BACKGROUND
acute ischemic stroke is characterised by the immediate depletion of oxygen and glucose in brain tissue.
a residual cerebral blood flow  of ≤  <dig> cm <dig> ×  <dig> g- <dig> × min- <dig> representing severe ischemia is associated with a nearly total loss of energy on vulnerable neurons.
ischemia therefore rapidly culminates in the formation of a necrotic core  <cit> .
in the penumbra, mild ischemia  leads to the activation of complex neurochemical cascades of cell death, mainly apoptosis.
in principle these apoptotic cascades are reversible and form an important aspect of the penumbra concept, which is the major target of therapeutic interventions  <cit> .
recent findings indicate that transplantation of external cell fractions could accompany established therapeutic procedures limited by narrow time windows  <cit> , but the underlying processes are still rather unclear.
our insights into pathophysiological processes and new therapeutic strategies have mostly been obtained from animal models of focal cerebral ischemia  <cit>  and rodent organotypic hippocampal slice cultures  <cit> .
however, the complexity of those systems has limited the detailed understanding of mechanisms related to ischemic brain injury  <cit>  and possible interfering effects of cellular therapies  <cit>  so far.
furthermore, results obtained from rodent models are not completely and unobjectionably transferable to human therapy  <cit> .
consequently, experimental expenditure and ethical considerations demand in vitro models representing the main properties of stroke-related processes as neuronal apoptosis to accompany more complex model systems.
this would allow to answer explicit questions concerning the role of cell-cell interactions and production of metabolites to verify observations made in in vivo models.
it furthermore offers the possibility to precisely manipulate extra cellular environments.
well described human neuronal cell lines exhibit a multitude of characteristics of typical central-nervous-system  neurons, overall cell material can be achieved in large quantities.
therefore, human neuronal cell lines, such as the teratocarcinoma nt- <dig> cell line, became useful tools to study the effects of hypoxic conditions on neurons  <cit> .
however, the utilisation of nt- <dig> neuronal cultures is restricted by time-consuming and expensive differentiation periods of up to 44– <dig> days  <cit>  that are also sensitive to environmental disturbances.
in contrast, the sh-sy5y neuroblastoma cell line was shown to be differentiated into neuronal cells within a comparatively short time of  <dig> days  <cit> .
furthermore, the cell line fits major relevant criteria  to serve as a model of hypoxic injury of central neurons  <cit> .
hence, our exclusive human model of neuronal hypoxia forms the basis to identify possible anti-apoptotic neuroprotective potentials of therapeutic supplements.
mononuclear cells  from human umbilical cord blood  were shown to improve functional outcome of animals after focal cerebral ischemia.
the cellular effects causing the observed benefits are not fully understood for these cells  <cit> .
in this context we investigated whether injured post-hypoxic neuronal cells or mnc initiated an anti-apoptotic response mediated by cytokines or chemokines.
RESULTS
after differentiation sh-sy5y exhibited neuronal morphology and specific neuronal markers
sixteen days of differentiation yielded cultures of fully matured neuronal cells shown in fig.
1a, b. following the seventh day of differentiation, the majority of sh-sy5y cells were stained positive for the specific neuronal markers .
at day  <dig> all cells exhibited all of these markers.
time course of marker expression showing continuously increasing stages of differentiation is given in fig.
1c.
markers showed typical localisation to cytoplasm and dendrites.
the explicit majority of differentiated sh-sy5y cells  resembled typical neuronal morphology with round phase-bright somata and long, terminal- branched dendrites forming a dense network .
however, the neuronal culture is also characterised by a cell type  which shows abundant cytoplasm and a lack of axonal extensions .
remarkably, no differences were noted with regard to expression of neuronal markers.
furthermore, the number of this type of cells remained constant during the culture time.
absence of proliferation after termination of differentiation
at day  <dig> of differentiation the total number of sh-sy5y cells, counted by nuclear staining with dapi, increased by twofold  since seeding.
beginning on the seventh day of culture numbers of nuclei remained nearly stable .
subsequent to hypoxia, there was no significant alteration in numbers of counted nuclei.
hence, the influence of oxygen deprivation induced no further proliferation and also no significant loss of cells in comparison to normoxic control cultures .
therefore all following results should be seen in the context of the nearly constant numbers of neuronal cells under normoxic and hypoxic cultivation conditions.
it is of note that the amount of stained nuclei as the equivalent of adherent cells gives no information about the physiologic status of these cells.
on the whole the cultures include cells in a viable, apoptotic or necrotic state.
hypoxia induced apoptosis in the majority of neuronal cells
post-hypoxic and normoxic control cultures exhibited pronounced differences in the quantity of apoptotic cells as well as in cell morphology.
hypoxic conditions for  <dig> hours induced an initial apoptosis rate of 26% ± 13%.
there was a continuous increase in the apoptotic cell fraction to 85% ± 11% within  <dig> days post-hypoxia as compared to control cultures .
the clearest effects of oxygen deficiency were seen three days following induction of hypoxia as shown by annexin-v staining .
additionally, post-hypoxic cultures were characterised by retracted dendrites, indicating a loss of multiple cell-cell contacts.
debris and apoptotic bodies were found in most culture dish areas, evidencing late stage of apoptosis .
in contrast, normoxic cultures displayed a much more reduced amount of apoptotic cells .
over the whole time, apoptosis in these control cultures remained stable at about 7% ± 3% .
after hypoxia, on day  <dig> and day  <dig>  number of late apoptotic/necrotic cells significantly increased up to 27% ± 13%.
further progression of propidium iodide  positive cells in post-hypoxic cultures resulted in  <dig> ± 16% on day  <dig> .
by comparison, in normoxic control cultures, necrosis levels remained stable below 17% and therefore did not statistically differ from post-hypoxic cultures on this time point.
this fact was corroborated by images that show no observable deviation in the amount of necrotic and late apoptotic cells, as indicated by pi staining .
in general, an increased release of calcein-am also indicated a strong decrease of neuronal cell viability which verifies the results of the apoptosis and necrosis rates .
hypoxia increased quantities of caspase- <dig> and cleaved parp immediately and secretion of vegf in delay
bar charts in figure  <dig> show the concentrations of caspase- <dig> and cleaved poly polymerase .
in post-hypoxic cultures apoptotic proteins drastically rose after oxygen deprivation.
the highest concentration of caspase- <dig> was measured directly after hypoxia on days  <dig> and  <dig> .
from day  <dig> on, the level of active caspase- <dig> sharply decreased.
the distribution of cleaved parp levels showed patterns similar to caspase- <dig>  this decline was accompanied by a secretion of vegf on day  <dig> after hypoxia .
adhesion molecules  were upregulated after hypoxia
independent of cultivation conditions immunofluorescence analysis revealed that nearly all  of the cells were positive for neurite cell adhesion molecule  and neural cell adhesion molecule  at day  <dig>  l <dig> and ncam were detected on somata and multiple dendrites.
furthermore, a few of the neuronal cells also expressed vascular cell adhesion molecule  and intercellular adhesion molecule  on their somata and dendrites.
there was no change in relative numbers of marked cells and expression patterns in post-hypoxic cultures.
to quantify the density of adhesion molecule expression, highly sensitive fluorescence measurement of supernatants of lysed cultures was performed.
here, comparative studies between cultures directly after  <dig> hours of hypoxia and normoxic control cultures showed a significant upregulation of l <dig>  ncam and icam- <dig> .
hypoxic conditions determined a considerable increase in l <dig> and ncam protein expressions of up to 189% ± 74% and 155% ± 50%, respectively.
hypoxia also strongly upregulated the icam- <dig> expression up to 424% ± 251%.
the level of expression of vcam- <dig> was not altered by the hypoxic environment.
the continued degradation of the dendritic networks did not allow assaying the specific expression of adhesion molecules at late stages of post-hypoxic cultures.
co-culturing with hucb mnc strongly reduced apoptosis in post-hypoxic neuronal cell cultures
we found noticeable neuroprotective properties of hucb mnc in the co-culture experiments.
untreated post-hypoxic mono-cultures of neuronal cells showed 85% ± 11% of apoptosis after three days, whereas in co-cultures with mnc the rate of apoptosis was stable at a level of  <dig> % ± 1% .
this is comparable to normoxic control cultures .
photographs of co-cultures with mnc revealed clear effort of mnc to localise next to somata and branches of post-hypoxic neuronal cells.
in the course of clustering mnc mostly avoided areas that were not settled by neuronal cells .
furthermore, the administration of mnc showed positive influence on the conservation of neuronal networks as compared to cultures that did not receive any cell therapy .
mnc increased ccl <dig>  ccl <dig>  ccl <dig> and cxcl <dig> in post-hypoxic neuronal cultures
the investigation of neuroprotective cytokines  , inflammatory cytokines   and chemokines   in supernatants of normoxic neuronal cultures revealed considerable amounts of cxcl <dig>  ccl <dig> and vegf.
remarkably, after three days under hypoxic conditions only vegf was strongly upregulated .
after administration of mnc, vegf was downregulated regarding to the total concentration measured in mono-cultures of mnc and in post-hypoxic neuronal cells.
inflammatory cytokines as il-1β, il- <dig> and cxcl <dig> were produced by mnc.
the secretion of cytokines such as il-1β and il- <dig> was not altered in post-hypoxic co-cultures, whereas cxcl <dig> was suppressed.
interestingly, the majority of chemokines was clearly upregulated in co-cultures with mnc.
we could show that ccl <dig>  ccl <dig>  ccl <dig> and cxcl <dig> were increased up to the tenfold , whereas the concentration ccl <dig> was not regulated.
discussion
in this study, we introduce an experimental human in vitro model to investigate  the mechanisms of neuronal hypoxia and  the interaction of neuronal cells with external stem cell-containing fractions as possible therapeutic tools.
according to the differentiation protocol of encinas et al.
<cit>  we obtained fully matured neuronal cells after  <dig> days.
previous, orientating experiments showed typical response of differentiated sh-sy5y cells to n-methyl-d-aspartate  application and most pronounced vulnerability after  <dig> hours hypoxic incubation .
these post-hypoxic neuronal cultures can be employed already  <dig> days after the seeding of naive cells.
therefore the time period until the model of hypoxic neuronal cells is available is very short.
in comparison to other approaches that provide assimilable hypoxic models using teratocarcinoma cell lines  <cit>  the generation of our model is shortened by at least  <dig> days  <cit> .
the proved expression of specific neuronal markers and the absence of any proliferation gave clear evidence of matured neuronal cells in the g0-phase of the cell cycle.
this steady state allows making direct conclusions about the influence of any external manipulation because any change will most probably be due to these procedures.
hence, the model of neuronal hypoxia affords the possibility to investigate manifold questions concerning mechanisms triggered in response to hypoxia and therapeutic interventions e.g.
application of hucb mnc.
following hypoxia, neuronal cultures displayed a correlation between morphological changes and increase of annexin-v positive cells as well as changes in adhesion molecule expression.
the specific apoptosis marker annexin-v indicated that hypoxic cultivation preferentially induced apoptosis.
moreover, there was a continuous enlargement in number of apoptotic cells from initial 26% ± 13% until cultures almost completely consisted of apoptotic cells  within three days.
conforming to changes of morphology in post-hypoxic cultures, there was an enhanced release of calcein-am also observable due to the loss of the integrity of membrane.
this is typical for late stages of apoptosis as well as of necrosis.
the model of neuronal hypoxia was approved by moderate rates of pi positive cells .
therefore the overrun of 100% by the summation of annexin-v positive and pi positive cells is due to a proportion of cells in a late state of apoptosis that are positive for annexin-v and pi  <cit> .
we focused on apoptosis because it is a reversible process which could be modulate by external stimulation  <cit> .
moreover after stroke cell death in the penumbra is predominantly considered to be apoptosis  <cit> .
therefore apoptosis is a therapeutic target of anti-apoptotic therapies like stem cells or cytokines  <cit> .
apoptosis in the penumbra takes place in a time span of about  <dig> hours after vessel occlusion in the rat after middle cerebral artery occlusion   <cit> .
this time course is represented by the model system introduced in this study.
the fact that there was no major alteration in the number in dapi-positive nuclei underlines that the influence of hypoxia did not lead to an immediate destruction of the cells but allows investigating apoptosis and subsequent therapeutic interventions within  <dig> hours.
these facts were also confirmed by characteristic changes in the morphology of post-hypoxic cells.
there was a total retraction of dendrites and a resulting destruction of neuronal networks, grained cell surfaces and extended cell degradation.
this damage was accompanied by an upregulation of neuronal adhesion molecules.
we hypothesize that this upregulation indicates a cellular answer to compensate for a loss of direct cell-cell interaction as a consequence of the preceding hypoxic stress as described by several authors  <cit> .
however, the final loss of intercellular networks could not be compensated by the increased expression of neuronal adhesion molecules alone.
neuronal cell death after hypoxia is caused by membrane depolarisation subsequent to energy failure  <cit> .
the resulting calcium overload is the initial point of the activation of manifold biochemical pathways that affect caspases, free radicals and gene expression  <cit> .
the specific apoptotic proteins caspase- <dig> and cleaved parp were quantified to prove whether our microscopic observations are corroborated by biochemical pathways  <cit> .
after  <dig> hours post-hypoxia, both marker proteins extensively increased.
this was followed by a sharp drop in their expression level.
this reduced amount of cleaved parp might be due to an aggravating energy failure in the cell.
this in turn is attributed to the high levels of cleaved parp, evidenced above, which caused adenosintriphosphate  depletion  <cit>  and which in this context prevents the enzymatic activity of caspases.
furthermore, an increase of the expression of vegf, a key mediator of angiogenesis  <cit> , was observed in higher concentrations in late post-hypoxic cultures.
this might be a reflection of compensatory in vivo processes of revascularisation in our model  <cit> .
vegf is also known to be involved in neuronal protection through inhibition of caspase- <dig>  <cit> .
the time-delayed increased release of vegf as an effect of hypoxia does not seem to influence the number of apoptotic cells in our cultures.
this may be a result of concentrations of vegf that did not reach efficient thresholds.
in this context the measured reduction of caspases on days  <dig> and  <dig> after hypoxia is caused by the already described ongoing energy failure in the post-hypoxic cells.
however, the regulation of those apoptotic proteins is probably not related to vegf mediated-effects.
the therapeutic benefit of cell administration after stroke has been demonstrated in numerous animal studies  <cit> .
however, important questions in basic cellular and molecular mechanisms of neuronal cell death and its prevention after inadequate oxygen supply still remain unanswered.
to understand the mechanisms of cellular neuroprotection after stroke we employed the model of neuronal hypoxia and applied mnc from hucb.
in vivo mnc are supposed to differentiate into neuronal cells  <cit>  to trophically support neuronal tissue through the production of growth factors  <cit> , to support the new formation of synapses and migration as well as the differentiation of endogenous neuronal progenitors  <cit> .
in our experiments, the administration of mnc from hucb to post-hypoxic neuronal cultures showed remarkable beneficial effects.
over three days we could show a clear reduction of neuronal apoptosis, even to the level of normoxic control cultures .
there are two observations that contribute to an explanation of neuroprotective mechanisms.
first, mnc were preferentially located close to hypoxically injured neuronal cells.
this phenomenon was facilitated through an intensive upregulation of icam- <dig> on neuronal cells which is the specific ligand for leukocyte cell adhesion molecule  expressed by all immune cell subsets  <cit> .
however, the formation of cell chimera was not observed, as there were no cfse-stained cells with neuronal morphology.
in future experiments we will investigate whether blocking of adhesion molecules on the surface of neuronal cells can inhibit the co-localisation of mnc.
further it will be proved whether this influences the rate of apoptosis.
an increase of apoptosis would be an evidence for the significance of direct cell-cell-contacts in context of therapeutic mechanisms of mnc.
the second observation would be concomitance of an mnc indicated specific alteration in levels of soluble factors.
that change might be held responsible for neuroprotection.
pronounced upregulation of chemokines  might be causal for the enhanced effort of mnc to localise near neuronal structures.
high concentrations of vegf are known to be neuroprotective  <cit> .
in contrast to post-hypoxic mono-cultures, we found no increase of vegf after administration of mnc.
it seems that presence of mnc inhibited the induction of elevated levels of vegf and compensated its neuroprotective effects by other mechanisms.
cord blood contains distinct cell types capable to differentiate into neuronal cells  <cit> .
however, the differentiation of stem cells  was not expected because of the short time of co-cultivation that clearly undershoots time frames responsible for neuronal differentiation.
so far, it is unclear whether neuroprotection resulted from one of the observed effects or from a combination of spatial proximity and specific cytokine patterns.
consequently, further studies in indirect co-cultures will be necessary.
CONCLUSIONS
pathophysiological models developed from animal studies form the basis of our understanding of the development of stroke.
in vivo data display a perfusion-related dependency of neuronal cell damage.
residual energy supply in the penumbra induces apoptosis, the early phases of which are reversible.
consequently, rescue of the penumbra is a major target of experimental stroke therapy.
data obtained from complex animal models of stroke strongly suggest that transplanted cells enhance neuronal survival.
we established a standardized human in vitro model of neuronal apoptotic cell death after hypoxia that can facilitate to address specific pathophysiological processes underlying hypoxic damage and cell-mediated neuroprotection more precisely.
interestingly, transplanted mnc not only strongly decreased the ratio of apoptosis in neuronal cells but also triggered retaining neuronal characteristics such as forming networks.
mnc clustered around post-hypoxic neuronal cells and induced an alteration in cytokine and chemokine concentrations.
our data suggest that the neuroprotective effects of mnc might result from direct cell-cell contacts and/or the adjustment of specific soluble mediators.

 acute ischemic stroke is characterised by the immediate depletion of oxygen and glucose in brain tissue. a residual cerebral blood flow of ≤ cm × g- × min- representing severe ischemia is associated with a nearly total loss of energy on vulnerable neurons. ischemia therefore rapidly culminates in the formation of a necrotic core. in the penumbra, mild ischemia leads to the activation of complex neurochemical cascades of cell death, mainly apoptosis. in principle these apoptotic cascades are reversible and form an important aspect of the penumbra concept, which is the major target of therapeutic interventions. recent findings indicate that transplantation of external cell fractions could accompany established therapeutic procedures limited by narrow time windows, but the underlying processes are still rather unclear. our insights into pathophysiological processes and new therapeutic strategies have mostly been obtained from animal models of focal cerebral ischemia and rodent organotypic hippocampal slice cultures. however, the complexity of those systems has limited the detailed understanding of mechanisms related to ischemic brain injury and possible interfering effects of cellular therapies so far. furthermore, results obtained from rodent models are not completely and unobjectionably transferable to human therapy. consequently, experimental expenditure and ethical considerations demand in vitro models representing the main properties of stroke-related processes as neuronal apoptosis to accompany more complex model systems. this would allow to answer explicit questions concerning the role of cell-cell interactions and production of metabolites to verify observations made in in vivo models. it furthermore offers the possibility to precisely manipulate extra cellular environments. well described human neuronal cell lines exhibit a multitude of characteristics of typical central-nervous-system neurons, overall cell material can be achieved in large quantities. therefore, human neuronal cell lines, such as the teratocarcinoma nt- cell line, became useful tools to study the effects of hypoxic conditions on neurons. however, the utilisation of nt- neuronal cultures is restricted by time-consuming and expensive differentiation periods of up to 44– days that are also sensitive to environmental disturbances. in contrast, the sh-sy5y neuroblastoma cell line was shown to be differentiated into neuronal cells within a comparatively short time of days. furthermore, the cell line fits major relevant criteria to serve as a model of hypoxic injury of central neurons. hence, our exclusive human model of neuronal hypoxia forms the basis to identify possible anti-apoptotic neuroprotective potentials of therapeutic supplements. mononuclear cells from human umbilical cord blood were shown to improve functional outcome of animals after focal cerebral ischemia. the cellular effects causing the observed benefits are not fully understood for these cells. in this context we investigated whether injured post-hypoxic neuronal cells or mnc initiated an anti-apoptotic response mediated by cytokines or chemokines.  after differentiation sh-sy5y exhibited neuronal morphology and specific neuronal markers sixteen days of differentiation yielded cultures of fully matured neuronal cells shown in fig. 1a, b. following the seventh day of differentiation, the majority of sh-sy5y cells were stained positive for the specific neuronal markers. at day all cells exhibited all of these markers. time course of marker expression showing continuously increasing stages of differentiation is given in fig. 1c. markers showed typical localisation to cytoplasm and dendrites. the explicit majority of differentiated sh-sy5y cells resembled typical neuronal morphology with round phase-bright somata and long, terminal- branched dendrites forming a dense network. however, the neuronal culture is also characterised by a cell type which shows abundant cytoplasm and a lack of axonal extensions. remarkably, no differences were noted with regard to expression of neuronal markers. furthermore, the number of this type of cells remained constant during the culture time. absence of proliferation after termination of differentiation at day of differentiation the total number of sh-sy5y cells, counted by nuclear staining with dapi, increased by twofold since seeding. beginning on the seventh day of culture numbers of nuclei remained nearly stable. subsequent to hypoxia, there was no significant alteration in numbers of counted nuclei. hence, the influence of oxygen deprivation induced no further proliferation and also no significant loss of cells in comparison to normoxic control cultures. therefore all following results should be seen in the context of the nearly constant numbers of neuronal cells under normoxic and hypoxic cultivation conditions. it is of note that the amount of stained nuclei as the equivalent of adherent cells gives no information about the physiologic status of these cells. on the whole the cultures include cells in a viable, apoptotic or necrotic state. hypoxia induced apoptosis in the majority of neuronal cells post-hypoxic and normoxic control cultures exhibited pronounced differences in the quantity of apoptotic cells as well as in cell morphology. hypoxic conditions for hours induced an initial apoptosis rate of 26% ± 13%. there was a continuous increase in the apoptotic cell fraction to 85% ± 11% within days post-hypoxia as compared to control cultures. the clearest effects of oxygen deficiency were seen three days following induction of hypoxia as shown by annexin-v staining. additionally, post-hypoxic cultures were characterised by retracted dendrites, indicating a loss of multiple cell-cell contacts. debris and apoptotic bodies were found in most culture dish areas, evidencing late stage of apoptosis. in contrast, normoxic cultures displayed a much more reduced amount of apoptotic cells. over the whole time, apoptosis in these control cultures remained stable at about 7% ± 3%. after hypoxia, on day and day number of late apoptotic/necrotic cells significantly increased up to 27% ± 13%. further progression of propidium iodide positive cells in post-hypoxic cultures resulted in ± 16% on day. by comparison, in normoxic control cultures, necrosis levels remained stable below 17% and therefore did not statistically differ from post-hypoxic cultures on this time point. this fact was corroborated by images that show no observable deviation in the amount of necrotic and late apoptotic cells, as indicated by pi staining. in general, an increased release of calcein-am also indicated a strong decrease of neuronal cell viability which verifies the results of the apoptosis and necrosis rates. hypoxia increased quantities of caspase- and cleaved parp immediately and secretion of vegf in delay bar charts in figure show the concentrations of caspase- and cleaved poly polymerase. in post-hypoxic cultures apoptotic proteins drastically rose after oxygen deprivation. the highest concentration of caspase- was measured directly after hypoxia on days and. from day on, the level of active caspase- sharply decreased. the distribution of cleaved parp levels showed patterns similar to caspase- this decline was accompanied by a secretion of vegf on day after hypoxia. adhesion molecules were upregulated after hypoxia independent of cultivation conditions immunofluorescence analysis revealed that nearly all of the cells were positive for neurite cell adhesion molecule and neural cell adhesion molecule at day l and ncam were detected on somata and multiple dendrites. furthermore, a few of the neuronal cells also expressed vascular cell adhesion molecule and intercellular adhesion molecule on their somata and dendrites. there was no change in relative numbers of marked cells and expression patterns in post-hypoxic cultures. to quantify the density of adhesion molecule expression, highly sensitive fluorescence measurement of supernatants of lysed cultures was performed. here, comparative studies between cultures directly after hours of hypoxia and normoxic control cultures showed a significant upregulation of l ncam and icam-. hypoxic conditions determined a considerable increase in l and ncam protein expressions of up to 189% ± 74% and 155% ± 50%, respectively. hypoxia also strongly upregulated the icam- expression up to 424% ± 251%. the level of expression of vcam- was not altered by the hypoxic environment. the continued degradation of the dendritic networks did not allow assaying the specific expression of adhesion molecules at late stages of post-hypoxic cultures. co-culturing with hucb mnc strongly reduced apoptosis in post-hypoxic neuronal cell cultures we found noticeable neuroprotective properties of hucb mnc in the co-culture experiments. untreated post-hypoxic mono-cultures of neuronal cells showed 85% ± 11% of apoptosis after three days, whereas in co-cultures with mnc the rate of apoptosis was stable at a level of % ± 1%. this is comparable to normoxic control cultures. photographs of co-cultures with mnc revealed clear effort of mnc to localise next to somata and branches of post-hypoxic neuronal cells. in the course of clustering mnc mostly avoided areas that were not settled by neuronal cells. furthermore, the administration of mnc showed positive influence on the conservation of neuronal networks as compared to cultures that did not receive any cell therapy. mnc increased ccl ccl ccl and cxcl in post-hypoxic neuronal cultures the investigation of neuroprotective cytokines, inflammatory cytokines and chemokines in supernatants of normoxic neuronal cultures revealed considerable amounts of cxcl ccl and vegf. remarkably, after three days under hypoxic conditions only vegf was strongly upregulated. after administration of mnc, vegf was downregulated regarding to the total concentration measured in mono-cultures of mnc and in post-hypoxic neuronal cells. inflammatory cytokines as il-1β, il- and cxcl were produced by mnc. the secretion of cytokines such as il-1β and il- was not altered in post-hypoxic co-cultures, whereas cxcl was suppressed. interestingly, the majority of chemokines was clearly upregulated in co-cultures with mnc. we could show that ccl ccl ccl and cxcl were increased up to the tenfold, whereas the concentration ccl was not regulated. discussion in this study, we introduce an experimental human in vitro model to investigate the mechanisms of neuronal hypoxia and the interaction of neuronal cells with external stem cell-containing fractions as possible therapeutic tools. according to the differentiation protocol of encinas et al. we obtained fully matured neuronal cells after days. previous, orientating experiments showed typical response of differentiated sh-sy5y cells to n-methyl-d-aspartate application and most pronounced vulnerability after hours hypoxic incubation. these post-hypoxic neuronal cultures can be employed already days after the seeding of naive cells. therefore the time period until the model of hypoxic neuronal cells is available is very short. in comparison to other approaches that provide assimilable hypoxic models using teratocarcinoma cell lines the generation of our model is shortened by at least days. the proved expression of specific neuronal markers and the absence of any proliferation gave clear evidence of matured neuronal cells in the g0-phase of the cell cycle. this steady state allows making direct conclusions about the influence of any external manipulation because any change will most probably be due to these procedures. hence, the model of neuronal hypoxia affords the possibility to investigate manifold questions concerning mechanisms triggered in response to hypoxia and therapeutic interventions e.g. application of hucb mnc. following hypoxia, neuronal cultures displayed a correlation between morphological changes and increase of annexin-v positive cells as well as changes in adhesion molecule expression. the specific apoptosis marker annexin-v indicated that hypoxic cultivation preferentially induced apoptosis. moreover, there was a continuous enlargement in number of apoptotic cells from initial 26% ± 13% until cultures almost completely consisted of apoptotic cells within three days. conforming to changes of morphology in post-hypoxic cultures, there was an enhanced release of calcein-am also observable due to the loss of the integrity of membrane. this is typical for late stages of apoptosis as well as of necrosis. the model of neuronal hypoxia was approved by moderate rates of pi positive cells. therefore the overrun of 100% by the summation of annexin-v positive and pi positive cells is due to a proportion of cells in a late state of apoptosis that are positive for annexin-v and pi. we focused on apoptosis because it is a reversible process which could be modulate by external stimulation. moreover after stroke cell death in the penumbra is predominantly considered to be apoptosis. therefore apoptosis is a therapeutic target of anti-apoptotic therapies like stem cells or cytokines. apoptosis in the penumbra takes place in a time span of about hours after vessel occlusion in the rat after middle cerebral artery occlusion. this time course is represented by the model system introduced in this study. the fact that there was no major alteration in the number in dapi-positive nuclei underlines that the influence of hypoxia did not lead to an immediate destruction of the cells but allows investigating apoptosis and subsequent therapeutic interventions within hours. these facts were also confirmed by characteristic changes in the morphology of post-hypoxic cells. there was a total retraction of dendrites and a resulting destruction of neuronal networks, grained cell surfaces and extended cell degradation. this damage was accompanied by an upregulation of neuronal adhesion molecules. we hypothesize that this upregulation indicates a cellular answer to compensate for a loss of direct cell-cell interaction as a consequence of the preceding hypoxic stress as described by several authors. however, the final loss of intercellular networks could not be compensated by the increased expression of neuronal adhesion molecules alone. neuronal cell death after hypoxia is caused by membrane depolarisation subsequent to energy failure. the resulting calcium overload is the initial point of the activation of manifold biochemical pathways that affect caspases, free radicals and gene expression. the specific apoptotic proteins caspase- and cleaved parp were quantified to prove whether our microscopic observations are corroborated by biochemical pathways. after hours post-hypoxia, both marker proteins extensively increased. this was followed by a sharp drop in their expression level. this reduced amount of cleaved parp might be due to an aggravating energy failure in the cell. this in turn is attributed to the high levels of cleaved parp, evidenced above, which caused adenosintriphosphate depletion and which in this context prevents the enzymatic activity of caspases. furthermore, an increase of the expression of vegf, a key mediator of angiogenesis, was observed in higher concentrations in late post-hypoxic cultures. this might be a reflection of compensatory in vivo processes of revascularisation in our model. vegf is also known to be involved in neuronal protection through inhibition of caspase-. the time-delayed increased release of vegf as an effect of hypoxia does not seem to influence the number of apoptotic cells in our cultures. this may be a result of concentrations of vegf that did not reach efficient thresholds. in this context the measured reduction of caspases on days and after hypoxia is caused by the already described ongoing energy failure in the post-hypoxic cells. however, the regulation of those apoptotic proteins is probably not related to vegf mediated-effects. the therapeutic benefit of cell administration after stroke has been demonstrated in numerous animal studies. however, important questions in basic cellular and molecular mechanisms of neuronal cell death and its prevention after inadequate oxygen supply still remain unanswered. to understand the mechanisms of cellular neuroprotection after stroke we employed the model of neuronal hypoxia and applied mnc from hucb. in vivo mnc are supposed to differentiate into neuronal cells to trophically support neuronal tissue through the production of growth factors, to support the new formation of synapses and migration as well as the differentiation of endogenous neuronal progenitors. in our experiments, the administration of mnc from hucb to post-hypoxic neuronal cultures showed remarkable beneficial effects. over three days we could show a clear reduction of neuronal apoptosis, even to the level of normoxic control cultures. there are two observations that contribute to an explanation of neuroprotective mechanisms. first, mnc were preferentially located close to hypoxically injured neuronal cells. this phenomenon was facilitated through an intensive upregulation of icam- on neuronal cells which is the specific ligand for leukocyte cell adhesion molecule expressed by all immune cell subsets. however, the formation of cell chimera was not observed, as there were no cfse-stained cells with neuronal morphology. in future experiments we will investigate whether blocking of adhesion molecules on the surface of neuronal cells can inhibit the co-localisation of mnc. further it will be proved whether this influences the rate of apoptosis. an increase of apoptosis would be an evidence for the significance of direct cell-cell-contacts in context of therapeutic mechanisms of mnc. the second observation would be concomitance of an mnc indicated specific alteration in levels of soluble factors. that change might be held responsible for neuroprotection. pronounced upregulation of chemokines might be causal for the enhanced effort of mnc to localise near neuronal structures. high concentrations of vegf are known to be neuroprotective. in contrast to post-hypoxic mono-cultures, we found no increase of vegf after administration of mnc. it seems that presence of mnc inhibited the induction of elevated levels of vegf and compensated its neuroprotective effects by other mechanisms. cord blood contains distinct cell types capable to differentiate into neuronal cells. however, the differentiation of stem cells was not expected because of the short time of co-cultivation that clearly undershoots time frames responsible for neuronal differentiation. so far, it is unclear whether neuroprotection resulted from one of the observed effects or from a combination of spatial proximity and specific cytokine patterns. consequently, further studies in indirect co-cultures will be necessary.  pathophysiological models developed from animal studies form the basis of our understanding of the development of stroke. in vivo data display a perfusion-related dependency of neuronal cell damage. residual energy supply in the penumbra induces apoptosis, the early phases of which are reversible. consequently, rescue of the penumbra is a major target of experimental stroke therapy. data obtained from complex animal models of stroke strongly suggest that transplanted cells enhance neuronal survival. we established a standardized human in vitro model of neuronal apoptotic cell death after hypoxia that can facilitate to address specific pathophysiological processes underlying hypoxic damage and cell-mediated neuroprotection more precisely. interestingly, transplanted mnc not only strongly decreased the ratio of apoptosis in neuronal cells but also triggered retaining neuronal characteristics such as forming networks. mnc clustered around post-hypoxic neuronal cells and induced an alteration in cytokine and chemokine concentrations. our data suggest that the neuroprotective effects of mnc might result from direct cell-cell contacts and/or the adjustment of specific soluble mediators. 
to address the influence of hucb cells on neuronal tissue after stroke we established and employed a human in vitro model of neuronal hypoxia using fully differentiated vulnerable sh-sy5y cells. we established an in vitro model of neuronal hypoxia that affords the possibility to investigate both, apoptotic neuronal cell death and neuroprotective therapies. subsequently, hucb mononuclear cells were added to post-hypoxic neuronal cultures. the impact of hucb cells and hypoxia on secretion of neuroprotective and inflammatory cytokines, chemokines and expression of adhesion molecules was proved. specific cytokine patterns also suggest anti-apoptotic strategies of neuronal cells. in parallel, clustering of administered mnc next to axons and somata of neuronal cells was observed. we hypothesize that the neuroprotective effect of mnc was due to anti-apoptotic mechanisms related to direct cell-cell contacts with injured neuronal cells and distinct changes in neuroprotective, inflammatory cytokines as well as to the upregulation of chemokines within the co-cultures. hypoxic cultivation of neurons initially induced a rate of 26% ± 13% of apoptosis. based on this we investigated the therapeutic influence of hucb mnc on the progression of apoptotic cell death. however, complexity of animal models has thus far limited the understanding of beneficial cellular mechanisms. remarkably, the administration of mnc showed a noticeable reduction of apoptosis rates to levels of normoxic control cultures. within the next three days rate of apoptosis in untreated hypoxic cultures cumulated to 85% ± 11%.
 one of the most promising options for treatment of stroke using adult stem cells are human umbilical cord blood cells that were already approved for therapeutic efficacy in vivo. however, complexity of animal models has thus far limited the understanding of beneficial cellular mechanisms. to address the influence of hucb cells on neuronal tissue after stroke we established and employed a human in vitro model of neuronal hypoxia using fully differentiated vulnerable sh-sy5y cells. these cells were incubated under an oxygen-reduced atmosphere for hours. subsequently, hucb mononuclear cells were added to post-hypoxic neuronal cultures. these cultures were characterized regarding to the development of apoptosis and necrosis over three days. based on this we investigated the therapeutic influence of hucb mnc on the progression of apoptotic cell death. the impact of hucb cells and hypoxia on secretion of neuroprotective and inflammatory cytokines, chemokines and expression of adhesion molecules was proved.  hypoxic cultivation of neurons initially induced a rate of 26% ± 13% of apoptosis. hypoxia also caused an enhanced expression of caspase- and cleaved poly polymerase. necrosis was only detected in low amounts. within the next three days rate of apoptosis in untreated hypoxic cultures cumulated to 85% ± 11%. specific cytokine patterns also suggest anti-apoptotic strategies of neuronal cells. remarkably, the administration of mnc showed a noticeable reduction of apoptosis rates to levels of normoxic control cultures. in parallel, clustering of administered mnc next to axons and somata of neuronal cells was observed. furthermore, mnc caused a pronounced increase of chemokines.  we established an in vitro model of neuronal hypoxia that affords the possibility to investigate both, apoptotic neuronal cell death and neuroprotective therapies. here we employed the therapeutic model to study neuroprotective properties of hucb cells. we hypothesize that the neuroprotective effect of mnc was due to anti-apoptotic mechanisms related to direct cell-cell contacts with injured neuronal cells and distinct changes in neuroprotective, inflammatory cytokines as well as to the upregulation of chemokines within the co-cultures. 