**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. [74–81], 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 [84–89].


**217**

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

Hypoxia E5-E20 White matter cysts in offspring P0–P7,

CCAL + hypoxia Selective vulnerability of late OL

CCAL + hypoxia Unilateral ischemic injury in the cortex,

survivors.

effect with pure HI.

CCAL + hypoxia Reduced injury in GPx1-Tg mice but not in

CCAL + hypoxia Increased BBB permeability 2–24 h,

96 h.

Transient MCAO, 3 h Severe unilateral perfusion deficits,

Transient MCAO, 1.5 h Time resolved cell-type specific increase in HIF-1a and

activation.

to control

VEGF expression, gliosis.

macrophages.

increased lipid peroxidation, WMI and

progenitors, independent of age.

Death of sub-plate neurons, motor deficits, altered thalamocortical connections to somatosensory and visual cortex normal.

hippocampus, basal ganglia in >90% of

Blocking lymphocyte trafficking reduced brain inflammation, BBB damage, and improved LPS-induced HI brain injury. No

SOD1-Tg or GPx1/SOD1. NOS inhibition did not improve outcome in SOD-Tg.

reduced BBB protein expression. Infarct volume reduction in Gal-3 KO mice.

Injury linked to inflammatory response &

Infarcts in frontoparietal cortex at 3-month recovery. DNA fragmentation from 6 to

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.

Focal ischemia–reperfusion, increased injury and caspase-3 cleavage associated with apoptotic neuronal debris in CD36 KO. Effects independent of NFκB

Significant morphological cell changes

Significantly increased 2- NBDG uptake by about 1.2 to 2.5 times in cells compared

Increased infarct volume and WMI,

prevented in TRIF KO.

decrease in M2-like microglia.

**Reference Species Animal model Outcomes**

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

Sprague– Dawley rats, embryonic

Rodent models with hypoxia-ischemia [98, 99] Sprague Dawley rats, P1–P3

Dawley rats, P7

Tg SOD1, GPx1 over-expressing P7 mice

and Gal-3 KO,

TRIF KO mice, P8–9

[105] Wistar rat, P7 Permanent MCAO +1 h

CCAO

[100] Wistar rat, P7 LPS, 4 h prior to CCAL

+ hypoxia

Poly I:C, 14 h prior to CCAL + hypoxia

Transient MCAO, 1.5 h

and 3 h

Reference Cell line Experimental model Outcomes

reperfusion

6 h OGD/ 0, 12, 24, 48 h reperfusion

[97] Pregnant

[65] Sprague–

[101] C57Bl/6 WT,

[102, 103] C57BL/6 WT

[104] C57BL/6 J and

Focal ischemia rodent models

[106–108] Sprague Dawley rats, P7

[109] Sprague Dawley rats, P10

[110] C57/Bl6 mice,

[75] Primary cortical astrocyte

In vitro models

CD36 KO and WT, P9

[74] PC12 cells 48 h OGD/ 2 h

P9


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

*Neuroprotection - New Approaches and Prospects*

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. [74–81], making this

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

> UCO Poor weight gain and cerebellar growth, abnormal brain

Bilateral CCAO Shorter HI (<30 min): selective neuronal loss. Longer HI:

related to insult

hypothermia.

final EEG amplitude.

Bilateral CCAO Necrosis of subcortical white matter,

UCO Hippocampal neuronal loss only in near

UCO.

Uterine ischemia P1 pups: overt posture and tone after

MRI: WMI in IC.

Ibotenate, i.c.v. Laminar neuronal depopulation of layer V–

[94] Pigs, <24 h old CCAO + hypoxia Secondary energy failure. Energy

[95] Pigs, P9 Hypotension + hypoxia ~60% fall in CBF, reduced cerebral O2

CP.

DTI, behavioral impairment, 43% develop

cortical necrosis. Post-HI EEG suppression

severity and pathology; prevented by

neuronal loss in thalamus and striatum similar to near term fetus. Little loss of

term group. Degree of injury associated with the severity of hypotension during

metabolism ameliorated by hypothermia

uptake, phosphorylated metabolites and pH and increased inorganic phosphate.

VIa. P5: neuronal loss in all cortical layers, formation of porencephalic cysts.

ischemia >37 min, correlates with microgliosis in basal ganglia and thalamus.

(35°C for 12 h) at 24 h–48 h.

model dependent on the specific conditions of the tissue or cells used [7].

involved in several cellular processes that trigger hypoxia [84–89].

**Reference Species Animal model Outcomes**

**3.1 In vitro approaches**

Large animal models [73] *Macaca* 

*nemestrina*, near

term

near term

midgestation

midgestation and near Term

Rodent models with global hypoxic or excitotoxic component

[90, 91] Fetal sheep,

[92] Fetal sheep,

[93] Fetal sheep,

[71] Rabbits, 21–22d gestation

[96] Mice at E8, P0 or P5

**216**


**219**

**4. Neuroactive steroids**

*Experimental models for HI.*

cytokines and inflammatory mediators [139].

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

[120] C57BL/6 mice DFO DFO up-regulated the expression of

DFO pretreatment/3 h

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)

45% reduction in cell death

cytokine erythropoietin.

neurons.

neuronal death

CCA/DFO treatment Neural-protective and angiogenesis effects

DFO preconditioning Restored neovascularization potential of ADSCs

MCA/DFO treatment Preserved brain volumes, upregulation of HIF1a

cortex.

DFO-induced increase in HIF-1 protein level and activity exerts significant attenuation of BA vasospasm

Cobalt induced the transcription of the

Cobalt and DFO, enhanced survival of

through regulating the levels of HIF-1α

Reduced the number of cleaved caspase 3-positive cells in the ipsilateral cerebral

DMOG exacerbates OGD-induced

**Reference Species Animal model Outcomes**

OGD

Subarachnoid hemorrhage/DFO treatment

Ppreconditioning CoCl2, DFO or dimethyloxylalyglycine (DMOG), 3 h OGD

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

DFO + Erythropoietin

treatment

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

[121] Hippocampal neurons

[123] Hippocampal cultures

Dawley rats

Dawley rats

Dawley rats

[127] Wistar rats MCAO/

[122] Sprague–

[124] Sprague–

[126] Sprague –

*Modified from [7].*

**Table 2.**

[125] Adipose-derived stem cells


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