**3.2 Inflammation as a consequence of seizures**

## *3.2.1 Kainic acid (KA) injection*

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.

Kainic acid injection into hippocampus of rats increases the number of IL-1ß, IL-6 and TNF-α positive cells in the hippocampus, indicating inflammatory response [49, 50]. There is also a higher number of GFAP-positive cells which shows that kainic acid promotes gliosis, also an indicator of neuroinflammation. Chen, Zhu [49] found that kainic acid administration causes swelling and deformities of endothelial cells and their nuclei in cerebromicrovessels. The BBB integrity is also destroyed following kainic acid injection with signs of vacuolation, perivascular edema and membrane damage [49].

KA is widely used in epilepsy studies and models include rodents as well as zebrafish. In rodents, KA works to induce seizures and SE in neonatal and adult rat and mice. In rat neonates, 2 mg/kg of KA injected intraperitoneally was found to induce seizures without mortality while 4 mg/kg of KA was shown to induce mortality in 60% of pups [51]. For adult rats and mice, a single dose of KA at15mg/ kg for rats and 20 mg/kg for mice can be used for inducing seizures [52].

In rat neonates, the phases of convulsions that are generated by KA are automatism (forelimb/hind-limb scratching) and continuous generalized tonic-clonic seizures, with loss of righting reflex, indicating tonic extension and SE. In this model, SE is defined as continuous clonic seizures involving both forelimbs and hind-limbs and continual loss of the righting reflex [51].

In adult rats and mice, seizures are characterized by a seizure scoring scale devised by Racine as follows:

Stage 1: Wet dog shakes, facial clonus and staring.

Stage 2: Head nodding.

Stage 3: Forelimb clonus.

Stage 4: Forelimb clonus with rearing.

Stage 5: Rearing, jumping, falling and SE.

Despite being a relatively new model, zebrafish is acknowledged to be a fairly popular animal model in pre-clinical researches and as a suitable alternative to rodents and other animal models in epilepsy research [53]. KA is used to induce seizures in zebrafish at a dose of 6 mg/kg, injected intraperitoneally. The seizures induced by KA are characterized as follows [54]:

Stage 1: Immobility and hyperventilation.

Stage 2: Whirlpool-like swimming behavior.

Stage 3: Rapid left-to-right movements.

Stage 4: Abnormal and spasmodic muscular contractions.

Stage 5: Rapid, whole-body, clonus-like convulsions.

Stage 6: Death.

SE is represented by seizure scores fluctuating between 4 and 6 for a period of 30 minutes or more [55].

### *3.2.2 Pilocarpine injection*

Pilocarpine is a cholinergic (muscarinic) agonist which induces SE, followed by recurrent spontaneous seizures (RSS), in animal models and is widely used to study the mechanisms of SE. It acts on the endothelial muscarinic receptors which compromises the integrity of the BBB [56]. It subsequently causes the influx of proinflammatory cytokines into the brain, which results in neuroinflammation [35]. The hippocampus is notably more vulnerable to pilocarpine-induced neuronal injury because it possesses numerous distinct neuronal circuits which are involved in the generation of seizures [57]. Pilocarpine causes extensive neuronal cell loss in CA1 and CA3 pyramidal cell layers, astrogliosis, and mossy fiber sprouting in the hippocampus [57, 58].

Upon administration of pilocarpine into rats, the levels of inflammatory biomarkers, IL-1β, TNF-α, NF-κβ and COX-2, are elevated in the hippocampus,

**25**

K+

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

indicating the presence of neuroinflammation [59]. During pilocarpine-induced seizures, ROS formation increases and glutathione (GSH) redox status becomes impaired in the hippocampus [60, 61]. The overproduction of ROS leads to an increase in oxidative stress which contributes to cell apoptosis in the brain [62]. Ali, Mahdy [59] reported that pilocarpine injection induces a significant elevation of hippocampal cytochrome c and caspase 3 levels which contributes to apoptosis. This apoptotic cell death is a key feature of hippocampal cell loss induced by SE [59]. Furthermore, apoptosis related proteins such as Bax, Bcl-2 family and caspase-3 can modify the neurotransmission pathways that are independent of cell death in the

Seizures can be induced in adult mice or rats with a subcutaneous or intraperitoneal injection of 340–350 mg/kg of pilocarpine hydrochloride [58, 65]. Pilocarpine treatment sequentially induces the following behavioral changes: akinesia, facial automatisms, forelimb clonus with rearing, salivation, masticatory jaw movements and falling [65, 66]. These behaviors build up progressively into motor limbic seizures that recur repeatedly and rapidly develop into SE, similar to that described in patients of temporal lobe epilepsy (TLE). EEG findings showed a significant surge of theta rhythms and isolated spikes in the hippocampus, synchronization of the hippocampal and cortical activities, isolated electrographic seizures and SE [65]. The electroencephalographical, behavioral, as well as anatomical alterations

Besides the rodent models, pilocarpine is also used to induce seizures in zebrafish larvae for anticonvulsant studies. A final concentration of 30 mM of pilocarpine is used with a 1-minute incubation before quantification of larval locomotor activity. Exposure to pilocarpine results in a more subtle convulsive behavior compared to PTZ, such as lurching/head banging, head-to-tail undulations, increased mouth movements, tremor, body contortions and loss of posture [67]. Eddins, Cerutti [68] reported the use of pilocarpine to induce seizures in zebrafish embryos to compare its effects on early exposure to developmental exposure to toxicants. Zebrafish embryos (2-hours post-fertilization) exposed to 100 μM pilocarpine exhibit very little to zero dose–response relationship of developmental pilocarpine exposure

Extravasation of serum albumin into the brain provokes prominent BBB dysfunction through the activation of transforming growth factor beta (TGF-β) receptor (RII) signaling. This causes the astrocytes to fail in buffering extracellular

which causes BBB dysfunction [69–71]. BBB dysfunction is a commonly found

Frigerio et al. [72] described a model using albumin to provoke BBB breakdown, mimicking brain excitability after SE. They showed that a single intracerebroventricular injection of albumin to rats causes the diffusion of albumin into the hippocampus before conveyed into principal neurons. The extravasation of albumin by parenchymal cells at pathological concentration causes the following conditions: (1) down-regulation of Kir4.1 channels and neuroinflammation in glial fibrillary acidic protein(GFAP)-positive astrocytes; (2) brief neuronal hyperexcitability manifested as involuntary epileptic spikes, and amplified KA–induced epileptic activity; and (3) chronic reduction in seizure threshold without causing cell loss or spontaneous

BBB disruption was induced using a single dose of intracerebroventricular injection of 1.9 mM rat albumin into deeply anesthetized adult rats. After the injection, GFAP-positive glial cells and IL-1β staining in the hippocampus were evaluated. It

CNS and have significant contribution in epileptogenesis [63, 64].

and characteristics of human TLE are emulated by this model [58].

with regard to the startle response [68].

following seizures or epileptogenic brain injuries [7].

*3.2.3 Albumin injection*

epileptic activity [72].

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

*Epilepsy - Advances in Diagnosis and Therapy*

edema and membrane damage [49].

devised by Racine as follows:

Stage 2: Head nodding. Stage 3: Forelimb clonus.

Stage 6: Death.

30 minutes or more [55].

*3.2.2 Pilocarpine injection*

Kainic acid injection into hippocampus of rats increases the number of IL-1ß,

response [49, 50]. There is also a higher number of GFAP-positive cells which shows that kainic acid promotes gliosis, also an indicator of neuroinflammation. Chen, Zhu [49] found that kainic acid administration causes swelling and deformities of endothelial cells and their nuclei in cerebromicrovessels. The BBB integrity is also destroyed following kainic acid injection with signs of vacuolation, perivascular

KA is widely used in epilepsy studies and models include rodents as well as zebrafish. In rodents, KA works to induce seizures and SE in neonatal and adult rat and mice. In rat neonates, 2 mg/kg of KA injected intraperitoneally was found to induce seizures without mortality while 4 mg/kg of KA was shown to induce mortality in 60% of pups [51]. For adult rats and mice, a single dose of KA at15mg/

In rat neonates, the phases of convulsions that are generated by KA are automatism (forelimb/hind-limb scratching) and continuous generalized tonic-clonic seizures, with loss of righting reflex, indicating tonic extension and SE. In this model, SE is defined as continuous clonic seizures involving both forelimbs and

In adult rats and mice, seizures are characterized by a seizure scoring scale

Despite being a relatively new model, zebrafish is acknowledged to be a fairly popular animal model in pre-clinical researches and as a suitable alternative to rodents and other animal models in epilepsy research [53]. KA is used to induce seizures in zebrafish at a dose of 6 mg/kg, injected intraperitoneally. The seizures

SE is represented by seizure scores fluctuating between 4 and 6 for a period of

Pilocarpine is a cholinergic (muscarinic) agonist which induces SE, followed by recurrent spontaneous seizures (RSS), in animal models and is widely used to study the mechanisms of SE. It acts on the endothelial muscarinic receptors which compromises the integrity of the BBB [56]. It subsequently causes the influx of proinflammatory cytokines into the brain, which results in neuroinflammation [35]. The hippocampus is notably more vulnerable to pilocarpine-induced neuronal injury because it possesses numerous distinct neuronal circuits which are involved in the generation of seizures [57]. Pilocarpine causes extensive neuronal cell loss in CA1 and CA3 pyramidal cell

layers, astrogliosis, and mossy fiber sprouting in the hippocampus [57, 58].

Upon administration of pilocarpine into rats, the levels of inflammatory biomarkers, IL-1β, TNF-α, NF-κβ and COX-2, are elevated in the hippocampus,

kg for rats and 20 mg/kg for mice can be used for inducing seizures [52].

hind-limbs and continual loss of the righting reflex [51].

Stage 1: Wet dog shakes, facial clonus and staring.

Stage 4: Forelimb clonus with rearing. Stage 5: Rearing, jumping, falling and SE.

induced by KA are characterized as follows [54]: Stage 1: Immobility and hyperventilation. Stage 2: Whirlpool-like swimming behavior. Stage 3: Rapid left-to-right movements.

Stage 4: Abnormal and spasmodic muscular contractions. Stage 5: Rapid, whole-body, clonus-like convulsions.

IL-6 and TNF-α positive cells in the hippocampus, indicating inflammatory

**24**

indicating the presence of neuroinflammation [59]. During pilocarpine-induced seizures, ROS formation increases and glutathione (GSH) redox status becomes impaired in the hippocampus [60, 61]. The overproduction of ROS leads to an increase in oxidative stress which contributes to cell apoptosis in the brain [62]. Ali, Mahdy [59] reported that pilocarpine injection induces a significant elevation of hippocampal cytochrome c and caspase 3 levels which contributes to apoptosis. This apoptotic cell death is a key feature of hippocampal cell loss induced by SE [59]. Furthermore, apoptosis related proteins such as Bax, Bcl-2 family and caspase-3 can modify the neurotransmission pathways that are independent of cell death in the CNS and have significant contribution in epileptogenesis [63, 64].

Seizures can be induced in adult mice or rats with a subcutaneous or intraperitoneal injection of 340–350 mg/kg of pilocarpine hydrochloride [58, 65]. Pilocarpine treatment sequentially induces the following behavioral changes: akinesia, facial automatisms, forelimb clonus with rearing, salivation, masticatory jaw movements and falling [65, 66]. These behaviors build up progressively into motor limbic seizures that recur repeatedly and rapidly develop into SE, similar to that described in patients of temporal lobe epilepsy (TLE). EEG findings showed a significant surge of theta rhythms and isolated spikes in the hippocampus, synchronization of the hippocampal and cortical activities, isolated electrographic seizures and SE [65]. The electroencephalographical, behavioral, as well as anatomical alterations and characteristics of human TLE are emulated by this model [58].

Besides the rodent models, pilocarpine is also used to induce seizures in zebrafish larvae for anticonvulsant studies. A final concentration of 30 mM of pilocarpine is used with a 1-minute incubation before quantification of larval locomotor activity. Exposure to pilocarpine results in a more subtle convulsive behavior compared to PTZ, such as lurching/head banging, head-to-tail undulations, increased mouth movements, tremor, body contortions and loss of posture [67]. Eddins, Cerutti [68] reported the use of pilocarpine to induce seizures in zebrafish embryos to compare its effects on early exposure to developmental exposure to toxicants. Zebrafish embryos (2-hours post-fertilization) exposed to 100 μM pilocarpine exhibit very little to zero dose–response relationship of developmental pilocarpine exposure with regard to the startle response [68].

### *3.2.3 Albumin injection*

Extravasation of serum albumin into the brain provokes prominent BBB dysfunction through the activation of transforming growth factor beta (TGF-β) receptor (RII) signaling. This causes the astrocytes to fail in buffering extracellular K+ which causes BBB dysfunction [69–71]. BBB dysfunction is a commonly found following seizures or epileptogenic brain injuries [7].

Frigerio et al. [72] described a model using albumin to provoke BBB breakdown, mimicking brain excitability after SE. They showed that a single intracerebroventricular injection of albumin to rats causes the diffusion of albumin into the hippocampus before conveyed into principal neurons. The extravasation of albumin by parenchymal cells at pathological concentration causes the following conditions: (1) down-regulation of Kir4.1 channels and neuroinflammation in glial fibrillary acidic protein(GFAP)-positive astrocytes; (2) brief neuronal hyperexcitability manifested as involuntary epileptic spikes, and amplified KA–induced epileptic activity; and (3) chronic reduction in seizure threshold without causing cell loss or spontaneous epileptic activity [72].

BBB disruption was induced using a single dose of intracerebroventricular injection of 1.9 mM rat albumin into deeply anesthetized adult rats. After the injection, GFAP-positive glial cells and IL-1β staining in the hippocampus were evaluated. It

was found that albumin injection prominently increases IL-1β immunoreactivity in GFAP-positive astrocytes and the number of IL-1β immunopositive cells, indicating the presence of inflammation. Besides that, the production of rapid onset and transient spiking activity in the hippocampus can be found on the EEG analysis of rats injected with rat albumin. This means that the injection of albumin provokes the increase in neuronal excitability. Interestingly, rats presented a significant decline in seizure threshold 3 months after albumin injection. This suggests that acute tissue exposure to albumin induces a long-lasting increase in brain excitability [72].

In short, this model is able to show the pro-ictogenic effect of serum albumin in the brain, mimicking those attained after prolonged seizures and BBB dysfunction. Albumin induces the production of inflammatory molecules and together, they significantly increase brain excitability and seizure susceptibility although insufficient to trigger spontaneous seizures.
