Introductory Chapter: Role of Fenton and Haber-Weiss Reaction in Epilepsy

*Kaneez Fatima Shad and Tushar Kanti Das*

#### **1. Introduction**

Epilepsy is one of the most widespread brain diseases worldwide. It has high morbidity and mortality rates and affects around 70 million people globally. Epileptic seizures have several cognitive impairments and psychosocial consequences in patients. The occurrence of epilepsy is a self-facilitated pathological process triggered by the initial brain damage, ultimately leading to the loss of excitatory and inhibitory neurons in specific areas of the brain. Decades of research failed to fully illuminate its etiology, whereas preventive or disease-modifying therapies are still missing. New insights into the mechanisms of epilepsy are required to create the effective treatments.

Oxidative stress is a contributing factor to the onset and evolution of epilepsy. The association between free radical production and oxidative stress is regarded as a possible mechanism involved in epileptogenesis.

In the following pages of this chapter, we will be looking at the role of Fenton and Haber-Weiss reaction in triggering epilepsy leading to the loss of excitatory and inhibitory neurons in specific regions of the brain.

Brain is the most vulnerable organ to oxidative stress due to its high oxygen intake and reduced antioxidative protection [1]. Excessive oxidative stress is one of the main causes of epileptic seizures. The association between free radical production and oxidative stress is regarded as a possible mechanism involved in epileptogenic seizures [2, 3].

The Fenton and Haber-Weiss reaction has a significant role in the generation of free radicals such as superoxide and highly toxic hydroxyl ions causing epileptic episodes [4, 5].

In this chapter, we discussed the role of the Fenton reaction and Haber-Weiss reaction in ferroptosis and epilepsy that provide a new direction for understanding the underlying mechanisms of epilepsy leading to new therapeutic targets.

#### **2. Chemistry of Fenton and Haber-Weiss reaction**

H.J. Fenton first described the oxidation of tartaric acid by hydrogen peroxide in the presence of ferrous irons, and then, it is known as the Fenton reaction [6, 7]. Fenton reaction can be carried out by two pathways: radical and non-radical systems for Fenton reaction [8, 9].

#### **2.1 Radical system for Fenton reaction**

Hydroxyl radical (OH**.** ) is mainly produced by the reaction between ferrous iron and hydrogen peroxide. The Fenton reaction requires a set of conditions such as pH, temperature, concentration of hydrogen peroxide, and iron [10].

The Fenton reaction needs acidic conditions (pH = 3–4) for its catalytic activities, which gradually decreases due to the precipitation of iron as Fe (OH)3 and the degradation of H2O2 into O2 and H2O [8, 11]. At the higher temperature, the rate of the reaction is increased, along with the increased decomposition rate of hydrogen peroxide [10, 12]. Increasing the concentration of Fe2+ leads to higher reaction rates until it reaches a certain concentration above which all rate increases appear to be marginal [13]. An adequate concentration of H2O2 is also needed for the reaction. Moreover, the Haber-Weiss reaction was first proposed by F. Haber and J J Weiss in 1932. Fe3+ is reduced to Fe2+ through the reaction with superoxide (O2 .�) and H2O2. Finally, OH. , OH�, and oxygen are produced:

$$\begin{aligned} \text{Fe}^{2+} + \text{H}\_{2}\text{O} &\xrightarrow{\text{-}} \text{Fe}^{3+} + \text{OH} + \text{OH}^{-} \text{ [Fenton reaction]}\\ \text{OH} + \text{H}\_{2}\text{O}\_{2} &\xrightarrow{\text{-}} \text{O}\_{2}^{-} + \text{H}^{+} + \text{H}\_{2}\text{O} \\ \text{O}\_{2}^{-} + \text{H}\_{2}\text{O}\_{2} &\xrightarrow{\text{Fe}^{3+}/\text{Fe}^{2+}} \text{OH} + \text{OH}^{-} + \text{O}\_{2} \text{ [Haber-Weiss reaction]} \end{aligned} \tag{1}$$

Due to the presence of multivalency of iron, iron can react with H2O2 by oneor two-electron transfer. Several studies indicate that as a classical Fenton reaction, Fe2+ reacts with H2O2 by the outer sphere electron transfer with no direct bonding interactions between the electron donor and the acceptor [8]. Other studies also indicate that metal-centered Fenton reaction occurs by the direct bonding between iron and H2O2 by inner sphere electron transfer mechanisms. This interaction could produce a metal-peroxo complex, Fe (II)HOO, which may react further to generate either HO� radicals (one-electron oxidant) or Fe (IV)O (two-electron oxidant) (**Figure 1**).

#### **2.2 Non-radical system for Fenton reaction**

Non-radical Fenton reaction begins with the reversible reaction between Fe2+ and H2O2. [Fe2+. H2O2], [FeO2+], and [FeOFe]5+ are the most important intermediate products of this reaction. The reaction is carried out either through oxidation or reduction reaction of iron ions and addition or subtraction of H2O2 reaction [8, 9]. The overall reaction is represented in **Figure 2**.

$$\begin{array}{c|c} \mathsf{Fe^{2+}} \star \mathsf{H\_{2}O\_{2}} & \xrightarrow{\text{Electrochemical transfer}} & \mathsf{Fe(III)}\mathrm{O(O)} + \mathrm{H^{+}} & \xrightarrow{\text{from linear super-sphere}} & \mathsf{Fe(III)}\mathrm{O} + \mathrm{H^{+}} \\ & & \mathsf{Fe(IV)} = \mathrm{O} + \mathrm{H\_{2}O} & \xrightarrow{\text{from linear super-sphere}} & \mathsf{Fe^{2+}} + \mathrm{OH^{-}} \star \mathrm{OH^{-}} \\ \end{array}$$

**Figure 1.**

*The reaction mechanism of the classical and metal-centered Fenton reaction [8].*

*Introductory Chapter: Role of Fenton and Haber-Weiss Reaction in Epilepsy DOI: http://dx.doi.org/10.5772/intechopen.108727*

**Figure 2.** *Reaction mechanism of non-radical Fenton reaction [8, 9].*

In addition, the Fenton reaction is catalyzed by several transition metals such as iron, zinc, copper, cobalt, manganese [14] The overall reaction is represented as follows:

$$\text{Mn}^+ + \text{H}\_2\text{O}\_2 = \text{Mn}^+ + \text{OH}^- + \text{OH} \tag{2}$$

The capacity of metal ions to induce epilepsy is well known. The concentration of Fe2+, Zn2+, Cu2+, and CO2+ are higher in the epileptic human brain compared with the healthy brain. The abnormal levels of trace metals may be epileptogenic, and they enhance excitatory synaptic mechanisms and reduce inhibitory processes. These metal ions also produce higher concentrations of hydroxyl radicals by the Fenton and Haber-Weiss reaction in epilepsy [5, 15, 16].

#### **3. Fenton and Haber-Weiss reaction in epilepsy**

Reactive oxygen species (ROS) play a major role in epilepsy [2, 16]. ROS is generated by many cellular processes such as mitochondrial metabolism, cellular respiration, metabolism of organic matter through a redox reaction, and tissue homeostasis. High-reactive hydroxyl radical is produced by the Fenton and Haber-Weiss reaction in the presence of suitable transition metal. Iron is abundant in the epileptic brain and is involved in the formation of hydroxyl radicals [5, 15].

In 2012, Dixon et al. first discovered iron-dependent cell death by the accumulation of iron-dependent free radicals, and this process is known as "ferroptosis" [17]. Therefore, the imbalance of ROS production is an important factor in ferroptosis. Burdened iron is a common cause of hemorrhagic post-stroke epilepsy and posttraumatic epilepsy [18, 19]. Enormous evidence suggests that a chronic epileptic animal model is created by the injection of hemoglobin or iron into the cortex of an animal [20, 21]. Higher levels of intracellular superoxide and hydroxyl radicals are found in the cerebral cortex after ferric chloride injection [22]. Other studies showed

**Figure 3.** *The association between the Fenton and Haber-Weiss reaction and epilepsy [5, 15].*

that concentrations of transferrin are markedly higher in patients with epilepsy [23]. It boosts iron intake into the cell and accelerates ferroptosis. In addition, sudden unexpected death in epilepsy is caused by cardiomyocyte ferroptosis in the heart through excess production of ROS [24]. Therefore, activation of the ferroptosis pathway is implicated in the pathogenesis of epilepsy and epileptic neuronal death. Excess iron ions generate hydroxyl radicals, which have high reactivity with proteins, lipids, and nucleic acids, leading to lipid peroxidation and promoting ferroptosis in epilepsy (**Figure 3**).

#### **3.1 Fenton and Heber-Weiss reactions and Ferroptosis**

Iron is a crucial element in cellular metabolism, energy generation, and growth in organisms. It participates in various oxidation-reduction reactions. Iron tends to be stored and transported in the Fe3+ form. In the blood, Fe3+ binds to transferrin (Tf) to form a complex which can be delivered into the cells by binding to transferrin receptor-1 (TFR1) in the cell membrane and then transported to the endosome [25]. Then, Fe3+ is converted to Fe2+ by an oxidation-reduction process with the help of sixtransmembrane epithelial antigen of prostate 3 (STEAP3) and divalent metal transporter 1 (DMT1) and then released into the labile iron pool of mitochondria, lysosome, cytosol, and the nucleus [26]. Iron can also be exported by ferroprotein, an iron efflux pump in the cellular membrane, which can oxidize Fe2+ to Fe3+. Excess Fe2+ reacts with H2O2 and produces OH anion and OH. radical by the Fenton reaction in ferroptosis. Moreover, the Haber-Weiss cycle showed that Fe3+ is reduced to Fe2+ through the reaction with superoxide (O2 . ), and Fe2+ reacts with H2O2 and forms OH. , OH, and Fe3+. Thus, Fe2+ is conducive to the production of ROS and promotes ferroptosis [15]. Autophagy can modulate the sensitivity to ferroptosis *via* the selective autophagy of ferritin; this process is called "ferritinophagy." Nuclear receptor

*Introductory Chapter: Role of Fenton and Haber-Weiss Reaction in Epilepsy DOI: http://dx.doi.org/10.5772/intechopen.108727*

**Figure 4.** *Schematic representation of iron metabolism and ferroptosis associated with Fenton and Haber-Weiss reaction [15].*

coactivator 4 (NCOA4) binds to ferritin and then delivers it to autophagosomes for lysosomal degradation. Fe2+ is released in the cell by degradation, which promotes ferroptosis (**Figure 4**).

#### **3.2 Fenton and Haber-Weiss reaction and lipid peroxidation**

Numerous lipid species are distributed in intra- or extra-cellular areas and play important roles in the energy supply and structural components of the intracellular membrane system. Cell membranes are sensitive to radical damage due to the presence of polyunsaturated fatty acids (PUFAs). Free radical oxidizes PUFAs, leading to the formation of hydroperoxides lipid and alkyl radical. This lipoperoxidation alters membrane structure, damages its fluidity integrity, and finally causes ferroptosis [27]. Due to the presence of double bonds, PUFAs are one of the most reactive substrates toward free radicals mainly hydroxyl radicals. Hydroxyl-dependent and hydroxylindependent pathways are the main routes for the lipid peroxidation process [28]. The Fenton reaction and Haber-Weiss reaction are involved in a hydroxyl-dependent pathway, whereas Fe2+ accelerates hydroxyl-independent lipid peroxidation. As a result, ferroptosis also gets accelerated [5, 15, 28].

The lipid peroxidation process is initiated by the attack of hydroxyl radicals at bisallelic positions in the fatty acid side chains, leading to generating of an alkyl radical. The radical is stabilized by the resonance with the double bond. Then, a chain reaction occurs with the extension of the damage and formation of further radical spices, and this process is known as the propagation phase. A newly formed radical reacts with oxygen and forms a peroxyl radical (LOO**.** ), which can react with other adjacent PUFAs to form a hydroperoxide and an alkyl radical, and it causes a chain reaction and

**Figure 5.** *Lipid peroxidation mechanism by hydroxyl radical [28].*

damages more fatty acids [28]. In lipid peroxidation, the fatty acid undergoes a further reaction with oxygen and produces hydroperoxynonenal and then hydroxynonenal (**Figure 5**).

In a nutshell, in the presence of excess iron ions, lipid peroxidation forms more lipid-free radicals and serves as a trigger for ferroptosis.

#### **3.3 Fenton and Haber-Weiss reaction and DNA damage**

Mitochondrial DNA (mtDNA) is mainly susceptible to ROS due to its proximity, despite being packaged with proteins as protective covering. Its mutation can lead to a variety of diseases such as epilepsy.

In DNA, ROS reacts with nitrogenous bases and deoxyribose. This can lead to mutations, carcinogenesis, apoptosis, and necrosis. Hydroxyl radical causes direct damage to DNA, mainly by standard excision, and causes oxidative damage to the pyrimidine and purine bases. This process starts with the radical-induced abstraction of a proton from any position of the deoxyribose and can result in many products (**Figure 6**). In thymine, the abstraction of methyl hydrogen from the 5-position by the hydroxyl radical generates a resonance-stabilized carbon radical, which provides the hydroxymethylene derivative, after treatment with oxygen and followed by reduction.

#### **3.4 Protein oxidation and Fenton Haber-Weiss reaction**

Proteins are encoded by nuclear and mitochondrial DNA, which have numerous functions in the cells. Their function and regulation depend on their structures. Oxidative stress damages their structural integrity, causes loss of catalytic activity, and dysregulates the metabolic pathways [28, 29]. The protein oxidation is initiated by the abstraction of hydrogen from the protein by the hydroxyl radical and generates the

*Introductory Chapter: Role of Fenton and Haber-Weiss Reaction in Epilepsy DOI: http://dx.doi.org/10.5772/intechopen.108727*

**Figure 6.** *Mechanism of oxidative damage to DNA-deoxyribose by Fenton and Haber-Weiss reaction [28].*

protein radicals. It is stabilized by the resonance with the carboxyl group of protein. Then this protein radical reacts with oxygen and forms the protein peroxyl radical.

#### **4. Conclusion**

In summary, we described the regulatory mechanism of ROS production (mainly hydroxyl radicals) by Fenton and Haber-Weiss reaction. Created hydroxyl radicals facilitate ferroptosis, lipid peroxidation, DNA damage, and protein oxidation leading to epileptic episodes. Increasing evidence demonstrated that epilepsy is closely related to ferroptosis and iron metabolism. Ferroptosis is also accelerated by hydroxyl radical, which is mainly formed by Fenton and Haber-Weiss reaction. Therefore, antioxidant therapy, free radical scavenger therapy, and metal chelator therapy may be novel approaches to slow the progression of epilepsy. However, further investigation is needed for understanding new treatment strategies based on Fenton and Haber-Weiss's reaction to neurological diseases such as epilepsy.

## **Author details**

Kaneez Fatima Shad1,2,3\* and Tushar Kanti Das4

1 Ingham Institute for Applied Medical Research, Liverpool, NSW, Australia

2 School of Life Sciences, University of Technology Sydney, NSW, Australia

3 Faculty of Health Sciences, School of Behavioral and Health Sciences, Australian Catholic University, NSW, Australia

4 Department of Neurology, McGovern Medical School at the University of Texas Health Science Center at Houston, Houston, TX, USA

\*Address all correspondence to: kaneez.Fatima-Shad@uts.edu.au; ftmshad@gmail.com

© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*Introductory Chapter: Role of Fenton and Haber-Weiss Reaction in Epilepsy DOI: http://dx.doi.org/10.5772/intechopen.108727*

#### **References**

[1] Lee KH, Cha M, Lee BH. Neuroprotective effect of antioxidants in the brain. International Journal of Molecular Sciences. 2020;**21**(19):7152

[2] Aguiar CC, Almeida AB, Araújo PV, de Abreu RN, Chaves EM, do Vale OC, et al. Oxidative stress and epilepsy: Literature review. Oxidative Medicine and Cellular Longevity. 2012;**2012**: 795259

[3] Borowicz-Reutt KK, Czuczwar SJ. Role of oxidative stress in epileptogenesis and potential implications for therapy. Pharmacological Reports. 2020;**72**(5): 1218-1226

[4] Puttachary S, Sharma S, Stark S, Thippeswamy T. Seizure-induced oxidative stress in temporal lobe epilepsy. BioMed Research International. 2015;**2015**:745613

[5] Chen S, Chen Y, Zhang Y, Kuang X, Liu Y, Guo M, et al. Iron metabolism and Ferroptosis in epilepsy. Frontiers in Neuroscience. 2020;**14**:601193

[6] Fenton HJH. Oxidation of tartaric acid in presence of iron. Journal of the Chemical Society, Transactions. 1894;**65**: 899-910

[7] Ovalle R. A history of the Fenton reactions (Fenton chemistry for beginners). In: Reactive Oxygen Species. London, United Kingdom: IntechOpen Limited; 2022

[8] Barbusinski K. Fenton reactioncontroversy concerning the chemistry. Ecological Chemistry and Engineering. 2009;**16**:347-358

[9] Kremer ML. New kinetic analysis of the Fenton reaction: Critical examination of the free radical – Chain reaction concept. Progress in Reaction Kinetics and Mechanism. 2019;**44**(4):289-299

[10] Ruben VM, Dorian PG, Michel V. Chapter 4 - Ferrioxalate-mediated processes. In: Ameta SC, Ameta R, editors. Advanced Oxidation Processes for Waste Water Treatment. Amsterdam, The Netherlands: Academic Press; 2018. pp. 89-113

[11] Pignatello JJ, Oliveros E, MacKay A. Advanced oxidation process for organic contaminant destruction based on the Fenton reaction. Critical Reviews in Environmental Science and Technology. 2006;**36**:1-84

[12] Paulina P, Filip M, Dawid Z, Kinga M. Agnieszka B decomposition of hydrogen perodixe - kinetics and review of chosen catalysts. Acta Innovations. 2018;**26**:45-52

[13] Rivas FJ, Beltran FJ, Frades J, Buxeda P. Oxidation of p-hydroxybenzoic acid by Fenton's reagent. Water Research. 2001;**35**:387-396

[14] Das TK, Wati MR, Shad KF. Oxidative stress gated by Fenton and Haber Weiss reactions and its association with Alzheimer's disease. Archives of Neuroscience. 2014;**2**(3):1-8

[15] Cai Y, Yang Z. Ferroptosis and its role in epilepsy. Frontiers in Cellular Neuroscience. 2021;**15**:1-10

[16] Geronzi U, Lotti F, Grosso S. Oxidative stress in epilepsy. Expert Review of Neurotherapeutics. 2018; **18**(5):427-434

[17] Dixon SJ, Lemberg KM, Lamprecht MR, Skouta R, Zaitsev EM, Gleason CE, et al. Ferroptosis: An irondependent form of nonapoptotic cell death. Cell. 2012;**149**(5):1060-1072

[18] Zhao Y, Li X, Zhang K, Tong T, Cui R. The Progress of epilepsy after stroke. Current Neuropharmacology. 2018;**16**(1):71-78

[19] Myint PK, Staufenberg EF, Sabanathan K. Post-stroke seizure and post-stroke epilepsy. Postgraduate Medical Journal. 2006;**82**(971):568-572

[20] Wang Y, Wei P, Yan F, Luo Y, Zhao G. Animal models of epilepsy: A phenotype-oriented review. Aging and Disease. 2022;**13**(1):215-231

[21] Kundap UP, Paudel YN, Shaikh MF. Animal models of metabolic epilepsy and epilepsy associated metabolic dysfunction: A systematic review. Pharmaceuticals (Basel). 2020;**13**(6):106

[22] Zou X, Jiang S, Wu Z, Shi Y, Cai S, Zhu R, et al. Effectiveness of deferoxamine on ferric chloride-induced epilepsy in rats. Brain Research. 2017;**1658**:25-30

[23] Zimmer TS, David B, Broekaart DWM, et al. Seizure-mediated iron accumulation and dysregulated iron metabolism after status epilepticus and in temporal lobe epilepsy. Acta Neuropathologica. 2021;**142**:729-759

[24] Akyuz E, Doganyigit Z, Eroglu E, Moscovicz F, Merelli A, Lazarowski A, et al. Myocardial iron overload in an experimental model of sudden unexpected death in epilepsy. Frontiers in Neurology. 2021;**2021**(12):609236

[25] Andrews NC, Schmidt PJ. Iron homeostasis. Annual Review of Physiology. 2007;**69**:69-85

[26] Galaris D, Barbouti A, Pantopoulos K. Iron homeostasis and oxidative stress: An intimate relationship. Biochimica et Biophysica Acta Molecular Cell Research. 2019; **1866**:118535

[27] Dixon SJ, Winter GE, Musavi LS, Lee ED, Snijder B, Rebsamen M, et al. Human haploid cell genetics reveals roles for lipid metabolism genes in nonapoptotic cell death. ACS Chemical Biology. 2015;**2015**(10):1604-1609

[28] Juan CA, Pérez de la Lastra JM, Plou FJ, Pérez-Lebeña E. The chemistry of reactive oxygen species (ROS) revisited: Outlining their role in biological macromolecules (DNA, lipids and proteins) and induced pathologies. International Journal of Molecular Sciences. 2021;**22**(9):4642

[29] Iakovou E, Kourti M. A comprehensive overview of the complex role of oxidative stress in aging, the contributing environmental stressors and emerging antioxidant therapeutic interventions. Frontiers in Aging Neuroscience. 2022;**14**:827900

#### **Chapter 2**

## The Role of Microglia in Neuroinflammation

*Shao-Wen Hung, Chia-Chi Chen, Hsiao-Yun Chen, Ying-Ching Hung, Ping-Min Huang and Chia-Yu Lin*

#### **Abstract**

Microglia typically exist in a resting state of a mature brain and monitors the brain environment. In response to brain injuries or immunological stimuli, however, microglia are readily activated. In their activated state, they can serve diverse beneficial functions essential for enhancing neuron survival through the release of trophic and anti-inflammatory factors. Under certain circumstances, such as sustained epilepsy, however, microglia become overactivated and can induce significant and highly detrimental neurotoxic effects by the excessive production of a large array of cytotoxic factors, such as nitric oxide and proinflammatory cytokines. Neuroinflammation has been identified in epileptogenic tissue and is suspected of participating in epileptogenesis. Recent evidence has shown the effects of antiinflammation and protection against ischemic brain injury by inhibiting soluble epoxide hydrolase (sEH) pharmacologically and genetically. We assume that sEH inhibition might be also beneficial to prevent inflammatory processes caused by seizures and subsequent chronic epilepsy. In the present study, we investigated whether sEH is involved in overactivated microglia-induced neuroinflammation and subsequent epileptogenesis in a mouse model of temporal lobe epilepsy. Overactivated microglia will be detected by using imaging techniques. It is hoped that the results of the present study would provide a better understanding of the roles of sEH and microglia in epileptogenesis.

**Keywords:** epilepsy, epileptogenesis, microglia, neuroinflammation, soluble epoxide hydrolase

#### **1. Introduction**

Neuroinflammation has been identified in epilepsy-related tissue from both experimental and clinical evidence and is suspected to participate in the formation of neuronal cell death, reactive gliosis, and neuroplastic changes in the hippocampus, which may contribute to epileptogenesis [1–4]. The role of active microglia in neuroinflammation is tightly regulated under neurodegenerative processes. Therefore, the microglial regulation of neuroinflammation may provide a therapeutic target for the treatment of severe or chronic neuroinflammation (**Figure 1**).

#### **Figure 1.**

*The relationship of neuroinflammation and epileptogenesis. Pilocarpine induces epilepsy. AUDA suppress epilepsy. Microglia plays an important role between epilepsy and neuroinflammation.*

#### **Figure 2.**

*The mechanism of pilocarpine (pilo) and sEH in the activation and inflammation in microglia. Muscarinic acetylcholine receptor (M3), PLC (phospholipase C), PLA2 (phospholipases A2), PKC (protein kinase C), DAG (diacylglycerol), COX (cyclooxygenase), LOX (lipoxygenase), sEH (soluble epoxide hydrolase), sEHI (soluble epoxide hydrolase inhibitors), AA (arachidonic acid), EETs (epoxyeicosatrienoic acids), DHET (dihydroxyeicosatrienoic acids), and NF-kB (nuclear factor kappa-light-chain-enhancer of activated B cells) play in the neuroinflammation. Pilo causes the neuroinflammation and epileptogenesis. 12-(3-Adamantan-1-ylureido)-dodecanoic acid (AUDA) suppress epilepsy.*

During neuroinflammation, the pro-inflammatory-related cytokines, such as tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and interleukin-1β (IL-1β), are produced by active microglia or astrocytes and next provoked pathological signaling cascades through phospholipase C and phospholipase A2 activations [5, 6]. Finally, the oxidized enzymes released the non-esterified arachidonic acid (AA) from cellular phospholipids and the formation of lysophospholipids and bioactive eicosanoids (**Figure 2**).

#### **2. Epilepsy and treatments in people**

In total, 1–3% of people in the world approximately suffer from epilepsy. Pharmacotherapy is the main treatment for most epileptic patients [7–10]. Moreover, the surgery is another option for epileptic patients according to the clinical doctors' diagnosis by referring to brain imaging and seizure mapping techniques. When

#### *The Role of Microglia in Neuroinflammation DOI: http://dx.doi.org/10.5772/intechopen.105865*

epileptic patients cannot control seizures, by treatment with antiepileptic drugs (AEDs) or are not viable for surgery, vagal nerve stimulation will be a third possible option [11–16]. Unfortunately, a number of epileptic patients cannot control seizures. Herein, it is needed to research and develop more efficacious therapies for these epileptic patients with uncontrolled seizures [17–20].

#### **3. The role of enzyme systems in the neuroinflammation**

The cyclooxygenases, lipoxygenases, and cytochrome P450 (CYP) epoxygenases participated in metabolizing released AA to lipid metabolites as leukotrienes, epoxyeicosatrienoic acids (EETs), and prostaglandins (**Figure 2**). Brain parenchymal tissue metabolizes AA to EETs via the CYP epoxygenase, which regulates cerebral blood flow (CBF) and against neuroinflammation and apoptosis. Recently, hypoxia and ischemic preconditioning experiments have shown that the increased expression of CYP epoxygenase and EETs in the brain may confer protection from an ischemic stroke induced by middle cerebral artery occlusion (MCAO) in the animal model. It also suggests that EETs signaling may suppress the ischemia-evoked inflammatory cytokine response in the brain, supporting an anti-neuroinflammatory role for EETs in the brain circulation [21–28].

Iba1 is specifically expressed in microglia or macrophages and is up-regulated during the activation of these cells following nerve injury, central nervous system ischemia, and several other brain diseases. Additionally, whether soluble epoxide hydrolase (sEH) expression in the microglia should be verified? sEH can perform the metabolic conversion of EETs into their less active form as dihydroxyeicosatrienoic acids. Currently, the inhibition of sEH has been used to increase systemic EETs level and bioactivity. Through applying the pharmacologic inhibitors or genetic deletion, the inhibition of sEH attenuated the cerebral ischemia-induced vascular and neural injury, suggesting sEH might be a novel target for stroke treatment [29–37].

#### **4. Experimental design** *in vitro* **for evaluating the role of microglia in Neuroinflammation**

The reagents were ordered and prepared to perform *in vitro* experiment. The 12-(3-Adamantan-1-yl-ureido)-dodecanoic acid (AUDA) was ordered from Cayman Chemical (Ann Arbor, MI, USA) and dissolved in dimethyl sulfoxide (DMSO; Cat No. 472301; Sigma-Aldrich, MO, USA). Pilocarpine was ordered and dissolved in 0.9% saline. The 90% ethanol (Sigma-Aldrich), Liu's stain (ASK, Taoyuan, and Taiwan), and Griess reagent system (Promega, Madison, and WI) were ordered. Cytofix/ Cytoperm™ (BD Biosciences, CA, USA), Perm/Wash™ (BD Biosciences), mouse anti- Iba1 monoclonal antibody (sc-52,328; Santa Cruz Biotechnology), mouse antisEH monoclonal antibody (sc-6260; Santa Cruz Biotechnology), and FITC-labeled goat anti-mouse IgG antibody (1:1000) (sc-2010; Santa Cruz Biotechnology) were ordered for the determination of activated microglial marker and sEH expression by flow cytometric assay. The 3-(4,5-Dimethyl- 2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT, MERCK, Darmstadt, Germany) was ordered and dissolved in 1× phosphate-buffered saline (PBS; Sigma-Aldrich).

Mouse retroviral immortalized microglia BV-2 cells belonging to the C57BL/6 background were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS; Gibco®), 2 mM L-glutamine, 100 U/mL penicillin (Sigma-Aldrich), and 100 μg/mL streptomycin (Sigma-Aldrich) in 5% CO2 atmosphere at 37°C. Cells were treated with 100 μM pilocarpine and/or 100 μM AUDA and cultured for 24 hrs in 10% FBS-DMEM on glass coverslips. Observation of cell morphology with/ without treatment was done by light microscope (Olympus CKX41, Olympus Optical Co. Ltd.). Cells were grown to 90% confluency before the experiments.

Measurement of cell viability was measured by MTT assay according to the manufacturer's instructions. At the experimental points, cell viability was detected by MTT assay. The reduced purple dye intensity of color was estimated by reading at an optical density of 570 nm in a spectrophotometer. Moreover, *In vitro* migration assays (scratch assay and transwell migration assay) were performed. Scratch assays were performed following the previously described. Briefly, the BV-2 microglia in six well plates were performed with serum-free DMEM for three wash times. A line down the center of each well was scraped with a sterile p200 pipette tip and followed by a wash step to remove debris with serum-free DMEM. Images were taken at 10× magnification of the light microscopy (Olympus BX43, Olympus Optical Co. Ltd.). The scratch widths were measured and wound closure was calculated by dividing widths measured after 8 hours of incubation by the initial scraped width. Each experiment was carried out in triplicate and three fields were blindly counted per well by scorers. Transwell migration assays were performed by using Boyden chambers (BD Bioscience). The 4 × 104 BV-2 microglia (200 μL serum-free DMEM) were added to the upper chamber of Boyden chambers and allowed to adhere to the polycarbonate filters (8 μm pore) for 30 mins at 37°C in a humidified atmosphere of 95% air and 5% CO2. Following this, BV-2 microglia were treated with 100 μM pilocarpine at 37°C for 30 mins prior to AUDA treatment. The 100 μM AUDA was then placed in the upper chamber and the lower chamber was added with 10% fetal bovine serum (FBS)-DMEM to attract BV-2 microglia migration. BV-2 cells did not migrate and remained on the upper surface of the Boyden chambers' filter were removed. BV-2 cells that had migrated to the lower surface were fixed with 90% ethanol, stained with Liu's stain (ASK, Taoyuan, Taiwan), and counted. In at least three independent experiments, three wells per treatment were blindly counted in nine random fields at 40× magnification per well by scorers.

A phagocytosis assay was performed in this experiment. BV-2 microglia seeded in six well plates were incubated with 100 μM pilocarpine at 37°C for 30 mins prior to 100 μM AUDA treatment for 24 hrs. After 24hrs treatment, the phagocytic ability of the microglia was measured by using FITC-labeled dextran (MW 40,000) as a tracer. Briefly, microglia were exposed to 30μg/mL FITC-labeled dextran for 30 mins. Later, three washing times with cold PBS (pH 7.4) were performed prior to measuring fluorescence at 480nm excitation and 520nm emission on a flow cytometer (FACS Calibular, BD Biosciences) or fluorescence microscopy (Olympus BX43, Olympus Optical). As a background, the cultures without FITC-dextran were used. Each culture condition was performed in quadruplicate, and three independent experiments were performed.

Measurement of extracellular nitric oxide production was performed. The nitrite, a stable breakdown product of nitric oxide, was measured with a Griess Reagent System (Promega, Madison, WI). Determination of activated microglial marker and sEH expression by flow cytometric assay was performed. First, BV-2 cells were pretreated with 100 μM pilocarpine for 30 mins then were treated with 100 μM AUDA for 24 hrs in 10% FBS with DMEM. After pilocarpine-AUDA co-treatment, these cells were harvested and fixed in Cytofix/Cytoperm™ (BD Biosciences) at 4°C for 15 mins and washed twice with 1× Perm/Wash™ (BD Biosciences). Fixed cells were stained with various primary antibodies [mouse anti-Iba1 monoclonal antibody (1:100

#### *The Role of Microglia in Neuroinflammation DOI: http://dx.doi.org/10.5772/intechopen.105865*

dilution) (sc-52,328; Santa Cruz Biotechnology) and mouse anti-sEH monoclonal antibody (1:100 dilution) (sc-6260; Santa Cruz Biotechnology)] at 4°C for 30 mins and then washed twice with 1× Perm/Wash™ (BD Biosciences). Secondary antibodies [FITC-labeled goat anti-mouse IgG antibody (1:1000 dilution) (sc-2010; Santa Cruz Biotechnology)] were subsequently stained at 4°C for 30 mins. Finally, cells were stained with 5 μg/mL PI (propidium iodide; BD Biosciences) at room temperature for 5 mins. Cells were analyzed by a flow cytometer (FACSCalibur, BD Biosciences) and WinMDI software (version 2.9). Statistical analysis was performed in this study. The values are reported as mean ± SE. All statistical comparisons were made with two-tailed tests. Statistical evaluation was performed using Student's *t*-test, one-way ANOVA, and/or Dunnett's post hoc test. Differences between groups were considered statistically significant at \*\**p* < 0.01 and \*\*\**p* < 0.001.

#### **5. AUDA significantly inhibited pilocarpine-induce BV-2 microglial activation and cytokine expressions**

The 100 μM pilocarpine did not affect cell viability and the half-maximal inhibitory concentration (IC50) was 17.5 mM. The 100 μM AUDA did not affect cell viability and the

#### **Figure 3.**

*BV-2 microglial cell viability for the pilocarpine and/or AUDA treatment. (A) BV-2 cells treated with the serial two-fold diluted concentration of pilocarpine (0 to 100,000 μM) at 37°C for 24 hrs in 10% serum-DMEM. Cell viability was determined by using MTT assay. The half maximal inhibitory concentration (IC50) of pilocarpine was 17.5 mM. (B) BV-2 cells treated with the serial two-fold diluted concentration of AUDA (0 to 16,000 μM) at 37°C for 24 hrs in 10% serum-DMEM. Cell viability was determined by using MTT assay. 100 μM pilocarpine did not affect cell viability and the half maximal inhibitory concentration (IC50) of AUDA was 0.35 mM. (C) Non-cytotoxic concentration (100 μM) of pilocarpine and AUDA were used in this study. Non-cytotoxic effect was presented after 100 μM pilocarpine combined with 100 μM AUDA treatment at 37°C for 24 hrs in 10% serum-DMEM. Values are reported as mean ± SE.*

half-maximal inhibitory concentration (IC50) was 0.35 mM. Non-cytotoxic concentration (100 μM) of pilocarpine and AUDA were used in this study (**Figure 3A** and **B**). The non-cytotoxic effect was presented after 100 μM pilocarpine combined with 100 μM AUDA treatment (**Figure 3C**). The mean fluorescence intensity (MFI) of Iba1 expression was significantly increased in the BV-2 microglial cells under direct 100 μM pilocarpine

#### **Figure 4.**

*AUDA significantly inhibited pilocarpine-induce BV-2 microglial activation and cytokine expressions. (A) MFI of Iba1 expression was significantly increased in the BV-2 microglial cells under the direct 100 μM pilocarpine stimulation. AUDA significantly decreased Iba1 expression. (B) sEH expression was presented in the BV-2 microglia. 100 μM AUDA significantly decreased sEH expression in BV-2 microglia. (C, D) AUDA significantly suppressed pilocarpine-active BV-2 cell migration by using wound-healing assay. (E, F) AUDA significantly suppressed pilocarpine-active BV-2 cell migration by using Boyden chamber assay. (G, H) histogram showed 100% phagocytosis in all groups. AUDA significantly suppressed phagocytic abilities of pilocarpine-active BV-2 cells by using flow cytometry. (I) No effects of nitric oxide production were presented in all groups. Values are reported as mean ± SE. All statistical comparisons were made with two-tailed tests. Statistical evaluation was performed using Student's t-test. Differences between groups were considered statistically significant at \*\*p < 0.01; \*\*\*p < 0.001.* stimulation (**Figure 4A**). The sEH expression was presented in the BV-2 microglia (**Figure 4B**). C-terminal inhibitor of she, AUDA (100 μM), significantly decreased Iba1 and sEH expressions in the active BV2 microglia (**Figure 4A** and **B**). After microglial activation, cell migration, phagocytosis, and cytotoxicity were enhanced. According to these results, AUDA significantly suppressed cell migration, and phagocytosis (**Figure 4C**–**H**). Additionally, alone or combined pilocarpine or AUDA treatment did not affect extracellular nitric oxide production in BV-2 microglia (**Figure 4I**).

#### **6. Discussion**

Epilepsy affects approximately 1–3% population of the world, and temporal lobe epilepsy (TLE) is the most common localized epilepsy disorder, accounting for approximately 40% of adults with epilepsy [38]. However, the exact mechanism for the formation of TLE remains unclear. According to the engulfment-promoted cell death theory, nerve cells have special receptors. When nerve cells are injured, activated microglia will recognize this receptor and contact nerve cells, indirectly causing nerve cell death [39]. In addition, some studies have confirmed that microglia can also be directly activated by some activating factors, thereby affecting the function and survival of nerve cells [39]. Previous studies have confirmed that the EETs-sEH pathway is associated with brain inflammation, but whether the EETssEH pathway is involved in the formation of TLE remains to be clarified. For these reasons, studying the molecular and cellular mechanisms of the brain's transition from normal to epilepsy can be used to understand the neurobiological changes in epilepsy formation and provide a new therapeutic strategy. Therefore, this study hopes to find a new treatment by understanding the performance and function of sEH microglia in the resting state and the microglia in the activated state, and using the functional inhibitor of sEH to find out how to regulate the activation of microglia. The method of epilepsy is expected to provide clinicians with a reference for the treatment of epilepsy and the use of AEDs in the future. This study demonstrated that AUDA, an inhibitor of sEH, significantly inhibited sEH expression and pilocarpine-induced microglia activation, including phagocytosis and migration. From these results, pilocarpine can directly activate microglia, and inhibition of the EET-sEH pathway can inhibit activated microglia, including phagocytosis and migration. Based on these research results, it is hoped that in the future, it will be helpful to neuroscience researchers in molecular and cellular research on the pathogenic mechanism of TLE, and provide clinicians with a reference for treating epilepsy and the use of anti-epileptic drugs.

#### **7. Conclusions**

Neurological disorders are complicated in the brain and spinal cord and are caused by a loss of neurons and glial cells in these injured areas. Currently, neurological disorders can affect hundreds of millions of people worldwide. More than 50 million people have epilepsy worldwide. The microglia are a key causative factor in the process of neuroinflammation. Commonly, microglia are activated after the brain injury and the activated microglia can induce neurocytotoxic factors. At present, much evidence have demonstrated microglial activation following pilocarpineinduced seizures. Our results suggest a role for sEH in regulating epileptogenesis of

BV-2 microglia *in vitro*, whereas the effect of hydrolase inhibition on epileptogenesis may provide a novel therapeutic approach for approximately 20–40% of the clinically anti-epileptic drugs-uncontrolled epileptic patients.

#### **Acknowledgements**

The authors thank the Ministry of Science and Technology and the Council of Agriculture, Taiwan for support. The plan number of Council of Agriculture is 111AS-11.3.2-ST-a2.

### **Conflict of interest**

The authors declare no conflict of interest.

## **Author details**

Shao-Wen Hung\*, Chia-Chi Chen, Hsiao-Yun Chen, Ying-Ching Hung, Ping-Min Huang and Chia-Yu Lin Division of Animal Industry, Animal Technology Research Center, Agricultural Technology Research Institute, Hsinchu, Taiwan

\*Address all correspondence to: 1032169@mail.atri.org.tw

© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*The Role of Microglia in Neuroinflammation DOI: http://dx.doi.org/10.5772/intechopen.105865*

#### **References**

[1] Hung YW, Lai MT, Tseng YJ, Chou CC, Lin YY. Monocyte chemoattractant protein-1 affects migration of hippocampal neural progenitors following status epilepticus in rats. Journal of Neuroinflammation. 2013;**10**:11

[2] Hung YW, Yang DI, Huang PY, Lee TS, Kuo TBJ, Yiu CH, et al. The duration of sustained convulsive seizures determines the pattern of hippocampal neurogenesis and the development of spontaneous epilepsy in rats. Epilepsy Research. 2012;**98**:206-215

[3] DeLorenzo RJ, Hauser WA, Towne AR, Boggs JG, Pellock JM, Penberthy L, et al. A prospective, population-based epidemiologic study of status epilepticus in Richmond, Virginia. Neurology. 1996;**46**:1029-1035

[4] Hanna J, Nichol A. Status epilepticus: an intensive care medicine problem. Anaesthesia & Intensive Care Medicine. 2012;**13**:148-151

[5] Marchi N, Oby E, Batra A, Uva L, De Curtis M, Hernandez N, et al. *In vivo* and *in vitro* effects of pilocarpine: Relevance to ictogenesis. Epilepsia. 2007;**48**:1934-1946

[6] Wang CH, Hung CP, Chen MT, Shih YH, Lin YY. Hippocampal desynchronization of functional connectivity prior to the onset of status epilepticus in pilocarpine-treated rats. PLoS One. 2012;**7**:e39763

[7] Vissers C, Ming GL, Song H. Nanoparticle technology and stem cell therapy team up against neurodegenerative disorders. Advanced Drug Delivery Reviews. 2019;**148**:239-251 [8] Loscher W. Molecular mechanisms of drug resistance in status epilepticus. Epilepsia. 2009;**50**(Suppl. 12):19-21

[9] Alldredge BK, Gelb AM, Isaacs SM, Corry MD, Allen F, Ulrich S, et al. A comparison of lorazepam, diazepam, and placebo for the treatment of out-ofhospital status epilepticus. New England Journal of Medicine. 2001;**345**:631-637

[10] Alvarez V, Januel JM, Burnand B, Rossetti AO. Second-line status epilepticus treatment: Comparison of phenytoin, valproate, and levetiracetam. Epilepsia. 2011;**52**:1292-1296

[11] Claassen J, Hirsch LJ, Emerson RG, Mayer SA. Treatment of refractory status epilepticus with pentobarbital, propofol, or midazolam: A systematic review. Epilepsia. 2002;**43**:146-153

[12] French JA. Response to early AED therapy and its prognostic implications. Epilepsy Currents. 2002;**2**:69-71

[13] Wahab A, Albus K, Gabriel S, Heinemann U. In search of models of pharmacoresistant epilepsy. Epilepsia. 2010;**51**(Suppl. 3):154-159

[14] Song CG, Zhang YZ, Wu HN, Cao XL, Guo CJ, Li YQ, et al. Stem cells: A promising candidate to treat neurological disorders. Neural Regeneration Research. 2018;**13**:1294-1304

[15] Ravizza T, Gagliardi B, Noe F, Boer K, Aronica E, Vezzani A. Innate and adaptive immunity during epileptogenesis and spontaneous seizures: Evidence from experimental models and human temporal lobe epilepsy. Neurobiology of Disease. 2008;**29**:142-160

[16] Vezzani A, Granata T. Brain inflammation in epilepsy: Experimental and clinical evidence. Epilepsia. 2005;**46**:1724-1743

[17] Jakubs K, Bonde S, Iosif RE, Ekdahl CT, Kokaia Z, Kokaia M, et al. Inflammation regulates functional integration of neurons born in adult brain. Journal of Neuroscience. 2008;**28**:12477-12488

[18] Choi J, Koh S. Role of brain inflammation in epileptogenesis. Yonsei Medical Journal. 2008;**49**:1-18

[19] Turrin NP, Rivest S. Innate immune reaction in response to seizures: Implications for the neuropathology associated with epilepsy. Neurobiology of Disease. 2004;**16**:321-334

[20] Vezzani A, French J, Bartfai T, Baram TZ. The role of inflammation in epilepsy. Nature Reviews Neurology. 2011;**7**:31-40

[21] Fritschy JM. Epilepsy, E/I balance and GABA(a) receptor plasticity. Frontiers Research Foundation. 2008;**1**:5

[22] Scharfman HE, Smith KL, Goodman JH, Sollas AL. Survival of dentate hilar mossy cells after pilocarpine-induced seizures and their synchronized burst discharges with area CA3 pyramidal cells. Neuroscience. 2001;**104**:741-759

[23] Isokawa M. Decrement of GABAA receptor-mediated inhibitory postsynaptic currents in dentate granule cells in epileptic hippocampus. Journal of Neurophysiology. 1996;**75**:1901-1908

[24] Wiebe SP, Staubli UV. Recognition memory correlates of hippocampal theta cells. Journal of Neuroscience. 2001;**21**:3955-3967

[25] Williams PA, Wuarin JP, Dou P, Ferraro DJ, Dudek FE. Reassessment of the effects of cycloheximide on mossy fiber sprouting and epileptogenesis in the pilocarpine model of temporal lobe epilepsy. Journal of Neurophysiology. 2002;**88**:2075-2087

[26] Hung YW, Hung SW, Wu YC, Wong LK, Lai MT, Shih YH, et al. Soluble epoxide hydrolase activity regulates inflammatory responses and seizure generation in two mouse models of temporal lobe epilepsy. Brain Behavior and Immunity. 2015;**43**:118-129

[27] Vezzani A, Moneta D, Richichi C, Aliprandi M, Burrows SJ, Ravizza T, et al. Functional role of inflammatory cytokines and antiinflammatory molecules in seizures and epileptogenesis. Epilepsia. 2002;**43**(Suppl. 5):30-35

[28] Hopkins SJ, Rothwell NJ. Cytokines and the nervous system. I: Expression and recognition. Trends in Neurosciences. 1995;**18**:83-88

[29] Aisen PS, Davis KL. Inflammatory mechanisms in Alzheimer's disease: Implications for therapy. American Journal of Psychiatry. 1994;**151**:1105-1113

[30] Hauss-Wegrzyniak B, Dobrzanski P, Stoehr JD, Wenk GL. Chronic neuroinflammation in rats reproduces components of the neurobiology of Alzheimer's disease. Brain Research. 1998;**780**:294-303

[31] Lee SC, Han JS, Seo JK, Cha YN. Modulation of cyclooxygenase-2 expression by phosphatidylcholine specific phospholipase C and D in macrophages stimulated with lipopolysaccharide. Molecules and Cells. 2003;**15**:320-326

[32] Balsinde J, Winstead MV, Dennis EA. Phospholipase a(2) *The Role of Microglia in Neuroinflammation DOI: http://dx.doi.org/10.5772/intechopen.105865*

regulation of arachidonic acid mobilization. FEBS Letters. 2002;**531**:2-6

[33] Rosenberger TA, Villacreses NE, Hovda JT, Bosetti F, Weerasinghe G, Wine RN, et al. Rat brain arachidonic acid metabolism is increased by a 6-day intracerebral ventricular infusion of bacterial lipopolysaccharide. Journal of Neurochemistry. 2004;**88**:1168-1178

[34] Shimizu T, Wolfe LS. Arachidonic acid cascade and signal transduction. Journal of Neurochemistry. 1990;**55**:1-15

[35] Crespel A, Coubes P, Rousset MC, Brana C, Rougier A, Rondouin G, et al. Inflammatory reactions in human medial temporal lobe epilepsy with hippocampal sclerosis. Brain Research. 2002;**952**:159-169

[36] Jankowsky JL, Patterson PH. The role of cytokines and growth factors in seizures and their sequelae. Progress in Neurobiology. 2001;**63**:125-149

[37] Vezzani A, Balosso S, Ravizza T. The role of cytokines in the pathophysiology of epilepsy. Brain, Behavior, and Immunity. 2008;**22**:797-803

[38] Loo LS, McNamara JO. Impaired volume regulation is the mechanism of excitotoxic sensitization to complement. Journal of Biomedical Science. 2006;**26**:10177-10187

[39] Marín-Teva JL, Dusart I, Colin C, Gervais A, Rooijen NV, Mallat M. Microglia promote the death of developing Purkinje cells. Neuron. 2004;**41**:535-547

**Chapter 3**
