# OVERVIEW OF TIGHT JUNCTION PROTEINS IN BRAIN ENDOTHELIAL CELLS

The paracellular transport of molecules at the BBB is highly selective, and this feature is associated with the expression of high levels of TJ proteins. These proteins act as a biological adhesive, anchoring together adjacent BECs via transmembrane proteins attached to intracellular scaffolding proteins (Haseloff et al., 2015).

The transmembrane proteins occludin, claudins and Junctional Adhesion Molecules (JAMs) form complex strands that interact between cells, reducing paracellular diffusion (Haseloff et al., 2015; Keaney and Campbell, 2015). However, in order to maintain this restrictiveness, TJs are linked to the cytoplasmic zonula occludens (ZO) proteins that provide a structural bridge to the actin cytoskeleton. In zones where there is contact between three BECs, the TJ protein tricellulin (MARVELD2) plays a pivotal role in modulating paracellular permeability by reducing the passage of large molecules (Reinhold and Rittner, 2017).

Since the discovery that the permeability of the BBB could be regulated through reversible disruption of TJs, this principle has served as an approach for the delivery of therapeutics that would not cross this barrier by conventional means, e.g., passive diffusion, carrier-mediated transport (Dithmer et al., 2017; Sol et al., 2017).

### OVERVIEW OF TRANSPORTERS INVOLVED IN BRAIN DRUG DISPOSITION

Data from in vitro and animal models have helped establish which membrane transporters impact brain drug disposition. Furthermore, the International Transporter Consortium et al. (2010) has published and updated recommendations (Hillgren et al., 2013) for decision-making processes related to drugtransporter interactions that could be translated to clinical settings. In this regard, the ABC transporters P-glycoprotein (P-GP; Cordon-Cardo et al., 1989), Multidrug Resistance-associated Proteins (MRPs) MRP4 and MRP5 (Huai-Yun et al., 1998; Seetharaman et al., 1998), Breast Cancer Resistance Protein (BCRP; Eisenblatter and Galla, 2002; Eisenblatter et al., 2003), and the Organic Anion Transporting Polypeptides (OATPs) OATP1A2 and OATP2B1 SLC transporters (Roth et al., 2012), are considered the most clinically important transporters within the BBB. Although there is evidence (from pre-clinical models) that other SLC transporters expressed in the BBB, including members of the Monocarboxylate Transporter (MCT; Lee and Kang, 2016), Organic Anion Transporter (OAT) subfamilies (Hosoya and Tachikawa, 2011) are involved in the uptake of drugs, this review will exclusively focus on the transporter proteins expressed in human BECs. The characteristics of these protein families in human BECs are briefly summarized in **Table 1** and the following section.

### ATP-Binding Cassette Transporters

P-glycoprotein is a 170 kDa efflux transporter encoded by the ABCB1 gene in human and the abcb1a/abcb1b genes in rodent. This protein is located at the luminal side of BECs and is described as a phenotypical marker (Sugawara et al., 1990;

Dauchy et al., 2008; Cantrill et al., 2012). P-GP exhibits a broad substrate specificity that includes anticancer drugs (Mealey and Fidel, 2015), antidepressant drugs (O'Brien et al., 2012), antiepileptic drugs (Stepien et al., 2012), cardiotonic drugs (Ledwitch et al., 2016), HIV protease inhibitors (Liu et al., 2017) and immunosuppressants (Picchianti-Diamanti et al., 2014) among others. This characteristic implies the transporter has a predominant role in regulation the disposition of xenobiotics, including therapeutic drugs, thereby acting as a mechanism of detoxification and drug resistance. Some studies have also proposed a role for P-GP in the transport of endogenous mediators including steroids, bilirubin (Cascorbi, 2011) and amyloid-β, the peptide responsible of the formation of amyloid plaques in Alzheimer's disease (Zhong et al., 2016).

The BCRP is an ABC transporter encoded by the ABCG2 gene in human and abcg2 gene in rodent, and is expressed at the luminal domain of BBB endothelial cells (Eisenblatter et al., 2003). BCRP is a monomeric protein (70 kDa) that requires the formation of at least a homodimer (and can even form homotetramers) to be functionally active (Ni et al., 2010). There is significant overlap in the substrate specificity of P-GP and BCRP and, like P-GP, BCRP can significantly influence drug transport in the body (Poguntke et al., 2010). In human BECs, BCRP is expressed at higher levels than P-GP (Shawahna et al., 2011; Uchida et al., 2011), but its overall contribution to the transport of substrates is less well understood than P-GP. As well as transporting drug substrates, BCRP also participates in transport of hormones (and conjugated metabolites) (Grube et al., 2018) and urate, a product of purine metabolism whose accumulation causes gout (Woodward et al., 2009; Fujita and Ichida, 2018).

The MRP transporters are encoded by the ABCC class of genes in human and abcc genes in rodents. MRPs mediate the transport of a diverse array of drugs and endogenous molecules including hormones, prostaglandins, leukotrienes and their conjugates (glucuronides, sulfates, and glutathione) (Zhou et al., 2008; Zhang et al., 2015; Bloise et al., 2016). Members of the MRP family are not as highly expressed in BECs as P-GP and BCRP in human BECs, but the findings of proteomic (Shawahna et al., 2011; Uchida et al., 2011) and transcriptomic (Warren et al., 2009) analyses have demonstrated that MRP4, an isoform located at the luminal side, is expressed at detectable levels in human BECs. Luminal expression of MRP5 in human BECs has also been confirmed by means of fluorescent immunohistochemistry (Nies et al., 2004).

### Solute Carrier Transporters

The OATPs, a group of SLC transporters belonging to the SLCO subfamily, are ubiquitously expressed throughout the body, and at the human BBB, luminal expression of OATP1A2 (Lee et al., 2005) and OATP2B1 (Bronger et al., 2005), has been reported. OATPs mediate the uptake and efflux of endogenous and exogenous molecules, which tend to possess amphiphilic characteristics (Roth et al., 2012) and substrates include prostaglandins, steroid and thyroid hormone conjugates (Grube et al., 2018), bile acids and therapeutic drugs (Kalliokoski and Niemi, 2009; Roth et al., 2012).

### EXPRESSION AND FUNCTIONALITY OF BLOOD-BRAIN BARRIER TIGHT JUNCTIONS PROTEINS AND DRUG TRANSPORTERS IN PREGNANCY

During normal pregnancy there is an increase in blood levels of several endogenous mediators including hormones and their metabolites, pro-inflammatory cytokines, chemokines, matrix metalloproteinases and growth factors (Chavan et al., 2017; Chen and Khalil, 2017). For example, clinical studies report that pro-inflammatory cytokines including interleukin-6 (IL-6) and markers of cyclooxygenase-2 activity were increased in healthy women, suggesting that pregnancy is characterized

by a mild, sub-clinical systemic inflammatory state (Palm et al., 2013; Danielsen et al., 2014). Under this physiological proinflammatory condition, expression and functionality of BBB TJ proteins and ABC/SLC transporters involved in drug disposition could be modified as is observed in other conditions (Keaney and Campbell, 2015; Qosa et al., 2015). Although little is known of the influence of pregnancy on the expression of BBB transporters and TJ proteins, recent studies are addressing this issue.

### Tight Junction Proteins and Pregnancy

The influence of pregnancy on the integrity of TJs has primarily been studied in endothelial cells of the placenta (Marzioni et al., 2001; Ahn et al., 2015), and to date, we are not aware of any studies reporting the effects of a healthy pregnancy on the expression of TJ proteins in the maternal BBB. During normal pregnancy, high plasma levels of vascular endothelial growth factor (VEGF), an angiogenic mediator that increases BBB permeability through changes in the expression of TJ proteins (Lafuente et al., 2006) including claudin-5 and occludin (Argaw et al., 2012), have been reported (Evans et al., 1998). However, despite elevated plasma levels of VEGF, the permeability of the BBB remains unaltered in normal pregnancy (Cipolla, 2013). Indeed, studies have shown that the serum collected during late pregnancy attenuates the effects of VEGF on permeability of cerebral veins isolated from non-pregnant rats and rats in latepregnancy (Schreurs et al., 2012). The authors attributed this outcome to the fact that, in late pregnancy, high plasma levels of soluble Fms-like tyrosine kinase 1 (sFlt1), a splice variant of the VEGF receptor (VEGFR) lacking activity, counteracts the effects of VEGF (Cipolla, 2013).

### Membrane Transporters

Current evidence suggests that expression levels of ABC transporters within the maternal BBB vary throughout pregnancy, although this outcome has only been demonstrated in animal models. Studies report the expression of rodent P-gp and mrp1 in the BBB of pregnant mice is higher at midgestation and decreases in late-gestation (Coles et al., 2009), whilst positron emission tomography (PET) studies conducted on macaques (Chung et al., 2010) report that P-GP activity increased from mid-gestation to late-gestation, as evidenced by reduced accumulation of the radiolabelled P-GP substrate 11C-verapamil. However, the latter study did not confirm if this effect was a result of increased P-GP expression. No studies to date have reported the influence of pregnancy on the expression of SLC transporters in the maternal BBB, although it has been demonstrated expression of rodent Oatp1a4 (a rodent isoform that shows a high homology with human OATP1A2) in the BBB of the newborn increases with maturation (Harati et al., 2013).

ATP-binding cassette and SLC transporters govern movement of a whole array of endogenous and exogenous molecules through endothelial cells of the BBB. Consequently, this transcellular passage of substances may be significantly affected by pregnancy-dependent changes in transporter expression. However, despite the potential implications of modification of the barrier properties of the maternal BBB, to date, the precise mechanisms by which pregnancy could influence the expression of the above transporters are unclear and are only relatively recently being investigated.

A recent study reported that acute exposure of isolated hippocampal rat brain capillaries to serum obtained from pregnant rats reduced P-GP activity (Johnson et al., 2018). The authors hypothesized that this inhibition of P-GP, mediated by high levels of circulating serum factors, was associated with the increased incidence of seizures in normal pregnant rats. However, this study did not identify the molecules responsible for reduced P-GP activity. Furthermore, the findings are in contrast to those reported in studies investigating the relationship between seizures and P-GP activity, which suggest glutamatemediated induction of cyclooxygenase-2 activity is responsible for the up-regulation of P-GP expression and activity in BECs in animal models of epilepsy (Zibell et al., 2009; van Vliet et al., 2010) and in capillaries isolated from human brains (Avemary et al., 2013). These findings are particularly important since studies suggest that in normal pregnancies and in preeclampsia, the cerebral levels of glutamate are reduced when compared to non-pregnant women (Nelander et al., 2018).

The specific effects of endogenous factors on the expression and activity of ABC transporters during pregnancy have been studied more in-depth on the developing fetal BBB than in the maternal BBB. Studies have reported that primary cultures of guinea pig BECs, obtained from late-gestational fetuses and postnatal pups, are highly responsive to the effects of glucocorticoids and pro-inflammatory cytokines, with hydrocortisone and dexamethasone (Iqbal et al., 2011) increasing P-gp activity and IL-1β, IL-6 and TNFα (Iqbal et al., 2012) decreasing P-gp activity. However, despite the opposing effects exerted by glucocorticoids and cytokines on P-gp expression and activity, a later report demonstrated that co-treatment with the synthetic glucocorticoid dexamethasone, apart from increasing the expression of the transporter, enhanced the inhibitory actions of IL-1β, IL-6, and TNFα on P-GP activity (Iqbal et al., 2016). The authors suggested that this enhancement of cytokine inhibitory actions is the result of a dexamethasone-mediated increase in the expression of pro-inflammatory cytokines receptors.

Transforming Growth Factor β (TGFβ), a protein found at high levels in plasma during pregnancy (Forbes and Westwood, 2010), has also been reported to regulate the expression of P-GP in BECs, and Baello et al. (2016), have reported TGFβ-mediated up-regulation of P-GP expression and activity, through activation of the ALK1 and ALK5 signaling pathways, in BECs isolated from male fetuses and postnatal guinea pig pups.

# ENDOTHELIAL DYSFUNCTION AT THE BRAIN IN PREGNANCY-RELATED DISORDERS

Pregnancy alone can be considered as an inflammatory (but not pathological) state. One hallmark of pregnancy-related disorders, including preeclampsia, is the manifestation of endothelial dysfunction promoted by high levels of factors released from the placenta (Escudero et al., 2009; Myatt and Roberts, 2015).

The brain vasogenic edema present in later stages of preeclampsia is apparently the result of impaired autoregulation of cerebral blood flow and increased BBB permeability (Cipolla and Kraig, 2011; Cipolla, 2013; Hammer and Cipolla, 2015), but the pathophysiological mechanisms involved are still unclear. In vitro studies have demonstrated that when rat cerebral vasculature was exposed to plasma from normal and preeclamptic human pregnancies, there was an increase in BBB permeability. Interestingly, this effect was more marked following treatment with preeclamptic plasma (Amburgey et al., 2010). Furthermore, this study demonstrated that inhibition of VEGF receptor tyrosine kinase activity reversed the effect elicited by the treatment with preeclamptic plasma, suggesting that VEGF could be involved in modulating vascular permeability.

The pro-inflammatory cytokine TNFα is also believed to contribute to the increased BBB permeability in preeclampsia. TNFα infusion in healthy pregnant rats at gestational day 19 increased the water content in the anterior cerebrum without increasing the BBB permeability (Warrington et al., 2015). However, when pregnant rats were subjected to a reduction of

TABLE 2 | Drugs employed for treatment of chronic diseases in pregnancy and preeclampsia as substrates/inhibitors of ABC/SLC transporters expressed in human brain endothelial cells.

uterine perfusion pressure (RUPP), a model of placental ischemia that emulates preeclampsia and impairs the maternal cerebral blood flow, they exhibited an increase in both the water content at the anterior cerebrum and BBB permeability, which was counteracted by treatment with the TNFα inhibitor etanercept.

Findings from other studies conducted in the RUPP model have helped to elucidate how BECs respond to the circulating factors present in preeclampsia. When RUPP was performed in pregnant rats at gestational day 14, edema and increased maternal BBB permeability in the anterior cerebrum were observed, with increased expression of the protein aquaporin 4 and no changes in the expression of TJ proteins (Warrington et al., 2014). However, it has been reported the same procedure led to postpartum edema and increased maternal BBB permeability in the posterior cortex, probably due to reduced expression of the TJ protein occludin (Clayton et al., 2018).

The increased BBB permeability reported by both Warrington et al. (2014) and Clayton et al. (2018), is partly supported by clinical studies which demonstrated that in women developing preeclampsia, blood levels of S100B, neuronal specific enolase (NSE) and neurofilament light chain (NfL), three markers of cerebral injury, were higher than those observed in women with normal pregnancies (Bergman et al., 2018). Indeed, another report showed that in preeclamptic women, the levels of S100B and NSE were still high 1-year post-partum, suggesting that the alterations in the integrity of the BBB are manifest for a substantial period of time following delivery (Bergman et al., 2016).

### PHARMACOLOGICAL MANAGEMENT OF PREECLAMPSIA

Hypertension is one of the pathological features of preeclampsia and is routinely managed with the use of antihypertensive drugs including nifedipine, nicardipine, labetalol, hydralazine and methyldopa (Odigboegwu et al., 2018). Although it remains unclear whether the BBB permeability of these drugs is affected during preeclampsia, it is noteworthy that several antihypertensives, namely labetalol, nicardipine and nifedipine, are substrates of efflux transporters including P-GP (Thiel-Demby et al., 2009; Choi et al., 2013; Incecayir et al., 2013). The therapeutic management of neurological complications associated with preeclampsia, including seizures, primarily relies on the intravenous or intramuscular administration of magnesium sulfate, a drug with demonstrated ability to prevent the development of eclampsia and reduce maternal mortality (Altman et al., 2002).

The mechanism of action by which magnesium sulfate exerts its effects is unknown, but despite this limitation, it is widely considered a neuroprotective agent capable of reducing BBB permeability in animal models of brain injury (Li et al., 2017). Furthermore, a recent study demonstrated that magnesium sulfate reduced the water content in the anterior cerebrum, as well protein, cytokine, chemokine and VEGF levels in cerebrospinal fluid of rats subjected to the RUPP procedure (Zhang and Warrington, 2016). The above findings are important since the transport of drugs and endogenous mediators across the choroid plexus, which constitutes the blood-cerebrospinal fluid barrier, is a subject that is receiving increasing interest. A better characterization of the mechanisms that govern transport of molecules across this barrier will certainly help to understand brain drug disposition in both healthy and disease states such as preeclampsia.

To date, as the specific effects of magnesium sulfate on the expression/functionality of BBB TJ proteins and drug transporters are unknown, future studies addressing this subject will prove crucial in identifying potential therapeutic targets and in developing treatment strategies.

# HOW BLOOD-BRAIN BARRIER ENDOTHELIAL DYSFUNCTION COULD ALTER BRAIN DRUG DISPOSITION IN PREECLAMPSIA

The effect of endothelial dysfunction elicited by preeclampsia on brain drug disposition is unknown. A preeclampsiamediated increase in BBB permeability could potentially result in increased permeation of endogenous, blood-borne substances, including placental derived sFlt-1, and hormones, into the brain. Furthermore, a less restrictive BBB could allow more extensive penetration of therapeutic drugs into the CNS, resulting in increased side effects. In preeclamptic women, in addition to hypertension, which is treated with antihypertensive P-GP drug substrates, comorbidities, including epilepsy, depression and HIV infection, are often reported (Pariente et al., 2016). Since there is evidence that drugs belonging to these pharmacological groups are substrates and/or of ABC/SLC transporters (O'Brien et al., 2012; Stepien et al., 2012; Alam et al., 2016; Han et al., 2017), there is an obvious need to better understand the effect of preeclampsia on BBB transporter physiology and the effects of transporter modifications on brain drug disposition. A list of drugs used for treatment of chronic diseases in pregnancy and preeclampsia is presented in **Table 2**.

Although the precise mechanisms, and effects, of alterations in BBB permeability associated with preeclampsia have not yet been elucidated, it is possible to gain an insight into the potential consequences of BBB modification from clinical situations in which the expression of BBB drug transporters and BBB integrity are altered. In this regard, ischemic and hemorrhagic stroke are life-threatening conditions whose outcomes include severe BBB disruption (Knowland et al., 2014; Keep et al., 2018). In rodent models of ischemic stroke, based on middle cerebral artery occlusion (MCAO), an increase in brain water content and a decrease in the expression of TJ proteins, including occludin and claudin-5, have been observed (Huang et al., 2017). In functional terms, the cited reports demonstrated an increase in BBB permeability, i.e., increased brain levels of drugs transported through the paracellular pathway. Interestingly, MCAO also resulted in a time dependent up-regulation of P-GP expression (Cen et al., 2013; DeMars et al., 2017), which may serve as a compensatory protective mechanism, especially for drugs that are substrates of this transporter.

Pathological similarities are observed in both ischemic stroke and preeclampsia, including neuroinflammation, vasogenic edema and increased BBB permeability (**Figure 2**). To date, there is a lack of studies addressing the effect of preeclampsia on BBB physiology and, in particular, the effects of this disorder on

### REFERENCES

TJ complexes, which govern paracellular permeability, and on ABC/SLC transporters, which regulate transcellular permeability. However, there is potential to monitor ABC transporter functionality, particularly P-GP activity, in women who have a history of preeclamptic pregnancies using non-invasive PET studies employing 11C-verapamil as tracer (Shin et al., 2016). Future studies could employ this technique in women who had suffered preeclampsia or eclampsia, in order to measure P-GP activity and investigate whether there is a correlation between transporter activity and propensity of seizures.

# CONCLUDING REMARKS

The BBB is a highly restrictive but dynamic system that regulates the transport of ions and molecules into and out of the brain. Consequently, alterations in its function could result in an increased CNS exposure to potentially toxic xenobiotics, including therapeutic drugs, and endogenous factors. Given the findings that preeclampsia is associated with increased BBB permeability, there is an urgent and fundamental need to characterize the functionality of BBB ABC and SLC transporter proteins involved in CNS drug disposition through the use of appropriate pre-clinical models and execution of clinical studies.

A better understanding of preeclampsia-associated changes in BBB physiology would not only allow characterization of the processes responsible for pathophysiological changes, but could help improve the therapeutic management of women experiencing, or those who had experienced, preeclamptic pregnancies. Indeed, this knowledge will help to reduce the risk of acute and chronic complications caused by alterations in BBB function elicited by preeclampsia.

# AUTHOR CONTRIBUTIONS

PT-V designed and wrote the manuscript. CE and JP contributed to the writing of the manuscript and provided a critical revision of its contents.

# ACKNOWLEDGMENTS

The authors would like to acknowledge the support from the Chilean National Commission of Science and Technological Research (CONICYT), which fund the project "Red multidisciplinaria para el análisis de los mecanismos fisiopatológicos subyacentes al daño endotelial cerebral presente en preeclampsia" Grant No. REDI170373.

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**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Torres-Vergara, Escudero and Penny. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Are the Cognitive Alterations Present in Children Born From Preeclamptic Pregnancies the Result of Impaired Angiogenesis? Focus on the Potential Role of the VEGF Family

Evelyn Lara1,2, Jesenia Acurio1,2, José Leon1,2, Jeffrey Penny3 , Pablo Torres-Vergara2,4 and Carlos Escudero1,2,5 \*

1 Vascular Physiology Laboratory, Group of Investigation in Tumor Angiogenesis (LFV-GIANT), Department of Basic Sciences, Faculty of Basic Sciences, Universidad del Bío-Bío, Chillán, Chile, 2 Group of Research and Innovation in Vascular Health (GRIVAS Health), Chillán, Chile, 3 Division of Pharmacy and Optometry, School of Health Sciences, Faculty of Biology, Medicine and Health, The University of Manchester, Manchester, United Kingdom, 4 Department of Pharmacy, Faculty of Pharmacy, Universidad de Concepción, Concepción, Chile, 5 Red Iberoamericana de alteraciones Vasculares Asociadas a TRastornos del EMbarazo (RIVA-TREM), Chillán, Chile

### Edited by:

Vincenzo Lionetti, Scuola Sant'Anna di Studi Avanzati, Italy

### Reviewed by:

Siu-Lung Chan, The University of Vermont, United States Jingyan Han, Boston University, United States

> \*Correspondence: Carlos Escudero<EMAIL_ADDRESS>
### Specialty section:

This article was submitted to Vascular Physiology, a section of the journal Frontiers in Physiology

Received: 28 June 2018 Accepted: 23 October 2018 Published: 14 November 2018

### Citation:

Lara E, Acurio J, Leon J, Penny J, Torres-Vergara P and Escudero C (2018) Are the Cognitive Alterations Present in Children Born From Preeclamptic Pregnancies the Result of Impaired Angiogenesis? Focus on the Potential Role of the VEGF Family. Front. Physiol. 9:1591. doi: 10.3389/fphys.2018.01591 Evidence from clinical studies has proposed that children born from preeclamptic women have a higher risk of suffering neurological, psychological, or behavioral alterations. However, to date, the mechanisms behind these outcomes are poorly understood. Here, we speculate that the neurodevelopmental alterations in the children of preeclamptic pregnancies result from impaired angiogenesis. The pro-angiogenic factors vascular endothelial growth factor (VEGF) and placental growth factor (PlGF) are key regulators of both vascular and neurological development, and it has been widely demonstrated that umbilical blood of preeclamptic pregnancies contains high levels of soluble VEGF receptor type 1 (sFlt-1), a decoy receptor of VEGF. As a consequence, this anti-angiogenic state could lead to long-lasting neurological outcomes. In this non-systematic review, we propose that alterations in the circulating concentrations of VEGF, PlGF, and sFlt-1 in preeclamptic pregnancies will affect both fetal cerebrovascular function and neurodevelopment, which in turn may cause cognitive alterations in post-natal life.

Keywords: preeclampsia, angiogenesis, neurovascular, neurocognitive, vascular endothelial growth factor, placental growth factor, sFlt-1

# INTRODUCTION

Preeclampsia is a multisystemic syndrome of unknown etiology that affects pregnant women after 20 weeks of gestation. The clinical presentation of preeclampsia is characterized by hypertension with co-morbidities including proteinuria or alterations in coagulation and liver function, thrombocytopenia, pulmonary edema, and brain or visual impairments (ACOG, 2013). The global incidence of preeclampsia ranges from 2 to 8% of all pregnancies, with the condition being the leading cause of maternal morbidity and mortality, premature birth and perinatal death in both developed and developing countries (Duley, 2009). Furthermore, 30% of newborns to

preeclamptic mothers experience some form of adverse perinatal outcome, including prematurity and intrauterine growth retardation among others (Villar et al., 2006). Although the remaining percentage (∼70%) of newborns may be considered healthy, evidence suggests that children born to preeclampsia have a higher risk of suffering cardiovascular-related diseases compared to children born to normal pregnancies (Davis et al., 2012a).

The etiology of preeclampsia is not well known, but the most accepted hypothesis targets the placenta as a primary source of endogenous factors that, upon release to the systemic circulation, impair the functionality of endothelial cells. Previous reports have proposed that anti-angiogenic factors, including the soluble fms-1-like tyrosine kinase-1, (sFlt-1 or sVEGFR-1) and soluble endoglin (sENG) are responsible for the endothelial dysfunction present in preeclampsia (see details in Roberts and Escudero, 2012).

Furthermore, multiple studies have shown alterations in the balance of pro-angiogenic and anti-angiogenic factors in the placenta at term, in the fetal-placental circulation, and in the circulation of preeclamptic women and their children years after the preeclamptic incident (Maynard and Karumanchi, 2011; Rana et al., 2014; Karumanchi, 2016).

In addition to imbalances in pro and anti-angiogenic processes, endothelial dysfunction is a well-recognized feature in the offspring born to preeclampsia, the consequences of which include cardiovascular disease in adulthood (Davis et al., 2012a,b). In this manuscript, we extend this concept to propose that impaired angiogenesis affects the cognitive development of children born from preeclamptic women.

### EVIDENCE OF COGNITIVE ALTERATIONS IN THE OFFSPRING BORN TO PREECLAMPTIC PREGNANCIES

The pathophysiology of pregnancy-related disorders such as preeclampsia is associated with impaired placental function that compromises the interaction between mother and fetus (Barker, 1995; Krause et al., 2009; Escudero et al., 2014b; Thornburg, 2015). These alterations in the mother-fetus interaction apparently generate adaptive changes in the functionality of the endocrine, metabolic, vascular and immune systems of the fetus, with consequences in post-natal life. In this regard, several reports have proposed that the offspring of preeclamptic pregnancies have a higher risk of developing metabolic, neurological and cardiovascular disorders in adulthood (Tenhola et al., 2003; Wu et al., 2008, 2009, 2011; Robinson et al., 2009; Jayet et al., 2010; Davis et al., 2012a; Tuovinen et al., 2012; Escudero et al., 2014b; Alsnes et al., 2016; Pinheiro et al., 2016; Ratsep et al., 2016a,b; Figueiro-Filho et al., 2017a).

 In agreement with this body of evidence, a recent systematic review concluded that preeclampsia is associated with neurocognitive alterations in children (Figueiro-Filho et al., 2017b).

# BRAIN ALTERATIONS IN OFFSPRING BORN TO PREECLAMPSIA

Pre-clinical studies have attempted to establish an explanation for the variety of pathoneurological consequences associated with preeclampsia. Studies report that the brain weight at birth of the offspring born to hypertensive pregnant rats was significantly lower compared to the offspring born to their normotensive counterparts (Liu et al., 2016). Furthermore, reduced brain weight was associated with reduced expression of markers of neurogenesis, markers of neuroproliferation in the cortex and with severe impairments in spatial learning and memory.

In the clinical setting, Dr. Ana Croy's group have been conducting pioneering studies which attempt to correlate cognitive parameters with brain anatomy (Ratsep et al., 2016a,c) or neuronal networking (Mak et al., 2018). The findings of these magnetic resonance imaging (MRI) studies, in 10 children born to preeclampsia and 10 children born to normotensive pregnancies, demonstrated that at least five brain regions, including the cerebellum, temporal lobe, brainstem, and right and left amygdala, were larger in children born to preeclampsia compared with controls (Ratsep et al., 2016c). The study also demonstrated cerebral blood flow in both parietal and occipital lobes in children born to preeclamptic women was lower than in controls. The authors proposed that changes in brain vasculature may have preceded the structural alterations. Consistent with these findings, the MRI studies of Figueiro-Filho et al. (2017a) reported increased volumes of the tract for the superior longitudinal fasciculus and the caudate nucleus, amongst other alterations, in the brains of children born to preeclamptic pregnancies.

More recently, a study suggested that children born from preeclamptic pregnancies demonstrated higher levels of connectivity between the left amygdala and bilateral frontal pole; the right amygdala and the left frontal pole, and between the medial prefrontal cortex and precuneus, compared to matchedcontrol children (Mak et al., 2018). Furthermore, this study also found that children born from preeclamptic pregnancies exhibit decreased connectivity between the medial prefrontal cortex and the left occipital fusiform gyrus.

The body of findings available therefore provides strong, albeit preliminary, evidence for the hypothesis that the neurological and cognitive impairments present in children delivered from preeclamptic pregnancies are likely the result of neurodevelopmental alterations. Furthermore, complementary to this hypothesis is the fact that impaired vascular development

in the fetal brain could be a contributing factor to preeclampsiaassociated impairments.

Crucially, a more in-depth understanding of (i) the potential confounding factors which impact fetal development, such as altered placental functionality (hypoxia), (ii) subject-specific data, including the weight of the newborn, gestational age at birth and the socioeconomic and nutritional context in which the newborn develops, and (iii) the physiological and morphological modifications in the cerebral vasculature of children from preeclamptic women, will be key to strengthening this hypothesis.

## EVIDENCE OF ALTERATIONS IN THE VASCULATURE OF CHILDREN BORN TO PREECLAMPTIC WOMEN

Angiogenesis, defined as the formation of new vessels from an existing vessel, is widespread during fetal and neonatal development, although it is rarely observed in adults under normal conditions (Carmeliet and Ruiz de Almodovar, 2013; Watson et al., 2016, 2017). Although several processes drive vascular development, angiogenesis is one of the most studied in the context of preeclampsia. Conversely, vascular remodeling or "vascular pruning," the process by which pro-angiogenic signaling is suppressed, resulting in accelerated apoptosis of endothelial cells (Watson et al., 2017) and organized loss of blood vessels, is also observed in the fetus. Consequently, angiogenesis and vascular remodeling are complementary processes, the relative extents of which can influence vascular development not only in the fetuses of normotensive and preeclamptic pregnancies, but can impact vascular architecture in neonates, and in individuals in later life.

Although studies have investigated the characteristics of accessible vascular networks in individuals born to preeclamptic pregnancies, no studies report on how preeclampsia influences the anatomy and physiology of the blood brain barrier in neonates, children, and adults.

Our research group (Escudero et al., 2013, 2014a; Acurio et al., 2014, 2016) and others (Staff et al., 2005; Tsao et al., 2005; Stark et al., 2009; Antonios et al., 2012; Touwslager et al., 2012; Yu et al., 2016) have studied how angiogenesis may be impaired in the children born to preeclamptic pregnancies. Interestingly, it has been shown that full-term babies of hypertensive pregnancies (including preeclampsia) exhibited a reduction in the maximum capillary density per square millimeter on the plantar surface of the big toe compared with control infants (Antonios et al., 2012).

The above results are in agreement with previous findings (Stark et al., 2009; Touwslager et al., 2012) that demonstrated structural and functional alterations in the microvasculature of newborns and increased vascular remodeling in the skin of 3-month old babies born to hypertensive pregnancies (Yu et al., 2016). Also, it is reported (Yu et al., 2012) that children born to preeclampsia exhibited a 45% reduction in the risk of prematurity-associated retinopathy, a condition characterized by increased retinal angiogenesis. Furthermore, studies report the retinal arteriolar caliber, but not the retinal venular caliber, was narrower in school-age children from hypertensive pregnancies, including preeclamptic pregnancies, than in children born from normotensive pregnancies (Yesil et al., 2016). Since prematurityassociated retinopathy is a condition characterized by an increase in the formation of blood vessels in the retina, differences in retinal arteriolar caliber are suggestive of differences in vascular remodeling in the two cohorts.

However, as far as we are aware, although Ratsep et al. (2016c) demonstrated alterations in blood flow to the parietal and occipital lobes in the offspring of preeclamptic pregnancies, the effects of preeclampsia on blood brain barrier anatomy and physiology in neonates, children, and adults are yet to be established. In this manuscript we propose that cerebral angiogenesis is altered in children born to mothers with preeclampsia, thereby initially influencing the early neonatal stage and subsequently throughout life (**Figure 1**).

Since preeclampsia-associated alterations in vascular networks (Stark et al., 2009; Antonios et al., 2012; Touwslager et al., 2012; Yesil et al., 2016; Yu et al., 2016), neurocognition (Wu et al., 2009; Dang et al., 2016; Ratsep et al., 2016a) and neuroanatomy (Ratsep et al., 2016a,b,c; Figueiro-Filho et al., 2017a; Mak et al., 2018) have been reported, there is an obvious need for an improved understanding of the molecular mechanisms underpinning such physiological changes. Vascular endothelial growth factor (VEGF), placental growth factor (PlGF) and soluble fms-like tyrosine kinase (sFlt-1) play key roles in angiogenesis and neurogenesis (Ruiz de Almodovar et al., 2009; Carmeliet and Ruiz de Almodovar, 2013) and have therefore been extensively studied in preeclampsia. These important biomarkers are discussed in more detail in the next section.

# VEGF AND ANGIOGENESIS AND NEUROGENESIS

The VEGF family of proteins is one of the key regulators of both angiogenesis and neurogenesis (Carmeliet and Ruiz de Almodovar, 2013). This family is comprised of five members including VEGF-A, VEGF-B, VEGF-C, VEGF-D, and PlGF, which activate vascular endothelial growth factor receptors (VEGFRs), namely VEGFR-1, VEGFR-2, and VEGFR-3, a group of membrane receptors possessing tyrosine kinase activity. Once activated these receptors mediate several processes including cell migration, proliferation and survival (Shibuya, 2013). In addition, VEGFRs are involved in development of the morphological features of blood vessels and regulate vascular permeability (Olsson et al., 2006). The activity of VEGF and PlGF is regulated by sFlt-1, a circulating variant of VEGRF-1 that prevents their binding to their membrane receptors, thus decreasing their activity (Ahmad et al., 2011). Deletion of VEGF (Carmeliet et al., 1996), VEGFR1 (Fong et al., 1995) or VEGFR2 (Shalaby et al., 1995), or even tyrosine residues (Y1175) responsible for VEGFR2 activation (Sakurai et al., 2005) causes severe failure in angiogenesis, and are embryonic lethal in mice. Furthermore, mice deficient in VEGFR1 die at the embryonic stage due to hypertrophy and disorganization of blood vessels (Fong et al., 1995), rather than a lack of vessel formation.

Several reports have demonstrated that in preeclampsia there is an increase in circulating levels of placental derived sFlt-1 (and other anti-angiogenic molecules such as sENG), thereby generating a state of systemic vascular dysfunction in the mother (see details in Karumanchi, 2018).

VEGF-mediated activation of VEGFR2 leads to cell migration, proliferation and angiogenesis (Koch and Claesson-Welsh, 2012; **Figure 2A**), and activation is mediated by phosphorylation of the tyrosine 951 (Y951) and tyrosine 1175 (Y1175) residues. Consequently, alterations in angiogenesis may well be a multifactorial process, influenced not only by circulating levels of ligand and levels of receptor expression, but by actual kinase-mediated phosphorylation of the receptor itself. Few publications have described activation of VEGFR2 (i.e., tyrosine phosphorylation) in preeclampsia, the findings of which are conflicting (Ahmad and Ahmed, 2004; Escudero et al., 2014a).

Activation of VEGFRs has also been linked with neural development (Ruiz de Almodovar et al., 2009; Carmeliet and Ruiz de Almodovar, 2013) and in vitro and animal studies (see details in Carmeliet and Ruiz de Almodovar, 2013) report VEGF and PlGF stimulate multiple processes, including neurogenesis, enhanced neuronal survival, axonal growth, and migration and proliferation of glial cells.

Therefore, VEGFRs are excellent candidates to study to further understand the mechanism(s) of angiogenesis impairment and neural development in children born to preeclamptic pregnancies.

# VEGF FAMILY AND PREECLAMPSIA

Preeclamptic women exhibit high plasma levels of sFlt-1 (Levine et al., 2004), and circulating levels of anti-angiogenic sFlt-1 in the fetoplacental circulation and children born to preeclamptic mothers are summarized in **Table 1**. Since the discovery of elevated sFlt-1 plasma levels in preeclamptic women, several studies have attempted to characterize the role of sFlt-1, PlGF, and VEGF in the pathophysiology of the condition (see details in Karumanchi, 2016, 2018), and the roles of these factors in preeclampsia are discussed below.

# VEGF, sFlt-1 and Angiogenesis in the Offspring of Preeclamptic Pregnancies

Elevated levels of sFlt-1 have been reported in blood collected from umbilical cord (Staff et al., 2005; Tsao et al., 2005) and in adults (between 20 and 30 years old) originally born

FIGURE 2 | VEGFR1 and VEGFR2 in brain vessel formation. (A) The extent of perfusion of brain tissue depends on both angiogenesis and vascular remodeling processes, which are mediated by pro- and anti-angiogenic factors. (B) VEGF and PlGF activate VEGFR1 and VEGFR2. Phosphorylation of tyrosine 951 (Y951) or Y1175 is associated with activation of VEGFR2, leading preferentially to cell migration or cell proliferation, respectively. These regulatory mechanisms are involved in angiogenesis and neuronal survival. In contrast, sFlt-1 prevents the binding of VEGF and PlGF to VEGFRs, thus decreasing their pro-angiogenic and neuronal survival activity. In this manuscript it is postulated that children born to preeclampsia exhibit a reduced number of blood vessels in the brain cortex, through impaired angiogenesis and/or increased vascular remodeling. We also propose that the underling mechanism might involve dysregulation in VEGFR2 activation due to high sFlt-1 circulating levels in preeclampsia.

TABLE 1 | Summary of findings of angiogenic factors in children born to preeclampsia in comparison with matched normotensive controls.

sFLT-1: soluble receptor tyrosine kinase similar to fms-1; VEGF: Vascular endothelial growth factor; PlGF: Placental growth factor.

to preeclamptic pregnancies (Lewandowski et al., 2015). Also, studies have shown that neonates born from preeclamptic pregnancies exhibit lower levels of VEGF and higher levels of sFlt-1 when compared to children born to normotensive women (Olmos et al., 2013). In addition, it has been suggested that high levels of sFlt-1 in the umbilical cord are associated with impaired function of both endothelial cell progenitors (Xia et al., 2007) and human umbilical vein endothelial cells (Yu et al., 2016). Furthermore the impact of sFlt-1 on tissue function has been reported, and in the thyroid gland, a highly vascularized tissue, there is a negative association between thyroid hormone production and umbilical cord blood levels of sFlt-1 (Korevaar et al., 2014). Moreover, in the broader context of the systemic effects of sFlt-1 on infant growth and development, it has been reported that high umbilical levels of sFlt-1 are negatively associated with

growth in infants in uncomplicated pregnancies (Bergen et al., 2015), and sFlt-1 levels are also negatively correlated with body weight percentile in preterm babies (Voller et al., 2014).

At the central nervous system level, the precise role of sFlt-1 in modifying neuroanatomical and vascular architecture, and circulatory characteristics in the brain is unknown. Interestingly, high circulating levels of sFlt-1 are associated with adult psychiatric and neurological alterations (Kim et al., 2007; Fulzele and Pillai, 2009; Emanuele et al., 2010; Lee et al., 2010; Lizano et al., 2016, 2017; Pillai et al., 2016) and studies also reveal a reduction in total frontal lobe volume in patients with schizophrenia/schizoaffective disorder (Pillai et al., 2016). Whether this association is the result of alterations in microvascular development in those patients is unknown, however, gene transfer of sFlt-1 in rats leads to a reduction in brain edema and in blood brain barrier permeability, suggesting a direct effect on brain endothelium (Kumai et al., 2007).

A limited number of studies have investigated the effect of sFlt-1 on brain development, including those of Carver et al. (2014) which report that, in a mouse model of preeclampsia, adenoviral transfer of sFlt-1 to the mother was associated with sex-dependent neuroanatomical alterations in the offspring at 6 months of age, which were partly counteracted by treating mothers with pravastatin (Carver et al., 2014).

If high levels of sFlt-1 in the mother or in the fetus effectively impair the proper development of brain blood vessels, and potentially brain development, in the offspring born from preeclamptic pregnancies (**Figure 2B**), sFlt-1 would represent a reliable prognostic biomarker that would help to predict adverse outcomes in children born from preeclamptic pregnancies.

# PlGF and Cerebral Circulation in Offspring of Preeclampsia

PlGF is highly expressed at all brain development stages (Luna et al., 2016) and is considered the key cerebral angiogenic factor (Liu et al., 2006; Gaal et al., 2013). Mechanistically, PlGF stimulates growth of neurons (Dewerchin and Carmeliet, 2012; Carmeliet and Ruiz de Almodovar, 2013) and formation of new blood vessels in the brain (Gaal et al., 2013) although the influence of PlGF on brain vasculature in preeclampsia has not been investigated.

However, studies report low levels of PlGF in maternal and fetal blood (Schlembach et al., 2007) and low levels of PlGF mRNA in the placenta (Andraweera et al., 2012) of preeclamptic pregnancies compared to normotensive pregnancies. Furthermore, low maternal levels of PlGF in the second trimester of gestation were associated with a narrower retinal arteriolar caliber (but not with a narrower retinal venular caliber) in childhood, compared to children who had experienced a normotensive pregnancy (Gishti et al., 2015). These findings suggest a highly selective role for PlGF in retinal vascular development.

A more direct relationship between PlGF levels during pregnancy and brain angiogenesis in offspring has been documented by Lecuyer et al. (2017), who demonstrated low placental levels of PlGF were associated with a reduction in radial distribution of fetal cerebral cortical microvasculature. Studies in normotensive mice virally transfected with PlGF (Gaal et al., 2013) and in PlGF (Plgf−/−) deficient mice (Freitas-Andrade et al., 2012; Luna et al., 2016) highlight the key role PlGF in the development of brain vasculature. Furthermore, seminal studies from Dr. Ana Croy's group found that Plgf−/− mice showed anatomical alterations in the cerebral macrocirculation at the level of anterior communicating cerebral arteries and anterior collateral vessels forming Willis' polygon. These structural changes in cerebral circulation observed in Plgf−/− mice were associated with impaired cognitive function (Luna et al., 2016), however, the latter study did not characterize the brain microcirculation, nor the profile of pro or antiangiogenic factors in the Plgf−/− mice. More recently, the same group has reported that PlGF−/− mice also exhibited alterations in the development of neurons (Chaballe et al., 2011) and the retina (Kay et al., 2017).

# IS IT ALL ABOUT BRAIN ANGIOGENESIS?

Mechanistically, impaired brain angiogenesis is unlikely to be the unique alteration that fully explains the complexities of the adverse cognitive outcomes present in children born from preeclamptic pregnancies. Considering the hypothesis of fetal programming, it is feasible to speculate that other factors including oxidative stress, inflammation, neuronal structural modifications and epigenetic modifications driven by intrauterine hypoxia, among others, are involved. Furthermore, the contribution of many other post-natal factors, including socioeconomic environment, breastfeeding and social interactions to increase brain stimulation, to cognitive function in children born from preeclamptic pregnancies need to be established. Several excellent reports, including (Van den Bergh, 2011; Babenko et al., 2015) address the concept of fetal programming and alterations at brain level.

# CONCLUDING REMARKS

The syndrome of preeclampsia is a frequent complication of pregnancy, affecting both the mother and the fetus. This disorder is characterized by an imbalance between pro and antiangiogenic factors released from the placenta and may lead to a series of adverse consequences in individuals throughout their post-natal life.

The evidence discussed in this review suggests that children of mothers with preeclampsia have a higher risk of developing cognitive disorders. However, despite increased research in this field, it is still unclear whether other confounding factors, such as intrauterine growth restriction, prematurity or asphyxia, contribute to the cognitive disorders in children born to preeclamptic pregnancies, and further studies are required to establish the underlying mechanisms responsible for such cognitive disorders.

Herein, we present supporting evidence for alterations in the characteristics of brain microvasculature in the offspring born to preeclamptic women, although some studies include a small sample size that indirectly suggests structural alterations. While we highlight a potential key role of impaired brain vessel formation as an underling mechanism for cognitive alterations in children born to preeclampsia, it is also unlikely that this would be a unique mechanism for explaining the complex alterations in brain development and cognitive deficiencies observed. Furthermore, both inflammation and nutrient access (both over and undernutrition) represent potential contributory factors involved in brain alterations of offspring born to preeclamptic pregnancies.

We acknowledge that focusing on impaired VEGFR-mediated brain angiogenesis is an oversimplification of the complexity of the underling mechanisms of adverse cognitive outcomes in offspring born to preeclampsia. However, as indicated in this review, literature in this field is extremely limited and there is a substantial need for more focused research to establish the precise contribution of preeclampsia, independent of other confounding factors, on cognitive alterations in children. In particular, a better understanding of the roles of VEGF, PlGF, and sFLT-1 in impaired brain angiogenesis may aid in early diagnosis and management, and also in development of new treatment interventions.

### REFERENCES

ACOG (2013). Hypertension in Pregnancy. Washington, DC: ACOG.

# AUTHOR CONTRIBUTIONS

EL wrote the draft of the manuscript. JA, JL, JP, and PT-V critically revised the manuscript. CE generated the original hypothesis and proposed the development of the review article.

### FUNDING

This work was supported by DIUBB 184309 4/R.

### ACKNOWLEDGMENTS

We are deeply grateful to the midwives and medical staff of the Obstetrics and Gynecology Department of Hospital Clínico Herminda Martin from Chillán, Chile. We would like to thank all research staff at the Vascular Physiology Laboratory and the Group of Investigation of Tumor Angiogenesis (GIANT) of the Universidad del Bío-Bío for their technical support. We also thank the researchers in the GRIVAS Health group for outstanding discussion regarding hypothesis presented in this manuscript.

Yu, X. D., Branch, D. W., Karumanchi, S. A., and Zhang, J. (2012). Preeclampsia and retinopathy of prematurity in preterm births. Pediatrics 130, e101–e107. doi: 10.1542/peds.2011-3881

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Lara, Acurio, Leon, Penny, Torres-Vergara and Escudero. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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**1. Introduction**

Tungsten, a metal, is identified as one of the strategic and critical minerals by numerous countries, including The United States, Europe, Russia, Britain, and Australia due to its significant economic importance and supply instability [1–4]. Historically, the extraction of tungsten from ores relied on a series of hydrometallurgy processes, such as alkali digestion followed by solvent extraction and ion exchange, that consume large amounts of reagents, water, and energy [5–7]. Meanwhile, the difficulty of processing tungsten ores continuously raises due to the increased exploitation of primary tungsten resources, which results in a lower grade more complex in mineralogy [8]. These are key hindrances to the future of tungsten extraction processes.

Gomes et al. studied a two-phase extraction method to process tungsten minerals [9,10]. This method utilized the liquid–liquid immiscibility between sodium tungstate and silicon dioxide, which is a well-known phenomenon in glass-forming oxide systems [11]. The two-phase extraction method could light a new direction in the development of future sustainable tungsten extraction processes. Phase diagram is an essential tool to predict the crystallization path and control crystallization behaviour, which has a pivotal role in the understanding of the crystallization process [12–15]. To gain a better understanding of the two-phase extraction method, the phase diagram for the Na2O-SiO2-WO3 system needs to be studied comprehensively to provide fundamental understandings of its thermophysical properties.

Systems containing alkali tungstate have been studied extensively. In the Na2O-WO3 system, two congruently melting compounds, Na2WO4 and Na2W2O7, and one incongruently melting compound, Na2W4O13, were identified along with two eutectic reactions [16–18]. The presence of Na2W6O19 was a subject of controversy, which was reported by Sakka, Chang, and Sachdev; however, this was not found by

**Citation:** Zhao, B.; Su, K.; Ma, X. Experimental Determination of Phase Equilibria in the Na2O-SiO2-WO3 System. *Metals* **2021**, *11*, 2014. https://doi.org/10.3390/met11122014

Academic Editor: Mark E. Schlesinger

Received: 9 November 2021 Accepted: 9 December 2021 Published: 13 December 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

Caillet [16–18]. No information is available on the WO3-SiO2 binary system. Moreover, only limited literature relating to the ternary system Na2O-SiO2-WO3 has been published. Štemprok reported a Na2O-SiO2-WO3 ternary phase diagram at 1473 K, which presented a wide two-liquid immiscibility [19]. It was reported that the two-liquid field corresponds to a stable demixing of the system between silicate glass and sodium tungstate. The two liquids' immiscible region disappears in the Na2O rich sector. Furthermore, it was reported that the presence of WO3 reduces the liquidus temperature of sodium silicates. However, the region with higher silica contents was not investigated [19]. Various homogenization techniques were carried out in the study, including repeated firing with intermittent crushing and mixing, slow cooling, and quenching. The previous study provided initial information on the Na2O-SiO2-WO3 system. However, more comprehensive studies of the system are needed due to the lack of detailed methodology, microstructure analysis, and isotherms to fill the gap.

In the present study, the phase equilibria of the Na2O-SiO2-WO3 system was investigated using the equilibration/quenching/EPMA technique from 1073 to 1273 K in air. The technique used has been proven to be efficient in acquiring high temperature equilibrium information for metallurgical slags [20]. It also can resolve some uncertainties resulting from dynamic and static methods for studying molten oxides that contain volatile materials, such as sodium and silica [21]. By applying this methodology, the change of initial bulk composition caused by the evaporation of the sodium oxide during high temperature equilibration will not affect the final liquid and solid compositions. This is because the change in the initial bulk composition will only affect the proportion of liquid and solid phases when equilibrium is achieved. The determined phase equilibrium data will be beneficial to the future development of sustainable tungsten industries and for scientific interests, especially for thermodynamic modelling studies where limited thermodynamic data have been reported on WO3-containing systems.

#### **2. Experimental**

The phase equilibrium investigation was carried out using the typical melting-holdingquenching method [22,23]. The samples were prepared by mixing the required amounts of chemicals thoroughly in an agate mortar for 30 min. Chemicals used to prepare the sample were powders of SiO2 (99.9 wt% Sigma-Aldrich, Missouri, MO, USA), Na2CO3 (>99.0 wt%, Sigma-Aldrich, Missouri, MO, USA), and WO3 (99.9 wt%, Sigma-Aldrich, Missouri, MO, USA). The Na2CO3 powder was dried in an oven at 473 K for at least 48 h to remove any moisture prior to sample preparation. After mixing, the mixture was pelletized and placed in a platinum envelope. Approximately 0.2 g of pelletized sample was used in each equilibration experiment.

Equilibration experiments were carried out in a vertical tube furnace with lanthanum chromate heating elements (supplied by Pyrox Thermique Matériaux, Rambouille, France). A B-type thermocouple (Pt-30% Rh/Pt-6%) was placed in an alumina sheath next to the crucible located in the hot zone of the furnace to accurately measure and monitor the temperature during the experiments. The thermocouple was periodically calibrated against a standard thermocouple (supplied by the National Measurement Institute of Australia) to ensure the temperature accuracy within ±2 K. A schematic view of the vertical furnace setup is shown in Figure 1. The platinum envelope that contained the pelletized sample was held in the furnace using a FeCrAl wire (0.5 mm diameter). Samples were inserted from the bottom of the furnace, then raised and kept in the furnace hot zone for the desired period. After equilibration, the samples were dropped directly into mineral oil to achieve rapid quenching. The mineral oil was refrigerated at 263 K (−10 ◦C) for three hours to ensure the retaining of glassy phase by achieving rapid quenching. Water was not selected as quenching media due to the solubilities of sodium tungstate and other sodium oxide compounds. The quenched samples were washed carefully with ethanol, dried, and mounted in epoxy resin and polished for electron probe X-ray microanalysis (EPMA).

**Figure 1.** A schematic view of the experimental apparatus.

Carbon coater JEOL (Japan Electron Optics Ltd. \* JEOL is a trademark of Japan Electron Optics Ltd., Tokyo, Japan) was used to apply a carbon coat on the surface of the polished samples prior to electron microscopic examination. JEOL JXA-8200 EPMA (Japan Electron Optics Ltd., Tokyo, Japan) with wavelength dispersive detectors was used to analyse the microstructures and phase compositions of the quenched samples. EPMA was conducted at an accelerating voltage of 15 kV and a probe current of 15 nA. The ZAF (Z is atomic number correction factor, A is absorption correction factor, and F is fluorescence correction factor) correction procedure supplied with the electron-probe software was applied. Overall, the measurements of EPMA have an average accuracy within ±1 wt% [24]. In general, 10–20 and 5–10 measurements were performed for the liquid and solid phases, respectively, at different areas of the samples. The homogeneities of compositions in the phases indicate that the sample reached equilibrium. Only the compositions with standard deviations less than 1 wt% were used in the construction of the phase diagram.

## **3. Results and Discussion**

#### *3.1. Determination of Equilibration Time*

At the beginning of the study, suitable equilibration times were determined to ensure that the equilibrium state was achieved in all experiments. The time needed to reach equilibrium is affected by the mass transfer in the sample. Hence, achieving equilibrium in the liquid phase with high viscosity is directly influenced by the low molecule transportation rate. Both composition and temperature can influence liquid viscosity. In general, the liquid phase with high SiO2 content is considered as highly viscous. In the present study, we investigated the equilibration time for samples in selected primary phase fields with high SiO2 content in the liquid phase at relatively low temperatures. The appropriate

equilibration time was determined by examining the compositional homogeneity using EPMA when the equilibration time was prolonged. Samples with identical initial composition were held at different temperatures and durations. Three phases were observed from the microstructures of the quenched sample that was held for 24 h at 1073 K, indicating the 24 h was not sufficient to allow the samples to reach equilibrium. As a result, the equilibration time at 1073 K was increased to 72 and 168 h. Table 1. shows the comparisons of the samples held for 72 and 168 h at 1073 K, and 24 and 72 h at 1173 K and 1273 K respectively. It can be seen from the table that the phases present in the samples with the same initial composition and different equilibration time are close. The results of the samples in the SiO2 primary phase field demonstrated that equilibrium was achieved within 72 h at 1073 K, and 24 h at 1173 and 1273 K.

**Table 1.** Comparison of the samples with same compositions and experimental temperature equilibrated for different times in selected primary phase fields.

#### *3.2. Description of the Ternary Phase Diagram*

Over 50 experiments were conducted in the Na2O-SiO2-WO3 system targeting the two-phase immiscibility region and adjacent primary phase fields at fixed temperatures of 1073, 1173, and 1273 K. Some experimental results were found in fully liquid condition. Four primary phases were identified in the composition range investigated, including Na2SiO3, Na2Si2O5, SiO2, and Na2WO4. The present results indicate that the phase relationships in the SiO2-rich region appear to be more complex than that in the previous investigation [19]. Typical microstructures observed in the quenched samples are presented in Figure 2. Figure 2 shows the equilibrium of liquids with (a) sodium metasilicate (Na2SiO3), (b) sodium disilicate (Na2Si2O5), (c) tridymite (SiO2), and (d) liquid sodium tungstate (Na2WO4). It can be seen from Figure 2d that the sodium tungstate liquid separated from the silicate glass (liquid) and settled on the bottom of the Pt envelope, due to its high density. Furthermore, the phase boundaries in the phase diagram were identified by determining the composition of the liquid phase co-existing with the two solid/liquid phases. Figure 3 shows the microstructures where the liquid was in equilibrium with two other phases, (a) tridymite and sodium tungstate, and (b) sodium disilicate and sodium tungstate. From the microstructures of both samples, the formation of drop-like segregated sodium tungstate liquids against the silicate glass (liquid) and crystals, due to the high viscosity of the silicate glass, were observed.

**Figure 2.** Typical backscattered scanning electron micrographs of quenched samples in the Na2O-SiO2 -WO3 system, (**a**) WE5—sodium metasilicate with liquid phase; (**b**) WE6—sodium disilicate with liquid phase; (**c**) WE9—tridymite with liquid phase; (**d**) WE10—sodium tungstate with liquid phase.

**Figure 3.** Typical backscattered scanning electron micrographs of the quenched samples in the Na2O-SiO2 -WO3 system showing equilibrium of liquid with (**a**) WE13—tridymite and liquid sodium tungstate, and (**b**) WE11—sodium disilicate and liquid sodium tungstate.

The compositions of the solid and liquid phases in the quenched samples analysed by EPMA that were used to construct the isotherms and phase boundaries of the ternary phase diagram are listed in Table 2. The initial compositions of the experiments shown in Table 2 are summarized in Table 3. It can be seen from Table 2 that up to 0.5 wt% WO3 is present in the sodium metasilicate and tridymite. WO3 in the sodium disilicate is only 0.2 wt%. SiO2 in the sodium tungstate is less than 0.1 wt%. The melting points and crystal structures of the solids present in the phase diagram are summarized in Table 4. Measuring the compositions of the liquid and solid phases using the equilibration-quenching

technique has one of the greatest advantages: the measured phase compositions are not affected by starting compositions and changes during equilibration. The phase diagram of Na2O-SiO2-WO3 system is constructed from the experimental results, and the isotherms at 1073, 1173, and 1273 K are determined. The construction was based on the ascertained information on the phase equilibria. Information from the well-established binary Na2O-SiO2 system is also presented on the ternary phase diagram, and used to construct the diagram by connecting the liquidus to the Na2O-SiO2 binary. The hollow dots represent the liquidus obtained from the Na2O-SiO2 system reported by Santoso and Taskinen [21] and Zaitsev et al. [25]. In the targeting region of the phase diagram (40–100 wt% SiO2, 40–60 wt% Na2O, and 0–20 wt% WO3), four primary phase fields (sodium metasilicate, sodium disilicate, sodium tungstate and tridymite) were identified at the temperature ranging 1073–1273 K. It can be seen from Figure 4 that, sodium metasilicate and tridymite occupy extraordinarily large primary phase fields in the studied area. As the SiO2 content increased from 40 to 73.4 wt%, the sodium metasilicate gradually dissolves in the slag, and the sodium disilicate starts to precipitate. Meanwhile, the boundary of the demixing region of the two immiscible liquids enters into the low-WO3 region. The solubility of the WO3 in the liquid decreases until it reaches the tridymite primary phase field by continuously increasing the SiO2 content.

**Table 3.** Experimental conditions for investigating the Na2O-SiO2 -WO3 system.

**Table 4.** Properties of the crystalline solids present in the ternary phase diagram.

**Figure 4.** Newly determined isotherms at 1073 K (800 ◦C), 1173 K (900 ◦C), and 1273 K (1000 ◦C) for the ternary phase diagram in the Na2O-SiO2 -WO3 system 25; faint dotted line: estimated liquidus. *3.3. Comparisons with the Existing Ternary Phase Diagram*

The data reported by Štemprok [19] are extracted and shown in the phase diagram for comparison with the present results in Figure 5. It can be seen from the figure that

Štemprok reported the same primary phase fields. However, isotherms were only reported in the sodium metasilicate primary phase field. The isotherms in the other primary phase fields were not reported in his study. It can be seen from Figure 5 that the size of the Na2SiO3 primary phase field determined in the present study is larger than that reported by Štemprok. In contrast, the size of the Na2Si2O5 primary phase field determined in the present study is smaller than that reported by Štemprok. The temperature of the eutectic point joining the Na2SiO3, Na2Si2O5, and Na2WO4 primary phase fields is below 1073 K in the present study, which is lower than that (1098 K) reported by Štemprok. The experimental points and the isotherms in the primary phase fields of SiO2, Na2Si2O5, and Na2WO4 fill the gaps not reported by Štemprok [19].

**Figure 5.** Comparison of the phase diagram between the present study and previous results [19,25], temperature in K.

Due to the limitations in the methodologies and techniques used in previous studies, large deviations were observed while comparing previously reported data with the present measurements. To the best of our knowledge, FactSage cannot calculate the liquidus temperatures for the Na2O-WO3 containing systems due to a lack of reliable experimental data. It is believed that the present study is beneficial for the further construction and optimization of thermodynamic modelling by fulfilling the FactSage database with this Na2O-SiO2-WO3 ternary system.


**4. Investigation of WT application on islanding state and fault location**

In this section, the detailed analysis of two important application of wavelet analysis, carried on detection of the islanding state and fault location by the authors, will be illustrated.

#### **4.1 Islanding detection**

#### **4.1.1 Methodology**

234 Advances in Wavelet Theory and Their Applications in Engineering, Physics and Technology

wavelets become more localized and oscillate less due to the dilation nature of the WT analysis. Hence, fast and short transient disturbances will be detected at lower scales whereas slow and long transient disturbances will be detected at higher scales. Thus, both

Apart from the application of wavelets to introduce new identification, classification and analysis methods such as those presented previously, at the moment is also studied the application of wavelets to develop new components models; for example (Abur et al., 2001) extends the results of previous works (Magnago & Abur, 2000) and describes a transmission line model which is based on WT taking into account frequency dependence of modal transformation matrices into the transients simulation. This allows the use of accurate modal transformation matrices that vary with frequency and yet still remain in the time domain

Although wavelet analysis usually combined with a large number of neural networks provides efficient classification of PQ events, the time-domain featured disturbances, such as sags, swells, etc. may not easily be classified. In addition, some important disturbance frequency component may not be precisely extracted by WT. Therefore, (Reddy & Mohanta, 2004) presents a new transform by incorporating phase correction to WT and is known as S– transform. The S-transform separates the localizing-in-time aspect of the real valued Gaussian window with modulation (selection of frequency) so that the window translates, but does not get modulated. (Reddy & Mohanta, 2010) extends the use of S-transform for detection, localization and classification of impulsive transients. The results obtained from S-transform are compared with those obtained from WT to validate the superiority of Stransform for PQ and transients analysis of complex disturbances. To do a case study the technique proposed in (Liao, 2010) is again studied, but for capacitor switching leading to the oscillatory transient. The results obtained are shown in Fig. 7. The dominant features of capacitor switching event was obtained as Δ*Ed*3 to Δ*Ed*5 mapped to the 3th to 5th energy

Fig. 7. (a) Simulated data of oscillatory transient with noise. (b) Energy spectrum of the simulated signals without noise suppression. (c) Energy spectrum of the simulated signals

fast and slow transients can be detected with a single type of analyzing wavelets.

during the simulations.

spectrums of the WTCs.

with noise suppression.

The proposed algorithm is based on the study of disturbances existed in the waveform of terminal current of DGs. It should be noted that once the islanding event is occurred, a transient component continues only for a very short time after the switching operation and then it is removed. But in non-islanding events this transient component continues for longer time, so it should be distinguished. In the proposed method, after studies done by the authors, it was found out that third decomposition level with 20 samples as the length of data window and 17 samples as the moving size of data window is accurate leading to detect the islanding state within maximum 54 samples i.e. 5.4 *ms*. In the first step, the ratio of maximum current magnitude in *r*th window to the previous window is calculated as follows:

$$Ratio-I\_{\underline{t}}(r) = \frac{\min \, \text{and} \, \max I\_{\underline{t}}(r)}{\max I\_{\underline{t}}(r-1)} \tag{11}$$

where, the threshold values are:

$$0.98 \le \text{Ratio} - I\_{\text{f}}(\text{r}) \le 1.02 \tag{12}$$

These threshold values are selected according to simulate the different events. If the calculated ratio satisfies (12) then there is no problem and this means that islanding has not been occurred. For values out of range of (12), the following criteria could be used to check whether the islanding event is taken place or not:

$$\text{Ratio} - D\_{\text{3}}(r) = \frac{\max D\_{\text{3}}(r)}{\max D\_{\text{3}}(r - 2)} \tag{13}$$

Considering different studies done, threshold value chosen for (13) is 0.02. This condition can be expressed by:

$$\text{Ratio} \ -D\_{\mathfrak{z}}(r) \le 0.0 \,\, \text{2} \tag{14}$$

This threshold value is also adopted according to simulate the different events. If value of (14) is less than 0.02, then the islanding event is occurred and trip command should be issued for islanded DGs. The algorithm diagram is shown in (Shariatinasab & Akbari, 2010).

It is worth to point out that moving size of the data window in the proposed algorithm is an important parameter. As decreasing the moving size reduces total time of detection, so the moving size should be decreased as possible.

Application of Wavelet Analysis in Power Systems 237

Fig. 10. The waveform of terminal current of DG2, due to breaker opening on line 7-8, d1-d3

**Number of Samples**

20 samples length data window is considered. In this window, ratio of the changed current is 1.003 for DG1 which satisfies (12). So as it is expected the algorithm would not issued a trip command for DG1. It is important to point out that in order to get a conservative result; it was assumed that the generated power of DG2 is equal to the customer load at the connected bus. Therefore, DG2 is islanded, the difference between the generated and consumed power in bus 8 will be zero, while the ratio of the changed current in related data window for DG2 is 0.0971 that is less than 0.98 and therefore (12) is not satisfied. Then data window is twice shifted to the right, either one up to 17 samples. In this new window, the obtained value of (13) is nearly zero, in which this value satisfies (14). So the proposed algorithm detects the islanding event in maximum time within 54 samples of 10 *kHz* sampling frequency, i.e. 5.4 *ms,* and issues a trip

The more research is done for various combinations and conditions of islanding for both

In order to perform a comprehensive study to check the accuracy of the proposed method, motor starting and capacitor switching are also investigated; as they may cause a similar situation to islanding state and hence should be distinguished correctly. To perform the motor starting study, a 15 *kVA* induction motor starting is studied, and results are shown in Figs. 11-12. For DG1 the value of (11) obtained under this condition is 1.353 that is more than 1.02 and the value of (13) is 0.054 that is more than 0.02. Also, for DG2 the obtained value of (11) is 2.392 and the value of (13) is 0.079, in which both values are more than criteria adopted in the proposed algorithm. Hence, the proposed method distinguishes this situation correctly, i.e. an islanding state is not detected for DGs under motor starting

DG1 and DG2 available in (Shariatinasab & Akbari, 2010).

are detail components of main signal

command for DG2.

condition.

#### **4.1.2 Case study**

The study system consists of two synchronous DGs (DG1 and DG2) operating on PQ mode, and is a part of Iranian distribution network located in Tehran (Fig. 8). The data of the network are available in (Shariatinasab & Akbari, 2010).

Fig. 8. Test system for islanding detection study

#### **4.1.3 Simulation results**

To be ensured of accuracy of the proposed algorithm, all the cases affecting the terminal current of DGs are analyzed. Figs. 9 and 10, show RMS current form and related three decomposition levels using 'Haar' mother wavelet for non-islanded DG1 and islanded DG2, respectively.

Fig. 9. The waveform of terminal current of DG1, due to breaker opening on line 7-8, d1-d3 are detail components of main signal

The study system consists of two synchronous DGs (DG1 and DG2) operating on PQ mode, and is a part of Iranian distribution network located in Tehran (Fig. 8). The data of the

To be ensured of accuracy of the proposed algorithm, all the cases affecting the terminal current of DGs are analyzed. Figs. 9 and 10, show RMS current form and related three decomposition levels using 'Haar' mother wavelet for non-islanded DG1 and islanded DG2, respectively.

Fig. 9. The waveform of terminal current of DG1, due to breaker opening on line 7-8, d1-d3

**Number of Samples**

network are available in (Shariatinasab & Akbari, 2010).

Fig. 8. Test system for islanding detection study

**4.1.3 Simulation results**

are detail components of main signal

**4.1.2 Case study**

Fig. 10. The waveform of terminal current of DG2, due to breaker opening on line 7-8, d1-d3 are detail components of main signal

20 samples length data window is considered. In this window, ratio of the changed current is 1.003 for DG1 which satisfies (12). So as it is expected the algorithm would not issued a trip command for DG1. It is important to point out that in order to get a conservative result; it was assumed that the generated power of DG2 is equal to the customer load at the connected bus. Therefore, DG2 is islanded, the difference between the generated and consumed power in bus 8 will be zero, while the ratio of the changed current in related data window for DG2 is 0.0971 that is less than 0.98 and therefore (12) is not satisfied. Then data window is twice shifted to the right, either one up to 17 samples. In this new window, the obtained value of (13) is nearly zero, in which this value satisfies (14). So the proposed algorithm detects the islanding event in maximum time within 54 samples of 10 *kHz* sampling frequency, i.e. 5.4 *ms,* and issues a trip command for DG2.

The more research is done for various combinations and conditions of islanding for both DG1 and DG2 available in (Shariatinasab & Akbari, 2010).

In order to perform a comprehensive study to check the accuracy of the proposed method, motor starting and capacitor switching are also investigated; as they may cause a similar situation to islanding state and hence should be distinguished correctly. To perform the motor starting study, a 15 *kVA* induction motor starting is studied, and results are shown in Figs. 11-12. For DG1 the value of (11) obtained under this condition is 1.353 that is more than 1.02 and the value of (13) is 0.054 that is more than 0.02. Also, for DG2 the obtained value of (11) is 2.392 and the value of (13) is 0.079, in which both values are more than criteria adopted in the proposed algorithm. Hence, the proposed method distinguishes this situation correctly, i.e. an islanding state is not detected for DGs under motor starting condition.

Application of Wavelet Analysis in Power Systems 239

software is then transformed to MATLAB software in order to apply the wavelet analysis. Only 46 samples/10 *kHz* sampling rate (equal to 4.6 *ms*) of data are considered after fault time. According to the analyses done, db4 mother wavelet was selected as a suitable

After DWT analysis, it is necessary to extract the characteristics of this transform to provide inputs of NN. To this, 2nd norm (norm2) of signal details was considered as NN inputs.

To describe the work, norm2 of 3rd level details versus fault distance from a generator (G3) is illustrated in Fig. 14. As shown in Fig. 14, the more fault distance, the lower value of norm2 is reached. In this study, norm2 of details of five levels were used. The NN used in this study was consisted of 3 hidden layers either with 20 neurons. The optimal number of neurons was determined based on the trial and error approach. The transfer functions applied in input, hidden and output layers were considered **tansig**, **tansig** and **purelin**,

For study system, fault was applied in 120 points which 85 points was considered as

Also, the details of 5 levels were obtained as the optimal solution to train the NN.

respectively, and training algorithm was also considered as **trainlm**.

Fig. 13. Schematic diagram of test system for fault location study

training patterns of NN and 35 points was considered for testing.

Fig. 14. Norm2 of 3rd level details (d3) for G3

**4.2.2 Simulation results**

solution.

Fig. 11. The waveform of terminal current of DG1, due to motor starting at bus 3, d1-d3 are detail components of main signal

Fig. 12. The waveform of terminal current of DG2, due to motor starting at bus 3, d1-d3 are detail components of main signal

Results of the capacitor bank switching are also available in (Shariatinasab & Akbari, 2010).

#### **4.2 Fault location**

#### **4.2.1 Methodology and study system**

In this section, the fault location by DWT and a trained NN will be discussed. The case study is IEEE 9-bus test system as shown in Fig. 13. This system is a 400 *kV* transmission system included 3 generators and 6 lines. Each line is divided to 20 points and then a fault is separately applied in each point. Totally 120 faults is applied in 120 points. As the most of faults occurred in transmission systems have low fault impedance, so fault impedance was considered equal to zero in this study. Then the terminal current signal of G1, G2 and G3 during the fault is obtained with sampling rate 10 *kHz*. The fault signals collected in ETAP

Fig. 11. The waveform of terminal current of DG1, due to motor starting at bus 3, d1-d3 are

**Number of Samples**

Fig. 12. The waveform of terminal current of DG2, due to motor starting at bus 3, d1-d3 are

**Number of Samples**

Results of the capacitor bank switching are also available in (Shariatinasab & Akbari, 2010).

In this section, the fault location by DWT and a trained NN will be discussed. The case study is IEEE 9-bus test system as shown in Fig. 13. This system is a 400 *kV* transmission system included 3 generators and 6 lines. Each line is divided to 20 points and then a fault is separately applied in each point. Totally 120 faults is applied in 120 points. As the most of faults occurred in transmission systems have low fault impedance, so fault impedance was considered equal to zero in this study. Then the terminal current signal of G1, G2 and G3 during the fault is obtained with sampling rate 10 *kHz*. The fault signals collected in ETAP

detail components of main signal

detail components of main signal

**4.2.1 Methodology and study system**

**4.2 Fault location**

software is then transformed to MATLAB software in order to apply the wavelet analysis. Only 46 samples/10 *kHz* sampling rate (equal to 4.6 *ms*) of data are considered after fault time. According to the analyses done, db4 mother wavelet was selected as a suitable solution.

After DWT analysis, it is necessary to extract the characteristics of this transform to provide inputs of NN. To this, 2nd norm (norm2) of signal details was considered as NN inputs. Also, the details of 5 levels were obtained as the optimal solution to train the NN.

To describe the work, norm2 of 3rd level details versus fault distance from a generator (G3) is illustrated in Fig. 14. As shown in Fig. 14, the more fault distance, the lower value of norm2 is reached. In this study, norm2 of details of five levels were used. The NN used in this study was consisted of 3 hidden layers either with 20 neurons. The optimal number of neurons was determined based on the trial and error approach. The transfer functions applied in input, hidden and output layers were considered **tansig**, **tansig** and **purelin**, respectively, and training algorithm was also considered as **trainlm**.

Fig. 13. Schematic diagram of test system for fault location study

Fig. 14. Norm2 of 3rd level details (d3) for G3

#### **4.2.2 Simulation results**

For study system, fault was applied in 120 points which 85 points was considered as training patterns of NN and 35 points was considered for testing.

According to the definition in (IEEE Std. PC37.114, 2004), error percentage of fault location estimation is determined as follows:

$$error\,\,\%=\frac{error\,\,\,\,value}{line\,\,length} \tag{15}$$

Application of Wavelet Analysis in Power Systems 241

Further, Although there have been a great effort in references to prove that one wavelet is more suitable than another, there have not been a comprehensive analysis involving a number of wavelets to prove the point of view suggested. Also, the method of comparison

Therefore, in this chapter for each application in power systems, it was tried to introduce principles and algorithms in order to determine the optimal mother wavelet. According to the literature review, Daubechies family has been the most of applications in power systems analysis. Further, often db4 have been the satisfactory results than the other mother wavelets of Daubechies family. However, it is should be noted that the type of mother wavelet, the number of decomposition levels and etc, may be changed from one application

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Some results obtained from the proposed DWT-NN technique are shown in Table 2. As seen in results, the error values are reasonable values and satisfactory. According to 4.2.1, the time of the fault detection and location is 4.6 *ms* equal to 46 samples per 10 *kHz* sampling rate. Therefore, this technique can be well used to estimate the fault detection and location in a specific transmission system.

Table 2. The results of fault location under db4 mother wavelet and 5 decomposition levels