Cleaved caspase-3 is present in the majority of glial cells in the intact rat spinal cord during postnatal life.

Cell death is an essential process that occurs during the development of the central nervous system. Despite the availability of a wide range of commercially produced antibodies against various apoptotic markers, data regarding apoptosis in intact spinal cord during postnatal development and adulthood are mostly missing. We investigated apoptosis in rat spinal cord at different stages of ontogenesis (postnatal days 8, 29, and 90). For this purpose, we applied immunofluorescent detection of two widely used apoptotic markers, cleaved caspase-3 (cC3) and cleaved poly(ADP-ribose) polymerase (cPARP). Surprisingly, we found significant discrepancy between the number of cC3+ cells and PARP+ cells, with a ratio between 500:1 and 5000:1 in rat spinal cord at all postnatal time points. The majority of cC3+ cells were glial cells and did not exhibit an apoptotic phenotype. In contrast with in vivo results, in vitro analysis of primary cell cultures derived from neonatal rat spinal cord and treated with the apoptotic inductor staurosporine revealed a similar onset of occurrence of both cC3 and cPARP in cells subjected to apoptosis. Gene expression analysis of spinal cord revealed elevated expression of the Birc4 (XIAP), Birc2, and Birc5 (Survivin) genes, which are known potent inhibitors of apoptosis. Our data indicate that cC3 is not an exclusive marker of apoptosis, especially in glial cells, owing its possible presence in inhibited forms and/or its participation in other non-apoptotic roles. Therefore, cPARP appears to be a more appropriate marker to detect apoptosis.

"Neural stem cells puzzle" missing piece: telomerase reverse transcriptase expression patterns during nervous system ontogenesis in rats.

For almost three decades, neural stem cells remain still up-to-date and enigmatic topic. The main problem for their studying is the non-existence of an exclusive neural stem cell marker and the heterogeneity of them across the nervous system. As one of the novel markers of neural stem/progenitor cells may serve telomerase reverse transcriptase (TERT), a catalytic subunit of the telomerase enzyme, responsible for retaining the cell immortality. Thus, the aim of our study was to reveal if TERT, as an enzyme for ensuring the immortality of proliferating cells, could be used as a potential marker of neural stem/progenitor cells during the ontogenesis of the rat central nervous system. In this study, we used various markers related to neural stem or progenitor cells character and examined their co-localization with TERT expression. Our experiments were performed on the tissue of the brain and spinal cord during several stages of postnatal development and the neural tube during the 14th embryonal day. Cytoplasmatic TERT expression was found in alar plate progenitors and ventral horn neuroblasts of E14 rats. In the postnatal stages of spinal cord ontogenesis, a cytoplasmatic expression in neurons and nuclear expression in astrocytes was defined. In the brain, nuclear TERT expression was found in neural progenitor cells of neurogenic areas. This study provides the first comparative study of TERT expression across the central nervous system ontogenesis. The nuclear presence of TERT may be used as a potential marker of neural stem/progenitor cells, however, further studies are required to confirm these assumptions.

Flow cytometric method for estimation of 5-bromo-2´-deoxyuridine content in rat serum.

Labelling of DNA in replicating cells using 5-bromo-2´-deoxyuridine (BrdU) is widely used, however the rapid clearance and metabolisation of BrdU in the living organism is a critical issue. Although the pharmacokinetic of BrdU in experimental animals is empirically approximated, the exact time-curve remains unknown. Here we present novel method for estimation of the BrdU content in the blood serum. The application is based on the in vitro cocultivation of tumour cells with the examined serum and the subsequent quantification of the incorporated BrdU in the DNA using flow cytometry analysis. Our results demonstrate that this approach can quantify the BrdU concentration in serum at 1 micromol.dm(-3) and might represent an attractive alternative to conventional chromatographic analysis. The employment of tumour cells as "detectors" of the BrdU content in serum provides an advantage over high pressure liquid chromatography (HPLC), as this approach allows us to approximate not only the concentration of BrdU, but also to determine, whether BrdU is present in the blood serum in effective concentration to reliable label all cells undergoing the S-phase of the cell cycle. The presented application might be a helpful tool for studies on pharmacokinetics of BrdU or other thymidine analogues when testing various administration routes or protocols.

Praziquantel and liposomized glucan-treatment modulated liver fibrogenesis and mastocytosis in mice infected with Mesocestoides vogae (M. corti, Cestoda) tetrathyridia.

Beta-glucans are immunomodulators able to activate innate immunity and to potentiate acquired immune reactions. We investigated the impact of co-administration of liposomized beta-glucan on the larvicidal effect of the anthelmintic praziquantel (PZQ) in the livers and peritoneal cavities in mice infected with Mesocestoides vogae (M. corti). Also, within 2 weeks following therapy (up to day 29 p.i.) we examined collagen synthesis in the livers of mice by means of biochemical determination of hydroxyproline concentration, total mast cell counts and cell proliferative capacity using immunohistochemical and radiometrical methods. After co-administration of liposomized glucan (LG) and PZQ efficacy (%) was significantly higher than after treatment with either compound alone, particularly in the peritoneal cavity compared to the liver. In comparison with the control, more intense collagenesis was found in the B-liver parts (high intensity of infection) and lowering of collagen content in the A-parts (very weak infection). This effect was strongest after LG treatment and co-administration of PZQ abolished the pro-fibrotic effect of LG. In all groups, mast cell counts were higher in the B-liver parts than in the A-parts and the dynamics of mastocytosis was profoundly modulated following therapy. Whereas the effect of PZQ was only moderate, early and very strong onset was seen after LG treatment. Administration of PZQ suppressed LG induced-elevation of mast cells counts in both liver parts. Using DNA S-phase markers (BrdU and 3H-thymidine) the proliferative capacity was shown to be associated with several kinds of liver cells. Therapy significantly stimulated [3H]-thymidine incorporation (cell proliferation) only in the A-parts over that in control, the most after LG administration. In summary (i) the anthelmintic effect of PZQ could be enhanced after simultaneous administration of the immunomodulator beta-glucan entrapped in a liposomal carrier, (ii) intense mastocytosis seen after treatment with LG seems to have a direct role in the glucan's pro-fibrotic activity and can be abolished after co-administration of PZQ in a time-dependent manner, (iii) the pattern of cell proliferation indicates that in the case of PZQ treatment, the reparative processes of liver parenchyma are enhanced in an inverse correlation with the intensity of infection.

Papillomavirus detection: demographic and behavioral characteristics influencing the identification of cervical disease.

OBJECTIVE: This study was undertaken to assess the association between detection of high-risk types of human papillomavirus and various demographic and behavioral characteristics and to further relate this association to cervical histopathologic findings. STUDY DESIGN: A total of 1007 patients with a Papanicolaou test result reported as high-grade squamous intraepithelial lesion or with 2 results reported as atypical squamous cells of undetermined significance or low-grade squamous intraepithelial lesion were referred from city and county clinics to a colposcopic clinic. All women had a cervical smear obtained, underwent colposcopically directed biopsy and endocervical curettage, and had a specimen taken for human papillomavirus deoxyribonucleic acid detection by polymerase chain reaction. Demographic information was obtained from each patient. RESULTS: Human papillomavirus deoxyribonucleic acid was identified in 655 (66%) of the specimens. High-risk human papillomavirus types (16, 18, 31, 33, and 35) were detected in 463 (70.7%) of these specimens. The prevalence of evidence of human papillomavirus (koilocytosis) and grade 1 cervical intraepithelial neoplasia in the biopsy specimen decreased significantly with age, whereas the prevalence of grade 2 or 3 cervical intraepithelial neoplasia in the biopsy specimen increased with age. There was a significant age-dependent decreasing trend in detection of high-risk human papillomavirus deoxyribonucleic acid among women who had human papillomavirus-associated changes, grade 1 cervical intraepithelial neoplasia, and grade 2 or 3 cervical intraepithelial neoplasia in the biopsy specimen. The prevalences of high-risk human papillomavirus among patients with grade 1 cervical intraepithelial neoplasia and grade 2 or 3 cervical intraepithelial neoplasia were similar, and both were significantly higher than among women with no evidence of cervical intraepithelial neoplasia or koilocytosis in the biopsy specimen. Risk factors associated with grade 2 or 3 cervical intraepithelial neoplasia were different from those associated with human papillomavirus-associated changes and with grade 1 cervical intraepithelial neoplasia. CONCLUSION: The detection of high-risk human papillomavirus was age-dependent for all histologic categories. Patients with grade 2 or 3 cervical intraepithelial neoplasia had a prevalence of high-risk human papillomavirus that was similar to that among women with grade 1 cervical intraepithelial neoplasia but significantly higher than that among women whose biopsy specimens appeared normal or demonstrated only the presence of human papillomavirus-induced changes (koilocytosis). This suggests that separation of human papillomavirus-associated changes only from grade 1 cervical intraepithelial neoplasia may be of significance in tissue diagnosis.

Graded postischemic reoxygenation attenuates ischemia-reperfusion-induced nuclear and nucleolar damage in lumbosacral dorsal root ganglia neurons. A light and electron microscopic study in rabbit.

Ischemia and reperfusion-induced nuclear and nucleolar changes of the lumbosacral dorsal root ganglia neurons were studied in a spinal cord ischemic model of a rabbit. Twenty and forty min abdominal aorta ligation followed immediately by perfusion fixation, 20 min abdominal aorta ligation followed by 1 h of normoxic reoxygenation and 20 min abdominal aorta ligation followed by 1 h of graded postischemic reoxygenation were tested. In animals subjected only to a 20 and 40 min aorta ligation irregularly undulated nuclear membrane and occurrence of dark osmiophilic dense clumps often located close to interchromatin granules were found. The segregation of the nucleolar granular and fibrillar components became apparent but fibrillar centers were almost completely absent. In animals subjected to 20 min ligation followed by 1 h of normoxic reoxygenation a high number of dense clumps, loosely disseminated throughout the nucleoplasm was detected along with almost complete fragmentation and segregation of the nucleolus. The application of the graded postischemic reoxygenation has proven effective as a neuroprotective and antisegregatory adjunct whereby the nucleolar fibrillar and granular components regained almost normal appearance, and the occurrence of dense intranuclear clumps was greatly reduced.

The nucleolar channel system of nerve cell. Short communication.

The nucleolar channel system which belongs to less known nuclear components has been described in nerve cells of dog dorsal root ganglia. This structure is formed by a system of twisted tubules. Till now this structure was not observed in all mammalian cells.

Nucleus ultrastructure of dog dorsal root ganglia cells after partial ischemia.

The partial ischemia of the adult dogs, both males and females was induced by the ligature of aorta abdominalis above the branching of arteria coeliaca within 80 minutes and 120 minutes duration with the following preparation of L6 und L7 dorsal root ganglia. The nuclei and nucleoli of the dorsal root ganglia nerve cells were studied by the usual standard transmission electron microscopic procedures. Comparing the control and ischemized material we have observed the different reaction of the nuclei and nucleoli to the ischemia. Some of these structures remained unchanged, the others demonstrated a various degree of the alteration. After the partial ischemia some nucleoli were more compact, some nucleolonemata were less distinct but no karyoplasmic changes were noted. The nucleolar interstitial vacuoles were occasionally enlarged and the satellite bodies were observed scarcely in the proximity of the nucleolus.

Influence of the season on the circadian rhythm of serum lipids in male Wistar rats.

Young male Wistar rats were adapted to a 12:12 h light: dark regimen (7 a.m. to 7 p.m., 7 p.m. to 7 a.m.), with free access to food and water. In January, April, July and October they were killed at 3-hour intervals and non-esterified fatty acids, tri-glycerides, total cholesterol and phospholipids were determined in their serum. Serum cholesterol was the least influenced by the season; a rhythm was present in all the seasons, the acrophases came in approximately the same part of the day and the mean adjusted oscillation (mesor) values (in the cosinor test) did not differ the whole year round. Serum triglycerides displayed a rhythm except in the summer; their autumn values were lower and their curves attained the maximum in different parts of the photophase. Non-esterified fatty acids and phospholipids were found to be significantly influenced by the season; the shapes of their curves and their maxima varied during the year. For evaluation of the influence of the seasons on the circadian oscillations of lipid metabolism in the rat, information on the ratio of the formation and utilization/degradation of tissue lipids in the various parts of the year is still lacking.

Total content of RNA in individual nerve cells of spinal ganglion during ischemia in dogs.

The total RNA content in the individual nerve cells of the lumbal spinal ganglia in the dog after the 80 minutes lasting partial ischemia was followed. The spinal ganglion neurons did not react in ischemia in the same way. The total RNA content in the individual neurons of the control animals occurred in the range 132-990 arbitrary units, after the ischemia in the range 116-670 arbitrary units. In both groups the cells with the low or high RNA content were seen. The decreases in total RNA content after ischemia was at the higher level of the statistical significance in the group with the largest and middle large neurons giving the evidence about the greater sensitivity to the oxygen shortage in comparison with the group of the smallest spinal ganglion nerve cells.