Cross-tolerance to otherwise lethal N-methyl-D-aspartate and oxygen-glucose deprivation in preconditioned cortical cultures.

In vitro ischemic preconditioning induced by subjecting rat cortical cultures to nonlethal oxygen-glucose deprivation protects against a subsequent exposure to otherwise lethal oxygen-glucose deprivation. We provide evidence that attenuation of the postsynaptic N-methyl-D-aspartate (NMDA) receptor- and Ca(2+)-dependent neurotoxicity underlies oxygen-glucose deprivation tolerance. It is demonstrated that extended tolerance to otherwise lethal NMDA or oxygen-glucose deprivation can be induced by either of their sublethal forms of preconditioning. These four pathways are linked, since NMDA receptor blockade during preconditioning by oxygen-glucose deprivation eliminates tolerance. These results suggest that NMDA tolerance, induced by nonlethal activation of these receptors during oxygen-glucose deprivation preconditioning, underlies oxygen-glucose deprivation tolerance. Several neurotoxic downstream Ca(2+)-dependent signaling events specifically linked to NMDA receptor activation are attenuated during otherwise lethal oxygen-glucose deprivation in preconditioned cultures. Specifically, calpain activation, as well as degradation of microtubule-associated protein-2 and postsynaptic density-95, are attenuated 2 h following otherwise lethal NMDA treatment alone or oxygen-glucose deprivation in preconditioned cultures. Formation of microtubule-associated protein-2-labeled dendritic varicosities is also attenuated in preconditioned cultures within 1 h of lethal oxygen-glucose deprivation or NMDA application. Intracellular Ca(2+) levels, measured using the high- or low-affinity dyes Fluo-4 (K(d) approximately equal 345 nM) or Fluo-4FF (K(d) approximately equal 9.7 microM) respectively, are markedly attenuated during lethal oxygen-glucose deprivation in preconditioned cultures.Collectively, the results suggest the attenuation of the postsynaptic NMDA-mediated component of otherwise lethal oxygen-glucose deprivation through the suppression of Ca(2+)-dependent neurotoxic signaling, a mechanism that is initially induced by transient nonlethal activation of this receptor during ischemic preconditioning.

Increased expression of the transcription factor E2F1 during dopamine-evoked, caspase-3-mediated apoptosis in rat cortical neurons.

The transcription factor E2F1 mRNA and protein levels increased in rat cortical neurons in response to dopamine (DA)- or 6-hydroxydopamine (OHDA)-evoked apoptosis. Increased E2F1 protein was detected in the nucleus of neurons by double fluorescent immunocytochemistry using antibodies to E2F1 and NeuN. DA and 6-OHDA induced caspase-3-mediated apoptosis of cortical neurons which was attenuated by the addition of antioxidants or caspase-3 inhibitors to the cultures. Antioxidants prevented DA-evoked neuronal apoptosis, and also attenuated the increase in E2F1 expression. These findings suggest that increased expression of the transcription factor E2F1 may serve as a death signal during DA-evoked neuronal apoptosis.

Structural modifications to an N-methyl-D-aspartate receptor antagonist result in large differences in trapping block.

Differences in the degree of trapping of initial block by N-methyl-D-aspartate (NMDA) receptor antagonists may affect their safety and, hence, suitability for clinical trials. In this comparative study, 23 compounds structurally related to the low-affinity, use-dependent NMDA receptor antagonist (S)-alpha-phenyl-2-pyridineethanamine dihydrochloride (AR-R15896AR) were examined to determine the degree of trapping block they exhibit. Compounds were tested at concentrations that produced a comparable initial 80% block of NMDA-mediated whole-cell current in rat cortical cultures. A wide range of values of trapping block was found, indicating that trapping is not an all-or-none event. Fifteen of the compounds trapped significantly more than the 54 +/- 3% of initial block trapped by AR-R15896AR. The off-rates of these compounds were slower than that of AR-R15896AR. Only 2 of the 23 compounds trapped significantly less than AR-R15896AR. AR-R15808, the piperidine analog of AR-R15896AR, appeared to trap only 8 +/- 3% of its initial block, although its fast off-rate confounded accurate quantification of trapping. AR-R26952, which, like AR-R15896AR, contains a pyridine in place of a phenyl group, trapped 40 +/- 5% of its initial block and exhibited kinetics comparable with AR-R15896AR. Structure-activity analysis suggested that the presence of two basic nitrogen atoms and decreased hydrophobicity led to decreased trapping. There was no correlation between trapping and lipophilicity as would be expected if closed-channel egress was due to escape through the lipid bilayer. However, there was a positive correlation between off-rate and degree of trapping. Models that can account for partial trapping are presented.

Evaluation of glutathione-sensitive fluorescent dyes in cortical culture.

The sensitivity of six fluorophores to glutathione (GSH) was evaluated in living rat cortical neuronal/glial mixed cultures during the first 23 days in vitro (DIV). Four of the dyes require glutathione-S-transferase (GST) to form a fluorescent conjugate, potentially conferring specificity for GSH: these included t-butoxycarbonyl-Leu-Met-7-amino-4-chloromethylcoumarin (CMAC), 7-amino-4-chloromethylcoumarin (CMAC-blue), monochlorobimane (MCB), and 5-chloromethylfluorescein diacetate (CMFDA). The final two dyes examined, 2,3-naphthalenedicarboxaldehyde (NDA) and o-phthaldehyde (OPD), do not require GST for adduct formation with GSH. To examine the specificity of the dyes for GSH, cultures grown less than 6 DIV were pretreated with diethyl maleate or DL-buthionine-(S, R)-sulfoximine to deplete endogenous GSH. This resulted in a substantial loss of staining by CMAC, CMAC-blue, and MCB and partial loss of staining by OPD, indicating specificity for GSH, while staining by CMFDA or NDA was not altered, indicating a lack of specificity for GSH. Neurons experienced a dramatic decline in GSH levels relative to astrocytes between 5-6 DIV, as shown by a loss of neuronal staining with CMAC, CMAC-blue and MCB. This decrease in staining was not due to a decrease in GST activity, as neurons stained with the GST-insensitive OPD also exhibited a decline in GSH-sensitive staining. Immunolabeling experiments demonstrated that CMAC staining co-localized with GFAP-positive astrocytes, but not with MAP-2-positive neurons, in 18 DIV cultures. Finally, CMAC was exploited as a specific morphological marker of astrocytes in cultures aged >5 DIV. CMAC staining was employed to monitor astrocyte proliferation and to resolve astrocytes in living mixed cultures co-loaded with the Ca(2+)-sensitive dye, calcium green 5N-AM. GLIA 30:329-341, 2000. Published 2000 Wiley-Liss, Inc.

Glycolipid markers of astrocytomas and oligodendrogliomas.

Neutral glycolipids and gangliosides were analyzed in 149 astrocytomas (A), 46 oligodendrogliomas (O), and 21 oligoastrocytomas (OA) to determine if specific glycolipids correlate with histologic diagnosis and grade. Positivity for asialoGM1 (GA1) and negativity for paragloboside by immuno-TLC correlated with histological diagnosis of O and OA, whereas the reverse pattern correlated with A. High levels (over 5 microg hexose per mg dry weight) of CMH generally correlated with an O component, but the association was not as strong as for either GA1 presence or paragloboside absence. Pilocytic astrocytomas and pleomorphic xanthoastrocytomas had high proportions (> 15%) of globoside, low ratios (< 0.5) of GD1a: GD1b, and identifiable ceramide trihexoside (CTH). Three gangliosides of the 1b pathway were progressively lost with increasing grade of A, but a similar correlation with grade was not seen in O or OA. A high proportion of cases expressing sialosylparagloboside (3'LM1; 6'LM1) were grade 4 A. Glycolipids are synthesized by glycosyltransferases that add specific sugars to the nascent oligosaccharide. Correlation of specific glycolipids with histological diagnoses and grades indicate that these tumor types express specific patterns of glycosyltransferases, several of which have been cloned. It is possible that critical genes coding for these enzymes are deleted, overexpressed, or mutated in certain tumor types and grades, thus leading to the patterns of glycolipids that we found to be associated with these tumors.

Gangliosides and neutral glycolipids in ependymal, neuronal and primitive neuroectodermal tumors.

Neutral glycolipid and ganglioside compositions were determined on 11 ependymal tumors, 12 medulloblastomas, 6 other neuronal tumors of the brain, 4 peripheral neuroblastomas, 1 cerebral primitive neuroectodermal tumor (PNET), and 1 PNET of the thoracic wall. Within the group of tumors that can demonstrate neuronal phenotypes, there was an association between the degree of neuronal differentiation usually demonstrated by these tumors and the proportions of both GD1a and 1b-pathway gangliosides. The amount of globoside also correlated with the amount of 1b pathway gangliosides. Patients with medulloblastomas whose 1b gangliosides made up over 15% of the total gangliosides survived longer that those with lower proportions of 1b gangliosides. The only gangliosides in the choroid plexus papilloma were GM3 and GD1a, but other ependymal tumors had significant amounts of GD1b and its metabolic precursors. Ependymoma and anaplastic ependymoma had similar neutral glycolipid compositions, which were different from subependymoma, which lacked ceramide monohexoside and ceramide dihexoside. These differences in glycolipid compositions suggest that there may be fundamental biological differences between these types of ependymal tumors.

Loss of cyclin D1 in necrotic and apoptotic models of cortical neuronal degeneration.

Recent evidence suggests that apoptosis in post-mitotic neurons involves an aborted attempt of cells to re-enter the cell cycle and it is characterized by increased expression of cyclins, such as cyclin D1, prior to death. Cyclin D1 increases to permit transition from growth phase (G0/G1) to synthesis phase (S) during normal development but there is controversy as to which of the cyclins are activated prior to apoptotic cell death. We looked at the expression of cyclin D1 in cortical neuronal cultures treated with either staurosporine to produce apoptotic death, or with glutamate, to produce a non-apoptotic death. Cyclin D1 immunoreactivity was observed in the cytoplasm and nucleus of virtually all neurons under control conditions. Following the addition of either staurosporine or glutamate, cyclin D1 immunoreactivity did not change within 4 h. The cyclin D1 immunoreactivity was lost by 6 h with the appearance of either staurosporine-induced fragmented nuclei or glutamate-induced pyknotic nuclei. These immunocytochemical observations were confirmed with immunoblot analysis. Therefore, cyclin D1 is not a reliable indicator of apoptosis in cortical neuronal cultures and should not be used as an indicator of apoptotic cell death.

Evidence from cultured rat cortical neurons of differences in the mechanism of ischemic preconditioning of brain and heart.

Ca2+ influx and activation of protein kinase C (PKC) and mitogen-activated protein kinase (MAPK) during nonlethal ischemic preconditioning have been implicated in the protection of the heart against subsequent lethal ischemic injury. Thus, we determined if Ca2+ influx, PKC and MAPK also mediate ischemic preconditioning-induced protection in neurons. Preconditioning by exposure of E18 rat cortical cultures to 90 min of nonlethal oxygen-glucose deprivation (OGD) 24 h prior to 180-240 min of lethal OGD was neuroprotective. Exposure to nominally free Ca2+, or blockade of the alpha-amino-hydroxy-5-methyl-isoxazolepropionate (AMPA) receptor with CNQX did not eliminate protection. MAPK activity did not change and PKC activity decreased by 50% relative to normal baseline levels at 0 and 24 h following preconditioning. The sustained decrease in PKC activity was not due to a loss of enzyme as determined from immunoblots using pan and epsilon-, beta- and zeta-specific PKC antibodies. Neuroprotection was maintained with pharmacological inhibition of PKC activity by staurosporine, chelerythrine and calphostin C and MAPK activity by PD 98059 during preconditioning, indicating that activation of these enzymes during preconditioning was not necessary for protection. Therefore, in contrast to cardiac tissue, ischemic preconditioning of neurons does not require activation of PKC and MAP kinase, and protection is maintained with substantial removal of extracellular Ca2+ or blockade of the AMPA receptor.

Immunohistochemical staining for ganglioside GD1b as a diagnostic and prognostic marker for primary human brain tumors.

Immunohistochemical staining intensity for ganglioside GD1b was determined for 108 human neuroectodermal tumors. Most of the tissue elements that immunostained were tumor cells; only a few axons and occasional neurons reacted in some specimens. All pilocytic astrocytomas stained very positively, whereas none of the ependymomas and only 11% of primitive neuroectodermal tumors, 20% of glioblastomas, and 28% of anaplastic astrocytomas showed more than faint staining. A similar association between grade and immunostaining was seen in tumors containing an oligodendrogliomatous component, but reactivity was not as strong as in astrocytic tumors or primitive neuroectodermal tumors. Results of Cox regression showed significant associations between immunostaining intensity and survival for all cases taken together (P = 0.007); for the group consisting of astrocytomas, oligoastrocytomas, and oligodendrogliomas (P = 0.002); and for astrocytomas alone (P = 0.04). Results were also significant using a proportional hazards model controlling for patient age (all cases P = 0.005; astrocytomas only P = 0.02), but not when controlling for tumor grade. Our results indicate that immunohistochemical staining for GD1b is correlated with tumor grade and that it may be of prognostic utility in some primary human brain tumors, especially astrocytomas.

[VIGILA: a tool for the epidemiologic surveillance of transmissible diseases]. / VIGILA: un instrumento para la vigilancia epidemiológica de enfermedades transmisibles.

It is described an instrument for the epidemiological surveillance of communicable diseases (VIGILA) that is based on the generalization of a modification of the statistical model proposed by Serfling for the prognosis of the endemic levels of the rates of communicable diseases in a period of 52 weeks (1 year). The comparison made between the observed rates and the prognosticated rates allows to detect unusual patterns of this indicator. The observation of rates over the higher limit of the prognosis interval during successive periods is suggested as a signal of alert. The introduction of this method in a computer program allows its application in the epidemiological surveillance units at the different levels of the health system.

A computer system for epidemiological surveillance.

In epidemiological surveillance, it is imperative that any unusual increase in reported cases be detected as quickly as possible. In the surveillance unit at "Pedro Kourí," it was necessary to create a computer system for the surveillance of transmissible diseases in order to manage such variables as morbidity, mortality, and the circulation of causal agents. As usage flexibility is a fundamental requirement, we developed VIGILA as a system readily adaptable to any level of the country's health organizations (national, provincial, or municipal). VIGILA permits the storage, validation, and statistical analysis of morbidity and mortality data, and it also allows the user to apply these features to causal agent information. These manipulations are all performed in the context of a specific transmissible disease. The stored information can be displayed in different types of statistical formats, such as calculated rates, accumulates and medians. The user can also design his/her own tables for display. In addition, the program offers graphics that depict the endemic forecast for a specific region and age group. Reliable forecasts based on temporal models of an epidemiological indicator are necessary for the prediction of the non-epidemic indicator and for the elaboration of an alert threshold. The endemic forecast or endemic channel is calculated by means of a modified version of R. Serfling's model [1] which adapts to data series with stationary characteristics. In this case, the model is fixed for the medical attention protocol and the circulation of agents of a specific transmissible disease. The parameters of the model are estimated by the least square method. The confidence limits are calculated with the T-Student distribution [2]. To enter information about a specific disease and level of health concern, the user must define the disease under analysis, the principal territory under surveillance and its dependencies, the causal agents of the disease, and the frequency with which the disease is reported. This system stores the number of cases (morbidity) and death (mortality) that occurred in the territories and age groups defined during entry. To facilitate the calculation of morbidity and mortality rates, information pertaining to the number of territory habitants and their age groups is also stored. The laboratory data includes the number of analyzed samples in the territories and age groups defined in the data entry, and the samples that were positive to the defined agents. Among the principal uses of the system are: calculation of cumulative cases, rates, and cumulative rates for the specific period of analysis, territory, and age group; comparison of these health indicators with the previous year's indicators or with the median of the previous years' indicators' calculation of the endemic forecast that permits the user to observe the epidemiological situation by territories and age groups; and the early detection of an increase in disease occurrence. The system also offers a geographical representation of the epidemiological situation in the territories, showing a map with the evaluation of each territory with respect to four possible risk factors. VIGILA is available in both DOS and WINDOWS 3.1 formats. This system allows the evaluation of the situation at a specific point in time in the analyzed territories, and may alert the infection control team to an epidemic early enough to allow implementation of control measures.