**4. Neuroactive steroids**

Neuroactive Steroids were defined by Baulieu [128] as steroids synthesized in the nervous system capable of inducing neuronal excitability [129]. Compounds as dehydroepiandrosterone, androstenedione, and deoxycorticosterone meet the requirements to be categorized as neuroactive steroids. Interestingly, neuroactive steroids induce responses on GABA receptors and modulate the activity of 5α and 3α reductases affecting steroid synthesis [130–132]. In this regard, neuroactive steroids can be exogenously synthesized and produce similar effects on the CNS. In the current definition neuroactive steroids are molecules capable of inducing several effects on CNS including ion channel modulation, voltage-dependent calcium channels activation and AMPA-NMDA receptors activation [133–135]. Besides the neuroactive properties of steroids, there are a plethora of protective functions characterized on neurons, astrocyte and microglia [136–139]. The effects of neuroactive steroids on neurons include the increase of dendritic spines, viability, antioxidant capacity [140, 141]. On astrocytes, neuroactive steroids improve the mitochondrial function, modulate the synthesis of antioxidant molecules and growth factors and pro-survival factors as Bcl-2 [142–145]. Finally, on microglia, the effects include the modulation of immune response via regulation of the synthesis and secretion of cytokines and inflammatory mediators [139].

Neuroactive steroids may induce both genomic and non-genomic mechanisms associated with its protective effects [146]. The genomic mechanisms involve the modulation of pro-survival genes, anti-inflammatory [147] and anti-apoptotic functions [148]. For example, the activation of signaling pathways like Akt-PI3K and MAPK, and the upregulation of the anti-apoptotic mediators like Bcl-2 and antioxidant enzymes like SOD and GPx [149] are under control of Neuroactive steroids. Other mechanisms include the downregulation of pro-inflammatory cytokines such as IL-1β, IL-6 and TNF-α [150]. The non-genomic effects include the antioxidant properties of some neurosteroids, especially the ones that include an A-phenolic ring in their chemical structure [151]. Interestingly, some neuroactive steroids are capable of exerting its effects through G-protein coupled receptors, for example via GPR30 receptor [152]. Until now, there is a large body of evidence demonstrating the beneficial effects of neuroactive steroids following ischemia/ reperfusion and traumatic brain injury (TBI) in animal models (Liu et al., 2005; O'Connor et al., 2005) as well as steroid-demonstrated effectiveness in glucose deprivation and oxygen–glucose deprivation in in vitro models [148]. Despite this evidence, the direct use of estrogens is not fully recommended and still represents a potential risk for human health [153, 154] (For further evidence, see **Table 3**). In fact, it has been documented that the use of estrogen and progesterone increases the risk to develop breast and uterus cancer, as well as, vascular diseases, brain hemorrhage and clotting disorders [155–159]. To circumvent these issues, selective compounds that mimic the protective action of neuroactive steroid without the side effects were developed. These compounds were defined as selective estrogen receptor modulators (SERMs) and selective tissue-specific estrogenic activity regulators (STEARs). SERMs and STEARs exert their actions as estrogenic agonists or antagonists depending on the target organ [146, 160]. Tissue selective properties of SERM and STEAR are currently under investigation (**Figure 1**).


**221**

**Table 3.**

*Neuroactive Steroids in Hypoxic–Ischemic Brain Injury: Overview and Future Directions*

pathology.

investigated.

[208] Women study Women at both extremes of the weight spectrum have low

[211] Animal model DHEAS and progesterone were good predictors of HPA Axis

[212] Clinical study The first-episode antipsychotic-naive schizophrenic patients

caused by cortisol.

modulate anxiety-like behavior.

deficits in women compared to men.

[215] Aged human study We observed a significant difference in plasma concentration of

Nervous diabetic complications show sex dimorphic features. In this regard, sex-oriented therapies with neuroactive steroids might be aimed to counteract nervous damage observed in diabetic

Neuroactive steroids under pathological conditions may alter their levels involving sex differencies in the outcome. Neuroactive steroid may be considered as neuroprotective factors to be deeply

Some studies point to a lag between neuroactive steroid dysregulation and subsequent symptoms. The study also consider key interactions with other aspects of neuroactive steroid physiology, such as synthetic enzymes or receptor plasticity.

There is a very close link among neuroactive steroids and the control of metabolic axis to understand the biological basis of many pathologies based on metabolic alterations, for example the

mean serum allopregnanolone. Neuroactive steroids such as allopregnanolone may be potential therapeutic targets for depression and anxiety in traditionally treatment-resistant groups.

Several Compounds have completed a phase 1 single ascending dose (SAD) and multiple ascending dose (MAD) clinical trial and is currently being studied in parallel phase 2 clinical trials for the treatment of postpartum depression (PPD), major depressive

showed a significantly higher blood level of DHEA-S compared with healthy controls. On the other hand, serum DHEA-S level has an inverse relationship with aggression and may serve as a biological adaptive mechanism to antagonize the neuronal damage

Clinical trials designed to test neuroactive steroid therapeutics in PTSD may benefit from such considerations. However it is needed to validate clinically accessible methods for identifying specific neuroactive steroid system abnormalities at the individual level.

Strain variation in neuroactive steroid levels correlated with numerous behavioral phenotypes of anxiety sensitivity accessed in GeneNetwork, consistent with evidence that neuroactive steroids

cortisol and estradiol between experimental groups. In the AIS group, higher levels of these neuroactive steroids were associated with more pronounced neurological, cognitive and functional

Low levels of neuroactive steroids could have a part in development of depression, neuro-inflammation, multiple sclerosis, experimental autoimmune encephalitis, epilepsy, and schizophrenia. On the other hand, stress and attention deficit

metabolic syndrome, obesity or diabetes.

disorder could occur during high levels.

disorder (MDD), and essential tremor (ET).

dysfunction and outcome in hospitalized foals.

*DOI: http://dx.doi.org/10.5772/intechopen.93956*

[204] Animal and human studies

[205] Animal and human studies

[206] Animal and human studies

[207] Animal and human studies

[209] Animal and human studies

[210] Animal and human studies

[213] Animal and human studies

[214] Animal and human studies

*Neuroactive steroids used in experimental models and clinical studies.*

**Reference Type of study Outcomes**


*Neuroactive Steroids in Hypoxic–Ischemic Brain Injury: Overview and Future Directions DOI: http://dx.doi.org/10.5772/intechopen.93956*

#### **Table 3.**

*Neuroprotection - New Approaches and Prospects*

and STEAR are currently under investigation (**Figure 1**).

dysfunction

illnesses.

[201] Human study Individual domains of cognitive can be considered as an

disorder

states for psychosis [202] Animal model Exposure to neuroactive steroids induced a sustained elevation in

The evidence summarized supports the idea that MDD and PPD are psychiatric disorders involving neurosteroids and GABAergic

The study shows potential mechanisms that underlie sex-related differences in behavior and its implications for stress-related

The negative cognitive consequences of sleep deprivation may arise from the effort of the brain to counteract the detrimental effect of

Progesterone might be a promissory marker for identifying continuous endogenous production of neuroactive steroids in foals

endophenotype of psychosis. It is possible that higher levels of cortisol and testosterone in siblings are consistent with high-risk

tonic current in Fmr1 KO mice. Neuroactive steroids may act to reverse the deficits of tonic inhibition seen in FXS, and thereby reduce aberrant neuronal hyperexcitability associated to this

Cortical GABA+/Cr concentrations are associated with postpartum RSFC. It is possible that allopregnanolone may be associated with

sleep loss via compensatory mechanisms

with suspected NMS and other diseases

postpartum intra-DMPFC connectivity.

**Reference Type of study Outcomes**

and animal studies

[197]9 Human Psychiatric study

[198] Comparative human

[199] Animal and human studies

[200] Animal (neonatal foal) study

[203] Peripartum

depressed women

Neuroactive steroids may induce both genomic and non-genomic mechanisms associated with its protective effects [146]. The genomic mechanisms involve the modulation of pro-survival genes, anti-inflammatory [147] and anti-apoptotic functions [148]. For example, the activation of signaling pathways like Akt-PI3K and MAPK, and the upregulation of the anti-apoptotic mediators like Bcl-2 and antioxidant enzymes like SOD and GPx [149] are under control of Neuroactive steroids. Other mechanisms include the downregulation of pro-inflammatory cytokines such as IL-1β, IL-6 and TNF-α [150]. The non-genomic effects include the antioxidant properties of some neurosteroids, especially the ones that include an A-phenolic ring in their chemical structure [151]. Interestingly, some neuroactive steroids are capable of exerting its effects through G-protein coupled receptors, for example via GPR30 receptor [152]. Until now, there is a large body of evidence demonstrating the beneficial effects of neuroactive steroids following ischemia/ reperfusion and traumatic brain injury (TBI) in animal models (Liu et al., 2005; O'Connor et al., 2005) as well as steroid-demonstrated effectiveness in glucose deprivation and oxygen–glucose deprivation in in vitro models [148]. Despite this evidence, the direct use of estrogens is not fully recommended and still represents a potential risk for human health [153, 154] (For further evidence, see **Table 3**). In fact, it has been documented that the use of estrogen and progesterone increases the risk to develop breast and uterus cancer, as well as, vascular diseases, brain hemorrhage and clotting disorders [155–159]. To circumvent these issues, selective compounds that mimic the protective action of neuroactive steroid without the side effects were developed. These compounds were defined as selective estrogen receptor modulators (SERMs) and selective tissue-specific estrogenic activity regulators (STEARs). SERMs and STEARs exert their actions as estrogenic agonists or antagonists depending on the target organ [146, 160]. Tissue selective properties of SERM

**220**

*Neuroactive steroids used in experimental models and clinical studies.*

#### **Figure 1.**

*Potential Neurosteroids action mechanism. The effects of neurosteroids on neurons include the increase of dendritic spines, viability, and antioxidant capacity. The action mechanism is associated to classical (canonical) transduction pathway that includes the transactivation of estrogen receptor to dimerize and promote the transcription of estrogen response elements ERE. For tibolone, it is described the classical transduction pathway but also the transactivation of androgen response elements ARE and progesterone response elements PRE. It is possible that all together response elements explain the beneficial and protective properties of tibolone. Interestingly, the protective properties also has been observed on astrocytes and microglia.*

#### **4.1 Selective estrogen receptor modulators**

The activation or partial activation of Estrogen receptors (ER) trigger critical signal pathways due to complex molecular mechanisms. ER interact with several endogens and exogenous ligands promoting structural changes with the subsequent transactivation of estrogen response elements (ERE) in the DNA. ER interact also with co-activators, co-repressors and chaperones, affecting the way that the tissues exert their estrogenic response [161, 162]. ER show structural components that may be involved in their particular action mechanism. One of the most striking domain is the ligand binding domain (LBD) that interacts with specific ligands [163] (Cano et al., 2006). It is believed that the high or low affinity of the ligand with LBD plays a central role in the function of ER. Ligand interaction with LBD induces conformational changes that lead to specific bind to activators with co-activators and co-repressors modulating the estrogenic response [161, 164]. In this context, the conformational change is predetermined in part by the chemical nature of the ligand and its interaction with ER [165]. SERMs are capable of exploiting this advantage. A clear example is tamoxifen, a selective compound with estrogenic activity in the liver, but anti-estrogenic activity in breast tissue [166]. These compounds have been widely used in clinics for the treatment of breast cancer and as hormonal replacement therapy (HRT) strategies [167]. SERMs are defined as compounds that are capable of binding ER and produce several responses, ranging from a pure estrogenic agonism

**223**

*Neuroactive Steroids in Hypoxic–Ischemic Brain Injury: Overview and Future Directions*

to an anti-estrogen activity [146]. SERMs may protect nervous tissue following spinal cord and traumatic brain injuries [168, 169]. Gonzales-Burgos et al. (2012) demonstrated that SERMs increase the number of dendritic spines in hippocampal neurons [170]. Raloxifene, a second-generation SERM, demonstrated to improve sensory motor and working memory deficits following TBI [168], suggesting that SERMs may

SERMs action mechanisms include the activation of transcription factors such as NF-κB through the PI3K-P38-ERK1/2 pathway [146]. SERMs also induce the production of antioxidant enzymes such as manganese superoxide dismutase (MnSOD) [171] and the endothelial nitric oxide synthase (eNOS) [172]. Interestingly,

SERMs may induce the upregulation of anti-apoptotic proteins such as Bcl-2 [173]. Altogether, the activation of these multifactorial protective signaling cascades may improve the outcome of highly heterogeneous pathologies like TBI and HI Brain Injury (HIBI). Currently, SERM are used as primary treatments to counter osteoporosis and some kind of cancer. Compounds like raloxifen (Evista ®) and tamoxifen (Nolvadex®) are routinely prescribed for thousand women [174, 175]. Several reports have described the protective effects of SERMs on the CNS [176–178]. It is well known that tamoxifen is capable of preserving pyramidal neurons following penetrant lesion [179]. Furthermore, raloxifen exerts protective functions by increasing glutamate reuptake via induction of GLT-1 expression on primary astrocytes [180]. However, the complete action mechanism of several SERMs needs to be

fully elucidated, due in part, to the complex agonist–antagonist action [181].

The pharmacologic necessity to develop estrogenic safe compounds against climacteric symptoms in post-menopause women lead to synthesize a distinctive compound with selective estrogenic properties. As a result, STEARs are compounds capable of inducing an estrogenic, progestogenic and androgenic response. The most used STEAR compound is tibolone [160]- Tibolone has become a well-known treatment for climacteric symptoms than other HRT compounds, especially in women suffering low libido, persistent fatigue and blunted motivation [172, 182]. Tibolone has been used in the prevention of cardiovascular diseases and osteoporosis [183, 184] Tibolone exhibits weak estrogenic, progestogenic and androgenic

The selective action mechanism of tibolone and STEARs is currently under investigation. However, it is well known that tibolone acts as a pro-drug that has complex effects due to its particular mode of action on different steroid receptors. It has been demonstrated that the body metabolized tibolone via two-phase reacts to produce three different metabolites [186]: two hydroxyl-metabolites (3-alphahydroxy- and 3-beta-hydroxy tibolone) as a result of 3-alpha and 3-beta hydroxysteroid dehydrogenase enzymes (3α-HSD and 3ß-HSD), and one isomer (delta-4 tibolone) synthesized by 3-beta-hydroxysteroid dehydrogenase [160, 183, 185]. Interestingly, 3α-HSD is predominantly expressed in the liver, whereas 3ß-HSD is expressed in adrenal glands, ovary and placental tissue [160, 183, 185]. Tibolone metabolism is under liver control by α-ketoreductases including hepatic AKR1C1 and AKR1C2 [186]. STEARs like tibolone might be metabolized by the brain, due to brain cells, for example, astrocytes fully expressing all the needed enzymes to carry out the biochemical steps. Kloobsterboer et al. 2017 demonstrated in primates (cynomolgus) the occurrence of 3α OH tibolone and 3β OH tibolone metabolites in the brain. They also detected sulfated tibolone metabolites (inactive chemical compounds) in the brain and plasma. Each metabolite has different features. For example, tibolone perse and delta-4 tibolone are agonists for progesterone receptor PR and androgen

*DOI: http://dx.doi.org/10.5772/intechopen.93956*

act as potential therapeutic compounds after CNS injury.

**4.2 Selective tissue estrogenic activity regulators**

properties [160, 183, 185].

#### *Neuroactive Steroids in Hypoxic–Ischemic Brain Injury: Overview and Future Directions DOI: http://dx.doi.org/10.5772/intechopen.93956*

to an anti-estrogen activity [146]. SERMs may protect nervous tissue following spinal cord and traumatic brain injuries [168, 169]. Gonzales-Burgos et al. (2012) demonstrated that SERMs increase the number of dendritic spines in hippocampal neurons [170]. Raloxifene, a second-generation SERM, demonstrated to improve sensory motor and working memory deficits following TBI [168], suggesting that SERMs may act as potential therapeutic compounds after CNS injury.

SERMs action mechanisms include the activation of transcription factors such as NF-κB through the PI3K-P38-ERK1/2 pathway [146]. SERMs also induce the production of antioxidant enzymes such as manganese superoxide dismutase (MnSOD) [171] and the endothelial nitric oxide synthase (eNOS) [172]. Interestingly, SERMs may induce the upregulation of anti-apoptotic proteins such as Bcl-2 [173]. Altogether, the activation of these multifactorial protective signaling cascades may improve the outcome of highly heterogeneous pathologies like TBI and HI Brain Injury (HIBI). Currently, SERM are used as primary treatments to counter osteoporosis and some kind of cancer. Compounds like raloxifen (Evista ®) and tamoxifen (Nolvadex®) are routinely prescribed for thousand women [174, 175]. Several reports have described the protective effects of SERMs on the CNS [176–178]. It is well known that tamoxifen is capable of preserving pyramidal neurons following penetrant lesion [179]. Furthermore, raloxifen exerts protective functions by increasing glutamate reuptake via induction of GLT-1 expression on primary astrocytes [180]. However, the complete action mechanism of several SERMs needs to be fully elucidated, due in part, to the complex agonist–antagonist action [181].

#### **4.2 Selective tissue estrogenic activity regulators**

The pharmacologic necessity to develop estrogenic safe compounds against climacteric symptoms in post-menopause women lead to synthesize a distinctive compound with selective estrogenic properties. As a result, STEARs are compounds capable of inducing an estrogenic, progestogenic and androgenic response. The most used STEAR compound is tibolone [160]- Tibolone has become a well-known treatment for climacteric symptoms than other HRT compounds, especially in women suffering low libido, persistent fatigue and blunted motivation [172, 182]. Tibolone has been used in the prevention of cardiovascular diseases and osteoporosis [183, 184] Tibolone exhibits weak estrogenic, progestogenic and androgenic properties [160, 183, 185].

The selective action mechanism of tibolone and STEARs is currently under investigation. However, it is well known that tibolone acts as a pro-drug that has complex effects due to its particular mode of action on different steroid receptors. It has been demonstrated that the body metabolized tibolone via two-phase reacts to produce three different metabolites [186]: two hydroxyl-metabolites (3-alphahydroxy- and 3-beta-hydroxy tibolone) as a result of 3-alpha and 3-beta hydroxysteroid dehydrogenase enzymes (3α-HSD and 3ß-HSD), and one isomer (delta-4 tibolone) synthesized by 3-beta-hydroxysteroid dehydrogenase [160, 183, 185].

Interestingly, 3α-HSD is predominantly expressed in the liver, whereas 3ß-HSD is expressed in adrenal glands, ovary and placental tissue [160, 183, 185]. Tibolone metabolism is under liver control by α-ketoreductases including hepatic AKR1C1 and AKR1C2 [186]. STEARs like tibolone might be metabolized by the brain, due to brain cells, for example, astrocytes fully expressing all the needed enzymes to carry out the biochemical steps. Kloobsterboer et al. 2017 demonstrated in primates (cynomolgus) the occurrence of 3α OH tibolone and 3β OH tibolone metabolites in the brain. They also detected sulfated tibolone metabolites (inactive chemical compounds) in the brain and plasma. Each metabolite has different features. For example, tibolone perse and delta-4 tibolone are agonists for progesterone receptor PR and androgen

*Neuroprotection - New Approaches and Prospects*

**4.1 Selective estrogen receptor modulators**

The activation or partial activation of Estrogen receptors (ER) trigger critical signal pathways due to complex molecular mechanisms. ER interact with several endogens and exogenous ligands promoting structural changes with the subsequent transactivation of estrogen response elements (ERE) in the DNA. ER interact also with co-activators, co-repressors and chaperones, affecting the way that the tissues exert their estrogenic response [161, 162]. ER show structural components that may be involved in their particular action mechanism. One of the most striking domain is the ligand binding domain (LBD) that interacts with specific ligands [163] (Cano et al., 2006). It is believed that the high or low affinity of the ligand with LBD plays a central role in the function of ER. Ligand interaction with LBD induces conformational changes that lead to specific bind to activators with co-activators and co-repressors modulating the estrogenic response [161, 164]. In this context, the conformational change is predetermined in part by the chemical nature of the ligand and its interaction with ER [165]. SERMs are capable of exploiting this advantage. A clear example is tamoxifen, a selective compound with estrogenic activity in the liver, but anti-estrogenic activity in breast tissue [166]. These compounds have been widely used in clinics for the treatment of breast cancer and as hormonal replacement therapy (HRT) strategies [167]. SERMs are defined as compounds that are capable of binding ER and produce several responses, ranging from a pure estrogenic agonism

*Potential Neurosteroids action mechanism. The effects of neurosteroids on neurons include the increase of dendritic spines, viability, and antioxidant capacity. The action mechanism is associated to classical (canonical) transduction pathway that includes the transactivation of estrogen receptor to dimerize and promote the transcription of estrogen response elements ERE. For tibolone, it is described the classical transduction pathway but also the transactivation of androgen response elements ARE and progesterone response elements PRE. It is possible that all together response elements explain the beneficial and protective properties of tibolone. Interestingly, the protective properties also has been observed on astrocytes and microglia.*

**222**

**Figure 1.**

receptor AR [185], while 3-alpha and 3-beta hydroxy metabolites are agonists for ER, but antagonists for PR and AR [185, 187]. This tibolone-steroid receptor interaction and other regulatory mechanisms might explain the tissue-selective effects of tibolone [160, 186]. Belenichev et al. (2012) used cortical neurons from neonatal rats to evaluate the neuroprotective activity of tibolone in a model of glutathione depletion that produces oxidative stress and mitochondrial dysfunction. These authors found that tibolone prevented mitochondrial dysfunction and neuronal cell death. Additional studies account for the protective effects of tibolone in an ovariectomized rat model following cerebral ischemia injury [188]. Tibolone has also shown antiinflammatory effects tested in cardiovascular animal models [184].

Kloosterboer et al. 2007 propose an additional action mechanism of tibolone and STEARs that involves the control of sulfatase and sulfotransferase tissue-specific activity [189]. Since sulfatase and sulfotransferase activity is tissue-specific, it is possible that tibolone exerts its function according to cell type specificity and modulating nuclear receptors activity in the tissues [190]. For instance, it is needed to further investigate the tissue-specific role of tibolone in CNS, for example, in neurons, astrocytes, and microglia. Interestingly, tibolone protects the mitochondrial activity by the preservation of the mitochondrial membrane potential and by increasing the levels of proteins that control the opening of the mitochondrial permeability transition pore (mPTP), such as Bcl-2. Avila-Rodriguez et al. (2014) demonstrated that tibolone protects the mitochondria of T98G glial cells from glucose deprivation [141].

De Marinis' research group recently described and characterized a particular globin belonging to CNS called neuroglobin (Ngb1). Neuroglobin is under control of estrogenic response. In fact, the use of estradiol in several cellular models demonstrated the increase of neuroglobin levels [191–193]. Currently, it is known that neuroglobin is an 18 kDa protein that binds molecular oxygen with more affinity than hemoglobin, probably, increasing the availability of oxygen in the neural tissue [194]. Neuroglobin is expressed in neurons under basal conditions and is also expressed in astrocytes and microglia after brain injury [194]. Avila-Rodriguez et al. 2016 demonstrated that tibolone is capable of increasing the expression of neuroglobin producing a protective effect in a glucose deprivation astrocyte-like model. The action mechanism of tibolone may be associated with ERß receptor as demonstrated by several studies [191, 193].

Other studies demonstrated the protective effect of tibolone against lipid peroxidation and protein oxidation [195]. Tibolone is capable of increasing the density of dendritic spines in hippocampal neurons, indicating a potential role in synaptic plasticity and memory [196]. Guzmán et al. (2007), also showed that tibolone metabolites exert estrogenic activity on human astrocytes and oligodendrocyteslike cell lines [187]. Tibolone may become a promissory option to counter the detrimental effects of TBI and hypoxic injury due to its pleiotropic beneficial properties.

#### **4.3 Selective tissue-specific estrogenic activity regulators and neuroglobin**

Pathologic conditions like hypoxia and glucose deprivation, which may lead to neuroinflammation, reduce the expression of ER-α and increase the expression of ER-ß [216]. In this regard, De Marinis et al. (2013) showed that hypoxia may induce the production of pro-inflammatory mediators like IL-6, and INF-γ [193]. Interestingly, estrogen is capable of diminishing the secretion of those pro-inflammatory mediators. It was demonstrated in a pro-oxidant model induced by H2O2 and stimulated via lipopolysaccharide (LPS). Later, it was demonstrated that the anti-inflammatory effect was mediated by NF-κB modulation and ER-ß activation [191, 193]. Therefore, it is reasonable to assume that the activation of ER-ß in hypoxic and glucose deprivation models may be considered as beneficial for brain tissues. Tibolone is capable of inducing the activation of ERß and increasing neuroglobin expression. Avila-Rodriguez

**225**

**Figure 2.**

*Neuroactive Steroids in Hypoxic–Ischemic Brain Injury: Overview and Future Directions*

et al. (2016) demonstrated that neuroglobin expression depends on ER-ß activation and tibolone favors both mechanisms [217]. Originally, neuroglobin was reported in neurons but later it was detected in other cell types such as astrocytes [218]. Interestingly, neuroglobin has been associated with neuroprotective effects on several injury models including middle cerebral artery occlusion (MCAO), focal cerebral ischemia, ß-amyloid induced toxicity, oxygen and glucose deprivation [217, 219–221]. Neuroglobin may mediate the response against hypoxia by inducing signal pathways. It has also been documented as a reactive oxygen radical scavenger with NADH oxidase activity to favor anaerobic glycolytic metabolism [217]. Controversial studies based on low levels of neuroglobin and low relative oxygen affinity propose that neuroglobin may exert or participate in collateral roles other than solely oxygen store [217, 222] (See **Figure 2** for further illustration). Additionally, photoactivation (NADH/FMN) experiments demonstrated that neuroglobin participates in the ROS and RNS elimination, suggesting a critical role in removing dangerous highly reactive species [223]. The change in the hexacoordinated state of neuroglobin according to normoxic or hypoxic conditions also suggests oxygen sensor capabilities [222]. Proper neuroglobin activity protects neurons and astrocytes against cell death [191]. In this regard, overexpression or induction of neuroglobin may be considered as potential neuroprotective therapies. Interestingly, STEARs such as tibolone are capable of increasing and inducing neuroglobin activity, which have been proposed as potential action mechanisms in

*Neuroglobin exerts interesting beneficial properties. Neuroglobin includes in its protein structure a particular prosthetic haem group to store oxygen. However, it is reported for neuroglobin additional protective functions that include oxygen sensor capabilities and detoxification properties (against reactive oxygen species and reactive nitrogen species). Evidence shows that the protective functions of neuroglobin may be induced via signal transduction mediators including steroid hormones and neurosteroids. For example, some neurosteroids increase neuroglobin production improving mitochondrial functions and inducing anti-apoptotic mechanisms.*

*DOI: http://dx.doi.org/10.5772/intechopen.93956*

#### *Neuroactive Steroids in Hypoxic–Ischemic Brain Injury: Overview and Future Directions DOI: http://dx.doi.org/10.5772/intechopen.93956*

et al. (2016) demonstrated that neuroglobin expression depends on ER-ß activation and tibolone favors both mechanisms [217]. Originally, neuroglobin was reported in neurons but later it was detected in other cell types such as astrocytes [218]. Interestingly, neuroglobin has been associated with neuroprotective effects on several injury models including middle cerebral artery occlusion (MCAO), focal cerebral ischemia, ß-amyloid induced toxicity, oxygen and glucose deprivation [217, 219–221].

Neuroglobin may mediate the response against hypoxia by inducing signal pathways. It has also been documented as a reactive oxygen radical scavenger with NADH oxidase activity to favor anaerobic glycolytic metabolism [217]. Controversial studies based on low levels of neuroglobin and low relative oxygen affinity propose that neuroglobin may exert or participate in collateral roles other than solely oxygen store [217, 222] (See **Figure 2** for further illustration). Additionally, photoactivation (NADH/FMN) experiments demonstrated that neuroglobin participates in the ROS and RNS elimination, suggesting a critical role in removing dangerous highly reactive species [223]. The change in the hexacoordinated state of neuroglobin according to normoxic or hypoxic conditions also suggests oxygen sensor capabilities [222]. Proper neuroglobin activity protects neurons and astrocytes against cell death [191]. In this regard, overexpression or induction of neuroglobin may be considered as potential neuroprotective therapies. Interestingly, STEARs such as tibolone are capable of increasing and inducing neuroglobin activity, which have been proposed as potential action mechanisms in

#### **Figure 2.**

*Neuroprotection - New Approaches and Prospects*

receptor AR [185], while 3-alpha and 3-beta hydroxy metabolites are agonists for ER, but antagonists for PR and AR [185, 187]. This tibolone-steroid receptor interaction and other regulatory mechanisms might explain the tissue-selective effects of tibolone [160, 186]. Belenichev et al. (2012) used cortical neurons from neonatal rats to evaluate the neuroprotective activity of tibolone in a model of glutathione depletion that produces oxidative stress and mitochondrial dysfunction. These authors found that tibolone prevented mitochondrial dysfunction and neuronal cell death. Additional studies account for the protective effects of tibolone in an ovariectomized rat model following cerebral ischemia injury [188]. Tibolone has also shown anti-

Kloosterboer et al. 2007 propose an additional action mechanism of tibolone and STEARs that involves the control of sulfatase and sulfotransferase tissue-specific activity [189]. Since sulfatase and sulfotransferase activity is tissue-specific, it is possible that tibolone exerts its function according to cell type specificity and modulating nuclear receptors activity in the tissues [190]. For instance, it is needed to further investigate the tissue-specific role of tibolone in CNS, for example, in neurons, astrocytes, and microglia. Interestingly, tibolone protects the mitochondrial activity by the preservation of the mitochondrial membrane potential and by increasing the levels of proteins that control the opening of the mitochondrial permeability transition pore (mPTP), such as Bcl-2. Avila-Rodriguez et al. (2014) demonstrated that tibolone

De Marinis' research group recently described and characterized a particular globin belonging to CNS called neuroglobin (Ngb1). Neuroglobin is under control of estrogenic response. In fact, the use of estradiol in several cellular models demonstrated the increase of neuroglobin levels [191–193]. Currently, it is known that neuroglobin is an 18 kDa protein that binds molecular oxygen with more affinity than hemoglobin, probably, increasing the availability of oxygen in the neural tissue [194]. Neuroglobin is expressed in neurons under basal conditions and is also expressed in astrocytes and microglia after brain injury [194]. Avila-Rodriguez et al. 2016 demonstrated that tibolone is capable of increasing the expression of neuroglobin producing a protective effect in a glucose deprivation astrocyte-like model. The action mechanism of tibolone may be associated with ERß receptor as demonstrated by several studies [191, 193]. Other studies demonstrated the protective effect of tibolone against lipid peroxidation and protein oxidation [195]. Tibolone is capable of increasing the density of dendritic spines in hippocampal neurons, indicating a potential role in synaptic plasticity and memory [196]. Guzmán et al. (2007), also showed that tibolone metabolites exert estrogenic activity on human astrocytes and oligodendrocyteslike cell lines [187]. Tibolone may become a promissory option to counter the detrimental effects of TBI and hypoxic injury due to its pleiotropic beneficial properties.

inflammatory effects tested in cardiovascular animal models [184].

protects the mitochondria of T98G glial cells from glucose deprivation [141].

**4.3 Selective tissue-specific estrogenic activity regulators and neuroglobin**

Pathologic conditions like hypoxia and glucose deprivation, which may lead to neuroinflammation, reduce the expression of ER-α and increase the expression of ER-ß [216]. In this regard, De Marinis et al. (2013) showed that hypoxia may induce the production of pro-inflammatory mediators like IL-6, and INF-γ [193]. Interestingly, estrogen is capable of diminishing the secretion of those pro-inflammatory mediators. It was demonstrated in a pro-oxidant model induced by H2O2 and stimulated via lipopolysaccharide (LPS). Later, it was demonstrated that the anti-inflammatory effect was mediated by NF-κB modulation and ER-ß activation [191, 193]. Therefore, it is reasonable to assume that the activation of ER-ß in hypoxic and glucose deprivation models may be considered as beneficial for brain tissues. Tibolone is capable of inducing the activation of ERß and increasing neuroglobin expression. Avila-Rodriguez

**224**

*Neuroglobin exerts interesting beneficial properties. Neuroglobin includes in its protein structure a particular prosthetic haem group to store oxygen. However, it is reported for neuroglobin additional protective functions that include oxygen sensor capabilities and detoxification properties (against reactive oxygen species and reactive nitrogen species). Evidence shows that the protective functions of neuroglobin may be induced via signal transduction mediators including steroid hormones and neurosteroids. For example, some neurosteroids increase neuroglobin production improving mitochondrial functions and inducing anti-apoptotic mechanisms.*

brain tissue [191, 217, 222]. According to computations studies and simulations, it has been proposed the neuroglobin may interact with cytochrome c. This apparent interaction may explain the electronic transfer between neuroglobin (ferrous) and cytochrome c (ferric) [191, 224]. Potentially, neuroglobin may modulate cytoplasmic cytochrome c, resulting in diminished apoptotic processes in injured tissues. Surprisingly, De Marinis et al. (2013) showed that neuroglobin hijacks cytochrome c in a neuroblastoma cell model injured via hydrogen peroxide [191]. The estrogenic induction of neuroglobin (and eventually by tibolone) increased neuroglobin expression and diminished the apoptotic cell death mechanism [191].
