In humans with aSAH, possible interrelations between cerebral vasospasm and the steroids estradiol, progesterone and testosterone in peripheral compartments, e.g. blood, or in central compartments, e.g. CSF, have not yet been comprehensively assessed. Given numerous studies in rodents on steroids, predominantly estradiol, and its association with cerebral vasospasm after experimental SAH, the idea that steroids might be involved in processes linked to cerebral vasospasm or functional outcome remains to be investigated in more detail. This investigation analyzed the correlations between transcranial Doppler flow velocities in cerebral arteries and concentrations of estradiol, progesterone and testosterone in serum and CSF of patients with aSAH. The results showed very weak correlations between the transcranial Doppler flow velocities and the steroid concentrations in both compartments.
The neuroprotective influences attributed to estradiol in aSAH comprise vasodilatory effects and general neuroprotective mechanisms. With regard to the vasodilatory effects of estradiol, such as countermanding vasoconstriction or promoting vasodilation, studies focused on nitrogen monoxide (NO), reactive oxygen species (ROS) and endothelins (ET) since endothelial damage and inflammation after aSAH remain discussed as key determinants of cerebral vasospasm. Various nitric oxide synthase (NOS) enzymes are identified, there among endothelial NOS (eNOS), neuronal NOS (nNOS) and inducible NOS (iNOS) (8). Chen et al. demonstrated in studies on ovine endothelial cells that the vasodilatory effects of estradiol are mediated by estradiol’s capacity to activate eNOS and that the activation of eNOS was impeded by blocking the estrogen receptor (9). Likewise, an in vitro study by Nevzati et al. in human umbilical and brain endothelial cells treated with estradiol showed a rise in NO and eNOS concentrations presumably due to estradiol mediated pathways (10). Also, Selles et al. showed in endothelial cells of rat aorta that treatment with estradiol and progesterone enhanced NOS activity (11). In a canine SAH model, Khurana et al. found a protective effect of recombinant eNOS against cerebral vasospasm (12). Studies have also addressed the issue of inflammation as a critical factor for vasospasm and evaluated a potentially positive impact of steroids on these processes. SAH may entail formation of oxy-hemoglobin and inflammatory cascades with an increase of ROS thereby decreasing NO levels and thus facilitating cerebral vasoconstriction (3, 13, 14). Furthermore, Ding et al. discussed that vascular inflammation may enhance iNOS activity resulting in an increased ROS generation hence promoting inflammatory pathways and cerebral vasospasm (2). In this respect Handa et al. found in primates good correlations between angiographic vasospasm and inflammatory reactions in cerebral vessels (3). In a rodent model of SAH Lin et al. assessed the extent of vasospasm by measuring cross-section surfaces of the basilar artery and analyzing the expressions of eNOS and iNOS after treatment with estradiol (15). In the view of Lin et al., the findings indicate vasodilatory effects of estradiol on cerebral arteries by averting an increase of iNOS expression and maintaining regular eNOS levels (15). Zancan et al. demonstrated in smooth muscle cell cultures of rat aorta that estradiol decreased the quantity and activity of iNOS presumably via the estrogen receptor and may also attenuate a provoked iNOS rise (16). Likewise, in a rat model of subarachnoid hemorrhage, Shih et al. showed that the treatment with estradiol inhibited vasospasm as well as elevations of iNOS protein levels (17). In the context of cerebral vasospasm and endothelial damage other studies focused on endothelins (ET) with ET-1 being the isoform exerting strong vasoconstrictive properties (18, 19). As ET-1 may foster cerebral vasoconstriction in aSAH, Lin et al. investigated the effect of treatment with estradiol on ET-1 levels and cerebral vasospasm in a SAH rat model (20). Lin et al. found a significant correlation between cross-section surfaces of the basilar artery and concentrations of ET-1 with a decrease of ET-1 levels in the rodents treated with estradiol thus suggesting an advantageous impact of estradiol on vasospasm (20). Interestingly, Macia et al. analyzed the temporal relationship between increased ET levels and neurological deterioration in patients with aSAH and, however, found increased ET levels in patients with poor neurological condition apparently independent of the existence of cerebral vasospasm (21).
The neuroprotective influences of progesterone and testosterone have been investigated to a lesser extent. In a rat model with induced SAH Chang et al. showed that both the vasospasm and the lowering of eNOS protein levels in these rodents could be counteracted by therapy with progesterone (5). In a mice SAH model Turan et al. demonstrated that progesterone treatment decreased cerebral vasospasm and had beneficial effects on behavior patterns (22). However, as mentioned above, Zancan et al. demonstrated in cells cultures of rat aorta that estradiol decreased the content of iNOS but such an effect could not be shown with progesterone (16).
The relevance of testosterone to SAH remains rather undetermined as studies are scarce. In rabbits with induced SAH, Gurer et al. evaluated the vasodilatory and neuroprotective properties of testosterone and reasoned that intraperitoneal application of testosterone protected against cerebral vasospasm and neuronal damage (23). Selles et al., however, noted that the enhanced NOS activity in endothelial cells of rat aorta after treatment with estradiol and progesterone could not be shown for testosterone (11).
In the present study, in humans with aSAH, merely very weak correlations were found between transcranial Doppler flow velocities of cerebral arteries and concentrations of estradiol, progesterone and testosterone in serum or CSF. In contrast, the aforementioned studies in rodents with experimental SAH demonstrated beneficial effects of estradiol and progesterone on cerebral vasospasm, often using cross-section surfaces of cerebral arteries as a parameter (5, 15, 17, 20). In the view of these studies, the findings in rodents appear not to be in accordance with the results of our study in humans. However, the comparability of studies using different parameters to evaluate cerebral vasospasm, such as cross-section of arteries and Doppler flow velocities respectively, may be limited. To the best of our knowledge, there are no studies in rodents on flow velocities of cerebral arteries after experimental SAH. In addition, caution is required when comparing the findings in rodents with the findings in humans. Lin et al. also stated in their study in rodents on the attenuation of cerebral vasospasm after experimental SAH by estradiol that their findings in rats may not necessarily be transferred to humans (4). These considerations apply and complicate the interpretation of the results in the present study.
In that regard, the discussion of the results of the present study should include general considerations on steroid metabolism. In the periphery, the steroid hormones progesterone, testosterone and estradiol are synthesized in steroidogenic tissues pursuing effects in the periphery and may also cross the blood-brain barrier and act centrally (24, 25). Within the brain, steroids can also be synthesized de novo (26–28) although the mechanisms regulating this synthesis remain mostly unclear (25, 27, 29–31). In addition, a conversion of steroids by neural cells into metabolites also occurs (32, 33). In our previous study on males without neurological disorders or diseases only weak to very weak correlations were found for estradiol, progesterone and testosterone between the CSF and serum compartments thus suggesting that concentrations in the periphery do not parallel concentrations in the central compartments (34). Also, the synthesis and conversion of steroids further increases the complexity in which these molecules affect the function of the nervous system and related structures as for example blood vessels. It thus remains unclear, to what extent an administration of exogenous estradiol, as performed in the aforementioned rodent studies, may result in altered estradiol concentrations in the respective compartments, e.g. blood, CSF or brain parenchyma, thereby potentially influencing the course of cerebral vasospasm. Further research is needed to clarify these aspects.
It is understood that a poor neurological condition or a neurological deterioration may be due to cerebral vasospasm but a poor neurological condition can also be attributed to proapoptotic mechanisms unrelated to cerebral vasospasm (2, 35). Studies in rodents suggest that steroids may have favorable influences on apoptotic pathways, there among studies in rodents with traumatic and ischemic brain injury (36–39) as well as studies in rodents with experimental SAH (4, 40–43). However, in the present study, with regard to possible general neuroprotective effects of steroids, the group comparisons between steroid concentrations in the respective compartments and the grading of the Hunt & Hess scale, the grading of the Glascow Outcome scale and the presence of cerebral infarction revealed no significant differences. Interestingly, in our previous research evaluating cognitive performance and histological damage in rats with experimental SAH no differences were found between rodents with different hormonal status (44).
The strength of the present study is its clinical approach with a study cohort consisting of humans with aSAH as rodent models of experimental SAH may not reliably represent the pathophysiological processes of spontaneous aSAH. To the best of our knowledge, there are no investigations in humans with aSAH on correlations between transcranial Doppler flow velocities of cerebral arteries and concentrations of steroids in blood or CSF. In the present study, analyses of estradiol, progesterone and testosterone were performed in both CSF and blood thus not relying solely on measurements in the blood compartment which may not adequately describe concentrations in the CSF (34). A further strength lies in the multiple measurement points after the initial bleeding resulting in approximately 280 observations for the calculation of each correlation.
Limitations of the present study must be considered. Measurements of steroid levels have long been a matter of debate and various methods are described in the literature (45), e.g. radioimmunoassay, enzyme immunoassay, liquid chromatography, gas chromatography-mass spectrometry, competitive protein binding assay. The electro-chemiluminescence immunoassay used in the present study was also used by Schonknecht et al. for the measurement of estradiol levels in CSF of humans with Alzheimer`s disease (46) as well as in our previous study on males without neurological disorders for the measurement of estradiol, progesterone and testosterone in CSF and serum (34). Steroid concentrations in CSF were also determined with an enzyme immunoassay by Kawass et al. and Brundu et al. (47, 48). Still, methodological deficiencies cannot be excluded. Another limitation is that steroid concentrations representing the central compartment were measured in the CSF obtained via ventricular drainage; thus, these measurements are coupled with the uncertainty that they adequately reflect the respective brain steroid concentrations. Consequently, the findings of this study regarding correlations between flow velocities of cerebral arteries and central steroid concentrations rest on concentrations in the CSF but not on brain parenchyma levels.