Posted comment on ´Neuron-derived estrogen is critical for astrocyte activation and neuroprotection of the ischemic brain` written by Y. Lu, G.R. Sareddy, J. Wang, Q. Zhang, F.-L. Tang, U.P. Pratap, R.R. Tekmal, R.K. Vadlamudi and D. W. Brann and published in Journal of Neuroscience 2020 40 (38) p. 7355 doi 10.1523/JNEUROSCI.0115-20.2020
SUMMARY
The authors, Lu and colleagues, describe in the article their investigation into the role of neuron-derived 17 beta estradiol (E2) in astrocyte activation in the hippocampus CA1 region following global cerebral ischemia. They found that E2 is part of the reactive astrocyte neuroprotective response and is linked to the suppression of FGF2 signalling.
In their experiments, Lu and team used the FBN-ARO-KO mouse model where the forebrain aromatase is deficient and hence, E2 production in the brain is lacking. Intact males and bilateral ovariectomised females plus sham animals were used of the FBN-ARO-KO species compared to FLOX mice having normal forebrain aromatase activity. The global ischaemia condition was set-up by using the two-vessel occlusion global cerebral ischemia (GCI) model (ie. cardiac arrest and hypotensive shock). The mice were subjected to GCI and for most experiments the hippocampal CA1 tissue lysates and slides were prepared 7 days later. Various experiments were carried out. Western Blots, RNA sequencing, fluorescent imaging, GFAP staining and confocal single-plane imaging (for astrocyte 3D images) were performed on the different mouse groups and appropriate image processing and RNA sequencing analyses were carried out on the results obtained. E2 levels were measured using high-sensitivity ELISA kits. Experiments where the level of E2 was rescued by the administration of exogenous E2 were undertaken using 3 month old ovariectomised female mice and these were divided into 4 groups (FLOX+GCI, KO+GCI, KO+GCI+E2 and KO+GCI+placebo) with the control being the placebo group. The endogenous E2 was given at a dose known to effectively restore forebrain E2 levels in the FBN-ARO-KO mice. In the experiments where the effect of neuronal FGF2 was assessed, 4 groups of mice were constructed (FLOX+GCI, KO+GCI, KO+GCI+FGFR3-neutralising antibody and KO+GCI+vehicle). Bilateral intracerebroventricular microinjections of FGFR3 antibody at the time of the GCI were given where necessary. The experiments to measure astrocyte activation and astrocyte phenotypes were performed at 7 days after GCI. This was also the time frame used for the assessment of cognitive capability which was assessed using three different types of test: the Barnes Maze test for spatial reference learning and memory; the Novel Object Recognition Test for hippocampal dependent recognition memory; and the Open Field Test for testing locomotor function. Appropriate statistical analyses were carried out where necessary.
Lu and team`s experiments showed that ovariectomised female FBN-ARO-KO mice that had GCI had significantly increased astrocyte activation in the hippocampal CA1 region after 3, 7 and 14 days as shown by GFAP intensity. The level at 7 days was higher than both 3 days and 14 days suggesting that this was the highest point of activation. Compared to the FLOX mice after GCI, the FBN-ARO-KO mice showed lower levels of astrocytic activation in general (140% to 200%) which suggested to the authors that the loss of neuron-derived E2 causes astrocytes to be less activated after GCI compared to the control, FLOX+GCI mice.
With regards to aromatase, the aromatase double staining experiment and the GFAP measurement showed that aromatase was specifically located in the hippocampal CA1 neurons of the FLOX+SHAM mice. The expression of aromatase of neurons was strongly decreased in the FBN-ARO-KO+SHAM mice compared to the control, FLOX+SHAM mice. It was not detected in the resting astrocytes of either the FBN-ARO-KO+SHAM mice or the control FLOX+SHAM mice. However, GCI strongly induced aromatase in the astrocytes of the FLOX+GCI mice (approx. 250% of control) whereas in the astrocytes in the FBN-ARO-KO+GCI mice the level was significantly decreased (approx. 50% of the control value). Using the ELISA kits, E2 levels of the different groups were measured. It was found that hippocampal E2 levels in ovariectomised female FLOX-GCI mice were significantly increased 7 days later than FLOX-SHAM (approx. 120% of the control value). However, FBN-ARO-KO+SHAM mice had only approx. 33% of the FLOX+SHAM mice and there was no significant increase in E2 production either after the GCI.
The authors produced 3D images of the astrocytes of the hippocampus CA1 and showed that there were no differences in the volumes of the astrocyte cell bodies between the FLOX+SHAM and FBN-ARO-KO+SHAM mice. This confirmed that the resting states of the two species were the same. However, GCI was found to strongly increase the volume of the astrocytes of the FLOX mice indicating that the GCI produced a robust induction of reactive astrocytes. The volume of the astrocytes of the FBN-ARO-KO+GCI mice was found not to be significantly increased compared to the SHAM control and it was significantly lower than the FLOX+GCI equivalent. This indicated that the induction of reactive astrocytes in the FBN-ARO-KO mice after GCI did not occur. The results were confirmed with the investigation of the expression levels of the typical astrocyte markers, GFAP and vimentin. Both of these were robustly increased in the FLOX mice 7 days after GCI, but the increases were not observed in the FBN-ARO-KO mice. (Although increases were seen these were said to be insignificant – 20% GFAP, 18% for vimentin.) Therefore, reactive astrogliosis was said not occur in the FBN-ARO-KO model. By using immunostaining techniques for GFAP and S100beta cells where no differences were observed between the various models, the authors could relate the decrease seen in the FBN -ARO-KO mice to decreased astrocyte activation and not to astrocyte loss or proliferation.
Lu and team then continued their investigation by looking at the level of neuronal damage following GCI. Using double staining for NeuN (marker for neurons) and F-Jade C (marker for neuronal degeneration), the authors found that in ovariectomised female FBN-ARO-KO+GCI mice there was significantly increased levels of F-Jade C positive pyramidal hippocampal neurons 7 days after GCI compared to the control FLOX+GCI mice. This indicated a higher level of neuronal degeneration in the FBN-ARO-KO mice. There was no F-Jade C staining in either FLOX+SHAM or FBN-ARO-KO-SHAM mice showing that neither SHAM models exhibited neuronal damage. A study of MAP2 staining showed that the neuronal structural integrity of the FBN-ARO-KO+GCI mice was poorer for these mice than the control FLOX+GCI mice.
The authors continued their investigation of the role of neuron-derived E2 in hippocampal function following ischemic injury by looking at behavioural cognitive capability. This was carried out by using the following behavioural tests: Barnes Maze test for spatial memory; Novel Object Recognition Test for object recognition; and Open Field Test for locomotor function. The results of the Barnes Maze Test showed that the FLOX+GCI mice displayed a significant increase in escape latency, an increase in exploring errors and a decrease in quadrant occupancy compared to the FLOX+SHAM mice. This indicated impaired spatial reference memory recall following the ischemic event. The FLOX+SHAM mice performed in general better than the FBN-ARO-KO+SHAM mice showing that they too exhibited cognitive deficits. However, there were even greater increases in escape latency and in exploring errors and greater decreases in quadrant occupancy in the FBN-ARO-KO+GCI mice compared to the FLOX+GCI mice. The differences could not be attributed to speed variations since the escape velocities for all groups were identical. In the case of the Open Field Test, no differences were shown between the subject groups showing that loss of forebrain neuronal E2 does not affect locomotor function under normal or GCI conditions. Therefore, the authors concluded that spatial memory capability was decreased with their FBN-ARO-KO model and this decrease was amplified by GCI.
In order to explore the nature of the astrocytic reactivity observed following GCI, Lu and team performed RNA sequence analyses on the hippocampus CA1 tissue of ovariectomised female FLOX+GCI mice and FBN-ARO-KO+GCI mice. They found that the FBN-ARO-KO+GCI mice exhibited differences in key pathways regulating astrocytic reactivity (eg. RhoA signalling, actin-based motility by Rho signalling, signalling by Rho family GTPases and NRF2-mediated oxidative stress response). It was found that there was significant down regulation of top pan-reactive astrocyte transcripts and strong down regulation of most of the astrocyte A2 specific transcripts in the FBN-ARO-KO+GCI mice. In the case of astrocyte A1 specific transcription, several areas of transcription showed significant decrease in the FBN-ARO-KO+GCI mice whereas a few others showed significant increase. Using qRT-PCR it was possible to show that there was significant up regulation of genes involved in neuroinflammation and apoptosis, but significant down regulation of genes involved in regulating the astrocyte A2 phenotype and significant down regulation of genes involved in synapse maturation. From the combination of the RNA sequencing results, it was possible to conclude that A2 astrocytes were reduced in the FBN-ARO-GCI mice and neuron-derived E2 regulates transcription of genes and pathways involved in astrocyte activation and neuroprotection after global brain ischemia.
The study on astrocyte phenotype continued with imaging experiments using the markers GFAP, vimentin and S100beta. Lu and team found that 7 days after GCI, FLOX+GCI mice demonstrated significantly increased GFAP, vimentin and S100beta compared to the FLOX+SHAM mice. This indicated a robust astrocyte activation following the GCI event. However, in the case of the FBN-ARO-KO+GCI mice, these astrocytes showed a pronounced decrease in GFAP, vimentin and S100beta compared to the FLOX+GCI astrocytes indicating attenuated astrocyte reactivity after loss of neuron-derived E2. The level of the astrocyte A1 phenotype was measured by using the three A1 selective markers C3D, FKBP5 and GBP2. No level of expression was found in the FLOX+GCI and FBN-ARO-KO+GCI showing that A1 phenotype was not induced in the CA1 with ischemic injury. In the case of the A2 phenotype, the markers S100A10, PTX3 and TGM1 were used. These were found not to be expressed in the FLOX-SHAM astrocytes and the FBN-ARO-KO+SHAM astrocytes, but all three were strongly induced in the FLOX+GCI astrocytes and only S100A10 and PTX3 were robustly down regulated (approx. 30% of FLOX control for S100A10 and approx 20% for PTX3) in the FBN-ARO-KO+GCI mice. This indicated to the authors that the FBN-ARO-KO+GCI mice have strongly induced A2 astrocyte polarisation compared to FLOX+GCI mice. These results were supported by the use of IHC analysis. C3D, the selected marker for A1 astrocytes was found not to be detected in any of the groups of ovariectomised female mice at 7 days after GCI whereas S100A10 (the marker for A2 astrocytes) showed strong induction and colocalization with GFAP in the FLOX+GCI hippocampal astrocytes. The FBN-ARO-KO+GCI exhibited significant decrease of S100A10 level in the hippocampal CA1 compared to the FLOX-GCI mice. Hence, this indicated to the authors that A2 astrocyte induction following GCI is suppressed in mice deficient in forebrain neuron-derived E2.
The authors continued their study by looking at the expression of BDNF and IGF-1 in hippocampal CA1 tissue. Using both double staining and Western Blot analysis, the expression of BDNF was strongly increased in FLOX+GCI mice at 7 days after GCI (approx. 450%) compared to the FLOX-SHAM whereas in the less activated astrocytes of the FBN-ARO-KO+GCI mice the astrocytic BDNF levels were significantly lower (approx. 200%) compared to the FLOX+GCI mice. There was no difference between BDNF levels in the resting astrocytes of FLOX-SHAM and FBN-ARO-KO+SHAM. In the case of IGF-1, the astrocytic expression of IGF-1 in FLOX+GCI mice was strongly upregulated (approx. 450%) at 7 days after GCI compared to the FBN-ARO-KO+GCI mice (approx. 150%). Again, there was no difference between the FBN-ARO-KO+SHAM and FLOX+SHAM. The levels of GLT-1 and GFAP showed that they were robustly up-regulated in the FLOX-GCI reactive astrocytes (GLT-1 – approx. 450%) compared to the FLOX-SHAM whereas the level was markedly reduced in the FBN-ARO-KO+GCI mice (approx. 150%) compared to the FLOX+GCI mice. This confirmed to the authors that neuron-derived E2 is critical for hippocampal CA1 astrocyte activation and up-regulation of the neuroprotective astrocyte-derived neurotrophic factors and GLT-1 after GCI.
Further studies were carried out to see whether time between GCI and sampling made a difference to the BDNF, IGF-1 and GLT-1 levels. Samples were taken at 3 days, 7 days (the normal sampling time) and 14 days and analysis showed that expression of all three in the FBN-ARO-KO+GCI mice was robustly decreased at both 3 day and 14 day time points compared to the FLOX+GCI mice (approx. 50% of control for BDNF, approx. 65% of IGF-1 and approx. 40% of GLT-1 at 3 days whereas approx. 70% for all at 14 days). This indicated to the authors that the astrocyte dysfunction occurs at an early stage of GCI injury along with diminished astrocyte activation and may cause ischemic brain damage. Therefore, they examined the levels of F-Jade C and NeuN and found increased F-Jade C staining at 3 days in FBN-ARO-KO+GCI mice compared to FLOX+GCI mice and even higher values at 14 days. This confirmed the view that compromised astrocyte function after loss of neuron-derived E2 contributes to enhanced ischemic brain injury.
Since most of the experiments had been performed with female mice, the authors repeated their studies with male mice in order to address whether neuron-derived E2 has similar functions in males. In the case of aromatase, this was found to be specifically localised in hippocampal CA1 neurons in male FLOX-SHAM mice and the level was markedly decreased in the FBN-ARO-KO+SHAM male mice. No aromatase was found in astrocytes from either group. GCI led to a strong induction of aromatase in the FLOX+GCI male mice and in the FBN-ARO-KO+GCI male mice it was also strongly decreased compared to the FLOX+GCI mice. Therefore, it was concluded that there is diminished astrocyte activation and aromatisation in male FBN-ARO-KO mice after GCI in the same way as the ovariectomised female mice.
Similar findings for the male mice to the female mice were also found for the astrocyte phenotypes. No A1 astrocytes were induced in either the FLOX-GCI males or the FBN-ARO-KO+GCI males since there was no detectable C3D expression in either the FLOX-GCI or the FBN-ARO-KO+GCI hippocampus 7 days after GCI. In the case of A2 astrocytes, the A2 marker S100A10 was found to be markedly decreased in the FBN-ARO-KO+GCI male mice compared to the FLOX-GCI males. BDNF and GLT-1 were also found to be strongly reduced in the FBN-ARO-KO+GCI males compared to the FLOX-GCI mice whereas the level of neuronal damage shown by F-Jade C staining was shown to be significantly increased in the FBN-ARO-KO+GCI males compared to the control, FLOX-GCI mice. Behavioural studies also showed that the male FBN-ARO-KO+GCI mice exhibited greater memory deficits (eg. increased escape latency, decreased exploring time) compared with the FLOX+GCI mice 7 days after the GCI administration. The level of GFAP expression and aromatase induction in astrocytes was found to be strongly decreased in FBN-ARO-KO+GCI male mice at both 3 days and 14 days after GCI indicative of diminished astrocyte activation and aromatisation. The levels of BDNF, IGF-1 and GLT-1 were also down-regulated in male FBN-ARO-KO+GCI mice at both 3 days and 14 days after GCI whereas F-Jade C intensity was enhanced showing neuronal damage was increased. Therefore, the authors concluded that there were no differences between male and female mice subjects.
The study continued with an exploration of whether FGF2 signalling is a mechanism employed by neuron-derived E2 in the regulation of reactive astrocyte induction after GCI. Investigation of neuronal FGF2 signalling showed that the FGF2 transcript was strongly increased in the hippocampus CA1 region of the FBN-ARO-KO+GCI mice compared to the FLOX+GCI mice. This indicated to the authors a role of FGF2 in the mediation of suppression of reactive astrogliosis in FBN-ARO-KO mice following GCI. FGF2 levels were found to be increased in ovariectomised female FBN-ARO-KO+GCI mice compared to the other groups especially FLOX+GCI mice and levels in FLOX-SHAM were higher than FLOX-GCI. This supported the view that reduced neuronal FGF2 facilitates reactive astrogliosis.
An investigation of FGF2´s major receptor FGFR3 in hippocampal astrocytes showed weak intensity of FGFR3 in the reactive astrocytes of the FLOX+GCI mice corresponding to the low levels of FGF2. However, the FGFR3 was strongly expressed in both SHAM groups and the less activated astrocytes of FBN-ARO-KO+GCI mice suggesting that increased FGF2 signalling in the FBN-ARO-KO+GCI might contribute to the lower astrogliosis levels in the FBN-ARO-KO+GCI mice following the GCI event. Blocking cerebral FGF2 signalling in ovariectomised female FBN-ARO-KO+GCI mice by bilaterally infusing simultaneously with FGFR3 antibody led to a restoration of the various factors to approximately FLOX levels, eg. GFAP levels elevated to approx. 92% showing that there was an almost complete rescue of reactive astrogliosis; approx. 95% induction of aromatase also occurred; pSTAT3 levels (marker for neuroprotective astrocyte phenotype) also showed increases to approx. 91%; BDNF and GLT-1 levels were also rescued; and Jade C intensity was found to be significantly attenuated indicating that decreased neuronal degeneration had occurred. These observations indicated to the authors that the neuroprotective astrocyte activation and functions were significantly restored after FGFR3 neuralisation in the FBN-ARO-KO+GCI mice. Therefore, neuron-derived E2 regulation of neuroprotective astrogliosis after GCI is due, at least in part to the suppression of neuronal FGF2 signalling.
The authors continued to verify their findings by performing re-instatement of E2 levels by the administration of exogenous E2 in vivo to FBN-ARO-KO+GCI mice. The administration of E2 to the ovariectomised FBN-ARO-KO+GCI mice at a level known to fully restore E2 in FBN-ARO-KO mice showed that there was significant repression of the strongly increased neuronal FGF2 expression in the FBN-ARO-KO+GCI mice and that the FGFR3 levels were also now significantly repressed having been strongly increased in the FBN-ARO-KO+GCI mice. This confirmed the view that FGF2 signalling is inhibited by E2. Exogenous E2 replacement was also able to rescue the astrocyte derived BDNF level, IGF-1 level and GLT-1 level in the FBN-ARO-KO+GCI mice to almost FLOX+GCI levels. It was also able to increase GFAP levels to 92% in FBN-ARO-KO+GCI mice compared to 65% in FLOX+GCI mice and robustly decrease the neuronal damage as shown by F-Jade C intensity measurements and neuronal structural changes as given by MAP2 staining. Exogenous E2 replacement also significantly improved the performance of FBN-ARO-KO mice at the behavioural test of Barnes Maze test (ie. strongly decreased primary escape latency and exploring errors and increased quadrant occupancy). In FBN-ARO-KO+GCI mice the E2 levels which were not significantly different to those observed for FLOX+GCI mice led to improved performance at the Novel Object Recognition Test. Exogenous E2 replacement strongly reversed the deficits as shown by increased exploring time of novel object and elevated discrimination index. Therefore, cognitive capability appeared to be restored following exogenous E2 administration to the FBN-ARO-KO mouse model where neuron-derived E2 is absent.
The authors concluded their article by discussing their observations that neuron-derived E2 plays several key roles in the ischemic brain. The first role is that it is critical for astrocyte activation in the hippocampal CA1 region. Reactive astrocytes after ischemia become transiently hypertrophic and highly express the intermediate filaments, GFAP and vimentin. Mice deficient in GFAP and vimentin display attenuated astrocyte hypertrophy and reactive astrogliosis after brain injury. In this study FBN-ARO-KO mice displayed a significant decrease in astrocyte hypertropy and a significant decrease in GFAP and vimentin in the hippocampal CA1 region after GCI. There were also significant alterations in RhoA signalling in the FBN-ARO-KO mice which is shown to constrain actin motility and astrocyte reactivity and is tightly controlled by STAT3, a factor whose expression and activation are known to be critical for induction of the reactive astrocyte phenotype. The authors also explained their observed significant loss of elevated astrocyte aromatase and hippocampal E2 in the FBN-ARO-KO+GCI mice model as being due to the decreased activation of astrocytes in this mouse species. Aromatase induction is known to occur only in activated astrocytes and not in the resting state and therefore, reinstating the astrocyte activation in the FBN-ARO-KO mice by blocking the FGF2 signalling rescued the aromatase levels in the hippocampal astrocytes after GCI treatment.
The second role of the neuron-derived E2 was described by the authors as suppression of neuronal FGF2 signalling. The authors found that there was increased FGF2 signalling (increased mRNA levels and protein levels) in the FBN-ARO-KO mice after GCI. Neuron-derived FGF2 exerts an inhibitory effect on astrocyte activation by suppressing GFAP expression in astrocytes. Therefore, blocking the FGF2 receptor in astrocytes invokes reactive astrogliosis basally and strongly enhances it after injury. This indicated to the authors that FGF2 is an important signal to restrain induction of reactive astrocytes. In the FBN-ARO-KO mouse model, FGF2 signalling played an important role in the decreased astrocyte activation. Blocking the FGF2 signalling by administering its receptor`s inhibitor, a FGFR3 neutralising antibody, fully restored astrocyte activation after GCI. This is also supported by the observations that neuron-derived E2 also enhanced the expression of astrocyte derived neuroprotective neurotrophic factors, BDNF and IGF-1 as well as the glutamate transporter, GLT-1.
The third role of the neuron-derived E2 described by the authors was that it exerts neuroprotection and preserves hippocampus dependent cognitive functions following ischemic injury. The FBN-ARO-KO mice used in the study meant that the lack of aromatase resulted in E2 depletion in neurons causing the cognitive deficits observed after GCI. This was supported by the exogenous E2 replacement being able to rescue these deficits. The FBN-ARO-KO mice exhibited more serious neuronal damage in the hippocampus following GCI manifesting as greater cognitive function deficits. They also exhibited attenuated reactive astrogliosis after GCI which suggested that the neuroprotective effect of neuron-derived E2 could be mediated by reactive astrocytes after GCI. This was supported by the observation that the FBN-ARO-KO+GCI model produced only mild injury and no scar formation which is indicative of a beneficial effect of reactive astrocytes rather than the detrimental situation of where reactive astrocytes cause proliferation and scar formation leading to the inhibition of axonal regeneration after injury.
The FBN-ARO-KO+GCI model also showed strong attenuation of GFAP and vimentin and reduced astrocytic GLT-1 levels in addition to the increased neuronal damage and attenuated reactive astrogliosis. This was supported by the evidence that reactive astrocytes can protect neurons through several mechanisms including the release of the neuroprotective factors such as BDNF, IGF-1 as well as increased uptake of excess glutamate. Most of the glutamate uptake (90%) in the brain is carried out by the astrocyte specific Na+ dependent glutamate transporter GLT-1. Up-regulation of GLT-1 in astrocytes is reported as protecting hippocampal CA1 neurons after GCI since glutamate excitotoxicity is known to be a major mechanism of neuronal damage and death after ischemia. Therefore, it was suggested that up regulation of GLT-1 in astrocytes as well as up regulation of BDNF and IGF-1 could be mechanisms by which neuron-derived E2 could cause neuroprotection after GCI. The authors suggested that the attenuated astrocyte activation and reduced aromatase/E2 induction in astrocytes after GCI could contribute to the enhanced neuronal damage seen in the FBN-ARO-KO mouse model used.
The authors also considered whether E2 itself provides a neuroprotective effect since E2 added exogenously in vitro can show a positive protective effect. However, the effect appeared to be instigated via the reactive astrocytes since aromatase and E2 increases in reactive astrocytes after GCI. The reinstatement of astrocyte activation in the FBN-ARO-KO mice after GCI by blocking the FGF2 signalling rescued the attenuated aromatase levels in the astrocytes as well as reinstating the pSTAT3, BDNF and GLT-1 levels. Neuronal damage was also strongly reduced.
Therefore, the authors in their study showed that neuron-derived E2 is likely to play a beneficial role in astrocyte activation in the CA1 area of the mouse hippocampus following global ischemia and it does this by suppressing FGF2 signalling. This leads to neuroprotection and improved behavioural cognitive function following ischemic injury in the brain.
COMMENT
What makes this article interesting is that it describes the roles of estradiol (E2) as a modulatory influence on hippocampal firing during normal functioning and as an influence in response to damage of this area caused by an ischemic event. Therefore, this comment focuses on two main areas: the role of E2 in hippocampus CA1 functioning relating to memory capability; and the role of E2 and the astrocytic response to ischemic assault of this area.
With regards to E2 and hippocampus firing, the E2 is probably under normal circumstances in females sourced from both local neurons and the ovaries under hypothalamus control. It is assumed that independent of source the neurochemical action of E2 comes from binding of the ligand to the cell membrane receptors, ERs (both alpha and beta varieties) or GPR30 receptors with all types of receptors having the same affinity for estradiol. This binding is followed by inclusion and binding to the nuclear DNA binding sites (either the unique DNA sequences, estrogen response elements ERE or interaction with other DNA bound transcription factors such as AP-1). The response to the DNA binding in the case of the hippocampus is the excitatory effect on the overall firing of the cell as well as synaptic modulation which supports and aids that firing excitability. Estradiol binding (as well as other estrogens) can alter the excitability of neurons with latencies of a few seconds independent of whether ER or GPR30 binding and this can stimulate Akt, MAPK and cAMP production within minutes. Estradiol binding to GPR30 receptors in particular can stimulate not only the production of cAMP, but also leads to mobilization of calcium ions as well as activating the growth factor signalling pathways.
This leads onto the second function of E2 in the hippocampal area which is the positive effects on synaptic and dendritic density via advantageous growth of neurites and dendritic spine density. Estrogens can also promote the growth of more excitatory synapses. The new spines have been shown to have more NMDA Rs which can increase the long-term plasticity of the hippocampus which would have an overall beneficial effect on the firing of the area manifesting in positive cognitive behaviour performances, eg. spatial memory. The LTP role of E2 in the hippocampus appears to be via cholinergic input into this area and increased NMDA neurotransmission mediated in particular by NR2B containing receptors (Smith). It also has a direct effect on excitation by depressing inherent synaptic inhibition. The binding of E2 to ERs on the inhibitory interneurons leads to the inhibition of spine growth and therefore, the E2 causes these inhibitory cells to produce less GABA resulting in decreased inhibition and by default greater neural activity which somehow triggers an increase in spines and excitatory synapses on the excitatory pyramidal cells. Therefore, the overall effect of E2 in the hippocampus is a general aid to the excitation of firing via neuronal changes and advantageous modulation of the synapse which supports the general firing excitability in the area.
(Before we continue, we should mention the male equivalent to E2, the androgens. In fact, E2 is produced from testosterone and so it should be assumed that there is an equivalent aid to neuronal firing and synaptic modulation of the hippocampus in the male. This is found to be correct with the androgens being seen to act on brain cells to modify their functions and ultimately, behaviour. In the case of androgens there are also three categories of receptors. One receptor type preferentially binds testosterone and a second one preferentially binds dehydroxytestosterone (DHT). Both of these receptors are in equilibrium between the nucleus and cytoplasm and can be activated and transformed by the binding of the androgen steroid leading to concentration of the complex just like E2 in the nucleus. The third receptor type however, can bind both steroids with the same relative affinity, but can only be activated and is not transformed by the DNA binding and hence, does not concentrate in the nucleus. The androgens are reported to have profound effects on hippocampal structure and function and like estrogens their effects include the induction of spines and synapses on the dendritic spines of the CA1 pyramidal neurons, as well as alterations in LTP resulting in effects on hippocampally dependent cognitive behaviours (Atwi). However, the method for this is different to the estrogens. The effects of androgens appear to be carried out via modulation of brain-derived neurotrophic factor (BDNF) in the mossy fibre (MF) system of the hippocampus which is suppressed in the presence of testosterone. This is supported by observations that orchidectomy of male rats increases synaptic transmission and excitability in the MF pathway with exogenous testosterone reversing these effects. This suggests that testosterone exerts a tonic suppression on MF BDNF levels and that loss of testosterone as in age for example leads to an increase in BDNF dependent MF plasticity. Therefore, unlike E2 androgens appear not to affect the firing of the neurons, but more the synaptic modulation. From a behavioural perspective, definitive findings are problematic since some researchers report no change in cognitive capability when androgens are deficient and others like in the case of estradiol report loss of verbal and spatial memory performance.)
However, in this comment in response to Lu and team`s article we are concentrating on the effects of E2 on the mouse hippocampal CA1 region and the results of what happens when E2 is deficient in particular. Therefore, as expected using the aromatase knock-out mouse model, FBN-ARO-KO, the level of E2 in the hippocampal CA1 area of these females was lower than in the FLOX mouse control. Ovariectomy of the FBN-ARO-KO female meant that the ovarian contribution of E2 via the hypothalamus was also missing and therefore, the level of E2 in these mice was only 33% of the control FLOX. The physical consequences of the deficiency as reported by Lu and team were as expected in that the structural integrity of the CA1 neurons was lower in the ovariectomised FBN-ARO-KO mice than the control. This manifested as expected as deficits in cognitive functional capability with spatial memory reported as being lower in the ovariectomised female FBN-ARO-KO mice than the control.
Several things should be pointed out at this stage relating to the findings. The first is there is little or no E2 in glials under normal circumstances. This implies that the reduction in structural integrity observed with E2 deficiency in the ovariectomised FBN-ARO-KO mouse model is not seen by the neuronal cells as an ´assault` on its functionality and therefore, a response of increasing glial functionality is not instigated. This may mean that there is redundancy in the neuronal system under normal circumstances. The second point is that an increase in FGF2 level is observed in the ovariectomised FBN-ARO-KO mouse compared to the FLOX control. Since applied FGF2 to rat cortical neurons is reported to enhance neurite growth, axonal branching and LTP in hippocampal neurons it is possible that the E2 deficiency may be compensated for under normal circumstances to some extent by the increase in FGF2 signalling. E2 loss of structural integrity may then relate to factors other than neurite growth, eg. microfilament organisation. The third point relates to the natural human situations of estrogen and androgen deficiencies as seen in menopause and male ageing for example. The physical and behavioural effects observed in these situations are not always observed and therefore, may reflect the physiological complexity of the human brain in relation to the mouse brain. One factor for the differences may be the dominance of the mouse behaviour on spatial information input, processing and memory compared to the human`s preference for visual information. Therefore, loss of estrogens and androgens in humans may indicate the same types of changes, but not to the same extent and direct transfer of knowledge of one system to another may not be fully valid.
Now we have established the effects of E2 on excitatory firing and synaptic modulation we can look at how E2 in the hippocampal CA1 can affect the astrocytic response to an ischemic assault of that area. Lu and team`s study found that in mice E2 deficiency produced no expected astrocyte increase in response to this type of damage. Ischemia is a condition in which the blood flow and hence, oxygen is reduced so that metabolic demands cannot be met. With regards to neurons, the interruption of blood flow for as little as 20 seconds can result in local cortical activity ceasing with this cessation spreading outwards from the point of assault (spreading depression). Neurons which do not receive enough blood to communicate result in an area called a penumbra, but they do receive sufficient levels of oxygen to avoid cell death at this time and ischemia induced in brains for up to an hour may be at least partially recovered.
The shut-down of firing observed with ischemia is induced by the efflux of potassium ions from the neurons via initially the opening of voltage dependent potassium channels and later by ATP dependent potassium channels leading to a transient plasma membrane hyperpolarisation. This cascades into a redistribution of ions across the plasma membrane (influx of sodium, calcium and chloride ions) which results in the excessive release of neurotransmitters. In particular, the dramatic release of glutamate is the major cause of destruction elicited by the ischemic event at the synapse. In this glutamate excitatotoxicity, the glutamate receptors are overstimulated leading to sodium ion influx and potassium ion efflux via the glutamate receptor-activated membrane channels and calcium influx via the NMDA receptor–gated ion channels. This adds to the firing challenges faced by the cells with further spread of the neuronal depolarisation as well as depletion of energy stores and initiation of calcium ion dependent detrimental cascades, eg. release of zinc ions. Further responses to the ischemic assault at the intracellular level involve enzyme activation, eg. protein kinase rapid activation that can lead to enhancement of the neuronal excitotoxicity by increasing the release of glutamate, or DNA transcription and gene expression changes, eg. MAP kinases and p38 kinases activation. The outcome is enhanced neuronal damage and cell death.
Tissue damage as a result of ischemic assault is likely to be mediated via the formation of several reactive oxygen species (ROS) or by the activation of catabolic enzymes. Both of these stem from the increased intracellular calcium concentration. Research shows that nitric oxide radicals can cause DNA single strand breaks and when produced by inducible NO synthase expressed in macrophages neutrophils and microglia may contribute to late tissue damage whereas in other cases NO may be part of the anti-inflammatory response. Activation of calcium-dependent catabolic enzymes such as phospholipase A2 and C following NMDA R stimulation promote membrane phospholipid breakdown thus ameliorating the formation of free radicals and inflammation processes. Other calcium activated proteases or calpains contribute to the destruction of the structural and regulatory proteins adding to the cellular damage.
Tissue damage by the ischemia also induces an inflammatory response with the elevation of mRNA levels for the pro-inflammatory cytokines TNFalpha and IL-1beta observed as early as I hour after the GCI. There is also an induction of adhesion molecules on the endothelial cell surface (eg. CAM-1, P- and E-selectins) which enhance the adhesion of neutrophils and passage through the vascular wall into the brain parenchyma leading to the invasion of macrophages and monocytes. Detrimental damage can be visualised by some cellular swelling and at some level apoptosis is instigated with the translocation of cytochrome c from the mitochondria to cytosol and the activation of caspase 3 and the apoptotic pathway.
The responses to ischemia are not all negative though with the cell instigating processes to protect itself from the assault and to remove cell debris which is itself destructive. One response is to enhance vascular blood flow in order to limit the reduction in blood supply and oxygen caused by the ischemia and one method for this NO-mediated vasodilation. This is where one form of NO synthase present in endothelial cells leads to the relaxation of vascular smooth muscle cells and helps to preserve blood flow. At the cellular level, there are neuroprotective mechanisms activated in order to reduce the excessive excitation. For example, the activation of interneurons leading to release of the inhibitory neurotransmitter GABA; the down regulation of NMDA receptor function by blocking the zinc binding site; and the depletion of extracellular calcium and sodium ions in order to reduce the membrane concentration gradient which favours influx. The glutamate excitotoxicity is also counteracted by the action of several other neurotransmitters that are in ischemia neuroprotective, eg. serotonin, GABA and adenosine. In the case of adenosine, this accumulates rapidly in ischemia due to the breakdown of ATP. The beneficial effects come from the ability of the neuronal adenosine A1 receptor to reduce neurotransmitter release membrane excitability and from the A2 receptors where activation on vascular smooth muscle cells enhances blood flow and activation on neutrophils decreases inflammation.
Lu and team observed massive upregulation of the growth factors BDNF, IGF-1 and the glutamate transport molecule, GLT-1 after GCI and these too are part of the neuroprotective response to ischemia. Others include (in rat) nerve growth factor (NGF), neurotrophins 4 and 5 (NT-4/5) and basic fibroblast growth factor (FGF2). The aim of the up regulation of these factors is to minimise damage, block apoptosis and even enhance functional recovery (eg. via enhancing nerve fibre sprouting and synapse formation). However, the situation is not clear since some, eg. BDNF, NT-3, NT4/5 are also reported as enhancing the neuronal vulnerability to excitotoxic and cell death induced by the free radicals. This negative effect is explained by some as being caused by the enhanced NMDAR mediated calcium influx, increased production of free radicals and even by acute pro-excitatory effects that could increase the ischemia induced excitotoxicity.
However, the main neuroprotective mechanism to ischemia and with relation to the comment on this article, is the role of astrocytes and the formation of reactive astrocytes. Astrocytes appear to possess a high potential for regeneration and neuroprotection following ischemia, but it should also be noted that, just like the neurotrophic factors, there is also evidence that astrocytes can exacerbate ischemic injury. This is because the excessive accumulation of ions and glutamate can overload the buffering role of the astrocytes leading to the activation of the catabolic enzymes and eventually cell death.
In Lu and team`s article, the response of the CA1 to the ischemic injury is the massive up regulation of astrocytes and their functional capability. Astrocytes are highly responsive and dramatically change their characteristics to the damage and therefore, these ´injury-activated` cells are termed reactive astrocytes. There are two subtypes, A1 and A2, depending on the assault faced. For example, A1 reactive astrocytes are induced by inflammatory agents such as lipopolysaccharide and are proinflammatory and neurotoxic; whereas A2 are induced following cerebral ischemia and are neuroprotective. (In Lu and team`s study it is the A2 subtype which appear to be dominant.)
Ischemia leads to the up regulation of astrocytes and reactive protoplasmic astrocytes in the cortex began to proliferate (reactive astrogliosis) within 3 to 5 days after injury. This is mirrored by the increase in glial fibrillary acidic protein (GFAP) observed and whereas initial morphology shows a clear cytoplasm with many mitochondria and some processes, after 3 days there are many phagocytic inclusions visible within the cytoplasm. From a biochemical point of view, there are two advantages to astrocytes, one is its energy supply and the second is its ability to up regulate specific proteins beneficial to the synapse. In the case of energy supply, astrocytes can respond to an oxygen deficient situation by changing their energy supply in comparison to neurons. The inhibition of mitochondrial respiration induced by NO leads to astrocytes increasing their glucose metabolism via the glycolytic pathway which limits the fall in ATP levels and prevents apoptosis. In contrast, neurons cannot and a large ATP depletion results which can lead to apoptosis.
The other biochemical response of the astrocytes to ischemia is the change in gene expression and specific protein levels. The intermediate filament proteins such as vimentin (part of the Lu study) and nestin, synemin are involved in the reactivity of astrocytes and are highly expressed in ischemic injury as well as in response to oxidative stress. Another factor up regulated under these conditions is cyclin-dependent kinase 5 (CDK5) of astrocytes which is involved in a number of beneficial processes such as synthesis of neurites, synapse formation and synaptic transmission and axonal cytoskeleton (assembly, organization and stabilization with substrates mainly neurofilaments and MAPs) and apoptosis. It is also involved in the elongation and reactivity of astrocytes themselves. GFAP, CDK5 and p35 (activated when the astrocyte is subjected to stress and generating active CDK5) forms immunocomplexes. The biochemical changes elicited by the ischemic injury allow the astrocytes to carry out their various neuroprotective functions.
One such function is to reduce the level of extracellular glutamate increased by the excessive excitation of the neurons and released into the extracellular space and also by the microglials. This function is achieved by the astrocytes expressing a much larger amount of membrane localized EAATs excitatory amino acid transporters (EAATs) of which GLT-1 is one so that there is increased uptake of the glutamate. Lu and team saw in their experiment that there were much higher levels of this transporter in their experimental mouse models when subjected to GCI.
Another function of the reactive astrocytes following ischemia is in aiding the defence of the neurons against oxidative stress. There are three ways in which this is carried out: indirectly via the high abundancy of NADPH in astrocytes with NADPH being used as an electron donor for the regeneration of reduced glutathione, the ROS scavenger; directly via the astrocytes releasing in response to excess glutaminergic activity ascorbic acid which is an antioxidant (The ascorbic acid released is taken up by the neurons and modifies the local energy metabolism by inhibition of glucose consumption and increasing the uptake of lactate.); and thirdly, by activating the expression in the astrocytes of Nrf2 which is a redox-sensitive transcription factor which acts as in neuroprotection (This coordinates the expression of the cytoprotective enzymes that can also scavenge any ROS produced.).
However, a major function of reactive astrocytes in the response to ischemic injury is the removal from the local environment of debris caused by the oxygen and blood flow depletion. Reactive astrocytes are capable of phagocytizing multiple cellular components because they are observed to engulf myelin-like structures, synaptophysin1plus synapses and other unidentified components. Although traditionally linked to A1 subtype, A2 astrocytes are also believed to demonstrate the same phagocytic function in response to ischemic injury as the A1 and they assist microglia in the removal of debris even if at different spatial temporal characteristics. These phagocytic reactive astrocytes found in the penumbra region are enriched with genes involved in engulfment pathways, which includes phagocytic receptors and intracellular molecules. Phagocytic astrocytes demonstrate upregulated ABCA1 and its pathway molecules, MEGF10 and GULP1, which are required for phagocytosis. The reactive astrocytes also express several other phagocytic receptors, including BAI1 and integrin αvβ3 or 5, which appear to function as upstream signals of another beneficial pathway. The onset of the astrocytic phagocytosis begins after 3 days (reactive astrocytes exhibit many phagocytic inclusions within their cytoplasm at this time) and persists for 14 days. The spatiotemporal pattern of the phagocytosis suggests a relationship to neuronal remodelling in the penumbra region of the ischemic injury with substantial axonal, dendritic and synaptic losses and debris removal within the first week following the ischemic injury followed by an increased number of synapses and axonal connections as part of the remodelling.
Reactive astrocytes also play a role in the remodelling of the neuronal area following the damage and therefore, the phagocytic function of astrocytes may be seen positively since it removes the debris so that renewal of the synapses can take place. For the remodelling function, the reactive astrocytes release neurotrophic factors and synaptogenic factors which promote neuronal survival, synapse formation and plasticity. For example, astrocytes are known to release thrombospondin-1, which is a major regulator of synaptic maturation and tissue plasminogen activator which may be required for recovering neurons to remodel their dendritic arbors. Reactive astrocytes are also linked to the development of new neurons. One mechanism identified for this following ischemic injury is that specific astrocytes (possibly NPC-astroglial cells and Olig2PC-astroglial cells) can acquire stem cell traits. This is observed by the induction of neurogenesis from a GFAP-expressing progenitor cell in the SVZ. These give rise to a reactive astrocytes subpopulation in the cortex that contribute to astrogliosis and scar formation. These astrocytes in the SVZ can also be converted to neurons in vivo by forced expression of Ascl1 and hence, lead to the formation of and migration of newly born neurons into a unique neurovascular niche. This is of particular importance for the hippocampal area and cognitive functioning since the area relies on newly formed neurons for memory capability. It is also found that in the hippocampus, reactive astrocytes preserve the function of the hippocampal neural niches which play a part in adult neurogenesis. It is found that astrocytes can allow the synaptic integration of adult-born hippocampal neurons, allowing local dendritic spine maturation and NMDAR functional integration. Astrocytes also secrete certain proteins, eg. beta-arrestin-1 in the dentate gyrus area of the hippocampus which aids in neurogenesis via expansion of the neural precursors in this region.
Part of the astrocyte response to ischemic injury and characteristic of it is glial scar formation which can be both positive and negative. On the positive side glial scar formation in areas surrounding severe ischemic lesions is characterized by astrocyte proliferation and a considerable extension of astrocytic processes beyond the previous domains of individual astrocytes. Upregulation of GFAP and other genes and pronounced hypertrophy of cell bodies and processes and interaction with other types of glial cells all occur. However, on the negative side, glial scar formation has been considered an inhibitor of axon regeneration and a factor that can cause neurotoxicity, inflammation, or chronic pain and this is supported by the observed increases in inflammatory factors such as interleukins 1β, 6, 10, IFN, TGFbeta as well as inflammatory molecules such as ROS, NO, glutamate and calcium-binding protein B. The immune response instigated is indicated by the expression of neurotrophic factors and by the expression of class II major histocompatibility complex (MHC) molecules which play a critical role in the induction of immune responses through the presentation of processed antigens to CD41 T-helper cells. This molecule is normally expressed on the known antigen presenting cells (APCs), such as B cells, macrophages, dendritic cells, and other cell types which includes the more unusual astrocytes. Reactive astrocytes also release chemokines after ischemia. In this case, chemokines in vascular endothelial cells increase adhesion molecules levels, attracting immune cells. The reactive astrocytes also are capable of expressing pattern-recognition receptors (PRRs), such as TLRs, scavenger receptors, and complement proteins. They are also resistant to apoptosis induced after inflammation by activation of the death receptors (eg. apoptosis antigen 1 and tumor necrosis factor (TNF)-related apoptosis-inducing ligand (FAS, TRAIL). Therefore, the cells themselves are capable of surviving inflammatory insults as well as playing a complex role in the local regulation of immune reactivity.
The final function of reactive astrocytes and important because of the type of damage occurring in ischemic injury is that they can play a role in the restoration of the vascular system. This is important since ischemia causes disruption and damage to blood flow and oxygen supply. Under normal circumstances, astrocytes have very close interactions via membrane proteins such as ion channels and growth factor and cytokine receptors. They and endothelial cells can also release neurotrophins, vascular growth factors, glucose and amino acids in order to generate stability and maintenance of the blood brain barrier for example. In ischemic injury the signalling between the astrocytes, pericytes and endothelium becomes disrupted and hence, repair to the gliovascular system is required. Astrocytes following ischemic injury modulate the cytotoxic response and induces the necessary angiogenesis in order to re-establish blood flow. This astrocytic mechanism requires secretion of a number of factors including TGFbeta, glial-derived neurotrophic factor (GDNF), FGF2 and angiopoietin 1 (Ang1). These factors can stimulate the production of new blood vessels and the proliferation of endothelial progenitor cells. Therefore, another function of reactive astrocytes is the re-establishment of blood flow.
With reference to Lu and team`s article, their research supports the other observations reported that GCI leads to the activation of astrocytes in their control FLOX mouse model (200% max, as shown by GFAP/vimentin) with no change in astrocyte number, but an increase in cell body volume which could indicate phagocytic functionality. The astrocytic response showed the expected temporal characteristics following the GCI administration with reports at 3 days, the best response at 7 days and possible recovery at 14 days. However, their research looked at the question as to the role of E2 in response to ischemia particularly in relation to astrocytes. The role of E2 was deduced by using a mouse model where the neuronal derived contribution was absent (FBN-ARO-KO mouse) and even the ovarian contribution was removed (ovariectomised FBN-ARO-KO mice).
So, what did Lu and team find and how can we interpret their findings? They found that under normal circumstances the control mouse (FLOX) on GCI exhibited strong activation of astrocytes (A2 variety) and a slight increase in E2 level (FLOX to FLOX+GCI – 100 to 120%). However, in the ovariectomised FBN-ARO-KO mouse with GCI, there was little astrocyte activation (approx. 20% max) although what was observed was time sensitive even if low and the temporal changes matched that of the FLOX controls. Again, there was no change in astrocyte number, but there was also no change in astrocyte volume implying non-involvement of astrocyte reactivity in the FBN-ARO-KO response to GCI. There was also no change in E2 level (FBN to FBN+GCI – no change – 33% of FLOX level). Therefore, from the aspect of E2 level, in normal mice the GCI stimulates E2 production since aromatase is produced in the reactive astrocytes so E2 is present in the astrocytes following ischemic assault (aromatase production increases time sensitive 7 days 250%, 14 days 70). Therefore, aromatase appears to be produced in response to this type of adverse condition. However, in the case of the FBN-ARO-KO mice, no increase in E2 is seen so in the FBN mice no stimulation is observed with GCI or if stimulated then no production is possible because of the lack of the aromatase enzyme. The increase in ER in response to ischemia is also likely to be lacking in the FBN-ARO-KO mice model.
From the astrocyte aspect, the control FLOX mouse model produced the expected proliferation and reactivity astrocytic response identified for ischemic injury (200% rise and predominantly A2 subtype). It also appeared time sensitive as described above (time sensitive aromatase production increase 7 days 250%, 14 days 70; astrocyte reactivity – best at 7 days). However, there was little astrocyte reactivity in the ovariectomised FBN-ARO-KO mice which implies that the absence of E2 suppresses the induction of the astrocyte response following ischemic injury in the mouse model.
How does the absence of E2 do this in the mouse model? The first explanation is that the removal of the excitotoxic glutamate released as a result of the ischemic injury is lower in the FBN-ARO-KO model and hence, there is more damage. Lu saw in the experiments that the level of glutamate transporter GLT-1 was lower in the ovariectomised FBN-ARO-KO mice than the control FLOX mice following GCI. This would result in higher levels of glutamate and hence, increased neuroinflammation and apoptosis through other non-E2 controlled means resulting in higher levels of damage seen. The astrocyte proliferation and shift to reactivity occurs even in the FBN-ARO-KO mouse model, but like most biochemical processes it acts as an interacting cascade and is self-proliferating and therefore, one process not activated at an early stage can have major consequences for the initiation of dependent processes both simultaneously and later on.
The second explanation also relates to damage instigated through the ischemic assault. Structural integrity is already lower in the FBN-ARO-KO mouse model since E2 is deficient which would normally aid in the mouse model in the synaptic modelling and regeneration processes. This is important in the interpretation of Lu`s experiments because of the dynamic nature of the hippocampus CA1 area corresponding to its memory and information gathering functions. Therefore, lower structural integrity would imply a greater propensity to damage and cell death during destructive assaults and decreased capability for regeneration and renewal not only when under assault, but also under normal circumstances. Without the reactive astrocytes removing the cell debris and instigating remodelling processes following the GCI, the FBN-ARO-KO mice in particular are more likely to exhibit damage which is in itself, a signal for higher levels of apoptosis.
The third explanation again links back to the cascade like processes that occur in the neurons, glials and synaptic spaces. Neuronal and glial cells function is dependent on neurotrophic factor actions. Two in particular, IGF-1 and BDNF, are cited in the role of E2 and its aid to reactive astrocyte functioning in the areas of neuronal synapse growth and renewal following ischemic injury. Therefore, lack of E2 as in the case of the FBN-ARO-KO mice means that these important neurotrophic factors are lacking and hence, their functions are not elicited. In the case of IGF-1, this acts in the early post-ischemic period to decrease apoptosis cell death and to promote vascular remodelling. When E2 is present, the IGF-1 and ERalpha bound with E2 forms a complex which interacts at the DNA sites to promote ERK/MAPK and CREB signalling aiding neuronal survival. Ischemia promotes rapid dephosphorylation and inactivation of the ERK/MAPK and CREB site and therefore, E2 when present performs a ´protective` effect on this particular site. When E2 is absent this protective function is not possible and therefore, ischemia can lead to the switch off of the ERK/MAPK and CREB sites, hence reducing neuronal survival. Although pre-treatment with E2 in situations where E2 is absent can lead to a reversal of the ischemic injury responses, a blockade of the ERK/MAPK site does not prevent the ischemic down regulation of Bcl-2, one of the anti-apoptotic proteins. Bcl-2 is a target of CREB and hence, a target of the ERalpha-E2 complex and would be expected to be at normal levels following E2 pre-treatment. However, its continual down regulation in the presence of E2 means that another factor or factors must also control Bcl-2 levels in response to ischemia. This is highly likely and indicates the complexity of the neurotrophic factor functioning. With regards to Lu and team`s experiments, they found that IGF-1 greatly increased in the control mouse model following GCI compared to the FBN-ARO-KO model where there was a smaller increase. Therefore, the response to ischemia in the presence of E2 was as expected with the IGF-1 performing a neuroprotective partnership and IGF-1 being induced probably due to the reactive astrocyte induction. In the absence of E2 only a slight rise in IGF-1 was observed, probably attributed to the induction by the lower level of reactive astrocytes present in the FBN-ARO-KO model.
BDNF followed the same pattern as IGF-1 in Lu and team`s experiments with a massive upregulation in BDNF in the control FLOX model and a smaller increase in the E2 deficient FBN-ARO-KO model. This again, can probably be attributed to induction of BDNF by the reactive astrocytes and represents the level of reactive astrocytes present not only in the control, but also the FBN-ARO-KO mice. Therefore, the results confirm that the lack of astrocyte reactivity in response to the glutamate increase and ROS from the neural damage caused by the ischemic assault means that not only was the astrocyte dependent debris removal and remodelling processes not put into play, but the synaptic remodelling aided by the E2 processes was also not available.
However, Lu and team`s experiments did show a different effect with the neurotrophic factor, FGF2 and indicated a relationship between E2 and reactive astrocyte response to GCI assault. Lu and team saw in their experiments that in the absence of E2 there was a large increase in FGF2 levels in the ovariectomised FBN-ARO-KO model after GCI (220%) than with the control model, FLOX+GSI+ova (60) which decreases or stays relatively the same. The results were mirrored by the experiments looking at the ligand binding FGF3 receptors (FGF3Rs) which also confirmed that endogenous E2 would remedy the situation (ie. levels would decrease in the FBN-ARO-KO mouse model). (For the sake of the debate we have to assume that FGF2 level reflects overall FGF levels. This is because FGF signalling is interchangeable and that factors FGF1 to 4 can bind to more than one receptor. Therefore just because FGF2 signalling is decreased or increased does not mean that overall function of the FGF neurotrophic factors is increased or decreased.) One explanation for this rise is that it is a result of the increase in damage observed which is worsened by the lack of E2 rather than a direct effect of E2 itself. FGF2 signalling is normally suppressed by E2 in the mouse model or alternatively, in the presence of E2 there is less damage to the CA1 after ischemic assault. This is because FGF2 is reported as being secreted by neurons that are damaged by glutamate as in the case of glutamate excitotoxicity in ischemic injury (Noda). In this case, FGF2 enhances the microglial migration and phagocytosis of the cellular debris and is neuroprotective via the FGF3R extracellular signal regulated kinase ERK pathway which is directly controlled by the Wnt signalling in the microglia. Therefore, the FGF2 effect in ischemia is via microglial activation and therefore, in the case of the control mice FGF2 increases may not be so high where there is a high level of astrocyte functioning for the debris and re-modelling situation as in the FBN-ARO-KO mice where the level of reactive astrocytes is low.
However, FGF2 signalling is linked to astrocytes directly and therefore, the low level activation of astrocytes in the ovariectomised FBN-ARO-KO mouse model following GCI means that this contribution is reduced. This probably occurs by reduced levels of appropriate migration of newly formed neurons. This view comes from the observation that in the subventricular zone (SVZ) of the hippocampus DG region, FGF2 is highly expressed normally by GFAP-positive cells (ie. glials and astrocytes) which suggests a pro-proliferation role for the astrocytes in this area. It is found that acute stress leads to enhanced neurogenesis in the SGZ and astrocytic FGF2 plays a role. FGF2 deficient mice however, show normal neuronal progenitor proliferation during development, but the progenitor cells fail to colonise their target layers in the cortex. This implies that FGF2 controls fate, migration and development of neuronal progenitor cells rather than proliferation during development. In addition, FGF2 is believed to modulate synaptic plasticity (enhances neurite growth, promotes LTP) and axonal branching in the hippocampus. Therefore, in situations where remodelling is required and total astrocytic guidance of this is not possible, FGF2 signalling would compensate.
Therefore, to conclude it has been shown ischemic assault in the given mouse models has a detrimental physiological effect that manifests as structural and functional changes of the hippocampal CA1 area. These physiological changes lead to cognitive functional impairments. In response, there is reactive astrocyte proliferation which carries out a number of neuroprotective processes including neurite growth and synaptic remodelling in order to promote neuronal survival and restore cognitive behaviour. When this is not possible to the degree required, then neuronal degeneration and apoptosis follows. The reactive astrocytes are aided in their neuroprotective effects by the action of E2. The E2 help comes in the form of specific structural synaptic changes in the mouse models indicated, eg. synaptic growth and spine density. The suggested link between absence of E2 and superior FGF2 signalling as suggested by Lu and team may be only indirect. It is likely that the high levels of FGF2 observed come from the increased microglial activation observed due to the lower structural integrity of the neurons and synaptic area not only in the control mouse model, but also increased after the damage caused by the ischemic injury.
For this reason, two things should be noted. The first is that it cannot be assumed that there is a relationship between two factors in one condition just because both things change when that condition changes and secondly, that in the case of E2 (and probably other hormones) the situation in mouse models may not directly be translated to the situation in humans. This refers to the lack of E2 and estrogens reported in human female menopause having reported effects on physiology and cognitive functioning compared to the same lack in the mouse model. For example, lots of research in humans relates to E2 loss and pre-treatment of E2 before ischemic events, but there are disputed effects. Estradiol administered at levels used for hormone therapy in postmenopausal women is reported as intervening in apoptotic cascades via increased caspase 3 activity in male gerbils which leads to strong protection against the ischemic injury induced cell death in the CA1. However, not all human studies show the same cognitive advantage when missing hormones are replaced. Therefore, mouse models can provide an idea of what may happen, but the demands on brain functioning in mice are different to humans, eg. spatial memory is strongly required in mice whereas the dominant sensory system in humans is visual. Transfer of knowledge about structure and function from one species to another should always be carefully scrutinised for validity.
Since we`re talking about the topic………………………………………………
…..it has been shown that the hippocampal synapses of aged female rats respond differently to estradiol than the synapses of younger mice and that there is an age-related decline in IGF-1. Can we assume that if the above experiments were repeated with aged mice, the FLOX mice would show similar results to the FBN-ARO-KO mice since estradiol levels would be naturally reduced?
….it has been shown that ischemic tolerance develops in heart tissue and in the gerbil brain when the subject is subjected to a series of brief sublethal ischemic insults. If the experiment`s of Lu and team are repeated, but the GCI at a lower level and spaced over a number of days, would the CA1 area of the FLOX mice demonstrate this ischemic tolerance, but the FBN-ARO-KO mice still show extensive damage?
…if E2 has a neuroprotective effect, would the administration of the ER antagonist ICI 182 780 to the FLOX+GCI mice lead to a reduction in astrocyte reactivity and neuroprotection following GCI that is in line with that shown by the FBN-ARO-KO mice?
