**5. Neuroprotective properties of estrogen and its derivates on brain injury**

A derivate of estrogen, 17β-estradiol, is a female sex hormone and neuroactive steroid (NAS) related to the development of secondary sexual characteristics, fat storage and regulation of menstrual cycle [225]. 17β-estradiol, showed beneficial effects in verbal and visual memory performance, which was originally administered as a hormone replacement therapy in order to ameliorate climacteric symptoms [226]. The activity of 17β-estradiol depends on its union with ERs [43, 226, 227]. These receptors are classified in two subtypes: estrogen receptor-beta (ER-β) and estrogen receptor-alpha (ER-α). ERα has its locus in 6 chromosome, while the locus for the Erβ is in the 14 chromosome [226]. These ERs are transcription factors which present the peculiarity of being activated by a ligand. ER-α and ER-β have a similar structure, with a DNA-binding domain and a ligand-binding domain [228]. 17β-estradiol binds to ERs and induces the activation and the homodimerization or heterodimerization of these receptors. Then, the ERs bind to estrogen-responsive elements (EREs) in the promoter region of specific genes through the DNA-binding domain, recruiting transcriptional co-activators and co-repressors [228, 229]. Classical ERs may also regulate gene transcription by acting as transcriptional partners at non-ERE sites, such as activating protein 1 (AP1) sites [230]. 17β-estradiol can bind to membrane-associated non-classical ERs, such as G protein-coupled ERs (GPERs). GPER30, a member of the G protein-coupled receptor superfamily, regulates the activity of extracellular signal-regulated kinases (ERKs) and the phosphoinositide 3-kinase (PI3K) signaling pathway. This union allows the interaction with the signaling of other neuroprotective molecules [228, 231]. Another membrane-associated non-classical ER is Gαq proteincoupled membrane ER (Gq-mER), which was originally identified in hypothalamic neurons, modulating μ-opioid and GABA neurotransmission [228, 232].

These findings have led to research on the neuroprotective properties of estrogen and its derivates in brain injury. In HI brain injury 17β- estradiol has shown several neuroprotective effects, such as: reducing reactive gliosis, decreasing oxidative stress, ameliorating the release of pro inflammatory molecules, preventing cell death and mitochondrial dysfunction, releasing neurotrophic factors [7]. It has also been reported that 17β- estradiol produced significant protection against OGD-induced cell death in primary oligodendrocytes and against oxidative stress, having a potential role in attenuation of HI and oxidative injury [233]. In addition, in neonate rats subjected to HI, three doses of 17β-estradiol (using repeated dosing paradigm) provided approximately 70% protection of the hippocampus, basal ganglia, and amygdala. These results suggest 17β-estradiol acts as a potent neuroprotective agent against HI-induced damage to the developing brain, and that pretreating infants at risk for hypoxic ischemic injury may be advisable [234]. Moreover, treatment with estradiol after PA augmented the expression of IGF-1 and its receptor (IGF-IR). The PI3K/Akt/GSK3 signaling pathway was activated as an increase

**227**

**Figure 3.** *Ros production.*

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

in Akt and GSK3 phosphorylation [235]. However, it has been found that male sex is a well-established epidemiological risk factor for poor neurodevelopmental outcome after PA. While the mechanisms responsible for this gender difference are unknown, growing evidence has identified neuro-inflammation, oxidative stress

Using a mice model of MCAO with a mutant form of ER-α, neuroprotection was absent, showing that protective properties depend on Er-α [237]. Similarly, after emulating hypoxia in the neuroblastoma cell line SH-SY5Y by using CoCl2 (250 μg/mL), an hypoxic mimetic agent, treatment with 17β-estradiol (250 nM) exerted neuroprotection.

and cell death pathways as key players in these differences [236].

*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*

in Akt and GSK3 phosphorylation [235]. However, it has been found that male sex is a well-established epidemiological risk factor for poor neurodevelopmental outcome after PA. While the mechanisms responsible for this gender difference are unknown, growing evidence has identified neuro-inflammation, oxidative stress and cell death pathways as key players in these differences [236].

Using a mice model of MCAO with a mutant form of ER-α, neuroprotection was absent, showing that protective properties depend on Er-α [237]. Similarly, after emulating hypoxia in the neuroblastoma cell line SH-SY5Y by using CoCl2 (250 μg/mL), an hypoxic mimetic agent, treatment with 17β-estradiol (250 nM) exerted neuroprotection.

**Figure 3.** *Ros production.*

*Neuroprotection - New Approaches and Prospects*

**injury**

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].

neurons, modulating μ-opioid and GABA neurotransmission [228, 232].

These findings have led to research on the neuroprotective properties of estrogen and its derivates in brain injury. In HI brain injury 17β- estradiol has shown several neuroprotective effects, such as: reducing reactive gliosis, decreasing oxidative stress, ameliorating the release of pro inflammatory molecules, preventing cell death and mitochondrial dysfunction, releasing neurotrophic factors [7]. It has also been reported that 17β- estradiol produced significant protection against OGD-induced cell death in primary oligodendrocytes and against oxidative stress, having a potential role in attenuation of HI and oxidative injury [233]. In addition, in neonate rats subjected to HI, three doses of 17β-estradiol (using repeated dosing paradigm) provided approximately 70% protection of the hippocampus, basal ganglia, and amygdala. These results suggest 17β-estradiol acts as a potent neuroprotective agent against HI-induced damage to the developing brain, and that pretreating infants at risk for hypoxic ischemic injury may be advisable [234]. Moreover, treatment with estradiol after PA augmented the expression of IGF-1 and its receptor (IGF-IR). The PI3K/Akt/GSK3 signaling pathway was activated as an increase

**5. Neuroprotective properties of estrogen and its derivates on brain** 

A derivate of estrogen, 17β-estradiol, is a female sex hormone and neuroactive steroid (NAS) related to the development of secondary sexual characteristics, fat storage and regulation of menstrual cycle [225]. 17β-estradiol, showed beneficial effects in verbal and visual memory performance, which was originally administered as a hormone replacement therapy in order to ameliorate climacteric symptoms [226]. The activity of 17β-estradiol depends on its union with ERs [43, 226, 227]. These receptors are classified in two subtypes: estrogen receptor-beta (ER-β) and estrogen receptor-alpha (ER-α). ERα has its locus in 6 chromosome, while the locus for the Erβ is in the 14 chromosome [226]. These ERs are transcription factors which present the peculiarity of being activated by a ligand. ER-α and ER-β have a similar structure, with a DNA-binding domain and a ligand-binding domain [228]. 17β-estradiol binds to ERs and induces the activation and the homodimerization or heterodimerization of these receptors. Then, the ERs bind to estrogen-responsive elements (EREs) in the promoter region of specific genes through the DNA-binding domain, recruiting transcriptional co-activators and co-repressors [228, 229]. Classical ERs may also regulate gene transcription by acting as transcriptional partners at non-ERE sites, such as activating protein 1 (AP1) sites [230]. 17β-estradiol can bind to membrane-associated non-classical ERs, such as G protein-coupled ERs (GPERs). GPER30, a member of the G protein-coupled receptor superfamily, regulates the activity of extracellular signal-regulated kinases (ERKs) and the phosphoinositide 3-kinase (PI3K) signaling pathway. This union allows the interaction with the signaling of other neuroprotective molecules [228, 231]. Another membrane-associated non-classical ER is Gαq proteincoupled membrane ER (Gq-mER), which was originally identified in hypothalamic

**226**

Afterwards, using ER-α and ER-β agonist (PPT and DPN, respectively) without 17β-estradiol treatment, results showed neuroprotection was mimicked by PPT and suggested that ER-α regulates this protective effect [235]. Likewise, in a model of astrocytic cells it was found that estradiol improved in one of the HI conditions, parameters such as cell viability, mitochondrial membrane potential, reduced ROS production and prevented the loss of mitochondrial mass [38]. Nevertheless, estrogen use can have detrimental effects like the augment in the incidence of breast and uterus cancer [12–14]. In order to maintain the benefits and avoid these side effects, other drugs have been developed, mainly SERMs and STEARs [12–14]. The mechanism of regulation of the SERMs that determines either if they act as agonist or antagonist in an specific cell type depends on the predominant subtype of estrogen receptor alpha or beta. In addition, the co-activators, co- factors and helper proteins of each cell will determine the kind of the response of the tissue exposed to SERMs [238, 239].

In a MCAO rat model, neurogenesis in the ipsilateral subventricular zone (SVZ) after ischemia was significantly higher in estrogen and raloxifene-treated animals compared to rats treated with placebo. Otherwhise, tamoxifen did not show this enhancing effect on neurogenesis. However, both SERMs tamoxifen and raloxifene as well as estrogen, significantly reversed the spine density loss observed in the ischemic cortex at day-5 post ischemia [240]. On the other hand, tibolone action is given by the metabolization of the tibolone to three different metabolites (delta-4 tibolone; alpha-hydroxy tibolone and 3- beta-hydroxy tibolone). Each of them produces different responses. Delta-4 tibolone is an agonist to the androgen receptor and the progesterone receptor, meanwhile alpha-hidroxy and beta-hidroxy tibolone are antagonists of those receptors but agonists of the ER [241]. Keeping this in mind, Avila-Rodriguez et al. (2014) found out that tibolone ameliorates the effects of the GD on an in vitro model of astrocytes, making this molecules interesting for further research in a OGD model [12]. For this reason, in recent years we have been working on the implementation of these neuroprotection strategies in an astrocyte model using Raloxifene as a neuroprotector in the OGD model. **Figures 3** and **4** show the

**229**

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

steorid may decrease damage in different concentrations (unpublished data).

tation of new therapeutic strategies and novel approaches.

deleterious effect caused by glucose and oxygen deprivation, both in the production of ROS and in the loss of mitochondrial mass, respectively, and how this neuroactive

The different pathologies in which the HI events and with these, the oxygen and glucose deprivation are present, have been shown to exert a high impact on society. Over the years, a multitude of efforts have been directed towards the search for effective treatments that counteract the damage caused by these conditions. The different neuroprotection targets try to combat specific points of damage caused by hypoxia, including oxidative stress, dysregulation of the cell cycle and energy homeostasis [242]. Both in the initial damage phase and in the final one, the different neuroprotective agents may have anti-inflammatory, antioxidant, anti-excitotoxicity or anti-apoptotic capacities [243]. However, due to the complex network of factors that influence these pathologies, such as the cellular interactions (molecular, biochemical, protein, etc.) inherent to the CNS, as well as the genderdependent response [236] to the use of these neuroprotective agents, the success in the treatments has not been optimal [7]. Estradiol treatment not only prevents neuronal damage, but may also limit the neurodegenerative modifications induced by HI in the early stage of development. The development of SERMs and STEARs brings with it a range of possibilities for the treatment of HI, due to its advantages, focused on the nervous system without having side effects. However, it is necessary to develop new generations of these compounds to improve their neuroprotective effects. Further research is necessary to provide new alternatives in the implemen-

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

**6. Conclusion**

**Figure 4.** *Mitochondrial mass.*

deleterious effect caused by glucose and oxygen deprivation, both in the production of ROS and in the loss of mitochondrial mass, respectively, and how this neuroactive steorid may decrease damage in different concentrations (unpublished data).
