat to humans. In this study, the impact of A. hydrophila infection in the air-breathing catfish, Clarias gariepinus was analyzed using a multidimensional approach. Aeromonas hydrophila (1 × 107 cells) was injected into C. gariepinus intraperitoneally and maintained at an ambient temperature and photoperiod with periodical monitoring for morphological changes. After 7 days post-infection, tissue samples of the gills, liver, intestine, and kidney were subjected to biochemical, histological, transmission electron microscope (TEM) and proteomic analyses. Observed results indicated distinct morphological changes with the significant increase of ROS and oxidative stress enzymes (CAT and SOD) in tissues of the infected group when compared to the control. Histological analysis in infected fish revealed the presence of pyknotic nuclei, early stages of necrosis in the liver, degradation of renal tubules and widened sinusoidal space in kidneys along with enlargement of the epithelial region in the intestine. TEM analysis of the infected intestine showed degeneration of villi and the presence of multinucleated erythrocytes. Two-dimensional proteomic and mass spectrometry analysis of intestine and liver displayed up-regulation of several immune regulatory proteins such as proteasome subunit 3 protein, prolactin and intermediated filament protein; and down-regulation of proteins including actin, serine/arginine-rich splicing factor and carbonic anhydrase. Taken together, these results suggest that the identified proteins may have a role in immune regulation against A. hydrophila infection in C. gariepinus and support further investigations of host-pathogen interactions.

First Report of Marasmiellus mesosporus Causing Marasmiellus Blight on Seashore Paspalum.

Seashore paspalum (Paspalum vaginatum Sw.) is a newly cultivated C4 turfgrass that has exceptional salinity tolerance and is highly suited for use on golf courses in coastal areas. In October 2008 and June 2009, circular patches of blighted seashore paspalum ranging from 30 cm to >3 m in diameter were observed in fairways, tees, and roughs established with 'Supreme' seashore paspalum at Roco Ki Golf Club in Macao, Dominican Republic. Affected patches were initially chlorotic followed by reddish brown necrosis of leaves and leaf sheaths. Reddish brown-to-gray lesions were also observed on leaf sheaths during the early stages of necrosis. During periods of wet or humid weather from June through October, basidiocarps were produced on necrotic plant tissue and identified as Marasmiellus mesosporus Singer (2). Three isolates were obtained by plating symptomatic leaf sheaths that were surface sterilized with a 0.5% NaOCl solution on potato dextrose agar amended with 50 ppm each of streptomycin, chloramphenicol, and tetracycline (PDA+++). Sequences of the internal transcribed spacer (ITS) region of rDNA, obtained from these three isolates and three stipes of basidiocarps, were identical to each other and 99% similar to a M. mesosporus sequence deposited in the NCBI database (Accession No. AB517375). To confirm pathogenicity, a M. mesosporus isolate obtained from symptomatic plant tissue was inoculated onto 6-week-old P. vaginatum ('Seaspray') planted (0.5 mg seed/cm2) in 10-cm-diameter pots containing a mixture of 80% sand and 20% reed sedge peat. Two weeks prior to inoculation, the isolate was grown on a sterilized mixture of 100 cm3 of rye grain, 4.9 ml of CaCO3, and 100 ml of water. Infested grains were placed 0.5 cm below the soil surface for inoculation. Pots were inoculated with five infested grains or five sterilized, uninfested grains with three replications of each treatment. After inoculation, pots were placed in a growth chamber with a 12-h photoperiod set to 30°C during the day and 26°C at night. Approximately 20% of plants in inoculated pots were necrotic 7 days postinoculation and this increased to 75% by 21 days postinoculation. Diseased plants in inoculated pots exhibited symptoms similar to those observed in the field. Leaves were initially chlorotic with brown lesions on lower leaf sheaths and eventually turned necrotic, reddish brown, and collapsed. Pots receiving uninfested grains were healthy and showed no symptoms on all rating dates. At 21 days postinoculation, basidiocarps were observed emerging from three colonized plants at the base of the oldest leaf sheath near the crown. Three reisolations were made on PDA+++ from stem lesions surface sterilized with a 0.5% NaOCl solution. All reisolations were confirmed as M. mesosporus by culture morphology and ITS sequence data. M. mesosporus was previously reported causing disease on American beachgrass (Ammophila breviligulata Fernald) in North Carolina (1) and recently in Japan (3). The pathogen was initially placed in the genus Marasmius and reported as the cause of the disease Marasmius blight (1). Subsequent morphological observation found that the pathogen belonged in the genus Marasmiellus (2). To our knowledge, this is the first report of M. mesosporus causing Marasmiellus blight on seashore paspalum, a high-amenity turfgrass.

Contribution of a mitochondrial pathway to excitotoxic neuronal necrosis

It is traditionally thought that excitotoxic necrosis is a passive mechanism that does not require the activation of a cell death program. In this study, we examined the contribution of the cytochrome c-dependent mitochondrial death pathway to excitotoxic neuronal necrosis, induced by exposing cultured cortical neurons to 1 mM glutamate for 6 hr and blocked by the NMDA antagonist, dizocilpine. Glutamate treatment induced early cytochrome c release, followed by activation of caspase-9 and caspase-3. Preincubation with the caspase-9 inhibitor z-LEHD-fmk, the caspase-3 inhibitor z-DEVD-fmk, or the specific pan-caspase inhibitor Q-VD-oph decreased the percentage of propidium iodide-positive neurons (52.5% +/- 3.1%, 39.4% +/- 3.5%, 44.6% +/- 3%, respectively, vs. 65% +/- 3% in glutamate + vehicle). EM studies showed mitochondrial release of cytochrome c in neurons in the early stages of necrosis and cleaved caspase-3 immunoreactivity in morphologically necrotic neurons. These results suggest that an active mechanism contributes to the demise of a subpopulation of excitotoxic necrotic neurons.

Factors affecting fertilization of porcine oocytes following intracytoplasmic injection of sperm

The objective of this study was to optimize intracytoplasmic sperm injection (ICSI), and to assess the effects of membrane-damaged sperm on development of porcine oocytes following ICSI. For optimization of development following the ICSI process, sperm injected oocytes were activated 0.5-1.0 hr after ICSI with 1 x 30 micros pulse of 1.2, 1.7, 2.2, and 2.7 kV/cm DC in experiment 1. After 7-days of culture ICSI oocytes activated with [x1]2.2 kV/cm produced more blastocyts ([x2]34.4%, P < 0.05) than other treatment groups. In experiment 2, oocytes were activated with 1 x 30 micros pulse of 2.2 kV/cm at either 0, 1.5, 3, or 6 hr after ICSI. Oocytes activated 1.5 hr after [x3]ICSI yielded more blastocysts (27.6%)[M4] than in other treatments. In experiment 3, sperm were briefly exposed to 0.1% Triton X-100 to induce membrane damage. Live-dead staining of Percoll-sorted untreated spermatozoa (frozen-thawed) used in this study showed that over 96% were "alive" whereas none were "alive" after Triton X-100 treatment. The rate of development to blastocyst of oocytes injected with Triton X-100 treated sperm combined with electrical activation (EA) at 1 x 30 micros pulse of 2.2 kV/cm (EA, 40.0%) was the best, when compared with those injected with untreated sperm plus EA (P < 0.05). In experiment 4, the development rate of oocytes to the blastocyst stage ([x5]32.1%) following injection of a sperm head only was not significantly different from that of oocytes injected with whole sperm (31.0%). In conclusion, we found that an intact membrane and tail structures of pig spermatozoa are not essential for embryo development by ICSI, and furthermore, dead porcine spermatozoa, at an early stage of necrosis caused by plasma membrane damage, support better embryo development than do live non-damaged sperm.

Intraplaque hemorrhage and progression of coronary atheroma.

Intraplaque hemorrhage is common in advanced coronary atherosclerotic lesions. The relation between hemorrhage and the vulnerability of plaque to disruption may involve the accumulation of free cholesterol from erythrocyte membranes. We stained multiple coronary lesions from 24 randomly selected patients who had died suddenly of coronary causes with an antibody against glycophorin A (a protein specific to erythrocytes that facilitates anion exchange) and Mallory's stain for iron (hemosiderin), markers of previous intraplaque hemorrhage. Coronary lesions were classified as lesions with pathologic intimal thickening, fibrous-cap atheromas with cores in an early or late stage of necrosis, or thin-cap fibrous atheromas (vulnerable plaques). The arterial response to plaque hemorrhage was further defined in a rabbit model of atherosclerosis. Only traces of glycophorin A and iron were found in lesions with pathologic intimal thickening or fibrous-cap atheromas with cores in an early stage of necrosis. In contrast, fibroatheromas with cores in a late stage of necrosis or thin caps had a marked increase in glycophorin A in regions of cholesterol clefts surrounded by iron deposits. Larger amounts of both glycophorin A and iron were associated with larger necrotic cores and greater macrophage infiltration. Rabbit lesions with induced intramural hemorrhage consistently showed cholesterol crystals with erythrocyte fragments, foam cells, and iron deposits. In contrast, control lesions from the same animals had a marked reduction in macrophages and lipid content. By contributing to the deposition of free cholesterol, macrophage infiltration, and enlargement of the necrotic core, the accumulation of erythrocyte membranes within an atherosclerotic plaque may represent a potent atherogenic stimulus. These factors may increase the risk of plaque destabilization.

Editorial Comment

Editorial Comment

Fertilization and development of mouse oocytes injected with membrane-damaged spermatozoa.

The objective of this study was to investigate the influence of sperm plasma membrane on fertilization and development in a mouse model. The sperm plasma membrane was destroyed by exposure to Triton X-100 prior to intracytoplasmic sperm injection (ICSI). A single sperm curling (SSC) test was used to evaluate cell viability. The fertilization rates of oocytes obtained following ICSI of membrane-damaged sperm was not significantly higher than that of the control group (62.4 versus 59.6%). Rates of development to blastocyst were also not significantly different (51.7 and 50%). Inner cell mass (ICM) and total embryo cell numbers in the two groups were not statistically different (16 +/- 3.7 versus 14.73 +/- 3.35 and 45.8 +/- 12.5 versus 39.5 +/- 12 respectively). There were no differences in the implantation and live fetus rates between the two groups after transfer to pseudopregnant mice (61.5 and 35.9% respectively for the membrane-damaged group and 53.5 and 31.4% for the intact group). In conclusion, the present study clearly shows that destruction of the sperm plasma membrane does not affect fertilization and further development following injection of membrane-damaged spermatozoa into mouse oocytes. Fertilization and development can be achieved by dead spermatozoa at an early stage of necrosis when only the plasma membrane has been damaged.

Sperm head decondensation, pronuclear formation, cleavage and embryonic development following intracytoplasmic injection of mitochondria-damaged sperm in mammals.

The objective of this study was to investigate the influence of sperm mitochondrial destruction on sperm head decondensation, male pronuclear formation, cleavage and embryonic development. In the study two models were used: heterologous (hamster ICSI assay: human sperm injected into a hamster oocyte) for evaluation of sperm head decondensation and pronuclear formation, and homologous (mouse model) for the study of fertilisation and development. Destruction of mitochondria of the sperm was achieved by exposure to cyanide, a respiratory poison. Rhodamine 123 was used to evaluate the functional integrity of mitochondria. Sperm head decondensation was found to be not statistically significantly affected by mitochondrial damage (p = 0.8), with 62.8% and 67.9% condensation in the experimental and control groups respectively. Male pronucleus formation was seen in 40.2% and 44.4% of the injected oocytes in the experimental and control groups respectively. In the mouse experiments 45.5% and 49.7% of the injected oocytes were fertilised in the mitochondria-damaged and live-intact sperm groups respectively (p = 0.53). Development to blastocyst was achieved in 53.5% and 59.4% of the experimental and control groups respectively; the difference was not significant (p = 0.71). Inner cell mass (ICM) cell number were 15.7 +/- 4.12 and 43.1 +/- 11.3 respectively in the mitochondria-damaged group; the equivalent numbers were 14.12 +/- 4.02 and 39.3 +/- 12.6 in the control group. However, the differences in ICM and total cell counts between these two groups were not significant. Of the blastocysts transferred to pseudopregnant mice, 51.3% (20/36) implanted and 33.4% (12/36) developed to live fetuses in the mitochondria-damaged group. These rates were 60.5% (23/38) and 39.5% (15/38) in the control group. In conclusion, this study shows that functional integrity of the sperm mitochondria is not necessary in the process of fertilisation and development when the sperm is deposited into the ooplasm. Fertilisation and development can be achieved by injection of sperm at the very early stage of necrosis in which only the mitochondria have been destroyed and the rest of the cell including the plasma membrane is still intact.

Knochenmarködem - Frühform der Hüftkopfnekrose

We examined 15 patients (16 hips) with painful hips whose radiographs were either normal (n = 9) or showed a minimal decrease in radiodensity (n = 7). The available bone scintigrams of 9 cases were positive. T1-weighted images visualised a diffuse signal loss of the bone marrow in all hips, with various extensions in the head, neck, and intertrochanteric area. These regions were hyperintensive on T2-weighted images. Focal anomalies were not seen in any of the cases. All patients underwent core decompression treatment. Histology of 13 hips confirmed not only the presence of bone marrow oedema but of bone changes corresponding to those of avascular necrosis. Follow-up examinations with MR after core decompression showed normal signal intensity in all cases. Magnetic resonance represents a viable diagnostic tool for identifying bone marrow oedema. Due to our histological results bone marrow oedema should be included in the differential diagnosis as an early stage of necrosis of the hip.

Lectin binding and desmin staining during bupivicaine-induced necrosis and regeneration in rat skeletal muscle.

The processes of bupivicaine-induced necrosis and regeneration in rat skeletal muscle have been studied with a panel of biotinylated lectins and by immunohistochemical staining for desmin. The results indicate that the binding of lectins to the periphery of muscle fibres is not altered in necrotic and regenerating fibres at the light microscopic level although abnormal cytoplasmic staining by lectins occurs early in necrosis and is also present during regeneration. Desmin staining is lost at an early stage of necrosis and is present at an early stage of regeneration. Desmin staining in combination with morphometrical assessment of the rate of fusion and growth of regenerating fibres provides a powerful method for evaluating the processes of muscle regeneration in experimental situations.

Vesicular demyelination induced by raised intracellular calcium

Incubation of nerve with high concentrations of the divalent cation ionophore A23187 produces myelin vesiculation (Schlaepfer 1977). This observation has now been extended using segments of rat ventral or dorsal root incubated with high (19 μM, 10 μg/ml) or low (1–1.5 μM) concentrations of A23187, or another divalent ionophore, ionomycin. Low concentrations of A23187 induced no vesiculation within a 2-h period. However, subsequent incubation of these roots in fresh, ionophore-free medium for 20 h, resulted in a prominent vesicular demyelination at the Schmidt-Lanterman incisures and paranodes of many fibres. At this time (22 h) the Schwann cells associated with some demyelinating internodes appeared vital upon ultrastructural examination: the cells also excluded the nuclear dye nigrosin. High concentrations of A23187 induced a similar vesicular demyelination in affected fibres within only 15–20 min. While the Schwann cells continued to exclude nigrosin for a further 4 h, their ultrastructural appearance indicated that they were probably in the early stages of necrosis. Incubation of moribund root with the ionophore produced no myelin vesiculation. At all ionophore concentrations, the myelin vesiculation was dependent upon the presence of extracellular Ca 2+, and could be modulated in severity by varying this concentration. Other divalent cations (Ba 2+, Co 2+, Mg 2+, Mn 2+, Ni 2+, Sr 2+) could not substitute for Ca 2+. The vesiculation induced by A23187 could be entirely prevented by the addition of Zn 2+ (⩾ 1 μM), Ni 2+ (⩾ 1–10 μM), Co 2+ (⩾ 100 μM) or Mn 2+ (⩾ 100 μM) to the bathing medium. A23187 applied to only part of an isolated internode resulted in a localization of the myelin disruption to that region. Ionomycin (⩾ 1 μM), an ionophore with a greater selectivity for Ca 2+ than A23187, also induced a prompt Ca 2+-dependent myelin vesiculation. We conclude that vesicular demyelination can be initiated in vital Schwann cells by a raised intracellular Ca 2+ concentration. Such demyelination does not necessarily lead to Schwann cell death. The possible relevance of the findings to vesicular demyelinating neuropathies is discussed, and a hypothesis regarding the mechanism of demyelination is advanced.

Transtrochanteric Rotational Osteotomy of the Femoral Head for the Treatment of Osteonecrosis

Transtrochanteric rotational osteotomy of the femoral head was technically established as a surgical procedure in 1972. This procedure has been successfully performed on 250 hips in 204 patients. In the treatment of idiopathic and steroid-induced osteonecrosis of the femoral head, the results of 128 hips in 90 patients followed two to nine years were reviewed. In 98 of 128 hips, excellent results were obtained clinically and roentgenographically. Progressive collapse in the newly created weight-bearing area occurred in 25 hips where the lesions had been extensive. In 35 of 39 Grade I or II hips, excellent results were obtained in every respect (success rate: 90%). Following the analysis of results on the preoperative lateral roentgenograms of the femoral head, 76 of the 80 hips in the group in which the intact area was greater than one third of the total articular surface showed no collapse of the newly created weight-bearing area, thus accounting for a 95% success rate. In cases when a further collapse was prevented, the necrotic lesions were smaller at follow-up. Excellent results were obtained when the osteotomy was performed during the early stage of necrosis and in the absence of an advanced collapse, even in extensive lesions. In advanced cases with an extensive lesion, adequate anterior rotation is essential and intentional varus position should be designed. These results suggest that this procedure will inhibit progressive collapse of the femoral head and restore the joint more effectively than other surgical procedures.

Time course of ventricular vulnerability to sustained tachycardia and fibrillation during the early stage of necrosis following acute coronary occlusions in dogs

Time course of ventricular vulnerability to sustained tachycardia and fibrillation during the early stage of necrosis following acute coronary occlusions in dogs

Alterations of substances (DNA, myoglobin, myosin, protein) in experimentally induced cardiac hypertrophy and under the influence of drugs (isoproterenol, cytostatics, strophanthin).

In growing human hearts the biochemically estimated total amount of DNA increases from 20 mg to 50–100 mg, and in hypertrophied hearts the increase may be threefold compared with normal hearts. Whereas most animal hearts have nearly only diploid muscle cells, in human hearts most muscle cell nuclei are polyploid. According to the grade of hypertrophy a pronounced polyploidisation of heart muscle cells occurs. The quantitative ratio of noncondensed chromatin/condensed chromatin remains constant 4:1 in all grades of hypertrophy. The amount of myoglobin in hearts above 500 g weight is more than 100% compared with normal hearts and is correlated with the width of the heart muscle fibres. In hypertrophied hearts an irregular and higher double refraction of myosin can be seen indicating an early stage of necrosis. In concentric cardiac hypertrophy the dry weight of the muscle fibres is more increased than in eccentric hypertrophy. The protein concentration within the muscle fibres increases with rising heart weight. Some cytostatics cause a decrease of the myocardial DNA content, a reduction of the ploidy level and a diminishing of the number of connective tissue cells. Isoproterenol effects, within the first 3 weeks after administration, a severe reduction of the cell number; after 2 months a pronounced polyploidisation of the heart muscle cells occurs and the cell number increases again above the normal level indicating the regeneration capabilities of the myocardium.