**2.2 Excitotoxicity**

*Neuroprotection - New Approaches and Prospects*

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) [5–7]. 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 [12–14]. 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.

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

**2. Mechanisms of brain damage in hypoxia-ischemia**

responsible for the brain cells death after hypoxia [7, 18].

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

**210**

**2.1 Second phase of injury**

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

**213**

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

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

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

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,

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 perme-

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

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].

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

further expand of cerebral infarction area [48].

**2.8 Microglia and endothelial cells**

ability of the cEND monolayer [56].

**2.9 Selective vulnerability of the brain to HI**

types present a selective vulnerability to this injury [18].

to cerebral ischemic injury [50] and inflammation [50, 51].

creating a cytotoxic environment triggering cell death [49, 52, 53].

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

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**

*Neuroprotection - New Approaches and Prospects*

and calcium homeostasis [26, 30, 31, 33].

**2.5 Cardiolipin peroxidation**

content of fatty acids [39].

proinflammatory cytokines and ROS.

**2.6 Inflammation in HI**

**2.7 Astrocytes**

glutathione peroxidase 1, required downstream of SOD1 [35].

extrinsic pathway, following activation of death receptors [36].

cell proliferation [31, 32], also in critical processes such the maintenance of neuronal homeostasis, including autophagy, elimination of toxic metabolites like ROS,

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

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

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),

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

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

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

complex IV (cytochrome C oxidase) and complex V (ATP synthase) [39].

**212**

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].

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.


**215**

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

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 neurodevelop-

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].

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

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

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] . 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, 71–73].

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

**2.10 Oligodendrocytes and astrocytes**

**2.11 Blood–brain barrier and vascular fragility**

event, resulting more susceptible to HI injury [60].

entry of molecules into the brain [63].

**3. Experimental models**

mental impairment [62].

*from [58].*

#### **Table 1.**

*Lesions caused by HI in the developing brain.*

#### **2.10 Oligodendrocytes and astrocytes**

*Neuroprotection - New Approaches and Prospects*

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

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

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.

Germinal matrix (ganglionic eminence) (frequently associated with PVL)

Single cerebral artery–distribution infarcts (porencephaly) Hydranencephaly (bilateral large hemispheric infarcts)

Thalamic and basal ganglionic injury ("status marmoratus")

*PHI = periventricular hemorrhagic infarction; PVL = periventricular leukomalacia.*

Mobius syndrome (brainstem neuronal loss and gliosis)

Neuronal necrosis in basis pontis and subiculum (pontosubicular necrosis)

*Grade refers to clinical severity assigned based on transfontanelle ultrasonography or other neuroimaging. Adapted* 

and hippocampus are also found in the immature brain [18].

hemorrhage, or thalamocortical injury (**Table 1**) [58].

**214**

*†*

*from [58].*

**Table 1.**

Hemorrhagic lesions

Limited (grade I†

White-matter lesions

Gray-matter lesions

Cerebellar infarct

With focal necrosis

Subpial Cerebellar

With PHI (grade IV)

) With intraventricular hemorrhage (grade II) With ventricular expansion (grade III)

Periventricular leukomalacia (PVL)

Multicystic encephalomalacia

*Lesions caused by HI in the developing brain.*

With diffuse white-matter gliosis only Periventricular hemorrhagic infarction (PHI) Combined gray- and white-matter lesions

Subarachnoid space (temporal lobe and cerebellum)

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] .

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, 71–73].
