Nitric oxide-dependent damage to neuronal mitochondria involves the NMDA receptor

Cytokine-stimulated astrocytes produce nitric oxide, which can inhibit components of the mitochondrial respiratory chain. We have previously demonstrated that prolonged exposure (48 h) to rat astrocytic nitric oxide damages complexes II--III and IV of neighbouring rat neurons in coculture, resulting in neuronal death. Expanding on these observations, we have now shown that the NMDA receptor antagonist, MK-801, prevents this damage, suggesting involvement of glutamate. We postulate that astrocyte-derived nitric oxide stimulates release of neuronal glutamate. Indeed we demonstrate that neurons incubated with nitric oxide-generating astrocytes display enhanced glutamate release. Furthermore, direct exposure to the nitric oxide donor, DETA-NONOate resulted in a loss of activity of all the neuronal mitochondrial complexes, which was again prevented by MK-801. Thus, nitric oxide, generated by both cytokine-stimulated astrocytes and by a nitric oxide donor, causes activation of the NMDA receptor leading to damage to the neuronal mitochondrial respiratory chain. Glutamate exposure is known to damage the neuronal mitochondrial respiratory chain via neuronal nitric oxide synthase. Therefore, we propose that astrocyte-derived nitric oxide is capable of eliciting neuronal glutamate release, which in turn activates the neuronal NMDA receptor and stimulates further formation of reactive nitrogen species via neuronal nitric oxide synthases, leading to mitochondrial damage and neuronal death. Our findings support the hypothesis that glutamate, reactive nitrogen species and mitochondrial dysfunction may have a role in the neurodegenerative process.     

Clinical Assisted Reproduction: An Analysis of Spontaneous Hatching in a Human Endometrial Epithelial Coculture System: Is Assisted Hatching Justified?

Purpose: To evaluate spontaneous embryo hatching in an endometrial epithelial coculture system, and compare it with cases where coculture was performed because of maternal age, previous repeated implantation failures, or both. To clarify in which cases assisted hatching would be appropriate.Methods: Individual human embryos were cocultured on an endometrial epithelial cell monolayer until Day 6.Results: Blastocyst hatching rate at Day 6, depending on maternal age, was 9.1% (age <37 years) and 3.4% (age 37 years). However, blastocyst hatching rates depending on number of previous IVF failures were similar.Conclusions: Maternal age and previous implantation failures are factors affecting the ability of human embryos to reach the blastocyst stage in coculture. However, assisted hatching is not justified in these populations because of the absence of hatching rate differences between blastocysts obtained from these two groups and the control group.     

Effect of coculture on subsequent survival and implantation of cryopreserved human embryos

Purpose This retrospective analysis was designed to assess the performance of human embryos following cryopreservation based on whether they were originally developed in standard culture medium (65 cycles, 223 embryos) or cocultured on partial monolayers of bovine oviductal epithelial cells (63 cycles, 198 embryos). Embryo cryosurvival and implantation were compared between the study group and the contemporaneously matched controls.Results During a 2-year period when no factors of the cryopreservation program were altered, 63 transfers of 159 surviving thawed control cleavage-stage embryos (71.3% survival) that were 54% intact gave rise to 11 viable pregnancies (17.5%/ET), to yield an implantation rate of 6.9% per embryo. Sixty-three transfers of 147 thawed cocultured embryos (74.2% survival) that were 61% intact gave rise to 17 viable pregnancies (27%/ET), which gave an implantation rate of 13.6% per embryo that was significantly higher than the control group (P< 0.05).Conclusion Coculture of embryos prior to cryopreservation does not appear to improve cryosurvival; however, it does improve implantation postthaw compared with embryos following standard culture prior to cryopreservation.Presented at the IXth World Congress on In Vitro Fertilization and Alternate Assisted Reproduction, Vienna, Austria, April 3–7, 1995.     

Astrocytes protect neurons from ethanol-induced oxidative stress and apoptotic death

Ethanol induces oxidative stress in cultured fetal rat cortical neurons and this is followed by apoptotic death, which can be prevented by normalization of cell content of reduced glutathione (GSH). Because astrocytes can play a central role in maintenance of neuron GSH homeostasis, the following experiments utilized cocultures of neonatal rat cortical astrocytes and fetal cortical neurons to determine if astrocytes could protect neurons from ethanol-mediated apoptotic death via this mechanism. In cortical neurons cultured in the absence of astrocytes, ethanol (2.5 and 4 mg/ml; 6-, 12-, and 24-hr exposures) decreased trypan blue exclusion and the MTT viability measures by up to 45% (P < 0.05), increased levels of reactive oxygen species (ROS) by up to 81% (P < 0.05), and decreased GSH within 1 hr of treatment by 49 and 51% for 2.5 and 4 mg/ml, respectively (P < 0.05). This was followed by onset of apoptotic cell death as determined by increased Annexin V binding and DNA fragmentation by 12 hr of ethanol exposure. Coculturing neurons with astrocytes prevented GSH depletion by 2.5 mg/ml ethanol, whereas GSH content was increased over controls in neurons exposed to 4 mg/ml ethanol (by up to 341%; P < 0.05). Ethanol generated increases in neuron ROS and apoptosis; decreases in viability were also prevented by coculture. Astrocytes were largely insensitive to ethanol, using the same measures. Only exposure to 4.0 mg/ml ethanol decreased GSH content in astrocytes, concomitant with a 204% increase in GSH efflux (P < 0.05). These studies illustrate that astrocytes can protect neurons from ethanol-mediated apoptotic death and that this may be related to maintenance of neuron GSH.     

CAL72: a human osteosarcoma cell line with unique effects on hematopoietic cells

Permanent osteoblastic cell lines are potential tools to study the interactions between osteoblastic and hematopoietic cells in the bone marrow cavity. In a recent work we have shown that the osteosarcoma cell line CAL72 may be more closely related to normal osteoblasts than the osteosarcoma cells previously described. In the present work we continued the characterisation of the CAL72 cell line with regard to its effects on various hematopoietic cells, in coculture experiments. We show here that CAL72 cells, in contrast to MG-63 or SaOS-2 osteosarcoma cell lines, do not inhibit hematopoietic colony formation and sustain the limited expansion of hematopoietic progenitors in a similar way to that described for normal osteoblasts. We also demonstrate that CAL72 cells induce the monocytic differentiation of the promyelocytic HL-60 cell line like MG-63 and SaOS-2, but support a better maturation and a longer survival of the differentiated cells than the two other osteosarcoma cell lines. In order to better understand the differential effects observed between CAL72 and MG-63 or SaOS-2, we analysed the cytokine and chemokine mRNA expression of these cells using the RNase protection quantitative assay. We show here that the expression profile of CAL72 is clearly different from that of MG-63 or SaOS-2 and may explain, at least in part, its specific effects on hematopoietic cells. Taken together these experiments confirm that CAL72 has particular properties and is an interesting tool to study the role of osteoblastic cells in hematopoietic cell growth and differentiation.     

Bone marrow stromal cells enhance differentiation of cocultured neurosphere cells and promote regeneration of injured spinal cord

Transplantation of bone marrow stromal cells (MSCs) has been regarded as a potential approach for promoting nerve regeneration. In the present study, we investigated the influence of MSCs on spinal cord neurosphere cells in vitro and on the regeneration of injured spinal cord in vivo by grafting. MSCs from adult rats were cocultured with fetal spinal cord-derived neurosphere cells by either cell mixing or making monolayered-feeder cultures. In the mixed cell cultures, neuroshpere cells were stimulated to develop extensive processes. In the monolayered-feeder cultures, numerous processes from neurosphere cells appeared to be attracted to MSCs. In an in vivo experiment, grafted MSCs promoted the regeneration of injured spinal cord by enhancing tissue repair of the lesion, leaving apparently smaller cavities than in controls. Although the number of grafted MSCs gradually decreased, some treated animals showed remarkable functional recovery. These results suggest that MSCs might have profound effects on the differentiation of neurosphere cells and be able to promote regeneration of the spinal cord by means of grafting.     

Optimizing tubal epithelial cell growth promotes mouse embryo hatching in coculture

Purpose: This study investigates the relationship between human tubal epithelial cell growth characteristics and mouse embryonic development to determine which cellular requirements should be preferentially provided in a coculture system. Methods: Cell growth and viability were assessed for 5 days in -minimal essential medium or human tubal fluid supplemented with 10% human serum or 10% synthetic serum. Two-cell mouse embryo development to blastocyst and hatching blastocyst stages was also assessed with or without coculture. Results: Both epithelial cell growth and embryo development were dependent on serum supplementation with better cell viability and growth rates in human serum and better blastocyst development in synthetic serum. The highest proportion of hatching blastocysts was found in -minimal essential medium and human serum with coculture. Conclusions: Culture conditions which improve tubal epithelial cell growth also improve the hatching rate of mouse embryos in coculture. This indicates that by meeting the metabolic and nutritional demands for epithelial cell growth, the beneficial effects of coculture on embryo development may be optimized.     

Immunolocalization of GLUT1 and GLUT3 glucose transporters in primary cultured neurons and glia

Immunofluorescence analysis was used to study the cellular localization of glucose transporters 1 and 3 (GLUT1 and GLUT3) in primary rat neuronal and glial cultures. In primary cultured cerebellar granule neurons and cortical neurons, GLUT3 was detected in a pattern consistent with a generalized cell surface distribution. GLUT3 distribution corresponded most closely with the neural cell adhesion molecule (NCAM), and showed overlapping but distinct distributions compared to synaptophysin, microtubule-associated protein 2 (MAP2), neurofilament protein, and growth-associated protein (GAP43). Culture of neurons in the presence of glia did not alter the cellular localization of GLUT3. GLUT1 was detectable in primary cerebellar granule neurons both at the cell surface and in the cytoplasm, and appeared decreased in neurons cocultured with glia. GLUT1, but not GLUT3, was detected in glial fibrillary acidic protein (GFAP)-positive astrocytes present in mixed neuronal-glial cultures derived from cerebellum and cerebral cortex, as well as in cortical astrocyte cultures. GLUT1, but not GLUT3, was also detected in microglia and oligodendrocytes present in these cultures. This study indicates a generalized cell surface expression of the glucose transporters expressed in neurons and glia, rather than selective targeting to different cellular domains or subcellular locations. © 1995 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

Successful freezing of unfertilized mouse oocytes and effect of cocultures in oviducts on development of in vitro fertilized embryos after thawing

Purpose To establish a freeze-thawing method for unfertilized oocytes with a high success rate, we examined several conditions for freeze-thawing. The effects of EDTA and cocultures in oviducts on the development of embryos fertilized in vitro after thawing were also studied. Results In the first experiment, unfertilized oocytes that were frozen in 1.5 Mdimethylsulfoxide (DMSO) supplemented with 0.2 Msucrose by a slow freeze-thawing method showed the best results (fertilization rate, 71.9%; blastocyst rate per frozen oocyte, 18.8%). The proportion of embryos that developed to blastocysts was significantly higher when DMSO was added at 4‡C than at room temperature (39.4 vs 19.4%; P<0.01). The addition of EDTA (10 ΜM)to the culture medium did not promote embryo development after fertilization in vitro. However, the rate of development of in vitro fertilized embryos to blastocysts after thawing was significantly higher when the embryos were cultured in oviducts in vitro than the rates in control cultures and those cultured with EDTA (blastocyst rate from fertilized oocytes, 71.4 vs 51.0 and 52.8%, respectively; P<0.01). Conclusion Unfertilized mouse oocytes can be cryopreserved successfully by a slow freeze-thawing method with the addition of 1.5 MDMSO and 0.2 Msucrose at low temperatures, and coculture with oviducts enhances the development of embryos that are fertilized in vitro after thawing.