Number Of Pyknotic Cells Research Articles

Comparison of part-time and full-time mandibular advancement: enlightenment based on type H vessel coupling osteogenesis.

This study was aimed at resolving the controversy over the wearing time of mandibular advancement (MA) appliances by comparing the differences between part-time and full-time MA (PTMA and FTMA) from the perspective of promoting H-type vessel coupling osteogenesis in the condylar heads. Thirty 30-week-old male C57BL/6J mice were randomly divided into 3 groups: the control (Ctrl), PTMA, and FTMA groups. The mandibular condyles were analyzed by morphology, micro-computed tomography, histological staining, and immunofluorescence staining to investigate the changes of the condylar heads in the PTMA and FTMA groups after 31 days. Both PTMA and FTMA models promoted condylar growth and achieved stable mandibular advancement at day 31. However, compared with PTMA, FTMA has the following characteristics. First, new bone formation in the condylar head was detected in the retrocentral region in addition to the posterior region. Second, the condylar proliferative layer was thicker, and the number of pyknotic cells in the hypertrophic and erosive layers was higher. Moreover, endochondral osteogenesis of the condylar head was more active. Finally, the retrocentral and posterior regions of the condylar head had more vascular loops or arcuate H-type vessel coupling Osterix+ osteoprogenitors. While both PTMA and FTMA induced new bone formation in the condylar heads of middle-aged mice, FTMA promoted more osteogenesis by volume and region. Furthermore, FTMA presented more H-type vessel coupling Osterix+ osteoprogenitors in both the retrocentral and posterior regions of the condylar head. FTMA is better at promoting condylar osteogenesis, especially in non-growing patients. We suggest that enhancing H-type angiogenesis could be an effective strategy to achieve favorable MA outcomes, especially for patients unable to meet the FT-wearing requirement or being non-growing.

Pyknotic Cell Features of Buccal Mucosal Smear in Various Oral Lesions of Betel Nut Chewers

The habit of chewing betel nut is one of the predisposing factors that can trigger the formation of various oral lesions and develop into a malignant condition in the form of oral squamous cell carcinoma (OSCC). This malignancy has a low five-year survival rate as early detection is not widely carried out. Early detection can be conducted by examining pyknotic cells to describe the level of apoptosis. This study aims to examine the pyknotic cell features as a marker of apoptosis in various oral lesions found in betel nut chewers. This study is an analytic survey conducted once with a post-test control group design. The study was conducted on 17 betel nut chewers and 17 healthy people as controls. A buccal mucous smear was collected, and cytological slides were made with Papanicolaou stain. Pyknotic cells were counted using a light microscope and Optilab pro camera. This study found several oral lesions in betel nut chewers, namely exogenous pigmentation, lesions resembling leukoplakia, and lichenoid reaction. Furthermore, the result also showed that the frequency of pyknotic cells in betel nut chewers was lower than in the control group. Non-parametric Kruskal Wallis and post hoc Mann Whitney test showed significant differences in the number of pyknotic cells in various oral lesions with a p-value = 0,001. It can be concluded that the consumption of betel nut affected the level of apoptosis, characterized by a decrease in pyknotic cells and significantly different pyknotic cells depending on the type of oral lesions.

Pentoxifylline Protects Against Hippocampal Damage and Memory Impairment Induced by Trimethyltin

Background: Trimethyltin (TMT) is a toxic agent that causes oxidative stress, a laboratory model for inducing hippocampal injuries. Pentoxifylline (PTX) inhibits phosphodiesterase, inflammation and oxidative stress. This study evaluated the neuroprotective effects of PTX on injuries induced by TMT in the hippocampus. Methods: Sixty male Wistar rats were divided into five groups of 12 each. Group 1 received normal saline while Group 2 received a single dose of TMT (8 mg/kg). The other four groups received TMT at 8 mg/kg plus 60, 100 or 120 mg/kg PTX twice daily for six consecutive days. The rats’ working and reference memory were investigated, using radial arm maze tasks. At the end of the experiments, the rats’ brains were removed and processed for histological study of the hippocampus. Results: The TMT treatment prolonged the four baited arms tasks (P<0.001), while the PTX treatment at 60, 100 or 120 mg/kg significantly reduced the effects of TMT on the spatial memory (P<0.01). The working and reference memory errors significantly increased in the TMT group compared to the controls (P<0.001) while the PTX treatment significantly reduced the TMT effect (P<0.001). Also, TMT increased the number of pyknotic cells in the hippocampus (P<0.001), while PTX significantly decreased the mean number of pyknotic cells (P<0.05). Conclusion: The findings suggest that PTX can protect against the memory deficit and deleterious effects on rat hippocampus induced by TMT neurotoxicity. Thus, PTX is likely to be a potential agent to prevent against the neurotoxicity induced by TMT.

Elucidating the role of hypoxia/reoxygenation in hippocampus-dependent memory impairment: do SK channels play role?

Professionals and mountaineers often face the problem of reperfusion injury due to re-oxygenation, upon their return to sea-level after sojourn at high altitude. Small conductance calcium-activated potassium channels (SK channels) have a role in regulating hippocampal synaptic plasticity. However, the role of SK channels under hypoxia-reoxygenation (H/R) is unknown. The present study hypothesized that SK channels play a significant role in H/R induced cognitive dysfunction. Sprague-Dawley rats were exposed to simulated HH (25,000 ft) continuously for 7days followed by reoxygenation periods 3, 6, 24, 48, 72 and 120h. It was observed that H/R exposure caused impairment in spatial memory as indicated by increased latency (p < 0.001) and pathlength (p < 0.001). The SK1 channel expression increased upon HH exposure (102.89 ± 7.055), which abrogated upon reoxygenation. HH exposure results in an increase in SK2 (CA3, 297.67 ± 6.69) and SK3 (CA1, 246 ± 5.13) channels which continued to increase gradually upon reoxygenation. The number of pyknotic cells (24 ± 2.03) (p < 0.01) and the expression of caspase-3 increased with HH exposure, which continued in the reoxygenation group (177.795 ± 1.264). Similar pattern was observed in lipid peroxidation (p < 0.001), LDH activity (p < 0.001) and ROS production (p < 0.001). A positive correlation of memory, cell death and oxidative stress indicates that H/R exposure increases oxidative stress coupled with SK channel expression, which may play a role in H/R-induced cognitive decline and neurodegeneration.

Cell death in the male and female rat medial prefrontal cortex during early postnatal development.

Apoptosis, programmed cell death, is a critical component of neurodevelopment occurring in temporal, spatial, and at times, sex-specific, patterns across the cortex during the early postnatal period. During this time, the brain is particularly susceptible to environmental influences that are often used in animal models of neurodevelopmental disorders. In the present study, the timing of peak cell death was assessed by the presence of pyknotic cells in the male and female rat medial prefrontal cortex (mPFC), a cortical region that in humans, is often involved in developmental disorders. One male and one female rat per litter were sacrificed at the following ages: postnatal day (P)2, 4, 6, 8, 10, 12, 14, 16, 18, and 25. The mPFC was Nissl-stained, the densities of pyknotic cells and live neurons were stereologically collected, and the number of pyknotic cells per 100 live neurons, pyknotic cell density, and neuron density were analyzed. Males and females showed a significant peak in the ratio of pyknotic to live neurons on P8, and in females, this elevation persisted through P12. Likewise, the density of pyknotic cells peaked on P8 in both sexes and persisted through P12 in females. The timing of cell death within the rat mPFC will inform study design in experiments that employ early environmental manipulations that might disrupt this process.

Increased DNA Damage, Instability and Cytokinesis Defects in Occupationally Exposed Car Painters.

Car painting is considered an occupational exposure job with high risk for cancer development, due to the association with harmful chemicals and mutagens. This study aimed to profile car painters occupationally exposed and determine its association with DNA damage and genomic instability. We collected a questionnaire and buccal cells of 74 individuals (37 car painters and 37 non-exposed workers) paired by age, alcohol and smoking habits. The number of pyknotic cells, karyolitic cells, karyorrhetic cells, condensed chromatin, binucleated cells, basal cells, differentiated cells (DIFF), micronucleated cells and nuclear buds were evaluated using the Buccal Micronucleus Cytome Assay protocol. A statistically significant increase was observed in all parameters (p<0.05) in the exposed group, but DIFF showed a statistically significant decrease (p<0.001), compared to the control group. In association with the poor work environment and lack of personal and collective protective equipment, occupational exposure of car painters leads to high DNA damage, genomic instability and alterations in cellular kinetics.

Cytological multimarker screening using BMCyt test in waterpipe smokers: an integrative study of cell damage, toxicological and cancer risk

Waterpipe smoking is an ancient method of tobacco smoking practiced worldwide. There is a common belief that waterpipe smoking is a safer alternative to cigarette, but many studies showed that some toxicants were associated with cancer risk, significantly higher in waterpipe smoking. Thus, this study aimed to evaluate the status of waterpipe smoker's buccal cells and its cancer risk using the buccal micronucleus cytome test. Forty waterpipe smokers (noncigarette smokers) were recruited and paired by gender, age and alcoholic habits with 40 control subjects. One-thousand cells from each individual were analysed and the number of pyknotic cells (PYC), karyolitic cells (KYL), karyorrhetic cells (KHC), condensed chromatin (CC), binucleated cells (BN), basal cells (BC), nuclear buds (NBUD) and differentiated cells (DIFF) were counted. Additionally, 2000 differentiated cells were analysed counting micronucleated cells (MNi) and nuclear buds. We observed an increasing P< 0.05 in all waterpipe smoker's cell parameters, except DIFF (fold-decrease). Only CC showed no differences between groups. The interference in the cell cycle plus DNA damage observed in this study could be responsible for the high number of damaged cells and in death process, showing the importance of our study and the high risk in waterpipe smoking.

Coenzyme Q10 ameliorates cerebral ischemia reperfusion injury in hyperglycemic rats

The purpose of this study is to investigate the effect of coenzyme Q10 (CoQ10) on focal cerebral ischemia/reperfusion (I/R) injury in hyperglycemic rats and the possible involved mechanisms. In this study, we established the transient middle cerebral artery occlusion (MCAO) for 30min in the rats with diabetic hyperglycemia. The neurological deficit score, 2,3,5-triphenyltetrazolium chloride (TTC) staining and pathohistology are applied to detect the extent of the damage. The expression of Fis1, Mfn2 and Lc3 in the brain is investigated by immunohistochemical and Western blotting techniques. The results showed that the streptozotocin-induced diabetic hyperglycemia and MCAO-induced focal cerebral ischemia were successfully prepared in rats. In the hyperglycemic group, the neurological deficit scores, infarct volumes, and number of pyknotic cells were higher than that in the normalglycemic group at 24h and/or 72h reperfusion. Pretreated with CoQ10 (10mg/kg) for four weeks could significantly reduce the neurological scores, infarct volume, and pyknotic cells at 24h and/or 72h reperfusion of the hyperglycemic rats compared with non-CoQ10 pretreated hyperglycemic animals. Immunohistochemistry and Western blotting showed that pretreatment with CoQ10 or insulin could significantly reduce the expression of Fis1 protein in the brain at 24h and 72h reperfusion. Inversely, a significantly increased expression of Mfn2 was observed in the rats CoQ10 or insulin pretreated at 24h and/or 72h reperfusion when compared with matched hyperglycemic rats. These results demonstrated that hyperglycemia could aggravate ischemic brain injury. Pretreatment with CoQ10 might ameliorate the diabetic hyperglycemia aggravated I/R brain damage in the MCAO rats by maintain the balance between mitochondrial fission and fusion.

Survival of adult generated hippocampal neurons is altered in circadian arrhythmic mice.

The subgranular zone of the hippocampal formation gives rise to new neurons that populate the dentate gyrus throughout life. Cells in the hippocampus exhibit rhythmic clock gene expression and the circadian clock is known to regulate the cycle of cell division in other areas of the body. These facts suggest that the circadian clock may regulate adult neurogenesis in the hippocampus as well. In the present study, neurogenesis in the hippocampal subgranular zone was examined in arrhythmic Bmal1 knockout (-KO) mice and their rhythmic heterozygous and wildtype littermates. Proliferation and survival of newly generated subgranular zone cells were examined using bromodeoxyuridine labelling, while pyknosis (a measure of cell death) and hippocampal volume were examined in cresyl violet stained sections. There was no significant difference in cellular proliferation between any of the groups, yet survival of proliferating cells, 6 weeks after the bromodeoxyuridine injection, was significantly greater in the BMAL1-KO animals. The number of pyknotic cells was significantly decreased in Bmal1-KO animals, yet hippocampal volume remained the same across genotypes. These findings suggest that while a functional circadian clock is not necessary for normal proliferation of neuronal precursor cells, the normal pruning of newly generated neurons in the hippocampus may require a functional circadian clock.

N,N'-Bis(2-chloroethyl)- N-nitrosourea (BCNU)-induced Apoptosis of Neural Progenitor Cells in the Developing Fetal Rat Brain

N,N’-bis(2-chloroethyl)-N-nitrosourea (BCNU) is one of the major drugs used in chemotherapy against malignant gliomas due to its effects, such as induction of bifunctional alkylation of DNA and formation of interstrand DNA cross-linkages, and induces cortical malformations in the fetal and neonatal rat brain. In this study, pregnant rats were treated with 7.5 mg/kg of BCNU on gestational day 13 (GD 13), and their fetuses were collected from 12 to 72 hours after BCNU treatment in order to examine the timecourses of morphological and immunohistochemical changes in neural progenitor cells in the developing brain. The number of pyknotic cells in the telencephalon peaked at 24 h and then gradually decreased until 72 h. The majority of these pyknotic cells were positive for cleaved caspase-3, a key executioner of apoptosis. The pyknotic cells showed the ultrastructural characteristics of apoptosis. The number of p53-positive cells began to increase prior to the appearance of apoptotic cells and p21-positive cells. The number of phosphorylated-histone H3-positive cells (mitotic cells) decreased from 24 to 36 h. The number of Iba1-positive cells (microglial cells) in the telencephalon increased from 12 to 48 h. These results suggest that BCNU induces p53-dependent apoptosis and reduces proliferative activity, resulting in reduction of the weight of the telencephalon and the thickness of the telencephalic wall in the fetal brain. This study will help to clarify the mechanisms of BCNU-induced fetal brain toxicity.

No effect of running and laboratory housing on adult hippocampal neurogenesis in wild caught long-tailed wood mouse

BackgroundStudies of adult hippocampal neurogenesis (AHN) in laboratory rodents have raised hopes for therapeutic interventions in neurodegenerative diseases and mood disorders, as AHN can be modulated by physical exercise, stress and environmental changes in these animals. Since it is not known whether cell proliferation and neurogenesis in wild living mice can be experimentally changed, this study investigates the responsiveness of AHN to voluntary running and to environmental change in wild caught long-tailed wood mice (Apodemus sylvaticus).ResultsStatistical analyses show that running had no impact on cell proliferation (p = 0.44), neurogenesis (p = 0.94) or survival of newly born neurons (p = 0.58). Likewise, housing in the laboratory has no effect on AHN. In addition, interindividual differences in the level of neurogenesis are not related to interindividual differences of running wheel performance (rs = -0.09, p = 0.79). There is a correlation between the number of proliferating cells and the number of cells of neuronal lineage (rs = 0.63, p < 0.001) and the number of pyknotic cells (rs = 0.5, p = 0.009), respectively.ConclusionPlasticity of adult neurogenesis is an established feature in strains of house mice and brown rats. Here, we demonstrate that voluntary running and environmental changes which are effective in house mice and brown rats cannot influence AHN in long-tailed wood mice. This indicates that in wild long-tailed wood mice different regulatory mechanisms act on cell proliferation and neurogenesis. If this difference reflects a species-specific adaptation or a broader adaptive strategy to a natural vs. domestic environment is unknown.

Granule cell number, cell death and cell proliferation in the dentate gyrus of wild‐living rodents

Adult neurogenesis in the dentate gyrus occurs at species-specific levels. Wood mice (Apodemus flavicollis) show higher proliferation rates than laboratory mice and voles (Clethrionomys glareolus, Microtus subterraneus). We compare rates of cell death and proliferation and investigate if cell proliferation leads to the long-term recruitment of granule cells. Granule and pyknotic cell numbers were estimated in wild-living rodents in different age classes and compared with laboratory mice of mixed genetic background. All species differ significantly in their number of granule cells, except for the comparison of laboratory mice with European pine voles. Granule cell number is significantly higher in old bank voles and wood mice as compared to adults (23 and 37%, respectively). The number of pyknotic cells is highest in wood mice and lowest in laboratory mice. Across all species, the numbers of proliferating and pyknotic cells correlate. Despite differences in cell proliferation and cell death, the ratio of proliferating to pyknotic cells does not differ between adults of the wild-living species, but in laboratory mice a significantly lower proportion of cells die compared with the other species. In addition, the ratio of proliferating to pyknotic cells was significantly higher in old wood mice than in adults. We conclude (i) that cell proliferation can lead to an increase in granule cell number in wild-living rodents and (ii) that species- and age-specific changes of the ratio between proliferating and pyknotic cells occur as deviations from a close correlation of these two numbers across all species and age groups.

Morphological changes of the thymus under stress caused by water immersion and restraint in SAMP1 mice

We compared the time course of the stress-induced morphological changes in the thymus between SAMP1 and C3H/He mice. The mice were kept under restraint in a 50-ml plastic centrifuge tube in water at 24° for 4 h. This treatment induced atrophy of the thymus and reduced the thymus weight, although it did not affect the body weight or major organ weights in either strains of mice. Histological studies of the thymus revealed that the stress groups of both strains showed a transient increase in the number of pyknotic cells and macrophages as compared with each control group just after the stressor treatment. Only a slight increase in the macrophage number was found at 4 h after the treatment. Furthermore, we investigated the morphological changes of the thymus until 7 days after the treatment, in order to compare the time course of recovery of the thymus atrophy between the two mouse strains. The stress groups of both strains had decreased lymphocyte populations in the thymus. The thymic lymphocyte population in C3H/He mice started to recover at 24 h after the stressor treatment. In clear contrast, the lymphocyte population in the SAMP1 thymus did not recover but continued to decrease until 7 days after the treatment, being progressively replaced by fibroblasts. These results suggest that the regenerative mechanisms of the SAMP1 thymus have some defects, and that the T-cell compartment of the immune system in SAMP1 mice may be as sensitive as aged mice from the control strain.

Hydroxyurea (HU)-induced apoptosis in the mouse fetal tissues.

Hydroxyurea (HU), a ribonucleotide reductase inhibitor, induces morphological anomalies in the central nervous system (CNS), craniofacial tissues and limb buds in animals, and neonatal respiratory distress in humans. In the present study, pregnant mice were treated with 400 mg/kg of HU at day 13 of gestation, and their fetuses were examined from 1 to 48 hours after treatment (HAT) to find a clue to clarify the mechanisms of HU-induced fetotoxicity and teratogenecity. At 6 and 12 HAT, a moderate to marked increase in the number of pyknotic cells was detected in the CNS and lung. A mild increase in the number of pyknotic cells was also found in the craniofacial mesenchymal tissues, limb buds and so on. These pyknotic cells had nuclei positively stained by the TUNEL method, which is widely used for the detection of apoptotic nuclei, and they also showed electron microscopic characteristics identical to those of apoptotic cells. The present results suggest that the HU-induced fetotoxicity is characterized by excess apoptotic cell death in the fetal tissues, and that such excess cell death in the fetal CNS, lung, craniofacial tissue and limb bud may have a certain relation to the later occurrence of morphological or functional anomalies reported in these tissues following HU-administration.

Insulin Rescues Retinal Neurons from Apoptosis by a Phosphatidylinositol 3-Kinase/Akt-mediated Mechanism That Reduces the Activation of Caspase-3

The ability of insulin to protect neurons from apoptosis was examined in differentiated R28 cells, a neural cell line derived from the neonatal rat retina. Apoptosis was induced by serum deprivation, and the number of pyknotic cells was counted. p53 and Akt were examined by immunoblotting after serum deprivation and insulin treatment, and caspase-3 activation was examined by immunocytochemistry. Serum deprivation for 24 h caused approximately 20% of R28 cells to undergo apoptosis, detected by both pyknosis and activation of caspase-3. 10 nm insulin maximally reduced the amount of apoptosis with a similar potency as 1.3 nm (10 ng/ml) insulin-like growth factor 1, which acted as a positive control. Insulin induced serine phosphorylation of Akt, through the phosphatidylinositol (PI) 3-kinase pathway. Inhibition of PI 3-kinase with wortmannin or LY294002 blocked the ability of insulin to rescue the cells from apoptosis. SN50, a peptide inhibitor of NF-kappaB nuclear translocation, blocked the rescue effect of insulin, but neither insulin or serum deprivation induced phosphorylation of IkappaB. These results suggest that insulin is a survival factor for retinal neurons by activating the PI 3-kinase/Akt pathway and by reducing caspase-3 activation. The rescue effect of insulin does not appear to be mediated by NF-kappaB or p53. These data suggest that insulin provides trophic support for retinal neurons through a PI 3-kinase/Akt-dependent pathway.

Ethylnitrosourea (ENU)-induced apoptosis in the rat fetal tissues.

Ethylnitrosourea (ENU), a well known DNA alkylating agent, induces anomalies in the central nervous system (CNS), craniofacial tissues and male reproductive organs. In this study, pregnant rats were treated with 60 mg/kg ENU at day 13 of gestation, and their fetuses were examined from 1 to 48 hours after treatment (HAT) to find a clue for clarifying the mechanisms of the ENU fetotoxicity and teratogenicity. From 3 to 12 HAT, the moderate to marked increase in the number of pyknotic cells was detected in the fetal CNS, craniofacial mesenchymal tissues, gonads and so on. These pyknotic cells had nuclei positively stained by the TUNEL method, which is widely used for the detection of apoptotic nuclei, and they also showed electron microscopic characteristics identical to those of apoptotic cells. The present results strongly suggest that excess cell death by apoptosis in the fetal CNS, craniofacial tissues and gonads may have a close relation to the later occurrence of anomalies reported in these tissues following ENU-administration.

Sex and seasonal changes in the rate of cell proliferation in the dentate gyrus of adult wild meadow voles

In order to study the neurobiological basis of seasonal changes in hippocampal structure and function, the rate of cell proliferation was examined in male and female wild meadow voles captured during different seasons. We found that the number of [ 3H]thymidine-labeled cells varied across the seasons and across sex in the meadow vole. Non-breeding female meadow voles had a higher rate of cell proliferation and cell death than males captured during either season or breeding females. These seasonal changes in the female meadow vole were associated with both fluctuating levels of adrenal steroids and gonadal steroids. Estradiol level was highly correlated with both the number of [ 3H]thymidine-labeled cells and the number of pyknotic cells in female meadow voles, with high levels of estradiol being associated with low levels of cell proliferation and cell death. Corticosterone level was associated with the number of [ 3H]thymidine-labeled cells in the hilus of female meadow voles. This seasonal change in the number of [ 3H]thymidine-labeled cells was also related to the overall volume of the hippocampus. At variance with past literature, there was no statistically significant sex difference favoring males in hippocampal volume, although the means were in the predicted direction. In male meadow voles, the number of pyknotic cells was related to testosterone level, with high levels of testosterone being associated with greater levels of cell death in the granular cell layer. There was also a suggestion that the number of [ 3H]thymidine-labeled cells in the hilus varied seasonally in males, with higher rates of cell proliferation during the breeding season than during the non-breeding season. In summary, we found that there were large fluctuations across the season in the rate of cell proliferation in the dentate gyrus of adult female meadow voles. Females captured during the non-breeding season had higher rates of cell proliferation in the granule cell layer than females captured during the breeding season. This seasonal fluctuation was related to hormone levels, with high levels of corticosterone and estradiol being related to lower levels of cell proliferation. These seasonal changes in cell proliferation may be related to known changes in spatial learning in the meadow vole and provide insights into changes in the hippocampus that occur in other species, including primates.

The vulnerability of the fetal sheep brain to hypoxemia at mid-gestation

Our aim was to test the hypothesis that a brief episode of hypoxemia near mid-gestation in fetal sheep will result in damage to the fetal brain with the extent and type of damage in any particular region being related to the developmental processes occurring at the time of the insult. Hypoxemia was induced, sufficient to reduce arterial O 2 content by approximately 50%, by restricting utero–placental blood flow in 14 chronically catheterised fetuses for 6 h or 12 h at 84 days of gestation (term 145–8 days). Age-matched fetuses ( n=14; 4 operated and 10 unoperated) were used as controls. Fetuses were killed 7 days after being exposed to hypoxemia, and brains removed for histological analysis at the light and ultrastructural levels. Body weights of hypoxemic fetuses did not differ significantly from controls but brain weights were significantly reduced both in absolute terms and when expressed in relation to body weight ( P<0.05). Most fetuses exposed to hypoxemia sustained no gross brain damage. However, in one hypoxemic fetus from a multiple pregnancy there was extensive leucomalacia in the cortical white matter; mild focal damage was seen in another 8 hypoxemic fetuses. In the cerebral cortex (frontal lobe) the surface folding index was significantly reduced ( P<0.05) in hypoxemic fetuses compared to controls suggesting that gyral formation had been delayed. In these fetuses there were also degenerating neurons in the deeper cortical layers. In the hippocampus of hypoxemic fetuses there was a delay ( P<0.05), compared to controls, in the migration of cells from the germinal layer to the pyramidal layer in the CA1 region, and decreases ( P<0.05) in the density (areal) of neurons in the pyramidal layer and in the width of stratum oriens. In the cerebellum of hypoxemic fetuses there was a decrease ( P<0.05), compared to controls, in the density (areal) of mitotic bodies in the external granule cell layer. However, there were no significant differences in the number of pyknotic cells in this layer, in the density of Purkinje cells, in their somal area, or in the width of the external granule cell or molecular layers. There was an increase ( P<0.05) in the proportion of the brain parenchyma occupied by blood vessels in both the hippocampus and cortex of hypoxemic fetuses compared to controls. This study has shown that an hypoxemic insult near mid-gestation can result, one week later, in white matter damage and in neuronal death in the hippocampus and to a lesser extent in the cerebral cortex and cerebellum. It can also retard neuronal migration and the growth of neural processes in the hippocampus where development is well established at this age. Such brain damage could result in less than optimal neuronal connectivity and could affect function postnatally.

Effects of low-dose phenytoin administered to newborn mice on developing cerebellum.

To examine correlations between dose levels of phenytoin (PHT) and neurotoxic effects on cerebellar development, we administered 10, 17.5, 25, and 35 mg/kg PHT suspended in sesame oil orally to newborn Jcl:ICR mice once a day during postnatal days 2-4 and determined plasma PHT concentrations during the administration period. Mortality rates were 12.5% and 35.2% in males and 15.3% and 33.3% in females for the 25 and 35 mg/kg PHT-treated groups during the PHT treatment, respectively. In the 25 and 35 mg/kg PHT-treated groups, total brain weight, the size of the cerebellum, and cerebellar weight were significantly reduced on postnatal day 21. However, in the 10 and 17.5 mg/kg PHT-treated groups, total brain weight and the size and weight of the cerebellum did not differ from those of the control group. Histologically, the number of pyknotic cells in the external granular layer (EGL) in the 25 and 35 mg/kg PHT-treated groups was increased on postnatal day 5, and the EGL was thicker than in the control group on postnatal day 14. Some of the Purkinje cells in the 35 mg/kg PHT-treated group showed degeneration. Plasma PHT levels were 10.7 +/- 2.2 and 24.6 +/- 2.6 micrograms/ml in the 25 and 35 mg/kg PHT groups on the third day of PHT treatment, respectively. In the 25 mg/kg PHT group, plasma PHT level was found to be in the therapeutic range for humans, 10-20 micrograms/ml. Accordingly, during pregnancy, epileptic women should be carefully given PHT at the lowest effective dose while plasma PHT levels are monitored properly. These findings emphasize the importance of pharmacokinetics in evaluating of phenytoin-induced developmental neurotoxicity.

Influence of maternal grooming, sex and age on Fos immunoreactivity in the preoptic area of neonatal rats: implications for sexual differentiation.

The medial preoptic area (mPOA) of the hypothalamus contains a sexually dimorphic nucleus (SDN-POA) that is 5-7 times larger in males than females and which contributes to the development and expression of male-specific sex behaviors in adulthood. Aside from a critical role for estrogen, the mechanisms that establish and maintain this sex difference are largely unknown. Differences in the size of the SDN-POA are thought to be related to estrogen-associated effects on programmed cell death (apoptosis) during early neonatal development. The expression of male sex behavior is also influenced by maternal behavior during development. During the postnatal period, the dam grooms the anogenital region of the pups to stimulate urination and defecation; however, male pups are groomed significantly more often than females and this maternal attention influences the expression of normal male sexual behavior in adulthood. Based on these observations, we hypothesized that different amounts of anogenital sensory stimulation might contribute to the sexually dimorphic development of the SDN-POA, specifically by providing for different levels of neuronal activation in the SDN-POA resulting in different degrees of cell death. Two experiments were conducted to test this hypothesis. In the first experiment, male and female rat pups on postnatal day 3 (PN 3) received simulated anogenital grooming with a stiff bristle paint brush. One hour later, the brains were removed and sections through the POA were cut and processed for the immunocytochemical detection of Fos-like immunoreactivity (IR) as an indicator of neuronal activation. In the second experiment, male and female littermates were killed on PN 3, 5, 7 and 12 and the number of Fos-immunoreactive cells and pyknotic cells detected in the SDN-POA were counted and compared. Our data demonstrate that anogenital stimulation on PN 3 results in a rapid induction of Fos-immunoreactive in the POA of both males and females. However, the majority of Fos-immunoreactive cells were located in the ventral POA and were distinctly lacking in the SDN-POA. In experiment 2, again no Fos-immunoreactive cells were detected in the SDN-POA of animals examined on PN 5-12. However, there was an increase in the number of pyknotic cells in the area surrounding and including the SDN-POA of females relative to males at PN 5, 7 and 12. Collectively, the data suggest that (1) anogenital grooming during early postnatal development induces a rapid activation of cells in the ventral mPOA, but not in the SDN-POA of rats, (2) there is a greater incidence of cell death in and around the SDN-POA of females vs. males during neonatal development, particularly toward the end of the hormone-sensitive critical period, and (3) Fos expression does not appear to be correlated with the sexually dimorphic development of, and/or programmed cell death within, the developing SDN-POA.