Open access peer-reviewed chapter

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

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Nicolas Toro-Urrego, Marco Avila-Rodriguez, María Inés Herrera, Andrea Aguilar, Lucas Udovin and Juan P. Luaces

Submitted: June 16th, 2019 Reviewed: September 9th, 2020 Published: November 26th, 2020

DOI: 10.5772/intechopen.93956

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Edited by Matilde Otero-Losada, Francisco Capani and Santiago Perez Lloret

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Hypoxic–ischemic brain injury is a number one cause of long-term neurologic disability and death worldwide. This public health burden is mainly characterized by a decrease in oxygen concentration and blood flow to the tissues, which lead to an inefficient supply of nutrients to the brain. This condition induces cell death by energy depletion and increases free radical generation and inflammation. Hypoxic–ischemic brain injury may occur in ischemic-stroke and over perinatal asphyxia, being both leading causes of morbidity in adults and children, respectively. Currently, there are no effective pharmaceutical strategies to prevent the triggering of secondary injury cascades, including oxidative stress and metabolic dysfunction. Neuroactive steroids like selective estrogen receptor modulators, SERMs, and selective tissue estrogenic activity regulators, STEARs, exert several neuroprotective effects. These encompass mitochondrial survival, a decrease in reactive oxygen species, and maintenance of cell viability, among others. In this context, these neurosteroids constitute promising molecules, which could modify brain response to injury. Here we show an updated overview of the underlying mechanisms of hypoxic–ischemic brain injury. We also highlight the neuroprotective effects of neurosteroids and their future directions.


  • neuroactive steroids
  • hypoxia-ischemia
  • brain injury
  • oxidative stress
  • metabolic dysfunction

1. Introduction

Hypoxic–ischemic (HI) brain injury is a major cause of long-term neurologic disability and death worldwide. Brain damage caused by hypoxia-ischemia responds to a wide variety of factors, being the central nervous system (CNS) especially susceptible to changes in energy levels, mainly glucose concentrations and oxygen [1]. The brain has a 25% glucose and 20% oxygen consumption of total body weight [2, 3]. This high energy demand is attributed to the functions performed by brain cells such as synaptic activity, neurotransmitter recycling and ion transport [2]. Thus, ensuring correct brain metabolism results in optimal neuronal functioning. HI brain injury is mainly characterized by a decrease in the concentration of oxygen and blood flow, which causes an insufficient supply of nutrients to the brain. These pathological conditions lead to cell death due to the increase in free radical production and depletion of ATP [4]. This phenomenon is observed both in perinatal asphyxia (PA) and in ischemic stroke (IS) [5, 6, 7]. Around 15 to 20% of infants that suffer PA will die in the postnatal period and further 25% will develop severe and long-lasting neurological impairments such as cerebral palsy, epilepsy and neurodevelopmental disorders [8], also representing one of the main causes of morbidity in children and adults in the world [9, 10]. Similarly, at a structural level HI injury mainly affects the layers II, III and VI of the cortex, CA1 and CA3 hippocampal areas, striatum and cerebellum [11]. Therefore, the understanding of the underlying mechanisms of this pathology is essential for the establishment of efficient treatments.

Several neuroprotective strategies have been tested, including Selective Estrogen Receptor Modulators (SERMs) and Selective Tissue Estrogenic Activity Regulators (STEARs), which have shown the same benefits as estrogen, including the decrease of reactive oxygen species (ROS), maintenance of cell viability, mitochondrial survival, among others; without its negative side effects [12, 13, 14]. However, there are no effective pharmaceutical strategies to prevent the triggering of secondary injury cascades, including oxidative stress and metabolic dysfunction. In this sense, the present chapter summarizes the underlying mechanisms of HI brain injury and compiles several neuroprotective strategies, including SERMs and STEARs.


2. Mechanisms of brain damage in hypoxia-ischemia

Hypoxia is a condition that affects mainly the brain, and it is characterized by a low concentration of oxygen, affecting the proper functioning of the organs and tissues exposed to it. This insult causes a variety of responses in the brain. An initial response occurs immediately after the insult and is associated with a depletion of ATP, glucose and phosphocreatine inside the brain. This immediate reaction determines the patient’s outcome against injury, which in turn triggers a secondary response that occurs several hours later. A temporary energy recovery takes place almost to the initial physiological levels, providing a treatment window between 1 and 6 hours following injury [8, 15, 16]. A third phase of persistent effects lasts for several years [17]. In general terms, global hypoxia affects the cerebral cortex, the sensorimotor cortex, the talamo and the basal ganglia, causing damage in deep gray matter [18].While the complete pathogenic pathways of HI are not fully described, some mechanisms like apoptosis, increased glutamate, calcium overload, mitochondrial dysfunction and oxidative stress have been proposed to contribute to generate neuronal damage [19].

Primary response depends on the energetic failure, which is characterized by the reduction of the energy supply, generating the accumulation of Reactive Oxygen Species (ROS) via lactate production augment, making the cell susceptible to oxidative stress and mitochondrial dysfunction [18]. Besides this, restricted cerebral blood flow causes a switch to anaerobic respiration, reducing ATP and phosphocreatine, and increasing lactic acid production [16]. Low levels of ATP derived from this energetical failure affect the integrity of the cell membrane. Calcium enters easily to the cell causing the membrane depolarization, blocking calcium storage in the cell, which in turn accumulates in the extracellular space. In addition, the ion flux of sodium/potassium is altered by the Na+/K+ pump dysfunction [20]. The second phase of injury is related to the recovery of blood flow and the reestablishment of brain metabolism, characterized by an inflammatory response, excitotoxicity and oxidative stress, being the main responsible for the brain cells death after hypoxia [7, 18].

2.1 Second phase of injury

Apoptosis as necrosis are the death pathways of the cell. They are present in brain damage caused by hypoxia, being apoptosis the most common death pathway in the young brain unchained by mitochondrial failure [21]. Apoptosis can follow two pathways, being the extrinsic triggered by external signals like the tumor necrosis factor alpha (TNF-α), Fatty acid synthase (FAS), and the intrinsic path mediated by internal factors such as DNA damage or cell stress [22]. The extrinsic pathway is involved in the action of caspase 8 and 10, which activate caspase effectors directly, interacting with the intrinsic pathway, and triggering a permeabilization of the mitochondrial membrane [23].

The Intrinsic pathway is mediated by the release of apoptotic factors such as cytochrome-c, Serine protease HTRA2, mitochondrial (Omi/HtrA2), apoptosis inducing factor (AIF), endonuclease G (endoG), Second mitochondria-derived activator of caspase/direct inhibitor of apoptosis-binding protein with low pI (Smac / Diablo) after permeabilization of the membrane. These apoptotic factors can trigger cell death processes that can be mediated by caspase-dependent pathways. Each of these factors has a role in programmed death. Cytochrome c interacts with Apoptosis protease-activating factor-1 (Apaf-1), creating the apoptosome. Smac/Diablo interacts with apoptosis inhibitors, AIF and endoG act through a caspase- dependent pathway. These are translocated to the nucleus, causing nuclear fragmentation [24, 25]. Hence, the permeabilization of the mitochondrial membrane has been proposed as a marker of a point of no return in hypoxic injury.

2.2 Excitotoxicity

HI injury triggers responses at both the systemic and cellular levels. When the energy supply is interrupted, excitotoxicity occurs through an uncontrolled release of excitatory neurotransmitters such as glutamate, causing an acute cascade damaging neurons and glial cells at cytoplasmic and mitochondrial levels, and also causing disruption of the BBB [23]. Glutamate activates NMDA receptors, causing the accumulation of Ca ++ and nitric oxide (NO), which in turn cause production of ROS. The increased levels of intracellular calcium in neurons and glial cells in turn results in the activation of calcium-dependent proteases, reactive oxygen species (ROS) production, mitochondrial dysfunction, oxidative stress, cytotoxic edema, lipases and deoxyribonuclease (DNase), and the stimulation of pro-cell death pathways [23, 26, 27].

2.3 Oxidative stress

The balance between the oxidant and the antioxidant levels of the cell is called redox homeostasis. An imbalance in favor of the intracellular level of oxidants results in what is known as oxidative stress. This deregulation occurs mainly in two free radicals, the reactive oxygen species (ROS), and the reactive nitrogen species (RNS) [28, 29]. Oxidative stress plays a major role in the pathophysiology of HI, due to the significant damage to nucleic acids (DNA degeneration), lipids (lipid oxidation), proteins and different organelles such as the mitochondria [7]. There are different sources of free radicals (ROS and RNS) following HI, including mitochondrial electron transport chain (ETC), xanthine oxidase (XO), NADPH oxidases (NOX) and nitric oxide synthase (NOS), and arachidonic acid (12/15 lipoxygenase) [26, 28].

2.4 Mitochondria

Mitochondria plays a vital role in survival of the different cells of the CNS [30]. It is composed of two membranes, one internal and one external, each with different functions. Within these membranes is the matrix. There are enzymes responsible for the main metabolic processes to produce ATP, such as the Krebs cycle, β-oxidation, as well as the metabolism of aminoacids [31]. Additionally, the mitochondria is involved in moderating processes of death (apoptosis) and biogenesis or cell proliferation [31, 32], also in critical processes such the maintenance of neuronal homeostasis, including autophagy, elimination of toxic metabolites like ROS, and calcium homeostasis [26, 30, 31, 33].

Neonatal brain has increased vulnerability to damage by oxidative stress when compared with the adult brain, in part due to lower levels of antioxidants [34]. In adult brain, superoxide dismutase (SOD) 1 can scavenge ROS generating hydrogen peroxide (H2 O2), thus allowing further breakdown by catalases to H2 O. In contrast, neonatal SOD1, although expressed, can exacerbate brain injury caused by HI possibly due to the absence or downregulation of enzymes such as catalase and glutathione peroxidase 1, required downstream of SOD1 [35].

Mitochondria plays a key role in HI injury since the disturbances in energy metabolism trigger a number of pathophysiological responses converging at mitochondrial levels, such as the control of energy metabolism, production of ROS, and the release of apoptotic factors into the cytoplasm [36]. Mitochondria constitutes an important regulator of cell death due to its ability to release proapoptotic proteins following mitochondrial permeabilization. Apoptosis can occur through an intrinsic pathway, where DNA damage or cellular stressors activate apoptosis, or an extrinsic pathway, following activation of death receptors [36].

2.5 Cardiolipin peroxidation

Another consequence of cell death caused by ROS-induced oxidative stress is the peroxidation of a mitochondrial lipid, cardiolipin [37], one of the most critical targets in the components of the evolution of HI injury. This is a unique phospholipid, which is found mostly in the inner mitochondrial membrane, where it has a very close association with the components of oxidative phosphorylation [37, 38]. Cardiolipin plays a crucial role in the insertion into the membrane and the function of cytochrome C, cytochrome C oxidase and other phosphorylation complexes. This is required, therefore, for an optimal functioning of complexes I (NADH: ubiquinone reductase), complex III (NADH: ubiquinone cytochrome C oxidoreductase), complex IV (cytochrome C oxidase) and complex V (ATP synthase) [39].

When HI occurs, enzymatic and non-enzymatic processes induce lipid peroxidation. The non-enzymatic process is triggered by the interaction of ROS with the fatty acyds of the membranes, and the enzymatic process include the activation of lipoxygenases (LOX), cyclooxygenases (COX), phospholipase A2 (PLA2) and Cyt C [40, 41], which leads to an alteration in the structure of this phospholipid responsible for mitochondrial dysfunction. Hence, the release of cytochrome c depends on the integrity of itself. This severe sensitivity to ROS is due to its high content of fatty acids [39].

2.6 Inflammation in HI

Accompanied by the reactions mentioned above, there is a role played by different glial cells in the injury caused by hypoxia, mainly in inflammation. This injury initially triggers an immediate response in neuroglial cells, which contribute to the damage mechanisms mentioned above, due to the secretion of a large amount of proinflammatory cytokines and ROS.

2.7 Astrocytes

In the last 20 years, astrocytes have been granted multiple functions, such as providing support, helping in the maintenance of the cerebral microenvironment for an appropriate function, regulating the blood flow in the brain, which are essential for the adequate functioning of neurons [2, 42]. Another important astrocytic function is the contribution to brain metabolism [43]. Astrocytes takes glucose from blood vessels and provide energy metabolites to neurons [44]. In addition, through the lactate shuttle, astrocytes provide lactate to the neurons as a substrate for the citric acid cycle and can therefore supply their energy requirements [45].

However, the role of astrocytes in injuries such as hypoxia are not fully elucidated. Astrocytes as microglia, when subjected to insults such as hypoxia, may act differently depending on the severity of the injury. Immediately after hypoxia, astrocytes enter in an activated state, which eventually ends in a glial scar [46, 47]. Astrocytes plays important roles in the brain during HI. Because of the tight connection with brain capillaries, astrocytes suffer damage firstly after ischemia, and then, damaged astrocytes kill neighboring neurons. The number of apoptotic astrocytes increases gradually as the extension of ischemic time, which leads to further expand of cerebral infarction area [48].

Astrocytes can exacerbate cytotoxicity death due to secrete inflammatory cytokines such as IL-1, IL-6, interferon-γ, and TNF-α; and can also help the migration of immune cells to the CNS by the secretion of chemokines [49]. Likewise, there is also a protective effect exerted by astrocytes, which play an important role in tolerance to cerebral ischemic injury [50] and inflammation [50, 51].

2.8 Microglia and endothelial cells

Microglia, the immune cells of the CNS, are the first to be activated after hypoxia. They migrate to the place of injury and change their morphology to an amoeboid-lice functional cell, acting in conjunction with monocytes and macrophages [49, 52, 53]. Microglia M1 release proinflammatory agents to the environment such as ROS, cytokines ((IL) -1β, IL-6, tumor necrosis factor-alpha (TNF-α)), glutamate, nitric oxide, creating a cytotoxic environment triggering cell death [49, 52, 53].

The extent of injury noted in HI is not only determined by the biochemical cascades that trigger the apoptosis-necrosis continuum of cell death in the brain parenchyma, but also by the pro-inflammatory factors of the Blood Brain Barrier (BBB), such as the endothelial cells [54]. Endothelial cells can sense variation in the Parcial Oxygen Pressure (PO2) through different mechano-sensors. Then, they can adapt their metabolism to maintain ATP production, switching into an hypoxic metabolism. In this way, endothelial cells augment the production of ROS by making the respiratory chain slower, reduce the cytochrome-c capacity in order to trap O2, and alter the cellular redox potential [54, 55]. In cerebrovascular endothelial cells (cEND) OGD augmented the mRNA expression of IL-1 alpha, IL- 6 glut-1 transporter and total nitric oxide concentration increasing significantly the permeability of the cEND monolayer [56].

2.9 Selective vulnerability of the brain to HI

The pathophysiology of HI is complex. The damage on the developing brain is determined by several factors: timing of asphyxia, intensity, severity of HI and immaturity of the brain. Beside this, different areas of the brain and different cell types present a selective vulnerability to this injury [18].

The immaturity of brain represents a significant factor in the outcome of HI brain injury. Although risk factors of HI in term newborns are similar to those observed in preterm newborns, the immature brain in the last ones, especially those with a very low birth weight, is highly vulnerable to injury [18]. This, due to hypoperfusion caused by the defectively functioning lungs and hearts in preterm newborns, and the poor auto-regulatory capacity the immature brain possess [57]. HI injury induces white matter injury with noticeable oligodendroglia loss, due to the poorly vascularization in white matter compared with cerebral cortex. This injury, known as periventricular leukomalacia (PLV), triggers cognitive, sensory, and motor impairment in preterm infants. Abnormalities of cortical gray matter and hippocampus are also found in the immature brain [18].

In addition, in the developing brain there is a spectrum of lesions caused by HI. Alongside PVL, periventricular hemorrhagic infarction in association with geminal matrix (ganglionic eminence) hemorrhage, with or without intraventricular hemorrhage, or thalamocortical injury (Table 1) [58].

Hemorrhagic lesions
Germinal matrix (ganglionic eminence) (frequently associated with PVL)
Limited (grade I)
With intraventricular hemorrhage (grade II)
With ventricular expansion (grade III)
With PHI (grade IV)
Subarachnoid space (temporal lobe and cerebellum)
White-matter lesions
Periventricular leukomalacia (PVL)
With focal necrosis
With diffuse white-matter gliosis only
Periventricular hemorrhagic infarction (PHI)
Combined gray- and white-matter lesions
Single cerebral artery–distribution infarcts (porencephaly)
Hydranencephaly (bilateral large hemispheric infarcts)
Multicystic encephalomalacia
Gray-matter lesions
Thalamic and basal ganglionic injury (“status marmoratus”)
Neuronal necrosis in basis pontis and subiculum (pontosubicular necrosis)
Mobius syndrome (brainstem neuronal loss and gliosis)
Cerebellar infarct

Table 1.

Lesions caused by HI in the developing brain.

Grade refers to clinical severity assigned based on transfontanelle ultrasonography or other neuroimaging. Adapted from [58].

PHI = periventricular hemorrhagic infarction; PVL = periventricular leukomalacia.

The developing brain exhibits selective vulnerability. As it was mentioned above, certain cells and regions appear vulnerable depending on the severity and timing of injury. Projection neurons, especially in the deep gray nuclei, are at greatest risk during ischemic insults in the term brain [18]. Subplate neurons are the earliest and the most transient cell population of the neocortex. The subplate zone peaks at the onset of the developmental window of vulnerability to PVL (GW 24) and undergoes dissolution during the third trimester. Subplate neurons are largely absent at 6 months of postnatal age. HI injury leads to moderate to near-complete subplate neuron cell death, whereas most cortical neurons are intact. This selective vulnerability may be due to early cellular maturation and a developmentally related increase in glutamate receptor expression, including NMDA receptor 1, kainate and AMPA receptors [59]. On the other hand, in the preterm brain, subplate neurons and oligodendrocytes (OL) precursors are most vulnerable. Consequent abnormal thalamocortical connectivity may explain the somatosensory and visual impairment seen in prematurely born infants suffering HI brain injury [60, 61]. OL progenitors appear to be the most vulnerable, showing impaired maturation and development following injury.

2.10 Oligodendrocytes and astrocytes

Oligodendrocytes, the myelin-forming glia that ensheath axons in the CNS, exhibit four sequential stages of maturation. Oligodendroglial progenitors, the pre-OL (or late oligodendroglial progenitor), the immature OL, and the mature myelin-producing OL [60], are extremely susceptible to HI. The injury involves maturational delays in oligodendrocyte population inducing oxidative stress. Following HI, OLs fail to fully mature, leading to persistent aberrations in myelin ultrastructure, which are associated with permanent disability and neurodevelopmental impairment [62].

Astrocytes are the predominant glial population in the CNS. They play a crucial role in HI as mentioned above. However, sustained HI brain injury can lead to decreased astrocytic function and, thereby, greatly decreased neuronal regeneration [60].

2.11 Blood–brain barrier and vascular fragility

The brain evidences a high requirement of oxygenated blood. This demand has resulted in the development of specific cerebral blood vessel networks with arteriovenous hierarchy. The Blood–Brain Barrier (BBB) is a specific and unique component of the cerebrovascular network. It is a highly specialized biochemical and structural barrier at the interface between blood and brain. BBB is involved in preserving ionic homeostasis within cerebral microenvironment and regulating the entry of molecules into the brain [63].

HI injury in neonatal brain induces an increase in BBB permeability, affecting important cellular and functional components of this vessel network such as pericytes, the tight junctions of endothelial cells and astrocytes [60, 63, 64].

Delicate and thin vessels in the developing brain may not sustain the lack of blood flow to compensate the requirements of oxygen and nutrients that the brain needs, due to the underdeveloped distal arterial network and an immature cerebral auto regulatory capacity. Peripheral arteries in the growing brain lack collateral vessels and exhibit limited vasodilatory function in response to the hypoxic–ischemic event, resulting more susceptible to HI injury [60].


3. Experimental models

In vivo and in vitro models are used for studying hypoxia (Table 2). In the most used animal model, a unilateral ligation of the carotid artery (UCCAO) is performed, followed by an exposure to an oxygen atmosphere of 8% for 1–3 hours, mainly developed in rodents [65]. This reproduces the anatomical damage caused by HI in neonates, with gray matter damage in the hippocampus, thalamus and basal ganglia, as well as in white matter [65, 66]. Similarly, it reproduces metabolic damage in parameters such as: cerebral acidosis, decreased cerebral blood flow, and decreased glucose uptake [52] and has the ability to show the neuroprotective effect of different therapeutic approaches like hypothermia [67, 68]. Bilateral ligation of the carotid artery is also used to accentuate white matter damage [69, 70] .

ReferenceSpeciesAnimal modelOutcomes
Large animal models
[73]Macaca nemestrina, near termUCOPoor weight gain and cerebellar growth, abnormal brain
DTI, behavioral impairment, 43% develop CP.
[90, 91]Fetal sheep, near termBilateral CCAOShorter HI (<30 min): selective neuronal loss. Longer HI:
cortical necrosis. Post-HI EEG suppression related to insult
severity and pathology; prevented by hypothermia.
[92]Fetal sheep, midgestationBilateral CCAONecrosis of subcortical white matter, neuronal loss in thalamus and striatum similar to near term fetus. Little loss of final EEG amplitude.
[93]Fetal sheep, midgestation and near TermUCOHippocampal neuronal loss only in near term group. Degree of injury associated with the severity of hypotension during UCO.
[94]Pigs, <24 h oldCCAO + hypoxiaSecondary energy failure. Energy metabolism ameliorated by hypothermia (35°C for 12 h) at 24 h–48 h.
[95]Pigs, P9Hypotension + hypoxia~60% fall in CBF, reduced cerebral O2 uptake, phosphorylated metabolites and pH and increased inorganic phosphate.
[71]Rabbits, 21–22d gestationUterine ischemiaP1 pups: overt posture and tone after ischemia >37 min, correlates with microgliosis in basal ganglia and thalamus.
Rodent models with global hypoxic or excitotoxic component
[96]Mice at E8, P0 or P5Ibotenate, i.c.v.Laminar neuronal depopulation of layer V–VIa. P5: neuronal loss in all cortical layers, formation of porencephalic cysts.
[97]Pregnant Sprague–Dawley rats, embryonicHypoxia E5-E20White matter cysts in offspring P0–P7, increased lipid peroxidation, WMI and macrophages.
Rodent models with hypoxia-ischemia
[98, 99]Sprague Dawley rats, P1–P3CCAL + hypoxiaSelective vulnerability of late OL progenitors, independent of age.
Death of sub-plate neurons, motor deficits, altered thalamocortical connections to somatosensory and visual cortex normal.
[65]Sprague–Dawley rats, P7CCAL + hypoxiaUnilateral ischemic injury in the cortex, hippocampus, basal ganglia in >90% of survivors.
[100]Wistar rat, P7LPS, 4 h prior to CCAL + hypoxiaBlocking lymphocyte trafficking reduced brain inflammation, BBB damage, and improved LPS-induced HI brain injury. No effect with pure HI.
[101]C57Bl/6 WT, Tg SOD1, GPx1 over-expressing P7 miceCCAL + hypoxiaReduced injury in GPx1-Tg mice but not in SOD1-Tg or GPx1/SOD1. NOS inhibition did not improve outcome in SOD-Tg.
[102, 103]C57BL/6 WT and Gal-3 KO, P9CCAL + hypoxiaIncreased BBB permeability 2–24 h, reduced BBB protein expression. Infarct volume reduction in Gal-3 KO mice.
[104]C57BL/6 J and TRIF KO mice, P8–9Poly I:C, 14 h prior to CCAL + hypoxiaIncreased infarct volume and WMI, prevented in TRIF KO.
Injury linked to inflammatory response & decrease in M2-like microglia.
Focal ischemia rodent models
[105]Wistar rat, P7Permanent MCAO +1 h CCAOInfarcts in frontoparietal cortex at 3-month recovery. DNA fragmentation from 6 to 96 h.
[106, 107, 108]Sprague Dawley rats, P7Transient MCAO, 3 hSevere unilateral perfusion deficits, restoration of CBF upon suture removal. Decreased ADC associated with brain injury at 24 h reperfusion. Demonstrated endogenous neuroprotective role of microglial cells after acute injury.
[109]Sprague Dawley rats, P10Transient MCAO, 1.5 hTime resolved cell-type specific increase in HIF-1a and
VEGF expression, gliosis.
[110]C57/Bl6 mice, CD36 KO and WT, P9Transient MCAO, 1.5 h and 3 hFocal ischemia–reperfusion, increased injury and caspase-3 cleavage associated with apoptotic neuronal debris in CD36 KO. Effects independent of NFκB activation.
In vitro models
ReferenceCell lineExperimental modelOutcomes
[74]PC12 cells48 h OGD/ 2 h reperfusionSignificant morphological cell changes
[75]Primary cortical astrocyte6 h OGD/ 0, 12, 24, 48 h reperfusionSignificantly increased 2- NBDG uptake by about 1.2 to 2.5 times in cells compared to control
[76]Primary cerebral cortex neurons3 h OGD/ 48 h ReperfusionDamage to neuronal viability, dendrite branch number in neurons deceased significantly
[79]Primary astrocyte3, 5, 7 h OGD/ 24 h ReoxygenationIncreases in HMGB1 and TNF-a, induced phosphorylation of PI3K, promoted nuclear translocation of NF-kB
[111]Primary cortical neurons2 h OGDSuppressed significantly cortical neurons proliferation
[112]SH-SY-5Y cells6 h OGD/ 1 h reoxygenationCaused significant mitochondrial fragmentation, excessive mitochondrial fission
[77]Primary Cortical NeuronOGDDecrease in neurite outgrowth
[78]Neural progenitor cell6 h OGDIncreased apoptosis
[113]Mouse hippocampal neurons HT224 h OGD/ 24 h ReoxygenationmiR-144-3p expression was significantly downregulated in neurons following OGD/R treatment
[81]Neuro 2a cells4 h OGD/ 12 h ReoxygenationInhibited cell viability and cell proliferation, reduced phosphorylation levels of p38 MAPK and ERK1/2
[114]SH-SY5Y cells and primary murine cortical neurons,4 h OGDOGDR-induced mitochondrial depolarization, reactive oxygen species production, lipid peroxidation and DNA damages
[115]Primary astrocytes and microglial cells2 h OGD/ 48 h ReoxygenationInduced abnormally opened hemichannels with increased ATP release and EtBr uptake but reduced GJIC permeability.
Astrocytic Cx43, hemichannels, and GJIC play critical roles in OGD/R injury-induced neuroinflammatory responses.
[116]Primary astrocytes4 h OGD/ 3 h, 6 h, 12 h, 24 h reoxygenationExpression of Ski was proved to be up-regulated
[117]Primary hippocampal neurons2 h OGD/ 24 h reperfusionCaspase-3 activity and expression increased in the first 24 h,
HMAs models
ReferenceCell line/speciesExperimental modelOutcomes
[82]Multiple myeloma cell line U266CoCl2CoCl2-mediated hypoxia affects the expression profiles of genes that are functionally related to apoptosis and angiogenesis
[83]Myeloid leukemic cell lines NB4 and U937CoCl2 and DFOApoptosis with a loss of mitochondrial transmembrane potentials, activation of caspase-3/8 and cleavage of anti-apoptotic protein Mcl-1
[118]U251 human glioblastoma cell lineCoCl2Increases HIF-1a gene expression
[119]Glioblastoma cell lines U373MG and DBTRG05MGDFOActivation of factors associated with ECM degradation and invasion of glioma cells
[120]C57BL/6 miceDFODFO up-regulated the expression of vascular endothelial growth factor (VEGF), HIF-1α protein and growth associated protein 43 (GAP43) and down-regulated the expression of divalent metal transporter with iron-responsive element (DMT1 + IRE), α-synuclein, and transferrin receptor (TFR)
[121]Hippocampal neuronsDFO pretreatment/3 h OGD45% reduction in cell death
[122]Sprague–Dawley ratsSubarachnoid hemorrhage/DFO treatmentDFO-induced increase in HIF-1 protein level and activity exerts significant attenuation of BA vasospasm
[123]Hippocampal culturesPpreconditioning CoCl2, DFO or dimethyloxylalyglycine (DMOG), 3 h OGDCobalt induced the transcription of the cytokine erythropoietin.
Cobalt and DFO, enhanced survival of neurons.
DMOG exacerbates OGD-induced neuronal death
[124]Sprague–Dawley ratsCCA/DFO treatmentNeural-protective and angiogenesis effects through regulating the levels of HIF-1α
[125]Adipose-derived stem cellsDFO preconditioningRestored neovascularization potential of ADSCs
[126]Sprague – Dawley ratsMCA/DFO treatmentPreserved brain volumes, upregulation of HIF1a
[127]Wistar ratsMCAO/DFO + Erythropoietin treatmentReduced the number of cleaved caspase 3-positive cells in the ipsilateral cerebral cortex.

Table 2.

Experimental models for HI.

Modified from [7].

In another animal model, ligation of the common carotid is excluded and hypoxic damage is performed by oxygen deprivation. This experimental paradigm is used to describe milder lesions and to investigate the biochemical alterations of the brain [52]. On the other hand, this model has been used in larger animals such as primates, sheep, pigs and rabbits in order to better replicate the conditions of a human fetus with HI, with the disadvantage of not being able to perform behavioral tests and not having a methodological archetype between experiments [52, 71, 72, 73].

3.1 In vitro approaches

The different methodological limitations of in vivo models make in vitro models relevant. In order to replicate the conditions that occur in the presence of a deprivation or decrease in glucose and oxygen levels such as those present in HI, several studies have proposed a model of oxygen and glucose deprivation (OGD) (Table 2). This experimental model has the ability to adjust to specific research needs and the versatility of being able to use different cell lines, making possible the study of the bases of the molecular and biochemical mechanisms of HI injury. However, methodological differences have been found in the implementation of this model, especially in the exposure time of hypoxia and reoxygenation. [74, 75, 76, 77, 78, 79, 80, 81], making this model dependent on the specific conditions of the tissue or cells used [7].

Another methodological approach used to study the effects of hypoxia in vitro include chemical hypoxia-mimetic agents (HMAs) (Table 2). These are based on producing at molecular level the effects caused by low concentration of oxygen, mainly those involved in the expression of Hypoxia-inducible factor-1 (HIF-1) [82, 83]. The activation of this factor depends on oxygen concentration, and HIF-1 is involved in several cellular processes that trigger hypoxia [84, 85, 86, 87, 88, 89].


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, 131, 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, 134, 135]. Besides the neuroactive properties of steroids, there are a plethora of protective functions characterized on neurons, astrocyte and microglia [136, 137, 138, 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, 143, 144, 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, 156, 157, 158, 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).

ReferenceType of studyOutcomes
[197]9Human Psychiatric studyThe evidence summarized supports the idea that MDD and PPD are psychiatric disorders involving neurosteroids and GABAergic dysfunction
[198]Comparative human and animal studiesThe study shows potential mechanisms that underlie sex-related differences in behavior and its implications for stress-related illnesses.
[199]Animal and human studiesThe negative cognitive consequences of sleep deprivation may arise from the effort of the brain to counteract the detrimental effect of sleep loss via compensatory mechanisms
[200]Animal (neonatal foal) studyProgesterone might be a promissory marker for identifying continuous endogenous production of neuroactive steroids in foals with suspected NMS and other diseases
[201]Human studyIndividual domains of cognitive can be considered as an endophenotype of psychosis. It is possible that higher levels of cortisol and testosterone in siblings are consistent with high-risk states for psychosis
[202]Animal modelExposure to neuroactive steroids induced a sustained elevation in 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 disorder
[203]Peripartum depressed womenCortical GABA+/Cr concentrations are associated with postpartum RSFC. It is possible that allopregnanolone may be associated with postpartum intra-DMPFC connectivity.
[204]Animal and human studiesNervous 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 pathology.
[205]Animal and human studiesNeuroactive 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 investigated.
[206]Animal and human studiesSome 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.
[207]Animal and human studiesThere 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 metabolic syndrome, obesity or diabetes.
[208]Women studyWomen at both extremes of the weight spectrum have low mean serum allopregnanolone. Neuroactive steroids such as allopregnanolone may be potential therapeutic targets for depression and anxiety in traditionally treatment-resistant groups.
[209]Animal and human studiesLow 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 disorder could occur during high levels.
[210]Animal and human studiesSeveral 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 disorder (MDD), and essential tremor (ET).
[211]Animal modelDHEAS and progesterone were good predictors of HPA Axis dysfunction and outcome in hospitalized foals.
[212]Clinical studyThe first-episode antipsychotic-naive schizophrenic patients 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 caused by cortisol.
[213]Animal and human studiesClinical 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.
[214]Animal and human studiesStrain variation in neuroactive steroid levels correlated with numerous behavioral phenotypes of anxiety sensitivity accessed in GeneNetwork, consistent with evidence that neuroactive steroids modulate anxiety-like behavior.
[215]Aged human studyWe observed a significant difference in plasma concentration of 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 deficits in women compared to men.

Table 3.

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 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, 177, 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-alpha-hydroxy- 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 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-inflammatory 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, 192, 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 oligodendrocytes-like 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 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, 220, 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 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].

Figure 2.

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.


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 protein-coupled 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 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. 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, 13, 14]. In order to maintain the benefits and avoid these side effects, other drugs have been developed, mainly SERMs and STEARs [12, 13, 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 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).

Figure 3.

Ros production.

Figure 4.

Mitochondrial mass.


6. Conclusion

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 gender-dependent 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 implementation of new therapeutic strategies and novel approaches.


  1. 1. Bélanger M, Allaman I, Magistretti PJ. Brain energy metabolism: Focus on astrocyte-neuron metabolic cooperation. Cell Metabolism. 2011 Dec 7;14(6):724-738 Available from:
  2. 2. Allaman I, Bélanger M, Magistretti PJ, et al. Trends in Neurosciences. 2011 Feb;34(2):76-87. Available from
  3. 3. Dwyer DS, Vannucci SJ, Simpson IA. Expression, regulation, and functional role of glucose transporters (GLUTs) in brain. International Review of Neurobiology. 2002;51:159-188. Available from:
  4. 4. Vavilis T, Delivanoglou N, Aggelidou E, Stamoula E, Mellidis K, Kaidoglou A, et al. Oxygen–glucose deprivation (OGD) modulates the unfolded protein response (UPR) and inflicts autophagy in a PC12 hypoxia cell line model. Cellular and Molecular Neurobiology. 2016;36(5):701-712 Available from:
  5. 5. Tian T, Zeng J, Zhao G, Zhao W, Gao S, Liu L. Neuroprotective effects of orientin on oxygen-glucose deprivation/reperfusion-induced cell injury in primary culture of rat cortical neurons. Experimental Biology and Medicine. 2017;153537021773798Available from:
  6. 6. Yang X, Zheng T, Hong H, Cai N, Zhou X, Sun C, et al. Neuroprotective effects of Ginkgo biloba extract and Ginkgolide B against oxygen–glucose deprivation/reoxygenation and glucose injury in a new in vitro multicellular network model. Frontiers in Medicine. 2017:1-12
  7. 7. Toro-Urrego N, Vesga-Jiménez DJ, Herrera MI, Luaces JP, Capani F. Neuroprotective role of hypothermia in hypoxic-ischemic brain injury: Combined therapies using estrogen. Current Neuropharmacology. 2018 Dec;6:17 Available from:
  8. 8. Baburamani AA, Hurling C, Stolp H, Sobotka K, Gressens P, Hagberg H, et al. Mitochondrial optic atrophy (OPA) 1 processing is altered in response to neonatal hypoxic-ischemic brain injury. OPEN ACCESS Int J Mol Sci. 2015;16:16 Available
  9. 9. Mozaffarian D, Benjamin EJ, Go AS, Arnett DK, Blaha MJ, Cushman M, et al. Heart disease and stroke statistics—2015 update. Circulation. 2015 Jan 27;131(4):e29LP-ee322 Available from:
  10. 10. Northington FJ, Chavez-Valdez R, Martin LJ. Neuronal cell death in neonatal hypoxia-ischemia. Annals of Neurology. 2011 May;69(5):743-758 Available from:
  11. 11. Petito CK, Pulsinelli WA. Delayed neuronal recovery and neuronal death in rat Hippocampus following severe cerebral ischemia: Possible relationship to abnormalities in neuronal processes. Journal of Cerebral Blood Flow and Metabolism. 1984 Jun 28;4(2):194-205 Available from:
  12. 12. Ávila Rodriguez M, Garcia-Segura LM, Cabezas R, Torrente D, Capani F, Gonzalez J, et al. Tibolone protects T98G cells from glucose deprivation. The Journal of Steroid Biochemistry and Molecular Biology. 2014;144(PART B):294-303
  13. 13. Arevalo MA, Santos-Galindo M, Lagunas N, Azcoitia I, Garcia-Segura LM. Selective estrogen receptor modulators as brain therapeutic agents. Journal of Molecular Endocrinology. 2011;46(1)
  14. 14. Garzón D, Cabezas R, Vega N, Ávila-Rodriguez M, Gonzalez J. et al., Novel approaches in astrocyte protection: From experimental methods to computational approaches. Journal of Molecular Neuroscience. 2016;58(4):483-492
  15. 15. Blumberg RM, Cady EB, Wigglesworth JS, McKenzie JE, Edwards AD. Relation between delayed impairment of cerebral energy metabolism and infarction following transient focal hypoxia-ischaemia in the developing brain. Experimental Brain Research. 1997 Jan;113(1):130-137 Available from:
  16. 16. Thornton C, Jones A, Nair S, Aabdien A, Mallard C, Hagberg H. Mitochondrial dynamics, mitophagy and biogenesis in neonatal hypoxic-ischaemic brain injury. FEBS Letters. Wiley Blackwell. 2018;592:812-830
  17. 17. Fleiss B, Gressens P. Tertiary mechanisms of brain damage: A new hope for treatment of cerebral palsy? The Lancet Neurology. 2012;11:556-566 Available from:
  18. 18. Li B, Concepcion K, Meng X, Zhang L. Brain-immune interactions in perinatal hypoxic-ischemic brain injury. Progress in Neurobiology. 2017 Dec 1;159:50-68 Available from:
  19. 19. Noraberg J, Poulsen FR, Blaabjerg M, Kristensen BW, Bonde C, Montero M, et al. Organotypic Hippocampal Slice Cultures for Studies of Brain Damage, Neuroprotection and Neurorepair. Vol. 4. Current Drug Targets: CNS and Neurological Disorders; 2005. pp. 435-452
  20. 20. Berger HR, Brekke E, Widerøe M, Morken TS, Sund Morken T, Morken TS, et al. Neuroprotective treatments after perinatal hypoxic-ischemic brain injury evaluated with magnetic resonance spectroscopy. Developmental Neuroscience. 2017;39(1-4):36-48 Available from:
  21. 21. Wu Q, Chen W, Sinha B, Tu Y, Manning S, Thomas N, et al. Neuroprotective agents for neonatal hypoxic–ischemic brain injury. Drug Discovery Today. 2015 Nov;20(11):1372-1381 Available from:
  22. 22. Thornton C, Leaw B, Mallard C, Nair S, Jinnai M, Hagberg H. Cell death in the developing brain after hypoxia-ischemia. Frontiers in Cellular Neuroscience. 2017;11(August):1-19 Available from:
  23. 23. Edwards AB, Anderton RS, Knuckey NW, Meloni BP. Perinatal Hypoxic-Ischemic Encephalopathy and Neuroprotective Peptide Therapies: A Case for Cationic Arginine-Rich Peptides (CARPs). Vol. 8. MDPI AG: Brain Sciences; 2018
  24. 24. Descloux C, Ginet V, Clarke PGH, Puyal J, Truttmann AC. Neuronal death after perinatal cerebral hypoxia-ischemia: Focus on autophagy-mediated cell death. International Journal of Developmental Neuroscience. 2015 Oct;45:75-85 Available from:
  25. 25. Leaw B, Nair S, Lim R, Thornton C, Mallard C, Hagberg H. Mitochondria. Bioenergetics and Excitotoxicity: New Therapeutic Targets in Perinatal Brain Injury. Front Cell Neurosci. 2017 Jul 12;11:199 Available from:
  26. 26. Cornelius C, Crupi R, Calabrese V, Graziano A, Milone P, Pennisi G, et al. Traumatic brain injury: Oxidative stress and Neuroprotection. Antioxidants & Redox Signaling. 2013 Sep 10;19(8):836-853 Available from:
  27. 27. Mehta A, Prabhakar M, Kumar P, Deshmukh R, Sharma PL. Excitotoxicity: Bridge to various triggers in neurodegenerative disorders. European Journal of Pharmacology. 2013 Jan 5;698(1-3):6-18 Available from:
  28. 28. Dasuri K, Zhang L, Keller JN. Oxidative stress, neurodegeneration, and the balance of protein degradation and protein synthesis. Free Radical Biology & Medicine. 2013 Sep;62:170-185 Available from:
  29. 29. Schimmel S, Acosta S, Lozano D. Neuroinflammation in traumatic brain injury: A chronic response to an acute injury. Brain Circ. 2017;3(3):135 Available from:
  30. 30. Venegoni W, Shen Q, Thimmesch AR, Bell M, Hiebert JB, Pierce JD. The use of antioxidants in the treatment of traumatic brain injury. Journal of Advanced Nursing. 2017 Jun;73(6):1331-1338 Available from:
  31. 31. Hiebert JB, Shen Q, Thimmesch AR, Pierce JD. Traumatic brain injury and mitochondrial dysfunction. The American Journal of the Medical Sciences. 2015 Aug;350(2):132-138 Available from:
  32. 32. Whelan SP, Zuckerbraun BS. Mitochondrial Signaling: Forwards, backwards, and In between. Oxidative Medicine and Cellular Longevity. 2013;2013:1-10 Available from:
  33. 33. Zhang L, Wang H, Zhou X, Mao L, Ding K, Hu Z. Role of mitochondrial calcium uniporter-mediated Ca 2+ and iron accumulation in traumatic brain injury. Journal of Cellular and Molecular Medicine. 2019 Feb 12 Available from:
  34. 34. Weidinger A, Kozlov A, Weidinger A, Kozlov AV. Biological activities of reactive oxygen and nitrogen species: Oxidative stress versus signal transduction. Biomolecules. 2015 Apr 15;5(2):472-484 Available from:
  35. 35. Rodríguez-Rodríguez A, Egea-Guerrero JJ, Murillo-Cabezas F, Carrillo-Vico A. Oxidative stress in traumatic brain injury. Current Medicinal Chemistry. 2014 Apr;21(10):1201-1211 Available from:
  36. 36. Rousset CI, Baburamani AA, Thornton C, Hagberg H. Mitochondria and perinatal brain injury. In: Journal of Maternal-Fetal and Neonatal Medicine. 2012. pp. 35-38
  37. 37. Kagan VE, Chu CT, Tyurina YY, Cheikhi A, Bayir H. Cardiolipin asymmetry, oxidation and signaling. Chemistry and Physics of Lipids. 2014 Apr;179:64-69. Available from
  38. 38. Toro-Urrego N, Garcia-Segura LM, Echeverria V, Barreto GE. Testosterone protects mitochondrial function and regulates Neuroglobin expression in Astrocytic cells exposed to glucose deprivation. Frontiers in Aging Neuroscience. 2016 Jun;27(8):152 Available from:
  39. 39. Paradies G, Petrosillo G, Paradies V, Ruggiero FM. Role of cardiolipin peroxidation and Ca2+in mitochondrial dysfunction and disease. Cell Calcium. 2009;45:643-650
  40. 40. Anthonymuthu TS, Kenny EM, Bayır H. Therapies targeting lipid peroxidation in traumatic brain injury. Brain Research. 2016 Jun 1;1640(Pt A):57-76 Available from:
  41. 41. Cristofori L, Tavazzi B, Gambin R, Vagnozzi R, Vivenza C, Amorini AM, et al. Early onset of lipid peroxidation after human traumatic brain injury: A fatal limitation for the free radical scavenger pharmacological therapy? Journal of Investigative Medicine. 2001;49(5):450-458
  42. 42. Bélanger M, Magistretti PJ. The role of astroglia in neuroprotection. Dialogues in Clinical Neuroscience. 2009;11(3):281-295 Available from:
  43. 43. Karki P, Webb A, Zerguine A, Choi J, Son DS, Lee E. Mechanism of raloxifene-induced upregulation of glutamate transporters in rat primary astrocytes. Glia. 2014;62(8):1270-1283
  44. 44. Guillamón-Vivancos T, Gómez-Pinedo U, Matías-Guiu J. Astrocitos en las enfermedades neurodegenerativas (I): función y caracterización molecular. Neurología. 2015 Mar;30(2):119-129 Available from:
  45. 45. Fuller S, Steele M, Münch G. Activated astroglia during chronic inflammation in Alzheimer’s disease—Do they neglect their neurosupportive roles? Mutat Res Mol Mech Mutagen. 2010 Aug 7;690(1-2):40-49 Available from:
  46. 46. Lee KM, AG ML. New advances on glial activation in health and disease. World J Virol. 2015 May 12;4(2):42-55 Available from:
  47. 47. Sullivan SM, Björkman ST, Miller SM, Colditz PB, Pow DV. Morphological changes in white matter astrocytes in response to hypoxia/ischemia in the neonatal pig. Brain Research. 2010 Mar;1319:164-174 Available from:
  48. 48. Wei S, Tong J, Xue Q, Liu Y, Xu X. Effect of puerarin on transcriptome of astrocyte during oxygen-glucose deprivation/reoxygenation injury. Molecular and Cellular Biochemistry. 2017 Jan 1;425(1-2):113-123
  49. 49. Rocha-Ferreira E, Hristova M. Antimicrobial peptides and complement in neonatal hypoxia-ischemia induced brain damage. Frontiers in Immunology. 2015;6:56. Available from:[cited 12 March 2018]
  50. 50. Hirayama Y, Koizumi S. Astrocytes and ischemic tolerance. Neuroscience Research. 2018;126:53-59. Available from:[cited 12 March 2018]
  51. 51. Sofroniew M V. Astrocyte barriers to neurotoxic inflammation. Vol. 16, Nature Reviews Neuroscience. NIH Public Access; 2015. p. 249-263. Available from:[cited 12 March 2018]
  52. 52. Millar LJ, Shi L, Hoerder-Suabedissen A, Molnár Z. Neonatal hypoxia Ischaemia: Mechanisms, models, and therapeutic challenges. Frontiers in Cellular Neuroscience. 2017;11:78. Available from:[cited 12 March 2018]
  53. 53. Ziemka-Nalecz M, Jaworska J, Zalewska T. Insights into the Neuroinflammatory responses after neonatal hypoxia-ischemia. Journal of Neuropathology and Experimental Neurology. 2017 Aug;76(8):644-654. Available from:[cited 12 March 2018]
  54. 54. Lee WLA, Michael-Titus AT, Shah DK. Hypoxic-ischaemic encephalopathy and the blood-brain barrier in neonates. Developmental Neuroscience. 2017;39(1-4):49-58
  55. 55. Paternotte E, Gaucher C, Labrude P, Stoltz JF, Menu P. Review: Behaviour of endothelial cells faced with hypoxia. Bio-medical Materials and Engineering. 2008;18(4-5):295-299
  56. 56. Salvador E, Burek M, Förster CY. Stretch and/or oxygen glucose deprivation (OGD) in an in vitro traumatic brain injury (TBI) model induces calcium alteration and inflammatory cascade. Frontiers in Cellular Neuroscience. 2015 Aug 21;9:323 Available from:
  57. 57. Huang BY, Castillo M. Hypoxic-ischemic brain injury: Imaging findings from birth to adulthood. Radiographics. 2008 Mar;28(2):417-439. Available from:[cited 19 July 2020]
  58. 58. Folkerth RD. Neuropathologic substrate of cerebral palsy. Journal of Child Neurology. 2005 Dec 2;20(12):940-949. Available from:[cited 19 July 2020]
  59. 59. McQuillen PS, Sheldon RA, Shatz CJ, Ferriero DM. Selective vulnerability of subplate neurons after early neonatal hypoxia-ischemia. The Journal of Neuroscience. 2003 Apr 15;23(8):3308-3315. Available from: /pmc/articles/PMC6742293/?report=abstract [cited 19 July 2020]
  60. 60. Gopagondanahalli KR, Li J, Fahey MC, Hunt RW, Jenkin G, Miller SL, et al. Preterm hypoxic-ischemic encephalopathy. Vol. 4, Frontiers in Pediatrics. Frontiers Media S.A.; 2016. p. 1. Available from: /pmc/articles/PMC5071348/?report=abstract [cited 19 July 2020]
  61. 61. Hoon AH, Stashinko EE, Nagae LM, Lin DDM, Keller J, Bastian A, et al. Sensory and motor deficits in children with cerebral palsy born preterm correlate with diffusion tensor imaging abnormalities in thalamocortical pathways. Developmental Medicine and Child Neurology. 2009;51(9):697-704. Available from: /pmc/articles/PMC2908264/?report=abstract [cited 19 July 2020]
  62. 62. Forbes TA, Goldstein EZ, Dupree JL, Jablonska B, Scafidi J, Adams KL, et al. Environmental enrichment ameliorates perinatal brain injury and promotes functional white matter recovery. Nature Communications. 2020 Dec 1;11(1). Available from: /pmc/articles/PMC7031237/?report=abstract [cited 19 July 2020]
  63. 63. Andjelkovic A V., Stamatovic SM, Phillips CM, Martinez-Revollar G, Keep RF. Modeling blood–brain barrier pathology in cerebrovascular disease in vitro: Current and future paradigms. Fluids Barriers CNS. 2020 Dec 16;17(1):44. Available from:[cited 19 July 2020]
  64. 64. Disdier C, Stonestreet BS. Hypoxic‐ischemic‐related cerebrovascular changes and potential therapeutic strategies in the neonatal brain. Journal of Neuroscience Research. 2020 Jul 14;98(7):1468-1484. Available from:[cited 19 July 2020]
  65. 65. Rice JE, Vannucci RC, Brierley JB. The influence of immaturity on hypoxic-ischemic brain damage in the rat. Annals of Neurology 1981 Feb;9(2):131-141. Available from:[cited 15 April 2018]
  66. 66. Drobyshevsky A, Derrick M, Wyrwicz AM, Ji X, Englof I, Ullman LM, et al. White matter injury correlates with hypertonia in an animal model of cerebral palsy. Journal of Cerebral Blood Flow and Metabolism. 2007 Feb 17;27(2):270-281. Available from:[cited 16 April 2018]
  67. 67. Kida H, Nomura S, Shinoyama M, Ideguchi M, Owada Y, Suzuki M. The effect of hypothermia therapy on cortical laminar disruption following ischemic injury in neonatal mice. Borlongan C V., editor. PLoS One. 2013 Jul 23;8(7):e68877. Available from:[cited 16 April 2018]
  68. 68. Lin EP, Miles L, Hughes EA, McCann JC, Vorhees C V., McAuliffe JJ, et al. A combination of mild hypothermia and Sevoflurane affords long-term protection in a modified neonatal mouse model of cerebral hypoxia-ischemia. Anesthesia and Analgesia. 2014 Nov;119(5):1158-1173. Available from:[cited 16 April 2018]
  69. 69. Dominguez R, Zitting M, Liu Q, Patel A, Babadjouni R, Hodis DM, et al. Estradiol protects white matter of male C57BL6J mice against experimental chronic cerebral Hypoperfusion. Journal of Stroke and Cerebrovascular Diseases. 2018 Jul;27(7):1743-1751. Available from:[cited 2019 Jul 9]
  70. 70. Reddy K, Mallard C, Guan J, Marks K, Bennet L, Gunning M, et al. Maturational change in the cortical response to Hypoperfusion injury in the Fetal sheep. Pediatric Research. 1998 May;43(5):674-682. Available from:[cited 16 April 2018]
  71. 71. Derrick M, Drobyshevsky A, Ji X, Tan S. A model of cerebral palsy from Fetal hypoxia-ischemia. Stroke. 2007 Feb 1;38(2):731-735. Available from:[cited 4 March 2018]
  72. 72. Derrick M, Drobyshevsky A, Ji X, Chen L, Yang Y, Ji H, et al. Hypoxia–ischemia causes persistent movement deficits in a perinatal rabbit model of cerebral palsy: Assessed by a new swim test. International Journal of Developmental Neuroscience. 2009 Oct 1;27(6):549-557. Available from:[cited 17 April 2018]
  73. 73. Traudt CM, McPherson RJ, Bauer LA, Richards TL, Burbacher TM, McAdams RM, et al. Concurrent erythropoietin and hypothermia treatment improve outcomes in a term nonhuman primate model of perinatal asphyxia. Developmental Neuroscience 2013;35(6):491-503. Available from:[cited 17 April 2018]
  74. 74. Alaa E, Mishima E, Niizuma K, Akiyama Y, Fujimura M, Tominaga T, et al. Stress-induced tRNA cleavage and tiRNA generation in rat neuronal PC12 cells. Journal of Neurochemistry. 2018 Feb 12; Available from:[cited 18 April 2018]
  75. 75. Chen Y, Zhang J, Zhang X. 2-NBDG as a marker for detecting glucose uptake in reactive astrocytes exposed to oxygen-glucose deprivation In vitro. Journal of Molecular Neuroscience. 2015 Jan 6;55(1):126-130. Available from:[cited 18 April 2018]
  76. 76. Cui X, Fu Z, Wang M, Nan X, Zhang B. Pitavastatin treatment induces neuroprotection through the BDNF-TrkB signalling pathway in cultured cerebral neurons after oxygen-glucose deprivation. Neurological Research. 2018 Mar 16;1-7. Available from:[cited 18 April 2018]
  77. 77. He W, Liu Y, Tian X. Rosuvastatin improves Neurite outgrowth of cortical neurons against oxygen-glucose deprivation via Notch1-mediated mitochondrial biogenesis and functional improvement. Frontiers in Cellular Neuroscience. 2018 Jan 17;12:6. Available from:[cited 18 April 2018]
  78. 78. Kim M, Jung K, Kim I-S, Lee I-S, Ko Y, Shin JE, et al. TNF-α induces human neural progenitor cell survival after oxygen–glucose deprivation by activating the NF-κB pathway. Experimental & Molecular Medicine. 2018 Apr 6;50(4):14. Available from:[cited 18 April 2018]
  79. 79. Dong Y-F, Guo R-B, Ji J, Cao L-L, Zhang L, Chen Z-Z, et al. S1PR3 is essential for phosphorylated fingolimod to protect astrocytes against oxygen-glucose deprivation-induced neuroinflammation via inhibiting TLR2/4-NFκB signalling. Journal of Cellular and Molecular Medicine. 2018 Mar 13; Available from:[cited 18 April 2018]
  80. 80. Wang Z, Guo L, Wang Y, Zhou H, Wang S, Chen D, et al. Inhibition of HSP90α protects cultured neurons from oxygen-glucose deprivation induced necroptosis by decreasing RIP3 expression. Journal of Cellular Physiology. 2018 Jun;233(6):4864-4884 Available from:
  81. 81. Wang K, Zhu Y. Dexmedetomidine protects against oxygen-glucose deprivation/reoxygenation injury-induced apoptosis via the p38 MAPK/ERK signalling pathway. The Journal of International Medical Research. 2018 Feb 6;46(2):675-686 Available from:
  82. 82. BAE S, H-J JEONG, CHA HJ, KIM K, CHOI YM, I-S AN, et al. The hypoxia-mimetic agent cobalt chloride induces cell cycle arrest and alters gene expression in U266 multiple myeloma cells. International Journal of Molecular Medicine. 2012 Nov 1;30(5):1180-1186 Available from:
  83. 83. Guo M, Song L-P, Jiang Y, Liu W, Yu Y, Chen G-Q. Hypoxia-mimetic agents desferrioxamine and cobalt chloride induce leukemic cell apoptosis through different hypoxia-inducible factor-1α independent mechanisms. Apoptosis. 2006 Jan 13;11(1):67-77 Available from:
  84. 84. Bordt EA. The importance of controlling in vitro oxygen tension to accurately model in vivo neurophysiology. Neurotoxicology. 2018 May;66:213-220 Available from:
  85. 85. Khan M, Khan H, Singh I, Singh AK. Hypoxia inducible factor-1 alpha stabilization for regenerative therapy in traumatic brain injury. Neural Regeneration Research. 2017 May;12(5):696-701 Available from:
  86. 86. Semenza GL. Hypoxia-inducible factor 1: Master regulator of O2 homeostasis. Current Opinion in Genetics & Development. 1998 Oct;8(5):588-594 Available from:
  87. 87. Ke Q, Costa M. Hypoxia-Inducible Factor-1 (HIF-1). 2006; Available from:
  88. 88. Huang LE, Gu J, Schau M, Bunn HF. Regulation of hypoxia-inducible factor 1alpha is mediated by an O2-dependent degradation domain via the ubiquitin-proteasome pathway. Proceedings of the National Academy of Sciences of the United States of America. 1998 Jul 7;95(14):7987-7992 Available from:
  89. 89. Wenger RH, Gassmann M. Oxygen(es) and the hypoxia-inducible factor-1. Biological Chemistry. 1997 Jul;378(7):609-616 Available from:
  90. 90. Tan WKM, Williams CE, Gunn AJ, Mallard CE, Gluckman PD. Suppression of postischemic epileptiform activity with MK-801 improves neural outcome in fetal sheep. Annals of Neurology. 1992 Nov 1;32(5):677-682 Available from:
  91. 91. Gunn AJ, Gunn TR, de Haan HH, Williams CE, Gluckman PD. Dramatic neuronal rescue with prolonged selective head cooling after ischemia in fetal lambs. The Journal of Clinical Investigation. 1997 Jan 15;99(2):248-256. Available from
  92. 92. Reddy K, Mallard C, Guan J, Marks K, Bennet L, Gunning M, et al. Maturational change in the cortical response to Hypoperfusion injury in the Fetal sheep. Pediatric Research. 1998 May;43(5):674-682 Available from:
  93. 93. Mallard EC, Williams CE, Johnston BM, Gluckman PD. Increased vulnerability to neuronal damage after umbilical cord occlusion in fetal sheep with advancing gestation. American Journal of Obstetrics and Gynecology. 1994 Jan;170(1 Pt 1):206-214 Available from:
  94. 94. Thoresen M, Penrice J, Lorek A, Cady EB, Wylezinska M, Kirkbride V, et al. Mild hypothermia after severe transient hypoxia-ischemia ameliorates delayed cerebral energy failure in the Newborn piglet. Pediatric Research. 1995 May;37(5):667-670 Available from:
  95. 95. Laptook AR, Hassan A, Peterson J, Corbett RJ, Nunnally RL, et al. NMR in Biomedicine. 1988 Apr;1(2):74-79 Available from:
  96. 96. Gressens P, Marret S, Evrard P. Developmental spectrum of the excitotoxic cascade induced by ibotenate: A model of hypoxic insults in fetuses and neonates. Neuropathology and Applied Neurobiology. 1996 May 30;22(6):498-502 Available from:
  97. 97. Baud O, Daire J-L, Dalmaz Y, Fontaine RH, Krueger RC, Sebag G, et al. Gestational hypoxia induces white matter damage in neonatal rats: A new model of periventricular leukomalacia. Brain Pathology. 2004 Jan;14(1):1-10 Available from:
  98. 98. Sheldon A, Chuai J, Ferriero DM. A rat model for hypoxic-ischemic brain damage in very premature infants. Neonatology. 1996;69(5):327-341 Available from:
  99. 99. Back SA, Han BH, Luo NL, Chricton CA, Xanthoudakis S, Tam J, et al. Selective vulnerability of late oligodendrocyte progenitors to hypoxia-ischemia. The Journal of Neuroscience. 2002 Jan 15;22(2):455-463 Available from:
  100. 100. Yang D, Sun Y-Y, Bhaumik SK, Li Y, Baumann JM, Lin X, et al. Blocking lymphocyte trafficking with FTY720 prevents inflammation-sensitized hypoxic–ischemic brain injury in Newborns. The Journal of Neuroscience. 2014 Dec 3;34(49):16467-16481 Available from:
  101. 101. Sheldon RA, Jiang X, Francisco C, Christen S, Vexler ZS, Täuber MG, et al. Manipulation of antioxidant pathways in neonatal murine brain. Pediatric Research. 2004 Oct;56(4):656-662 Available from:
  102. 102. Doverhag C, Hedtjärn M, Poirier F, Mallard C, Hagberg H, Karlsson A, et al. Galectin-3 contributes to neonatal hypoxic–ischemic brain injury. Neurobiology of Disease. 2010 Apr;38(1):36-46 Available from:
  103. 103. Ek CJ, D’Angelo B, Baburamani AA, Lehner C, Leverin A-L, Smith PL, et al. Brain barrier properties and cerebral blood flow in neonatal mice exposed to cerebral hypoxia-ischemia. Journal of Cerebral Blood Flow and Metabolism. 2015 May 28;35(5):818-827 Available from:
  104. 104. Hagberg H, Mallard C, Ferriero DM, Vannucci SJ, Levison SW, Vexler ZS, et al. The role of inflammation in perinatal brain injury. Nature Reviews. Neurology. 2015 Feb 17;11(4):192-208 Available from:
  105. 105. Renolleau S, Aggoun-Zouaoui D, Ben-Ari Y, Charriaut-Marlangue C. A model of transient unilateral focal ischemia with reperfusion in the P7 neonatal rat: Morphological changes indicative of apoptosis. Stroke. 1998 Jul;29(7):1454-1460 discussion 1461. Available from:
  106. 106. Derugin N, Ferriero DM, Vexler ZS. Neonatal reversible focal cerebral ischemia: A new model. Neuroscience Research. 1998 Dec;32(4):349-353 Available from:
  107. 107. Fernandez-Lopez D, Faustino J, Daneman R, Zhou L, Lee SY, Derugin N, et al. Blood-brain barrier permeability is increased after acute adult stroke but not neonatal stroke in the rat. The Journal of Neuroscience. 2012 Jul 11;32(28):9588-9600 Available from:
  108. 108. Faustino JV, Wang X, Johnson CE, Klibanov A, Derugin N, Wendland MF, et al. Microglial cells contribute to endogenous brain Defenses after acute neonatal focal stroke. The Journal of Neuroscience. 2011 Sep 7;31(36):12992-13001 Available from:
  109. 109. Mu D, Jiang X, Sheldon RA, Fox CK, Hamrick SEG, Vexler ZS, et al. Regulation of hypoxia-inducible factor 1alpha and induction of vascular endothelial growth factor in a rat neonatal stroke model. Neurobiology of Disease. 2003 Dec;14(3):524-534 Available from:
  110. 110. Woo M-S, Wang X, Faustino JV, Derugin N, Wendland MF, Zhou P, et al. Genetic deletion of CD36 enhances injury after acute neonatal stroke. Annals of Neurology. 2012 Dec;72(6):961-970 Available from:
  111. 111. Feng S-J, Zhang X-Q, Li J-T, Dai X-M, Zhao F. miRNA-223 regulates ischemic neuronal injury by targeting the type 1 insulin-like growth factor receptor (IGF1R). Folia Neuropathologica. 2018;56(1):49-57 Available from:
  112. 112. Guo M, Wang X, Zhao Y, Yang Q, Ding H, Dong Q, et al. Ketogenic diet improves brain ischemic tolerance and inhibits NLRP3 Inflammasome activation by preventing Drp1-mediated mitochondrial fission and endoplasmic reticulum stress. Frontiers in Molecular Neuroscience. 2018 Mar 20;11:86 Available from:
  113. 113. Li Y, Zhao Y, Cheng M, Qiao Y, Wang Y, Xiong W, et al. Suppression of microRNA-144-3p attenuates oxygen-glucose deprivation/reoxygenation-induced neuronal injury by promoting Brg1/Nrf2/ARE signaling. Journal of Biochemical and Molecular Toxicology. 2018 Feb 19:e22044 Available from:
  114. 114. Weng Y, Lin J, Liu H, Wu H, Yan Z, Zhao J. AMPK activation by Tanshinone IIA protects neuronal cells from oxygen-glucose deprivation. Oncotarget. 2018 Jan 12;9(4):4511-4521 Available from:
  115. 115. Yin X, Feng L, Ma D, Yin P, Wang X, Hou S, et al. Roles of astrocytic connexin-43, hemichannels, and gap junctions in oxygen-glucose deprivation/reperfusion injury induced neuroinflammation and the possible regulatory mechanisms of salvianolic acid B and carbenoxolone. Journal of Neuroinflammation. 2018 Dec 27;15(1):97 Available from:
  116. 116. Zhao X, Zhou K-S, Li Z-H, Nan W, Wang J, Xia Y-Y, et al. Knockdown of ski decreased the reactive astrocytes proliferation in vitro induced by oxygen-glucose deprivation/reoxygenation. Journal of Cellular Biochemistry. 2018 Mar 1 Available from:
  117. 117. Zhou T, Lin H, Jiang L, Yu T, Zeng C, Liu J, et al. Mild hypothermia protects hippocampal neurons from oxygen-glucose deprivation injury through inhibiting caspase-3 activation. Cryobiology. 2018 Feb;80:55-61 Available from:
  118. 118. Al Okail MS. Cobalt chloride, a chemical inducer of hypoxia-inducible factor-1α in U251 human glioblastoma cell line. Journal of Saudi Chemical Society. 2010 Apr 1;14(2):197-201 Available from:
  119. 119. Elstner A, Holtkamp N, von Deimling A. Involvement of Hif-1 in desferrioxamine-induced invasion of glioblastoma cells. Clinical & Experimental Metastasis. 2007 Mar 15;24(1):57-66 Available from:
  120. 120. Guo C, Hao L-J, Yang Z-H, Chai R, Zhang S, Gu Y, et al. Deferoxamine-mediated up-regulation of HIF-1α prevents dopaminergic neuronal death via the activation of MAPK family proteins in MPTP-treated mice. Experimental Neurology. 2016 Jun;280:13-23 Available from:
  121. 121. Hamrick SEG, McQuillen PS, Jiang X, Mu D, Madan A, Ferriero DM. A role for hypoxia-inducible factor-1α in desferoxamine neuroprotection. Neuroscience Letters. 2005 May 6;379(2):96-100 Available from:
  122. 122. Hishikawa T, Ono S, Ogawa T, Tokunaga K, Sugiu K, Date I. Effects of DEFEROXAMINE-activated hypoxia-inducible FACTOR-1 on the brainstem after subarachnoid HEMORRHAGE IN rats. Neurosurgery. 2008 Jan 1;62(1):232-241 Available from:
  123. 123. Jones SM, Novak AE, Elliott JP. The role of HIF in cobalt-induced ischemic tolerance. Neuroscience. 2013 Nov 12;252:420-430 Available from:
  124. 124. Li L, Yin X, Ma N, Lin F, Kong X, Chi J, et al. Desferrioxamine regulates HIF-1 alpha expression in neonatal rat brain after hypoxia-ischemia. American Journal of Translational Research. 2014;6(4):377-383 Available from:
  125. 125. Mehrabani M, Najafi M, Kamarul T, Mansouri K, Iranpour M, Nematollahi MH, et al. Deferoxamine preconditioning to restore impaired HIF-1α-mediated angiogenic mechanisms in adipose-derived stem cells from STZ-induced type 1 diabetic rats. Cell Proliferation. 2015 Oct;48(5):532-549 Available from:
  126. 126. Mu D, Chang YS, Vexler ZS, Ferriero DM. Hypoxia-inducible factor 1α and erythropoietin upregulation with deferoxamine salvage after neonatal stroke. Experimental Neurology. 2005 Oct;195(2):407-415 Available from:
  127. 127. van der Kooij MA, Groenendaal F, Kavelaars A, Heijnen CJ, van Bel F. Combination of deferoxamine and erythropoietin: Therapy for hypoxia–ischemia-induced brain injury in the neonatal rat? Neuroscience Letters. 2009 Feb 20;451(2):109-113 Available from:
  128. 128. Baulieu EE, Robel P, Schumacher M. Neurosteroids: Beginning of the story. International Review of Neurobiology. 2001;46:1-32 Available from:
  129. 129. Reddy DS, Estes WA. Clinical potential of Neurosteroids for CNS disorders. Vol. 37, trends in pharmacological sciences. Elsevier Ltd. 2016:543-561
  130. 130. Dubrovsky B. Neurosteroids, neuroactive steroids, and symptoms of affective disorders. Pharmacology, Biochemistry, and Behavior. 2006 Aug;84(4):644-655
  131. 131. Ferando I, Mody I. GABA a receptor modulation by neurosteroids in models of temporal lobe epilepsies. Epilepsia. 2012 Dec;53:89-101
  132. 132. Gavrilova VA, Ivanova SA, Gusev SI, Trofimova MV, Bokhan NA. Neurosteroids dehydroepiandrosterone and its sulfate in individuals with personality disorders convicted of serious violent crimes. Bulletin of Experimental Biology and Medicine. 2012 Nov;154(1):89-91
  133. 133. Borovska J, Vyklicky V, Stastna E, Kapras V, Slavikova B, Horak M, et al. Access of inhibitory neurosteroids to the NMDA receptor. British Journal of Pharmacology. 2012 Jun;166(3):1069-1083
  134. 134. González-Usano A, Cauli O, Agustí A, Felipo V. Hyperammonemia alters the modulation by different neurosteroids of the glutamate-nitric oxide-cyclic GMP pathway through NMDA- GABAA- or sigma receptors in cerebellum in vivo. Journal of Neurochemistry. 2013 Apr;125(1):133-143
  135. 135. Omura Y, Lu D, Jones MK, Nihrane A, Duvvi H, Shimotsuura Y, et al. Early detection of autism (ASD) by a non-invasive quick measurement of markedly reduced acetylcholine & DHEA and increased β-amyloid (1-42), Asbestos (Chrysotile), titanium dioxide, Al, Hg & often coexisting virus infections (CMV, HPV 16 and 18), bacterial infections etc. in the brain and corresponding safe individualized effective treatment. Acupuncture and Electro-Therapeutics Research. Cognizant Communication Corporation. 2015;40:157-187
  136. 136. Biagini G, Marinelli C, Panuccio G, Puia G, Avoli M. Glia-neuron interactions: Neurosteroids and Epileptogenesis. Jasper’s Basic Mech Epilepsies. 2012:1-16 Available from:
  137. 137. Brinton RD. Neurosteroids as regenerative agents in the brain: Therapeutic implications. Nature Reviews Endocrinology. 2013;9:241-250
  138. 138. King SR. Neurosteroids and the Nervous System2013. pp. 1-122. Available from. DOI:
  139. 139. Hidalgo Lanussa O, Ávila-Rodriguez M, Miguel García-Segura L, González J, Echeverria V, Aliev G, et al. Microglial dependent protective effects of neuroactive steroids. CNS Neurol Disord - Drug Targets. 2016 Feb 9;15(2):242-249
  140. 140. Srivastava DP. Two-step wiring plasticity - a mechanism for estrogen-induced rewiring of cortical circuits. Journal of Steroid Biochemistry and Molecular Biology. 2012;131:17-23
  141. 141. Ávila Rodriguez M, Garcia-Segura LM, Cabezas R, Torrente D, Capani F, Gonzalez J, et al. Tibolone protects T98G cells from glucose deprivation. The Journal of Steroid Biochemistry and Molecular Biology. 2014;144(PART B):294-303
  142. 142. Wang S, Wang B, Feng Y, Mo M, Du F, Li H, et al. 17β-Estradiol ameliorates light-induced retinal damage in Sprague–Dawley rats by reducing oxidative stress. Journal of Molecular Neuroscience. 2014;55(1):141-151
  143. 143. Cardona-Gomez GP, Mendez P, Garcia-Segura LM. Synergistic interaction of estradiol and insulin-like growth factor-I in the activation of PI3K/Akt signaling in the adult rat hypothalamus. Brain Research. Molecular Brain Research. 2002 Oct 30;107(1):80-88 Available from:
  144. 144. Garcia-Segura LM, Arevalo MA, Azcoitia I. Interactions of estradiol and insulin-like growth factor-I signalling in the nervous system: New advances. Progress in Brain Research. 2010;181(C):251-272
  145. 145. Perillo B, Sasso A, Abbondanza C, Palumbo G. 17beta -Estradiol inhibits apoptosis in MCF-7 cells, inducing bcl-2 expression via two Estrogen-responsive elements present in the coding sequence. Molecular and Cellular Biology. 2000 Apr 15;20(8):2890-2901
  146. 146. Arevalo MA, Santos-Galindo M, Lagunas N, Azcoitia I, Garcia-Segura LM. Selective estrogen receptor modulators as brain therapeutic agents. Journal of Molecular Endocrinology. 2011 Feb 1;46(1):R1-R9 Available from:
  147. 147. Vegeto E, Benedusi V, Maggi A. Estrogen anti-inflammatory activity in brain: A therapeutic opportunity for menopause and neurodegenerative diseases. Frontiers in Neuroendocrinology. 2008;29:507-519
  148. 148. Guo J, Duckles SP, Weiss JH, Li X, Krause DN. 17β-Estradiol prevents cell death and mitochondrial dysfunction by an estrogen receptor-dependent mechanism in astrocytes after oxygen-glucose deprivation/reperfusion. Free Radical Biology & Medicine. 2012 Jun 1;52(11-12):2151-2160
  149. 149. Simpkins JW, Dykens JA. Mitochondrial mechanisms of estrogen neuroprotection. Brain Research Reviews. 2008;57:421-430
  150. 150. Mendez P, Garcia-Segura LM. Phosphatidylinositol 3-kinase and glycogen synthase kinase 3 regulate Estrogen receptor-mediated transcription in neuronal cells. Endocrinology. 2006 Jun 1;147(6):3027-3039 Available from:
  151. 151. Prokai L, Prokai-Tatrai K, Perjési P, Simpkins JW. Mechanistic insights into the direct antioxidant effects of estrogens. Drug Development Research. 2005 Oct;66(2):118-125 Available from:
  152. 152. Tang S. ERGDB: Estrogen Responsive Genes Database. Nucleic Acids Research. 2004 Jan 1;32(90001):533D-5536D
  153. 153. Colditz GA, Hankinson SE, Hunter DJ, Willett WC, Manson JE, Stampfer MJ, et al. The use of estrogens and progestins and the risk of breast cancer in postmenopausal women. The New England Journal of Medicine. 1995 Jun 15;332(24):1589-1593
  154. 154. Manolio TA, Furberg CD, Shemanski L, Psaty BM, O’Leary DH, Tracy RP, et al. Associations of postmenopausal estrogen use with cardiovascular disease and its risk factors in older women. The CHS collaborative research group 86. Circulation. 1993 Nov;88(0009-7322 (Print):2163-2171
  155. 155. Brynhildsen J, Hammar M. Lipids and clotting factors during low dose transdermal estradiol/norethisterone use. Maturitas. 2005 Apr 11;50(4):344-352
  156. 156. Lemini C, Franco Y, Avila ME, Jaimez R. Contrasting effects of estradiol and 17 beta-aminoestrogens on blood clotting time in rats and mice. European Journal of Pharmacology. 2005 Mar 14;510(3):229-233 Available from:
  157. 157. Taxel P, Luthra P, Fall PM, Dauser D. The effect of short-term estradiol therapy on clotting and inflammatory markers in older men receiving hormonal suppression therapy for prostate cancer. The Aging Male. 2008 Jun;11(2):71-75
  158. 158. Wise PM, Suzuki S, Brown CM. Estradiol: A hormone with diverse and contradictory neuroprotective actions. Dialogues in Clinical Neuroscience. 2009;11:297-303
  159. 159. Liu M, Kelley MH, Herson PS, Hurn PD. Neuroprotection of sex steroids. Minerva Endocrinologica. 2010;35:127-143
  160. 160. Kloosterboer HJ. Tissue-selectivity: The mechanism of action of tibolone. In: Maturitas. Elsevier Ireland Ltd; 2004. pp. 30-40
  161. 161. Sanchez AC, Alsina JCI, Dueñas-Díez JL. Selective Estrogen Receptor Modulators: A New Brand of Multitarget Drugs. Selective Estrogen Receptor Modulators: A New Brand of Multitarget Drugs. Springer Berlin Heidelberg; 2006. pp. 1-357
  162. 162. Brzozowski AM, Pike ACW, Dauter Z, Hubbard RE, Bonn T, Engström O, et al. Molecular basis of agonism and antagonism in the oestrogen receptor. Nature. 1997;389(6652):753-758
  163. 163. Cano A, Morcillo N, Lopez F, Marquina P, Parrilla JJ, Abad L. Cytoplasmic and nuclear estrogen binding capacity in the rat uterus during treatment with danazol and testosterone. European Journal of Obstetrics, Gynecology, and Reproductive Biology. 1986 Apr;21(4):245-252 Available from:
  164. 164. Díaz Chico BN, Bosch DN, Díaz Chico JC, Escriche EE. Molecular mechanisms of estrogen action in target tissues. In: Selective Estrogen Receptor Modulators: A New Brand of Multitarget Drugs. Berlin Heidelberg: Springer; 2006. pp. 2-47
  165. 165. McKenna NJ, Lanz RB, O’Malley BW. Nuclear receptor Coregulators: Cellular and molecular biology*. Endocrine Reviews. 1999 Jun 1;20(3):321-344
  166. 166. Shiau AK, Barstad D, Loria PM, Cheng L, Kushner PJ, Agard DA, et al. The structural basis of estrogen receptor/coactivator recognition and the antagonism of this interaction by tamoxifen. Cell. 1998 Dec 23;95(7):927-937 Available from:
  167. 167. Vogelvang TE, Van Der Mooren MJ, Mijatovic V. Hormone replacement therapy, selective Estrogen receptor modulators, and tissue-specific compounds: Cardiovascular effects and clinical implications. Treatments in Endocrinology. 2004;3:105-115
  168. 168. Kokiko ON, Murashov AK, Hoane MR. Administration of raloxifene reduces sensorimotor and working memory deficits following traumatic brain injury. Behavioural Brain Research. 2006 Jun 30;170(2):233-240
  169. 169. Mosquera L, Colón JM, Santiago JM, Torrado AI, Meléndez M, Segarra AC, et al. Tamoxifen and estradiol improved locomotor function and increased spared tissue in rats after spinal cord injury: Their antioxidant effect and role of estrogen receptor alpha. Brain Research. 2014 May 2;1561:11-22
  170. 170. González-Burgos I, Rivera-Cervantes MC, Velázquez-Zamora DA, Feria-Velasco A, Garcia-Segura LM. Selective estrogen receptor modulators regulate dendritic spine plasticity in the hippocampus of male rats. Neural Plasticity. 2012;2012
  171. 171. Wakade C, Khan MM, De Sevilla LM, Zhang QG, Mahesh VB, Brann DW. Tamoxifen neuroprotection in cerebral ischemia involves attenuation of kinase activation and superoxide production and potentiation of mitochondrial superoxide dismutase. Endocrinology. 2008 Jan;149(1):367-379
  172. 172. Simoncini T, Mannella P, Fornari L, Caruso A, Varone G, Genazzani AR. Genomic and non-genomic effects of estrogens on endothelial cells. Steroids. 2004;69(8):537-542 Available from:
  173. 173. Armagan G, Kanit L, Terek CM, Sozmen EY, Yalcin A. The levels of glutathione and nitrite-nitrate and the expression of BCL-2 mRNA in ovariectomized rats treated by raloxifene against kainic acid. The International Journal of Neuroscience. 2009 Feb;119(2):227-239
  174. 174. Martino S, Cauley JA, Barrett-Connor E, Powles TJ, Mershon J, Disch D, et al. Continuing outcomes relevant to Evista: Breast cancer incidence in postmenopausal osteoporotic women in a randomized trial of raloxifene. Journal of the National Cancer Institute. 2004 Dec 1;96(23):1751-1761
  175. 175. Ingle JN, Suman VJ, Mailliard JA, Kugler JW, Krook JE, Michalak JC, et al. Randomized trial of tamoxifen alone or combined with fluoxymesterone as adjuvant therapy in postmenopausal women with resected estrogen receptor positive breast cancer. North central Cancer treatment group trial 89-30-52. Breast Cancer Research and Treatment. 2006 Jul;98(2):217-222
  176. 176. Arevalo MA, Diz-Chaves Y, Santos-Galindo M, Bellini MJ, Garcia-Segura LM. Selective oestrogen receptor modulators decrease the inflammatory response of glial cells. Journal of Neuroendocrinology. 2012;24:183-190
  177. 177. Belenichev IF, Odnokoz OV, Pavlov SV, Belenicheva OI, Polyakova EN. The neuroprotective activity of tamoxifen and tibolone during glutathione depletion in vitro. Neurochemical Journal. 2012 Jul;6(3):202-212
  178. 178. Catalano S, Giordano C, Panza S, Chemi F, Bonofiglio D, Lanzino M, et al. Tamoxifen through GPER upregulates aromatase expression: A novel mechanism sustaining tamoxifen-resistant breast cancer cell growth. Breast Cancer Research and Treatment. 2014;146(2):273-285
  179. 179. López Ruiz JR, Osuna Carrasco LP, López Valenzuela CL, Franco Rodríguez NE, de la Torre VB, Jiménez Estrada I, et al. The hippocampus participates in the control of locomotion speed. Neuroscience. 2015 Dec 17;311:207-215
  180. 180. Karki P, Webb A, Zerguine A, Choi J, Son DS, Lee E. Mechanism of raloxifene-induced upregulation of glutamate transporters in rat primary astrocytes. Glia. 2014;62(8):1270-1283
  181. 181. Shang Y, Brown M. Molecular determinants for the tissue specificity of SERMs. Science (80- ). 2002 Mar 29;295(5564):2465-2468
  182. 182. Gupta B, Mittal P, Khuteta R, Bhargava A. A comparative study of CEE, tibolone, and DHEA as hormone replacement therapy for surgical menopause. J Obstet Gynecol India. 2013 Jun;63(3):194-198
  183. 183. Albertazzi P, Di Micco R, Zanardi E. Tibolone: A review. Maturitas. 1998 Nov 16;30(3):295-305 Available from:
  184. 184. Campisi R, Marengo FD. Cardiovascular effects of tibolone: A selective tissue estrogenic activity regulator. Cardiovascular Drug Reviews. 2007;25:132-145
  185. 185. Escande A, Servant N, Rabenoelina F, Auzou G, Kloosterboer H, Cavaillès V, et al. Regulation of activities of steroid hormone receptors by tibolone and its primary metabolites. The Journal of Steroid Biochemistry and Molecular Biology. 2009 Aug;116(1-2):8-14
  186. 186. Vos RME, Krebbers SFM, Verhoeven CHJ, Delbressine LPC. The in vivo human metabolism of tibolone. Drug Metabolism and Disposition. 2002;30(2):106-112
  187. 187. Guzmán CB, Zhao C, Deighton-collins S, Kleerekoper M, Benjamins JA, Skafar DF. Agonist activity of the 3-hydroxy metabolites of tibolone through the oestrogen receptor in the mouse N20.1 oligodendrocyte cell line and normal human astrocytes. Journal of Neuroendocrinology. 2007 Dec;19(12):958-965
  188. 188. Tian WY, Zhang HY, Su LK, Shen WZ. Effects of tibolone on apoptosis of neurons after cerebral ischemia-reperfusion injury in rats. Acad J Second Mil Med Univ. 2009 Jul;30(7):790-792
  189. 189. Kloosterboer HJ, Löfgren L, Von Schoulz E, Von Schoultz B, Verheul HAM. Estrogen and tibolone metabolite levels in blood and breast tissue of postmenopausal women recently diagnosed with early-stage breast cancer and treated with tibolone or placebo for 14 days. Reproductive Sciences. 2007 Feb;14(2):151-159
  190. 190. Falany JL, Falany CN. Interactions of the human cytosolic sulfotransferases and steroid sulfatase in the metabolism of tibolone and raloxifene. The Journal of Steroid Biochemistry and Molecular Biology. 2007 Nov;107(3-5):202-210
  191. 191. De Marinis E, Fiocchetti M, Acconcia F, Ascenzi P, Marino M. Neuroglobin upregulation induced by 17β-estradiol sequesters cytocrome c in the mitochondria preventing H2 O2 -induced apoptosis of neuroblastoma cells. Cell Death & Disease. 2013 Feb;4:2
  192. 192. De Marinis E, Ascenzi P, Pellegrini M, Galluzzo P, Bulzomi P, Arevalo MA, et al. 17β-Estradiol - a new modulator of neuroglobin levels in neurons: Role in neuroprotection against H 2O 2-induced toxicity. Neuro-Signals. 2011 Mar;18(4):223-235
  193. 193. De Marinis E, Acaz-Fonseca E, Arevalo MA, Ascenzi P, Fiocchetti M, Marino M, et al. 17β-Oestradiol anti-inflammatory effects in primary astrocytes require oestrogen receptor β-mediated neuroglobin up-regulation. Journal of Neuroendocrinology. 2013 Mar;25(3):260-270
  194. 194. Acaz-Fonseca E, Sanchez-Gonzalez R, Azcoitia I, Arevalo MA, Garcia-Segura LM. Role of astrocytes in the neuroprotective actions of 17β-estradiol and selective estrogen receptor modulators. Molecular and Cellular Endocrinology. 2014;389:48-57
  195. 195. Pinto Almazán R, Rivas Arancibia S, Farfán García ED, Rodríguez Martínez E, Guerra AC. Efecto neuroprotector de la tibolona contra el estrés oxidativo inducido por la exposición a ozono. Revista de Neurologia. 2014;58(10):441 Available from:
  196. 196. Beltrán-Campos V, Díaz-Ruiz A, Padilla-Gómez E, Aguilar Zavala H, Ríos C, Díaz Cintra S. Effect of tibolone on dendritic spine density in the rat hippocampus. Neurol(English Ed.). 2015 Sep;30(7):401-406
  197. 197. Maguire J. Neuroactive steroids and GABaergic involvement in the neuroendocrine dysfunction associated with major depressive disorder and postpartum depression. Frontiers in Cellular Neuroscience, Frontiers Media S.A. 2019;13
  198. 198. Hillerer KM, Slattery DA, Pletzer B. Neurobiological mechanisms underlying sex-related differences in stress-related disorders: Effects of neuroactive steroids on the hippocampus. Frontiers in Neuroendocrinology. 2019;55
  199. 199. Frau R, Traccis F, Bortolato M. Neurobehavioural complications of sleep deprivation: Shedding light on the emerging role of neuroactive steroids. Journal of Neuroendocrinology. 2019
  200. 200. Aleman M, McCue PM, Chigerwe M, Madigan JE. Plasma concentrations of steroid precursors, steroids, neuroactive steroids, and neurosteroids in healthy neonatal foals from birth to 7 days of age. Journal of Veterinary Internal Medicine. 2019 Sep 1;33(5):2286-2293
  201. 201. Knytl P, VoráCková V, Dorazilová A, Rodriguez M, CvrCková A, Kofronová E, et al. Neuroactive steroids and cognitive functions in first-episode psychosis patients and their healthy siblings. Frontiers in Psychiatry. 2019;10(Jun)
  202. 202. Modgil A, Parakala ML, Ackley MA, Doherty JJ, Moss SJ, Davies PA. Endogenous and synthetic neuroactive steroids evoke sustained increases in the efficacy of GABAergic inhibition via a protein kinase C-dependent mechanism. Neuropharmacology. 2017 Feb 1;113:314-322
  203. 203. Deligiannidis KM, Fales CL, Kroll-Desrosiers AR, Shaffer SA, Villamarin V, Tan Y, et al. Resting-state functional connectivity, cortical GABA, and neuroactive steroids in peripartum and peripartum depressed women: A functional magnetic resonance imaging and spectroscopy study. Neuropsychopharmacology. 2019 Feb 1;44(3):546-554
  204. 204. Giatti S, Diviccaro S, Melcangi RC. Neuroactive steroids and sex-dimorphic nervous damage induced by diabetes mellitus. Cellular and Molecular Neurobiology. 2019;39:493-502
  205. 205. Giatti S, Garcia-Segura LM, Barreto GE, Melcangi RC. Neuroactive steroids, neurosteroidogenesis and sex. Progress in Neurobiology. 2019;176:1-17
  206. 206. McEvoy K, Payne JL, Osborne LM. Neuroactive steroids and perinatal depression: A review of recent literature. Current Psychiatry Reports. 2018;20
  207. 207. Melcangi RC, Panzica GC. Neuroactive steroids and metabolic axis. Frontiers in Neuroendocrinology. 2018;48:1-2
  208. 208. Dichtel LE, Lawson EA, Schorr M, Meenaghan E, Paskal ML, Eddy KT, et al. Neuroactive steroids and affective symptoms in women across the weight Spectrum. Neuropsychopharmacology. 2018 May 1;43(6):1436-1444
  209. 209. Tuem KB, Atey TM. Neuroactive steroids: Receptor interactions and responses. Frontiers in Neurology. 2017 Aug;8(Aug):28
  210. 210. Martinez Botella G, Salituro FG, Harrison BL, Beresis RT, Bai Z, Blanco MJ, et al. Neuroactive steroids. 2. 3α-Hydroxy-3β-methyl-21-(4-cyano-1H-pyrazol-1′-yl)-19-nor-5β-pregnan-20-one (SAGE-217): A clinical next generation Neuroactive steroid positive allosteric modulator of the (γ-Aminobutyric acid) a receptor. Journal of Medicinal Chemistry. 2017;60(18):7810-7819
  211. 211. Dembek KA, Timko KJ, Johnson LM, Hart KA, Barr BS, David B, et al. Steroids, steroid precursors, and neuroactive steroids in critically ill equine neonates. Veterinary Journal. 2017 Jul 1;225:42-49
  212. 212. Solanki RK, Sharma P, Tyagi A, Singh C. Serum levels of neuroactive steroids in first-episode antipsychotic-na’ive schizophrenic patients and its correlation with aggression: A case-control study. East Asian Archives of Psychiatry. 2017 Jun 1;27(2):79-84
  213. 213. Rasmusson AM, Marx CE, Pineles SL, Locci A, Scioli-Salter ER, Nillni YI, et al. Neuroactive steroids and PTSD treatment. Neuroscience Letters. 2017;649:156-163
  214. 214. Porcu P, O’Buckley TK, Lopez MF, Becker HC, Miles MF, Williams RW, et al. Initial genetic dissection of serum neuroactive steroids following chronic intermittent ethanol across BXD mouse strains. Alcohol. 2017 Feb 1;58:107-125
  215. 215. Casas S, Gonzalez Deniselle MC, Gargiulo-Monachelli GM, Perez AF, Tourreilles M, Mattiazzi M, et al. Neuroactive steroids in acute ischemic stroke: Association with cognitive, functional, and neurological outcomes. Hormone and Metabolic Research. 2017 Jan 1;49(1):16-22
  216. 216. Straub RH. The complex role of estrogens in inflammation. Endocrine Reviews. 2007;28:521-574
  217. 217. Brunori M, Vallone B. Neuroglobin, seven years after. Cellular and Molecular Life Sciences. 2007 May;64(10):1259-1268 Available from:
  218. 218. Xiao QC, Lu YQ, Chen GZ, Li TY, Gao Z, Liu S, et al. Presence of neuroglobin in cultured astrocytes. Glia. 2005 Apr 15;50(2):182-186
  219. 219. Venis S. Neuroglobin might protect brain cells during stroke. Lancet. 2001;358(9298):2055
  220. 220. Gao XY, Huang JO, Hu YF, Gu Y, Zhu SZ, Bin HK, et al. Combination of mild hypothermia with neuroprotectants has greater neuroprotective effects during oxygen-glucose deprivation and reoxygenation-mediated neuronal injury. Scientific Reports. 2014;4
  221. 221. Li Y, Dai Y. Bing, Sun J yun, Xiang Y, Yang J, Dai S yang, et al. Neuroglobin attenuates Beta amyloid-induced apoptosis through inhibiting Caspases activity by activating PI3K/Akt Signaling pathway. Journal of Molecular Neuroscience. 2016 Jan 1;58(1):28-38
  222. 222. Trent JT, Watts RA, Hargrove MS. Human Neuroglobin, a Hexacoordinate Hemoglobin that reversibly binds oxygen. The Journal of Biological Chemistry. 2001 Aug 10;276(32):30106-30110
  223. 223. Hua S, Antao ST, Corbet A, Witting P. k. Retraction notice: The significance of Neuroglobin in the brain. Current Medicinal Chemistry. 2009 Dec 21;17(2):160-172
  224. 224. Brittain T, Skommer J, Raychaudhuri S, Birch N. An Antiapoptotic Neuroprotective Role for Neuroglobin. International Journal of Molecular Sciences. 2010 May 27;11(6):2306-2321 Available from:
  225. 225. Sadava DE, Hillis DM, Heller CH, Berenbaum M. Libro Life 10th.Pdf2014. pp. 893-894
  226. 226. Zhao L, O’Neill K, Diaz BR. Selective estrogen receptor modulators (SERMs) for the brain: Current status and remaining challenges for developing NeuroSERMs. Brain Research Reviews. 2005;49(3):472-493
  227. 227. Paterni I, Granchi C, Katzenellenbogen JA, Minutolo F. Estrogen receptors alpha (ERα) and Beta (ERβ): Subtype-selective ligands and clinical potential. Steroids. 2014 Nov 15:13-29 Available from:
  228. 228. Arevalo MA, Azcoitia I, Garcia-Segura LM. The neuroprotective actions of oestradiol and oestrogen receptors. Nature Reviews. Neuroscience. 2015;16(1):17-29 Available from:
  229. 229. Shang Y, Hu X, DiRenzo J, Lazar MA, Brown M. Cofactor dynamics and sufficiency in Estrogen receptor–regulated transcription. Cell. 2000 Dec 8;103(6):843-852 Available from:
  230. 230. Safe S, Kim K. NONCLASSICAL GENOMIC ER/Sp AND ER/AP-1 SIGNALING PATHWAYS. Journal of Molecular Endocrinology. 2008 Nov 4;41(5):263-275 Available from:
  231. 231. Ruiz-Palmero I, Hernando M, Garcia-Segura LM, Arevalo M-A. G protein-coupled estrogen receptor is required for the neuritogenic mechanism of 17β-estradiol in developing hippocampal neurons. Molecular and Cellular Endocrinology. 2013;372(1-2):105-115 Available from:
  232. 232. Qiu J, Bosch MA, Tobias SC, Grandy DK, Scanlan TS, Ronnekleiv OK, et al. Rapid signaling of estrogen in hypothalamic neurons involves a novel G-protein-coupled estrogen receptor that activates protein kinase C. The Journal of Neuroscience. 2003 Oct 22;23(29):9529-9540 Available from:
  233. 233. Gerstner B, Lee J, DeSilva TM, Jensen FE, Volpe JJ, Rosenberg PA. 17β-Estradiol protects against hypoxic/ischemic white matter damage in the neonatal rat brain. Journal of Neuroscience Research. 2009 Jul;87(9):2078-2086 Available from:
  234. 234. Nuñez J, Yang Z, Jiang Y, Grandys T, Mark I, Levison SW. 17β-Estradiol protects the neonatal brain from hypoxia-ischemia. Experimental Neurology. 2007 Dec 12;208(2):269-276 Available from:
  235. 235. Barreto G, Saraceno E, Gonzalez J, Kolliker R, Castilla R, Capani F. Chapter 8 - Neuroprotection with Estradiol in Experimental Perinatal Asphyxia: A New Approach A2 - Duncan, Kelli a. BT - Estrogen Effects on Traumatic Brain Injury. San Diego: Academic Press; 2015. pp. 113-124 Available from:
  236. 236. Charriaut-Marlangue C, Besson VC, Baud O. Sexually dimorphic outcomes after neonatal stroke and hypoxia-ischemia. International Journal of Molecular Sciences. 2018;19:1
  237. 237. Elzer JG, Muhammad S, Wintermantel TM, Regnier-Vigouroux A, Ludwig J, Schütz G, et al. Neuronal Estrogen receptor-α mediates Neuroprotection by 17β-Estradiol. Journal of Cerebral Blood Flow and Metabolism. 2009 Dec 16;30(5):935-942 Available from:
  238. 238. Nelson ER, Wardell SE, McDonnell DP. The molecular mechanisms underlying the pharmacological actions of estrogens, SERMs and oxysterols: Implications for the treatment and prevention of osteoporosis. Bone. 2013;53(1):42-50
  239. 239. Marín F, Barbancho MC. Action of selective Estrogen receptor modulators (SERMs) through the classical mechanism of Estrogen action. In: Selective Estrogen Receptor Modulators. Berlin Heidelberg: Springer; 2006. pp. 71-77. Available from. DOI:
  240. 240. Khan MM, Wakade C, de Sevilla L, Brann DW. Selective estrogen receptor modulators (SERMs) enhance neurogenesis and spine density following focal cerebral ischemia. The Journal of Steroid Biochemistry and Molecular Biology. 2015 Feb;146:38-47 Available from:
  241. 241. Lopez-Rodriguez AB, Ávila-Rodriguez M, Vega-vela NE, Capani F, Gonzalez J, Garciá-Segura LM, et al. Estrogen Effects on Traumatic Brain Injury. 2015.
  242. 242. Leaw B, Nair S, Lim R, Thornton C, Mallard C, Hagberg H. Mitochondria, bioenergetics and Excitotoxicity: New therapeutic targets in perinatal brain injury. Frontiers in Cellular Neuroscience. 2017 Jul 12;11:199 Available from:
  243. 243. Wu Q, Chen W, Sinha B, Tu Y, Manning S, Thomas N, et al. Neuroprotective agents for neonatal hypoxic-ischemic brain injury. Drug Discovery Today. 2015;20:1372-1381 Available from:

Written By

Nicolas Toro-Urrego, Marco Avila-Rodriguez, María Inés Herrera, Andrea Aguilar, Lucas Udovin and Juan P. Luaces

Submitted: June 16th, 2019 Reviewed: September 9th, 2020 Published: November 26th, 2020