**3. Experimental models**

*Epilepsy - Advances in Diagnosis and Therapy*

**2. Inflammatory response in epilepsy**

seizure recurrence in epilepsy models [19, 20].

which functions as a protector of the central nervous system, has an important role in regulating the transfer of blood constituents in the brain extracellular space [8]. Increased BBB permeability or BBB leakage is said to be one of the earliest characteristics of the pathophysiology of epileptogenesis [9, 10]. BBB dysfunction may contribute to epileptogenesis via a cascade of events triggered by leakage of inflammatory mediators into the CNS which causes neuroinflammation [11, 12]. Here, we discuss briefly how neuroinflammation is involved in epileptogenesis as well as the status of inflammation in post-epileptic conditions; whether it is the cause or

Considering inflammation as one of the culprits of epileptogenesis, neuroinflammation occurs as a result of a cascade of inflammatory pathways. This involves inflammatory and anti-inflammatory molecules as a response to noxious stimuli or immune stimulation; targeted to defend against pathogenic threats. The activation of inflammatory mediators such as interleukins (ILs), interferons (IFNs), cyclooxygenase (COX)-2 and nuclear factor kappa B (NF-κB), and the surplus of downstream inflammatory mediators including IL-1β, IL-6, tumor necrosis factor (TNF)-α and prostaglandin E2 (PGE2) contribute to seizure progression [13, 14]. Inflammatory mediators are produced by the glia, neurons, endothelial cells of the BBB and peripheral immune cells. In the presence of noxious stimuli, cytokines are secreted by immunocompetent and endothelial cells as well as glial and neuronal cells in the CNS. In the presence of noxious stimuli, cytokines are released which

enable effective communication between effector and target cells [7, 15].

Both innate and adaptive immunity is known to contribute in the generation of inflammation in the brain via the microglia, astrocytes and neurons [7]. In a non-epileptic condition, innate immunity activation occurs during infection and is instrumental for pathogen recognition as well as removal via homeostatic-type tissue inflammation [16]. In epileptic condition where pathogens are absent, innate immunity signaling is activated by damage-associated molecular patterns (DAMPs) which are secreted by injured or activated neurons, bringing about a phenomenon called 'sterile inflammation' [17]. The microglia and astrocytes recognize proteins such as high mobility group box 1 (HMGB1), S100 proteins, adenosine triphosphate (ATP), migration inhibitory factor-related protein 8 (MRP8), which makes are DAMPs, extracellular matrix degradation products and IL-1β to induce inflammation [17, 18]. On top of that, the inflammatory signaling disrupts the BBB integrity by inducing up-regulation of adhesion molecules as well as leukocyte recruitment. These processes reduces seizure threshold and contribute to epileptogenesis and

Clinically, it is observed that patients with autoimmune diseases such as systemic

Moreover, a number of reports suggest that the onset and perpetuation of epilepsy can be driven by inflammation and is not caused by the autoimmune process.

lupus erythematosus (SLE), Hashimoto's encephalopathy, Behcet's disease, and Sjogren's syndrome have an increased risk of developing epilepsy [5]. Another example of an autoimmune disease associated with a predisposition to seizures is Rasmussen encephalitis (RE), a rare inflammatory brain disease causing cerebral hemiatrophy, which progressively leads to severe seizures [21]. Patients of RE have higher levels of astrocytosis, proinflammatory mediators as well as lymphocytes and activated microglial cells in the brain [22, 23]. In these cases, usually, immunotherapies are more effective as compared to antiepileptic drugs in the management

consequence of epilepsy, together with experimental evidences.

**20**

of epilepsy [24].

Moving forward with the understanding on the clinical association of inflammation with epileptogenesis, researchers sought to decipher the role of inflammation and associated pathways in the genesis of a seizure in the brain. Experimental models of inflammation have been instrumental in understanding the role of inflammation in epilepsy. It is still an ongoing debate as there are two field of thoughts; (1) inflammation acts as the cause of seizures and (2) inflammation is the consequence of seizures [7]. Here, we discuss the different types of experimental models and the outcomes of the experimental work, summarized in **Table 1**.

### **3.1 Inflammation increases seizure susceptibility**

## *3.1.1 Hyperthermia-induced seizures*

Febrile seizures (FS) are common in children aged between 6 months and 5 years and occur in response to fever but without infection of the CNS. Fever is the elevation of the body temperature set point within the hypothalamus which results in an elevation of core temperature and is generated by inflammatory mediators such as cytokines and prostaglandins which then invokes a systemic inflammatory response [28, 29]. A widely used hyperthermia-induced seizure model for studying FS is one in which hyperthermia is induced using a regulated stream of mildly heated air to increase the body temperature of neonatal rats aged 10–13 days [30–32]. The brain development of rats between 10 and 15 postnatal days best corresponds to the development of brain in human infants when they are most susceptible to FS [30]. The 'ideal' increase of core temperature in the pups is around 2.9°C, which is reported to be parallel with the temperature increment observed in children experiencing FS [33].

In this model, seizures can be confirmed using electroencephalogram (EEG) [30]. The behaviors exhibited by the pups, such as biting tonic stiffening, and falling over, are similar to those observed after administration of convulsants. Generalized tonic seizures are rarely observed, however [30, 32]. In addition to biochemical analysis, behavioral tests such as the balance beam test and footprint test provide information on the severity and progression of seizures. Research has also shown that in this hyperthermic model, there is a remarkably high release of cytokines within the brain, specifically IL-1β within the hippocampus, and activation of astrocytes, which elevates the brain temperature. This finding is similar to those seen in children suffering from FS [31].

### *3.1.2 Systemic inflammation*

Systemic inflammation is believed to have several CNS manifestations, such as fever, locomotor activity reduction and behaviors that are associated with brain hyperactivity during peripheral inflammation [34]. In other words, the inflammatory response which can be observed during the manifestation of peripheral inflammatory diseases is similar to the inflammatory response generated in the


### **Table 1.**

*Animal models in epilepsy studies.*

periphery [35]. It is important to note that systemic inflammation alone is insufficient to induce seizures, and therefore, a double-hit with a proconvulsant is usually adopted in experiments to show that the first hit of existing inflammation predisposes the subject to increased seizure susceptibility in response to a second hit. We discuss two models of systemic inflammation which have been used for the study of epilepsy and seizures.

The first one is a model of inflammatory bowel disease [36–38]. Inflammatory colitis is induced in adult male rats by intracolonic administration of 2,4,6 trinitobenzene sulfonic acid (TNBS) to initiate a T helper-1 cell-mediated model of inflammatory bowel disease. A dose of 50 mg/mL, 50 mL per rat, invoked an

**23**

additional release of HMGB1.

*3.2.1 Kainic acid (KA) injection*

**3.2 Inflammation as a consequence of seizures**

*Inflammation: Cause or Consequence of Epilepsy? DOI: http://dx.doi.org/10.5772/intechopen.83428*

acute form of localized inflammatory colitis. To study the susceptibility to seizures, a convulsant, pentylenetetrazole (PTZ) was given through intravenous infusion to induce seizures. In this study by Riazi et al. [38], they found that TNBS-treated rats express increased susceptibility to PTZ-induced seizures that strongly correlates with the severity and progression of intestinal inflammation. The TNBS-treated rats present a prominent and reversible inflammatory response within the hippocampus along with microglial activation and TNF-α level elevation [38]. The inflammatory colitis model is also used by Rao, Medhi [39] to establish the correlation between systemic inflammation and seizures. They induced colitis using a method described by MacPherson and Pfeiffer [40], which is the application of acetic acid on the colonic lumen of adult rats. They too found that systemic inflammation can be

The second model of systemic inflammation is bacterial lipopolysaccharide (LPS) injection. In adult rats, an intraperitoneal injection of LPS results in an increase in body temperature elicited by an inflammatory response, which mimics febrile seizures, though a second-hit with a pro-convulsant drug, usually kainic acid, is usually required to generate febrile convulsions. LPS increases rat's body temperature by 1–1.5°C, which mimics fever and amplifies the convulsant actions of KA [41]. Single intraperitoneal injection of *Escherichia coli* LPS at 5 mg/kg or infusion of at a dose of 2.5 mg/kg/day into the peritoneal cavity of adult rats for 7 days via an osmotic mini-pump is sufficient to induce peripheral inflammation [42, 43]. In mice, a single dose of 1 mg/kg of LPS i.p. is sufficient to elicit effects on body temperature and seizure susceptibility [44]. Seizure susceptibility is then tested using an intraperitoneal injection of KA (10 mg/kg) or PTZ (10 mg/mL) after 2 hours [43, 44]. LPS infusion is reported to increase plasma levels of IL-1β, IL-6 and TNF-α. This means that the systemic inflammation induced by LPS infusion brings about the activation of microglia, enhancement of pro-inflammatory cytokines production and tissue oxidative stress in the hippocampus [43, 45]. As a result, LPS administration increases body temperature slightly and reduces PTZ-induced seizure susceptibility in a dose-dependent and time-dependent manner. Recent studies have shown that LPS acts as an activator for Toll-like receptor 4 (TLR 4) and induces seizures. The probable mechanism in explanation to this is that LPS mimics the actions stressed or damaged neurons which releases endogenous 'danger signals' via a protein called HMGB1. After being released from neurons, HMGB1 communicates with TLR4 to induce seizures, which activates a positive feedback cycle, by stimulating activated astrocytes and microglia for

KA is an agonist for α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) and KA receptor. AMPA is a subtype of ionotropic glutamate receptor. Systemic and intracerebral injections of KA induce progressive limbic seizures, which resemble human temporal lobe epilepsy, in rats [46]. These peak in status epilepticus (SE) where, in limbic structures (i.e. hippocampal CA1 and CA3, and the hilus of dentate gyrus) of the brain, reactive oxygen species (ROS) production and mitochondrial dysfunction lead to neuronal cell death [47]. Moreover, the delayed release of proinflammatory gene expressions, such as TNF-α, IL-1β, IL-6, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), is believed to promote prolonged neurodegeneration [48]. For these reasons, KA is used for various studies into neurological disorders including inflammation and epilepsy.

associated with a decreased threshold to PTZ-induced seizures [39].

*Inflammation: Cause or Consequence of Epilepsy? DOI: http://dx.doi.org/10.5772/intechopen.83428*

*Epilepsy - Advances in Diagnosis and Therapy*

Using a regulated stream of mildly heated air to increase core temperature.

Intracolonic administration of 2,4,6-trinitobenzene sulfonic acid (TNBS) at a dose of 50 mg/ml, 50mL per rat.

Intraperitoneal injection of 5 mg/kg or peritoneal infusion of 2.5 mg/kg/day for 7 days.

Intraperitoneal injection of 2 mg/kg in rat neonates, 15 mg/ kg in adult rats and 20 mg/kg in mice, 6 mg/kg in zebrafish.

Subcutaneous or intraperitoneal injection of 340–350 mg/kg in rats, 30 mM in zebrafish larvae.

Intracerebroventricular injection of 1.9 mM in rats.

**Dose/method Outcome References**

within the hippocampus. • Activation of microglial. • Increases levels of TNFα.

2.9°C.

seizures.

TNF-α.

0.9°C).

lobe epilepsy.

hippocampus.

• Astrocyte dysfunction.

neuronal excitability. • Reduction in seizure threshold.

• Mimics febrile seizures in children. • Core body temperature increase by around [28–31]

[32–35]

[36–38]

[39–44]

[45–50]

[51]

• Marked release of cytokines within the brain and activation of astrocytes.

• Induces significant inflammatory response

• Increases susceptibility to PTZ-induced

• Increases plasma levels of IL-1β, IL-6 and

• Increases body temperature slightly (by

• Induces limbic seizures characterized by a seizure scale devised by Racine.

• Seizures induced resemble human temporal

• Leads to neuronal cell death and induction of proinflammatory gene expression.

• Induces status epilepticus, followed by recurrent spontaneous seizures. • Causes extensive neuronal cell loss, astrogliosis, and mossy fiber sprouting in

• Developmental exposure to pilocarpine shows very little effect to startle response.

• Mimics BBB breakdown following seizures. • Increased IL-1β immunoreactivity.

• Significant increase in interictal spikes and

• Increases seizure susceptibility.

**Types of animal model**

Hyperthermicinduced seizure

Inflammatory bowel disease model

*Escherichia coli* LPS injection

Kainic acid injection

Pilocarpine injection

Serum albumin injection

**Table 1.**

**22**

epilepsy and seizures.

*Animal models in epilepsy studies.*

periphery [35]. It is important to note that systemic inflammation alone is insufficient to induce seizures, and therefore, a double-hit with a proconvulsant is usually adopted in experiments to show that the first hit of existing inflammation predisposes the subject to increased seizure susceptibility in response to a second hit. We discuss two models of systemic inflammation which have been used for the study of

The first one is a model of inflammatory bowel disease [36–38]. Inflammatory

colitis is induced in adult male rats by intracolonic administration of 2,4,6 trinitobenzene sulfonic acid (TNBS) to initiate a T helper-1 cell-mediated model of inflammatory bowel disease. A dose of 50 mg/mL, 50 mL per rat, invoked an

acute form of localized inflammatory colitis. To study the susceptibility to seizures, a convulsant, pentylenetetrazole (PTZ) was given through intravenous infusion to induce seizures. In this study by Riazi et al. [38], they found that TNBS-treated rats express increased susceptibility to PTZ-induced seizures that strongly correlates with the severity and progression of intestinal inflammation. The TNBS-treated rats present a prominent and reversible inflammatory response within the hippocampus along with microglial activation and TNF-α level elevation [38]. The inflammatory colitis model is also used by Rao, Medhi [39] to establish the correlation between systemic inflammation and seizures. They induced colitis using a method described by MacPherson and Pfeiffer [40], which is the application of acetic acid on the colonic lumen of adult rats. They too found that systemic inflammation can be associated with a decreased threshold to PTZ-induced seizures [39].

The second model of systemic inflammation is bacterial lipopolysaccharide (LPS) injection. In adult rats, an intraperitoneal injection of LPS results in an increase in body temperature elicited by an inflammatory response, which mimics febrile seizures, though a second-hit with a pro-convulsant drug, usually kainic acid, is usually required to generate febrile convulsions. LPS increases rat's body temperature by 1–1.5°C, which mimics fever and amplifies the convulsant actions of KA [41]. Single intraperitoneal injection of *Escherichia coli* LPS at 5 mg/kg or infusion of at a dose of 2.5 mg/kg/day into the peritoneal cavity of adult rats for 7 days via an osmotic mini-pump is sufficient to induce peripheral inflammation [42, 43]. In mice, a single dose of 1 mg/kg of LPS i.p. is sufficient to elicit effects on body temperature and seizure susceptibility [44]. Seizure susceptibility is then tested using an intraperitoneal injection of KA (10 mg/kg) or PTZ (10 mg/mL) after 2 hours [43, 44]. LPS infusion is reported to increase plasma levels of IL-1β, IL-6 and TNF-α. This means that the systemic inflammation induced by LPS infusion brings about the activation of microglia, enhancement of pro-inflammatory cytokines production and tissue oxidative stress in the hippocampus [43, 45]. As a result, LPS administration increases body temperature slightly and reduces PTZ-induced seizure susceptibility in a dose-dependent and time-dependent manner. Recent studies have shown that LPS acts as an activator for Toll-like receptor 4 (TLR 4) and induces seizures. The probable mechanism in explanation to this is that LPS mimics the actions stressed or damaged neurons which releases endogenous 'danger signals' via a protein called HMGB1. After being released from neurons, HMGB1 communicates with TLR4 to induce seizures, which activates a positive feedback cycle, by stimulating activated astrocytes and microglia for additional release of HMGB1.
