Number Of Pyknotic Cells Research Articles

Response of cultured human pulmonary artery endothelial cells to endotoxin

Endotoxemia is the leading cause of the adult respiratory distress syndrome. The effects of endotoxin on pulmonary endothelium, both in vivo and in culture, are diverse and complicated, and vary between species and cellular origin. Species such as sheep and cows are particularly sensitive to endotoxin, whereas rats and mice are more resistant. Studies using cultured pulmonary endothelial cells confirm these findings. Such species variations lead us to question whether human pulmonary artery endothelial cells (HPAEC) are directly affected by endotoxin. The present study examined the effects of endotoxin on HPAEC. Cells were exposed to endotoxin (0.001-10 micrograms/ml) for 24 h and were examined by phase-contrast microscopy, and measurements were made of lactate dehydrogenase, prostacyclin, and prostaglandin E2 release in the cell-free supernatant. In the presence of serum, endotoxin doses as small as 0.01 microgram/ml resulted in endothelial retraction and pyknosis compared with controls (P < 0.05). Exposure to 10 micrograms/ml of endotoxin resulted in a significant increase in the number of pyknotic cells (P < 0.05), and lactate dehydrogenase release paralleled this finding. Endotoxin also resulted in a gradual increase in prostaglandin E2 release, reaching significance at 1 and 10 micrograms/ml of endotoxin (P < 0.05). A similar trend was noted for prostacyclin release. We conclude that the direct cytotoxic effects elicited by endotoxin on HPAEC may contribute to the onset of pulmonary edema in patients with adult respiratory distress syndrome.

Nerve growth factor reduces apoptosis of axotomized retinal ganglion cells in the neonatal rat

It has recently been reported that the degeneration of retinal ganglion cells induced by transection of the optic nerve in the neonatal rat is due to an active process of apoptosis, as opposed to passive necrosis. Here we tested whether the administration of the trophic factor nerve growth factor could prevent the apoptotic death of the axotomized cells. We administered nerve growth factor by two intraocular injections, one immediately after the lesion and the second 12 h later. The retinas were taken at 24 h post-lesion and stained as whole mounts with Cresyl Violet. Pyknotic as well as surviving cells were counted in the retinal ganglion cell layer. In this layer at least 95% of the total cell population is composed by ganglion cells, as revealed by retrogradely labelling these cells with horseradish peroxidase injected in the superior colliculi. We found that intraocular administration of nerve growth factor diminishes the degeneration induced by optic nerve transection in the neonatal rat. After nerve growth factor injection, in fact, the number of pyknotic cells is reduced by 39% compared with controls (lesioned, injected with saline); in addition, nerve growth factor also increases the survival of retinal ganglion cells by 30% at 24 h post-lesion.

Naturally Occurring Cell Death during Postnatal Development of the Substantia Nigra Pars Compacta of Rat

We have quantified degenerating cells during the postnatal development of the substantia nigra pars compacta (SNpc) of normal rat. The cells are identified at the light microscope level by the characteristic morphologic appearance of large, rounded clumps of chromatin, stained intensely with both Nissl and silver stains. These cells were most numerous on Postnatal Day (PND) 2 and rapidly declined in number by PND 12. However, on PND 14, there was a second peak in the number of pyknotic cells. Their number again rapidly diminished to very few by PND 28. Many of these cells appear neuronal in origin. We conclude that naturally occurring cell death is a prominent feature of normal nigra development, and we hypothesize that the magnitude of this regressive event may be regulated by target (striatal)-derived trophic support.

Early Effects of Low Doses of Ionizing Radiation on the Fetal Cerebral Cortex in Rats

Pregnant rats were exposed to gamma radiation from a 137Cs irradiator on gestational Day 15. Fetuses that received 0.25, 0.5, 0.75, or 1.0 Gy were examined 24 h after irradiation for changes in the cells of the cerebral mantle of the developing brain. The extent of changes following 0.5 Gy was studied at 3, 6, 12, or 24 h after exposure. Cortical thickness of the cerebral mantle was not significantly altered. The number of pyknotic cells, number of macrophages, nuclear area, and number of mitotic cells were altered in a dose-related way. The number of pyknotic cells was significantly increased at all doses. A positive correlation between the number of pyknotic cells and the number of macrophages developed with time. At 3 h after irradiation about 60% of pyknotic cells were found in the subventricular zone and about 25% in the intermediate zone and cortical plate. The number of such cells in the upper layers of the cortex steadily increased up to 24 h, at which time about 70% of pyknotic cells were in these two layers. The relationship of the movement of pyknotic cells to migration of postmitotic neuroblasts is discussed.

Acute response of fetal rat telencephalon to ultrasound exposure in utero

Pregnant rats were exposed to ultrasound or microwaves from transducers located over one uterine horn. Ultrasound intensity (SPTA) was 0.78 W/cm 2 for 30 min at a frequency of 2.5 MHz. Microwave exposure was used to reproduce approximately the rate of rise of uterine temperature of the rats exposed to ultrasound. The average peak temperature was 40.1°C for ultrasound exposure and 42.2°C for microwave exposure. On Gestational Day 16, 24 h after exposure, fetuses were removed and prepared for morphological examination of the developing cerebral cortical mantle. Morphometric measurements were made of nuclear area of subventricular zone cells, number of mitoses per mm in the ventricular zone, number of pyknotic cells in the mantle per mm, and number of ventricular macrophages per mm. Both exposures increased nuclear size and numbers of pyknotic cells and macrophages, and decreased the number of mitotic figures. The data from the four measurements were evidence of damage from ultrasound similar to the effects produced by microwave heating. The thermal effects of ultrasound, even at relatively low levels of rise in temperature, may have been the cause of the damage to the fetal cortex in these experiments or may have interacted with other effects of ultrasound energies to produce damage to developing neurons.

Short-term glucocorticoid manipulations affect neuronal morphology and survival in the adult dentate gyrus

In order to determine whether short-term glucocorticoid manipulations influence the morphology and survival of neurons in the adult mammalian hippocampal formation, we performed quantitative analyses of Golgi-impregnated and Nissl-stained tissue from the brains of sham operated male rats, adrenalectomized male rats and adrenalectomized male rats which received corticosterone replacement. Three days after adrenalectomy, massive cell death, as detected by a dramatic increase in number of pyknotic cells, was observed in the granule cell layer of the dentate gyrus. By seven days following adrenalectomy, the numbers of pyknotic cells were even greater. Moreover, significant decreases in cross-sectional cell body area and numbers of dendritic branch points of Golgi-impregnated dentate gyrus granule cells were detected at seven days after adrenalectomy. Replacement of corticosterone to adrenalectomized rats prevented the appearance of large numbers of pyknotic cells as well as the decrease in granule cell cross-sectional cell body area and the numbers of dendritic branch points. In contrast, no obvious signs of degeneration were detected in the pyramidal cell layers of the CA1 and CA3 regions of the hippocampus at either three or seven days following adrenalectomy. In addition, no significant changes in morphological characteristics were observed in CA1 or CA3 pyramidal cells with adrenalectomy. These results show that dentate gyrus granule cells require glucocorticoids for their survival and for the maintenance of normal morphology and suggest that granule cell morphology and/or survival may undergo constant fluctuation in response to diurnal rhythms or stress-induced changes in glucocorticoid levels.

Comparative histomorphometric changes in SENCAR mouse epidermis in response to multiple treatments with complete and stage-specific tumor promoting agents.

Responses of various cells of the epidermis and dermis to topically applied agents have been implicated in the mechanism of multistage mouse tumorigenesis. These responses have been discussed almost entirely in the context of a single promoter treatment, although tumor expression is dependent on multiple applications. Responses of keratinocytes, epidermal dendritic non-keratinocytes and dermal leukocytes were therefore recorded following multiple topical applications of the potent complete tumor-promoting agent 12-O-tetradecanoylphorbol-13-acetate (TPA). In order to assess the importance of the response of individual cell types to the mechanisms and stages of promotion, responses to TPA were compared with those to agents with low complete promoting activity, but significant activity in individual stages of multistage promotion models. These included 4-O-methyl-TPA, a stage 1 promoting agent, mezerein and n-dodecane, stage 2 promoting agents of apparently different mechanism of action, and ethyl phenylpropiolate (EPP), a highly inflammatory stage 3 promoting agent. In agreement with previous findings, TPA induced a persistent epidermal hyperplasia and an increase in dark keratinocytes, although a similar finding was made for EPP and n-dodecane. The response to n-dodecane was significantly delayed, however, and that to EPP was accompanied by focal epidermal destruction and inflammation. The response to n-dodecane contrasted with that found for mezerein, supporting the suggestion that their mechanisms of action are distinct. Multiple treatments of 4-O-methylTPA caused no increase in dark cells, and mezerein induced no increase in numbers of pyknotic cells, whereas increases were expected in both cases on the basis of single dose experiments. Of the agents examined, only TPA induced a decrease in pale dendritic epidermal cells in the absence of marked toxicity, supporting the previous proposal that prolonged effects on this cell type are important in the promotion process. Some degree of persistent dermal leukocyte infiltration was observed with all agents excepting 4-O-methylTPA, although the extent of the response and its cellular characteristics appeared strongly dependent on the agent applied. In the case of TPA small mononuclear cells, neutrophils and macrophages all provided significant contributions to the total infiltrate. A similar phenomenon was observed with n-dodecane and EPP, with an additional increase in eosinophils which was not observed with TPA. Mezerein differed from both TPA and n-dodecane in inducing a significant increase only in eosinophils. As reported previously for single applications, prolonged TPA application caused a change in morphology and a considerable decrease in numbers of Thy-1+ epidermal dendritic cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of maternal hyperphenylalaninemia on fetal brain development: A morphological study

We examined the effects of maternal hyperphenylalaninemia on the morphological development of the fetal and neonatal rat brain. High concentrations of phenylalanine were induced in pregnant rats from embryonic days 14 through 21 by subcutaneous injections of α-methylphenylalanine (mPhe) (to inhibit maternal phenylalanine hydroxylase) at a dosage of 30 mg 100 g body weight plus phenylalanine (Phe) supplementation (to raise fetal plasma phenylalanine) at a dosage of 60 mg 100 g body weight two times daily at 12-h intervals. This treatment resulted in a significant hyperphenylalaninemia compared with vehicle-injected, pair-fed control rats. At embryonic day 21, mPhe Phe -treated embryos displayed a reduced thickness of the cortical plate and marginal zone, a decrease in the size of postmitotic neurons, and an increase in the packing density of cells in the cortical plate. There was an increase in the number of pyknotic cells (cell death) and in the reactive microglia in the mPhe Phe -treated group. During the postnatal period the differences between mPhe Phe -treated and control rats became fewer and by postnatal day 7 the morphology of the mPhe Phe -exposed brains was similar to the controls. These morphologic data, in conjunction with our previous biochemical finding of reduced cerebral DNA, RNA, and protein contents of mPhe Phe -treated rats, indicate that the induced hyperphenylalaninemia caused a significant delay in the development of the cerebral cortex which was able to undergo recovery during the postnatal period.

Electrical stimulation of hindlimb increases neuronal cell death in chick embryo.

The daily treatment of chick embryos with neuromuscular blocking agents during the period of massive naturally occurring death of spinal motoneurones (days 5–10) reduces the normal loss of these cells1,2. Because both pre- and postsynaptic blocking agents have this effect3, the cause seems to be related to a reduction in synaptic and/or muscular activity. If this interpretation is correct, an experimentally induced increase in nerve or muscle activity (and/or an increased activation of acetylcholine receptors, AChR), during the normal cell death period should accelerate or enhance the loss of motoneurones. Consistent with this prediction, we have found that daily treatment with the nicotinic receptor agonist, carbachol, or with the anti-cholinesterase (anti-ChE) agent, eserine, leads to a significant enhancement of cell death of spinal motoneurones3. However, because in the same study other related agents (for example nicotine) failed to alter normal cell death, we believed that a different approach might provide a better test of our prediction. We now report that short-term continuous electrical stimulation of the hindlimb (the musculature and nerve trunks) initiated at any time between stages 31 (day 7) and stage 34 (day 8) results in a significant reduction in the number of healthy motoneurones and an increase in the number of pyknotic (degenerating) motoneurones in the lumbar lateral motor column (LMC). As the number of pyknotic cells in the dorsal root ganglia (DRG) is unaltered, this effect seems to be specific to motoneurones.

Murine colonic mucosal metabolism and cytotoxicity of benzo [alpha] pyrene.

Homogenates of colonic mucosa from seven mouse strains metabolized 14 C-benzol [alpha] pyrene (BP) to alkali-soluble and water-phase products. In those strains in which liver aryl hydrocarbon hydroxylase activity is inducible by beta-naphthoflavone (beta-NF), the C57Bl/6, BALB/c, C3H and random-bred Swiss CD-1, colonic metabolism of BP was also induced by intraperitoneal treatment with 150 mg/kg beta-NF 24 h before assay. No increase in the colonic metabolism of BP was seen after beta-NF treatment of the noninducible strains, AKR, DBA and SWR. A detailed study of the products formed was carried out with C57BL/6 male mice. High-performance liquid-chromatography analysis of the products of BP metabolism revealed that phenolic derivatives predominated, but that a small percentage of the BP was converted into the proximate carcinogen, the BP-7,8-diol. Addition of uridine 5'-diphosphoglucuronic acid to the incubation mixture caused an increase in total product formation and a shift from alkali-soluble to water-phase products. Of the water-phase products formed by beta-NF-induced colonic mucosal homogenates, about 40% were nondialyzable and associated with precipitable material. In a study of the cellular effects of BP on the colonic mucosa, intrarectal administration of 1 mg BP had a significant cytotoxic effect, as indicated by the number of pyknotic cells and 3H-thymidine-labeling index, and prior treatment with beta-NF afforded significant protection against this cytotoxicity.

A morphological study of incubated slices of rat cerebellum in relation to postnatal age.

The morphology of incubated slices of cerebellum from rats of various ages between birth and 70-90 days was studied using light and electron microscopy. The slices were prepared using a mechanical chopper and were incubated in Krebs solution for 1 1/2-2 h. Up to 8 days of age, cellular and subcellular damage in the slices was only rarely encountered. At 14 days, the number of pyknotic cells had increased, particularly at the cut edges of the slices, but otherwise the morphology resembled that of the cerebellum fixed in situ. By 21 days, pyknosis and swelling was more extensive, the most vulnerable neurones appearing to be the Purkinje cells and the inhibitory interneurones in the molecular layer, the internal granule cell layer being relatively spared. In slices from the adult, damage had further increased to include more than 90% of the granule cells. This non-uniform preservation of the cells in the slices with respect to age is likely to have a considerable influence on results from developmental studies carried out using mechanically-chopped brain slices. When prepared by hand, using a thin blade and glass guide, slices from the adult cerebellum showed good morphological preservation.

A quantitative histological study of the effects of acute triethyl lead poisoning on the adult mouse brain.

The effects of a single injection of triethyl lead chloride on the mouse brain was studied 1, 3, 5, 7 and 30 days postinjection using quantitative histological techniques. The total number of glia in the anterior commissure was significantly reduced following injection but by 30 days postinjection had returned to normal. The number of neurons and the number of glia in the indusium griseum did not change significantly. The number of mitotic cells in the subependymal layer fell slightly from 1 to 3 days postinjection then returned to normal 5 days postinjection. The number of pyknotic cells in the subependymal layer did not appear to change following injection. In the anterior commissure the number of mitotic cells fell significantly from 1 to 3 days postinjection and then increased significantly at 5 days postinjection. A similar increase in mitosis was found at 5 days postinjection in the indusium griseum. At 7 days postinjection a significant decrease occurred in pyknotic cells in the anterior commissure and indusium griseum. Changes in the percentage of each type of glial cell present were found 30 days postinjection. This suggests that although the total number of glia may return to normal the number of each type of glial cell present changes following injection of triethyl lead. There was no evidence of cerebral oedema following triethyl lead injection either at the light or electron microscopic level.

Differential effect of cortisol on thymocytes from mice of different ages.

The influence of cortisol in vivo and in vitro was studied in thymus sections, suspensions, and smears obtained from new-born, 6 days old, and 30 days old mice. Histology as well as differential counts revealed that new-born thymocytes are far more sensitive to the cytotoxic effect of cortisol than thymocytes from older animals, and thymocytes from 30 days old mice are more sensitive than cells from 6 days old animals. Thus, the number of pyknotic cells in the thymus of new-born and 30 days old mice increased up to 20 per cent 6 h after injection of cortisol compared to 10 per cent in 6 days old animals. At 24 h after injection, the per cent of pyknotic cells in the thymus of new-born mice was 50 compared with about 1 in the two older age-groups. Supravital dye exclusion showed that thymocytes in 6 days old mice are less sensitive to the cytolytic action of cortisol in vivo than thymocytes from new-born and 30 days old mice. Incorporation of 3H-TdR and 3H-UdR into thymocyte DNA and RNA, respectively, was inhibited in all groups after treatment in vivo. DNA-synthesis of thymocytes in new-born animals was inhibited in all groups after treatment in vivo. DNA-synthesis of thymocytes in new born animals was inhibited more than DNA-synthesis in thymocytes of older animals, whereas RNA-synthesis was inhibited to nearly the same extend in the three groups studied. Incubation in vitro with 10(-6)M cortisol inhibited the DNA-synthesis to the same extent in thymocytes from the three age groups studied whereas the inhibiting effect of 10(-5)M cortisol on thymocytes from 6 and 30 days old animals was more intense than that on cells in new-born mice. The RNA-synthesis of thymocytes from 6 days old mice incubated in 10(-5)M cortisol was inhibited far less than RNA-synthesis in cells from new-born and 30 days old mice. The experiments reported, showing differential effects of cortisol on thymocytes obtained from mice of different ages, may reflect different stages of thymocytes maturation during the process of ontogenesis.

Rapid Radiation Cell Death and Cell Proliferation in Intestinal Epithelium after 1000-Rad Irradiation

Rapid cell death of mouse intestinal crypt cells was investigated after 1000 rads of x-irradiation by counting pyknotic cells in tissue sections. The number of normal crypt cells decreased with the appearance of pyknotic cells after irradiation. Pyknotic cells appeared within 30 min and the number of pyknotic cells reached a maximum 4 hr after irradiation. From the disappearance rate of the cells the mean life span of pyknotic cells was estimated to be 2 hr or less. Pyknotic cells observed within 4 hr after irradiation were produced without cell division, and this interphase death by pyknosis occurred in all phases of cell cycle. The tendency for the cells to develop into pyknotic cells was high in G2 phase, intermediate in S phase, low in G1 phase. The increase in mitotic index and the decrease in pyknotic cells from the second to the fifth day after irradiation were inversely related. After the disappearance of pyknotic cells, rapid recovery of the cell population took place with logarithmic cell proliferation and shortening of the cell cycle. Recovery to the normal cell population level was attained within 5 days after irradiation. (auth)