**3. Distinguishing pathological from normal oscillatory brain patterns**

The identification of physiologically relevant brain wave patterns is indispensable when doing EEG studies. In essence, oscillatory brain patterns can be classified as normal (nonpathological) or abnormal (pathological) brain oscillations. For example, normal brain oscillatory synchronization is highly correlated with mental process, perception, memory and behavioral states, such as sleep (Singer, 1999; Engel *et al*., 2001; Pareti and De Palma, 2004; Gross *et al*., 2004; Cantero and Atienza, 2005; Schnitzler and Gross, 2005). By comparison, abnormal brain oscillations are usually associated with dysfunctions, such as, cholinergic system imbalances and epilepsy (Traub, 2003; Timofeev and Steriade, 2004; Schnitzler and Gross, 2005). When spike/wave activity is present in the EEG, it is defined as an epileptiform pattern. It might not necessarily mean that the subject developed epilepsy, since this pathology is characterized by spontaneous recurrent seizures (SRS). The spike/wave activity occurs due to hypersynchronous firing in certain regions of the brain that are then called an "epileptic focus" (Engel, 1993). Depending on the affected area, the manifestations can be sensory, motor, or cognitive. The limbic regions are the most

neurological characterization of various transgenic mouse models and giving valuable information about epilepsies and sleep disorders in humans. They emphasized that without restraint from tethered EEG systems, the subjects can be observed without interference in

Williams and co-workers (2006) used a three EEG channel system (DSI; St. Paul, Minn.) to record interictal spikes and epileptiform activity in the cortex and hippocampus of rats. They studied the model of kainic acid-induced seizures and long-term telemetric EEG recording to investigate epileptogenesis. According to them, although the chance to perform prolonged recordings is a great advantage, the cost, surgical complexity and frequency resolution of the system are listed as disadvantages. Obviously, collecting the data is just the first step, and throughout the use of the same system, White and colleagues (2006) tested different algorithms to process very large EEG data files acquired over 13 days. They concluded that the quality of the EEG and the type of analysis method employed can affect the positive predictive value (PPV, or true positives divided by the sum of true positives and false positives) and sensitivity (true positives divided by the sum of true positives and false negatives). In that sense, both implantation surgery accuracy and telemetry device

Lapray and colleagues (2008) presented a cost-effective and reusable telemetry system to record EEG in rats. The system allows a sampling rate of 500 Hz (bi-directional) and a range of up to 3 meters. The data transmission rate is roughly 115 kbps and the receiver connected to a computer through the USB port. The software developed by the group allows the recording of simultaneous video, opening the possibility to efficiently correlate behavior and EEG patterns. Finally, the study not only of EEG, but also action potentials during normal behavior, can be benefited by telemetry. It is known that the activity of place cells is highly correlated with the animal's spatial position (O'Keefe and Speakman, 1987; O'Keefe *et al*., 1998). A very innovative system was created by Chen and co-workers (2008) that used telemetry to record brain potentials in 3D mazes to investigate the role of hippocampal place cells in rats. The wireless technology used was Bluetooth which allowed a range of 5 meters and sampling at up to 10 kHZ, drastically increasing the frequency resolution and satisfying

**3. Distinguishing pathological from normal oscillatory brain patterns** 

The identification of physiologically relevant brain wave patterns is indispensable when doing EEG studies. In essence, oscillatory brain patterns can be classified as normal (nonpathological) or abnormal (pathological) brain oscillations. For example, normal brain oscillatory synchronization is highly correlated with mental process, perception, memory and behavioral states, such as sleep (Singer, 1999; Engel *et al*., 2001; Pareti and De Palma, 2004; Gross *et al*., 2004; Cantero and Atienza, 2005; Schnitzler and Gross, 2005). By comparison, abnormal brain oscillations are usually associated with dysfunctions, such as, cholinergic system imbalances and epilepsy (Traub, 2003; Timofeev and Steriade, 2004; Schnitzler and Gross, 2005). When spike/wave activity is present in the EEG, it is defined as an epileptiform pattern. It might not necessarily mean that the subject developed epilepsy, since this pathology is characterized by spontaneous recurrent seizures (SRS). The spike/wave activity occurs due to hypersynchronous firing in certain regions of the brain that are then called an "epileptic focus" (Engel, 1993). Depending on the affected area, the manifestations can be sensory, motor, or cognitive. The limbic regions are the most

their physiology.

integrity may be very important factors.

the conditions to have single unit recordings.

frequently affected areas, including the hippocampus, amygdala, pyriform cortex, and cortex (Turski *et al*., 1983b; Carpentier *et al*., 1990; Petras, 1994; Scremin *et al*., 1998; Shih *et al*., 2003). The situation becomes critical if the seizure is sustained for a prolonged period without significant interruption or recovery. When such an event takes place, the subject is experiencing *status epilepticus* (SE) and can years later display SRS. Under these circumstances, appropriate treatment is anticonvulsant therapy and monitoring (*i.e*. continuous video-EEG) in order to try to interrupt the process of epileptogenesis.

#### **3.1 Normal brain oscillatory synchronization**

Various types of brain oscillations can be identified during the circadian cycle. A simplification of these types is exemplified in Fig. 1. Among normal function during the circadian cycle, sleep is of great importance and, obviously, sleep scoring or staging is fundamental as a tool in understanding normal and pathological situations. Gottesmann, (1992) described seven sleep-waking stages in the rat: 1 - attentive walking with dorsal hippocampus theta; 2 - quiet waking without theta pattern; 3 - sleep with cortical slow waves of increasing amplitude; 4 - deeper sleep with cortical spindles that progressively increase in number and amplitude; 5 - pre-paradoxal sleep events with high amplitude spindles that occur in parallel with thalamic sensory transmission to cortex; 6 - paradoxical sleep (eye movements are absent); 7 - paradoxical sleep with the characteristic rapid eye movements (REM). Since manual sleep scoring is laborious and time-consuming, several attempts have been made to automate this process. Gross and co-workers (2009) designed a MATLAB toolbox to perform semi-automated sleep scoring. The system is able to distinguish the states of waking, non-REM (NREM), transition-to-REM, and REM sleep if EEG and EMG are recorded simultaneously. Methods describing details for optimal EEG acquisition calibration, electrode application, signal filtering and power spectral analysis for sleep research were described by Campbell (2009).

The search for the substrates of normal brain oscillations and its correlation with cognitive function, neurochemistry and behavioral states has been studied for several decades. Graf & Kastin, (1984) pointed that peptides can play a role, for example, in sleep, EEG and circadian patterns. Neurons that secrete orexins (excitatory neuropeptide hormones) are likely to be very important in promoting wakefulness during the circadian cycle and in controlling the transition to REM sleep. Also, hormones, like estradiol, can decrease sleep and increase locomotion (Mong *et al*., 2003). Among the neuroanatomical areas that play a role on sleep, the locus coeruleus is very important, generating brain states such as alertness. Its activation changes the EEG activity from typical non-alert patterns to alert patterns. The locus coeruleus also has a role in attention processes by changing the sensory responses of neocortical neurons and participating in orienting responses occurring in the forebrain that are closely linked to event-related potentials (Foote *et al*., 1991). EEG studies indicated that the noradrenergic connections from the locus coeruleus excite the upper brain areas, while activation of serotonergic pathways inhibits the same areas. A population of cholinergic neurons can induce and maintain paradoxical sleep and also induce a rapid and transient elevation of alertness (Kayama and Koyama, 1998). In other words projections from the locus coeruleus work as the arousal system. The suprachiasmatic nucleus located in the hypothalamus can also modulate sleep (Dijk and Duffy, 1999). The hypothalamic ventrolateral preoptic area and pons/basal forebrain can play a role on both arousing and sleep-inducing neuronal networks. The mentioned structures could play a role as an ON/OFF switch or transition from sleep to awake state and vice-versa. During sleep, one

Use of Telemetric EEG in Brain Injury 205

reorganization in the human brain, particularly frontal cortex, may contribute to these EEG changes. As this period is crucial, errors in brain plasticity may induce mental illness, such

Several investigators have focused on the study of sleep patterns in different species. Immediately prior to hibernation, REM sleep is not present if temperature is below 25oC and during deep hibernation animals are preferentially in NREM sleep. The hibernation is not homogenous through time and the power of the signal in the delta band is higher after arousal from hibernation and then reduced over time (Canguilhem & Boissin, 1996). Birds are frequently used to investigate auditory processing through the analysis of multiunit electrophysiological responses (Terleph *et al*., 2006), but little is known about the occurrence of sleep in flying birds. Circumstantial evidence of sleep during flight indicates that similar to mammals, birds can exhibit slow-wave and REM sleep. Interestingly, slow wave sleep can occur in one or both hemispheres at a single time and REM sleep occurs only simultaneously in both hemispheres of the brain. Since the eye connected to the "awake" hemisphere remains open, it allows the bird to have navigation information during most of time during a flight (Rattenborg, 2006). In sum, the study of EEG sleep pattern in different

Fig. 1. Cortical electrocorticograms showing baseline electrical activity in Sprague-Dawley rats. The raw EEG (top), Morlet wavelet transform (bottom left), and FFT (bottom right) are being represented during the states of awake alert (exploratory behavior - A), awake non-alert (B resting), REM sleep (C) and non-REM sleep (D)*.* Note the sustained frequency on the theta band (4.1-8.0 Hz) during awake alert (scanning) and REM sleep. The dominant frequencies are

The abnormal changes found in EEG oscillations are highly linked to sleep disturbances, cognitive performance and syndromes like epilepsy. It is very important to keep regular

shifted to the left during awake non-alert and much more during non-REM sleep.

**3.2 Abnormal brain oscillatory synchronization** 

species could one day allow a better understanding of sleep disturbances.

as schizophrenia.

subpopulation of pontine neurons discharges during REM stage exclusively and another subpopulation stops its firing activity during REM (Sinton and McCarley, 2004)*.* Finally, the so-called sleep spindles occur due to cyclical interactions between thalamo-cortical and thalamo-reticular neurons (McCormick and Bal, 1997).

Several authors investigated the relationship between normal oscillations with cognitive function and behavioral states. The importance of "naps" is very well recognized in certain cultures and, indeed, brief periods of sleep (5-15 min) can improve cognitive performance. However, side naps that last longer than 30 min can result in a short period of impairment but produce better cognitive performance over longer periods. Early afternoon naps are most effective and can result in performance improvement revealing that the circadian time within which the nap occurs is very important (Lovato and Lack, 2010). Buzsáki, (1991) elaborated a model of memory trace formation based on neocortical–hippocampal interactions, proposing that during exploratory behavior, information is transmitted from neocortex to hippocampus through fast-firing granule cells projections to a specific population of CA3 pyramidal neurons. In fact, during the acquisition of memories (spatial and episodic), the hippocampus is initially engaged, but later the memory traces are migrated to the neocortex (Ribeiro *et al*., 2007). Indeed, the immediate early genes expression is upregulated during REM sleep in cortical areas but not in the hippocampus (Ribeiro *et al*., 2007). O'Neill and co-workers (2010) investigated the role of the hippocampus in episodic and spatial memories. The hippocampus is able to not only encode this type of memory, but also to consolidate it throughout interactions with the cortex during "reactivation" of the original network firing patterns during sleep and rest. These interactions could be coordinated by sharp wave/ripple events occurring in the hippocampus. There is a close relationship between sleep mechanisms and memory processes. During REM sleep, there is an increase on the transcription of genes linked to plasticity phenomena, allowing the occurrence of both long-term potentiation (LTP) and depotentiation in areas such as the hippocampus. Sleep spindles would be related to plasticity in the cortex, due to specific reactivation of hippocampal and cortical neuronal circuits. Interestingly, when there is a predominance of delta waves, a neuronal reactivation (in phase with delta activity) concomitant with high protein synthesis levels may have a crucial role to play in a longlasting LTP (Poe *et al*, 2010).

Other authors have been investigating the sleep/awake EEG patterns during several types of situations. Miller (1995) studied EEG data acquired from truck drivers during sleep and wake period (driving) with the purpose of creating a database available internationally. Pavy Le-Traon & Roussel (1993) reviewed several studies about sleep during manned space flights and found that the most important disturbances occur because of changes in phase due to tasks that are required during the flight*.* The authors consider that environmental factors, such as microgravity, light-dark cycles and psychological elements, play a role and must be studied. Using an interesting approach to investigate the link between genetics and neurophysiology, Linkowski (1999) studied sleep in twins. Recording EEG during three consecutive nights using a small sample of both monozygotic and dizygotic young male twins, they found out that the twins had a variance in sleep stages that could be genetically determined. However, REM sleep variances apparently did not have a relationship with genetics. Teenagers have peculiar sleep schedules that are likely linked to brain "maturation". According to Feinberg & Campbell, (2010) the power on the delta (1-4 Hz) band declines between ages 11 and 12 years and falls by 65% by age 17 years. Theta power during NREM is reduced earlier. The group hypothesizes that during adolescence, the

subpopulation of pontine neurons discharges during REM stage exclusively and another subpopulation stops its firing activity during REM (Sinton and McCarley, 2004)*.* Finally, the so-called sleep spindles occur due to cyclical interactions between thalamo-cortical and

Several authors investigated the relationship between normal oscillations with cognitive function and behavioral states. The importance of "naps" is very well recognized in certain cultures and, indeed, brief periods of sleep (5-15 min) can improve cognitive performance. However, side naps that last longer than 30 min can result in a short period of impairment but produce better cognitive performance over longer periods. Early afternoon naps are most effective and can result in performance improvement revealing that the circadian time within which the nap occurs is very important (Lovato and Lack, 2010). Buzsáki, (1991) elaborated a model of memory trace formation based on neocortical–hippocampal interactions, proposing that during exploratory behavior, information is transmitted from neocortex to hippocampus through fast-firing granule cells projections to a specific population of CA3 pyramidal neurons. In fact, during the acquisition of memories (spatial and episodic), the hippocampus is initially engaged, but later the memory traces are migrated to the neocortex (Ribeiro *et al*., 2007). Indeed, the immediate early genes expression is upregulated during REM sleep in cortical areas but not in the hippocampus (Ribeiro *et al*., 2007). O'Neill and co-workers (2010) investigated the role of the hippocampus in episodic and spatial memories. The hippocampus is able to not only encode this type of memory, but also to consolidate it throughout interactions with the cortex during "reactivation" of the original network firing patterns during sleep and rest. These interactions could be coordinated by sharp wave/ripple events occurring in the hippocampus. There is a close relationship between sleep mechanisms and memory processes. During REM sleep, there is an increase on the transcription of genes linked to plasticity phenomena, allowing the occurrence of both long-term potentiation (LTP) and depotentiation in areas such as the hippocampus. Sleep spindles would be related to plasticity in the cortex, due to specific reactivation of hippocampal and cortical neuronal circuits. Interestingly, when there is a predominance of delta waves, a neuronal reactivation (in phase with delta activity) concomitant with high protein synthesis levels may have a crucial role to play in a long-

Other authors have been investigating the sleep/awake EEG patterns during several types of situations. Miller (1995) studied EEG data acquired from truck drivers during sleep and wake period (driving) with the purpose of creating a database available internationally. Pavy Le-Traon & Roussel (1993) reviewed several studies about sleep during manned space flights and found that the most important disturbances occur because of changes in phase due to tasks that are required during the flight*.* The authors consider that environmental factors, such as microgravity, light-dark cycles and psychological elements, play a role and must be studied. Using an interesting approach to investigate the link between genetics and neurophysiology, Linkowski (1999) studied sleep in twins. Recording EEG during three consecutive nights using a small sample of both monozygotic and dizygotic young male twins, they found out that the twins had a variance in sleep stages that could be genetically determined. However, REM sleep variances apparently did not have a relationship with genetics. Teenagers have peculiar sleep schedules that are likely linked to brain "maturation". According to Feinberg & Campbell, (2010) the power on the delta (1-4 Hz) band declines between ages 11 and 12 years and falls by 65% by age 17 years. Theta power during NREM is reduced earlier. The group hypothesizes that during adolescence, the

thalamo-reticular neurons (McCormick and Bal, 1997).

lasting LTP (Poe *et al*, 2010).

reorganization in the human brain, particularly frontal cortex, may contribute to these EEG changes. As this period is crucial, errors in brain plasticity may induce mental illness, such as schizophrenia.

Several investigators have focused on the study of sleep patterns in different species. Immediately prior to hibernation, REM sleep is not present if temperature is below 25oC and during deep hibernation animals are preferentially in NREM sleep. The hibernation is not homogenous through time and the power of the signal in the delta band is higher after arousal from hibernation and then reduced over time (Canguilhem & Boissin, 1996). Birds are frequently used to investigate auditory processing through the analysis of multiunit electrophysiological responses (Terleph *et al*., 2006), but little is known about the occurrence of sleep in flying birds. Circumstantial evidence of sleep during flight indicates that similar to mammals, birds can exhibit slow-wave and REM sleep. Interestingly, slow wave sleep can occur in one or both hemispheres at a single time and REM sleep occurs only simultaneously in both hemispheres of the brain. Since the eye connected to the "awake" hemisphere remains open, it allows the bird to have navigation information during most of time during a flight (Rattenborg, 2006). In sum, the study of EEG sleep pattern in different species could one day allow a better understanding of sleep disturbances.

Fig. 1. Cortical electrocorticograms showing baseline electrical activity in Sprague-Dawley rats. The raw EEG (top), Morlet wavelet transform (bottom left), and FFT (bottom right) are being represented during the states of awake alert (exploratory behavior - A), awake non-alert (B resting), REM sleep (C) and non-REM sleep (D)*.* Note the sustained frequency on the theta band (4.1-8.0 Hz) during awake alert (scanning) and REM sleep. The dominant frequencies are shifted to the left during awake non-alert and much more during non-REM sleep.

#### **3.2 Abnormal brain oscillatory synchronization**

The abnormal changes found in EEG oscillations are highly linked to sleep disturbances, cognitive performance and syndromes like epilepsy. It is very important to keep regular

Use of Telemetric EEG in Brain Injury 207

Although prolonged seizures lasting 30 min or more are characterized as SE (Sloviter, 1999), recently, Chen and Wasterlain (2006) proposed the term ''impending status epilepticus'' for seizures that last at least 5 min, pointing out such seizures should be treated immediately. The use of animal models of SE is an excellent tool to study SE and its consequences. Approaches such as telemetry have greatly reduced the number of animals used and greatly refined such studies. Models such as seizures induced by systemic and intra-hippocampal pilocarpine (Turski *et al*., 1983a; Cavalheiro *et al*., 1991; Furtado *et al*., 2002; Furtado *et al*., 2011; Castro *et al*., 2011), soman (McDonough *et al*., 1986; Carpentier *et al*., 1990; Petras, 1994; Shih and McDonough, 1997; Myhrer, 2007; de Araujo Furtado *et al*., 2010; Figueiredo *et al*., 2011), kainic acid (Ben-Ari *et al*., 1979; Williams *et al*., 2007) and electrical stimulation of the amygdala (Nissinen *et al*., 2000) have brought answers to fundamental questions about the mechanisms of seizures and treatment options. SRS was found in animals experiencing SE induced by pilocarpine (Leite *et al*., 1990; Mello *et al*., 1993) and kainic acid (Pisa *et al*., 1980; Cronin and Dudek, 1988; Hellier and Dudek, 1999) after a latent period. Also, self-sustaining SE and SRS can be provoked by uninterrupted electrical hippocampal stimulation (Lothman *et al*., 1989; 1990), perforant path stimulation (Mazarati *et al*., 1998; Mazarati *et al*., 2002) and electrical stimulation of the amygdala (Nissinen *et al*., 2000). Brain damage caused by OP (such as soman) can also lead to SRS. There are reports in the literature implying the occurrence of recurrent seizures in rats (McDonough *et al*., 1986b) and a full characterization of soman-induced SRS is described by de Araujo Furtado *et al*., (2010) using long-term EEG

Regardless of the fact that the discussion continues as to which brain changes lead to SRS, the occurrence of an initial insult may likely induce SRS (Sloviter, 1999). However, recent reports have shown that subjects that are challenged to a convulsive stimulus, but do not display SE, still have a probability of developing SRS much later (Navarro Mora *et al*., 2009; Pernot *et al*., 2009) suggesting that long-term video-EEG monitoring may be necessary in

It is important to recognize that different patterns of seizures can be present after the brain receives a mechanical, electrical or chemical challenge. Although it is very complex, several seizure patterns have been found during the SE (Treiman *et al*., 1990). Fig. 2 shows characteristic EEG during SE and a summary of recurrent seizure patterns and SRS are

Fig. 2. Representative cortical electrocorticograms showing electrical activity during

different periods after soman exposure. (A) 13 min after exposure. (B) 33 min after exposure. SE can last for several hrs and, even after treatment, recurrent seizures may occur (see next

recording through telemetry.

presented in the next section.

section).

most studies in order to truly study epileptogenesis.

sleep periods and a reduction of as little as 1.3 hrs may result in reductions in alertness (Bonnet and Arand, 1995). According to Newmark & Clayton, (1995), headaches and sleep problems are probably overlooked during medical evaluations during active duty. Sleep disturbances can be associated with depression (Vanbemmel, 1997) and interestingly, sleep deprivation can function as an antidepressant treatment in 40-60% of patients that suffer from depression (Hemmeter *et al*., 2010). Although the mechanisms are still unclear, this phenomenon may help on the development of new antidepressants.

Among all situations that cause alterations of brain oscillatory patterns, brain damage is the most critical, leading to seizures and sleep disturbances. Shouse, da Silva, & Sammaritano (1996) pinpointed that seizures and inter-ictal events have circadian distribution, indicating that some arousal and sleep states are seizure-prone, while others are seizure resistant, both modulating seizure occurrence. Kotagal & Yardi (2008) pointed out that seizures during the sleep state are reported in approximately one third of epileptic patients. Both normal sleep pattern and sleep deprivation modulate the frequency of epileptiform discharges observed in the EEG and behavioral seizures do occur more frequently during NREM sleep.

Brain damage can be caused mechanically, chemically or even influenced by genetic factors. Blast is currently the major cause of battlefield injuries and death. Blast overpressure waves affect organs such the brain, auditory system, the gastrointestinal tract, and predominantly the lungs (Wightman and Gladish, 2001; DePalma *et al*, 2005; Garner and Brett, 2007; and Long *et al*, 2009). Unfortunately, there are no currently approved neuroprotective agents for use in ischemic stroke or traumatic brain injury. Recently, Vespa and co-workers (2010) showed that TBI can lead to electrographic SE, a state in which prolonged and uninterrupted seizures occur without recovery, for a period of 30 min or more. The identification of SE is essential in avoiding the development of epilepsy. Seizures are clinical manifestations of hypersynchronous and hyperexcitatory neuronal activity in a given neuronal network and can lead to brain damage and further "rewiring" that causes a chronic epileptic state, characterized by SRS (Shorvon, 2000). It is known that patients that suffer TBI may, at some point, develop SRS and latency to SRS is dependent on the degree of damage (Salazar *et al*., 1995; Chen *et al*., 2009; Lowenstein, 2009). It is very important to distinguish EEG traces characteristic of each state from seizures and seizure-like events. The clear identification of electrographic SE is essential to interfere and attempt to avoid the development of epilepsy.

Exposure to certain compounds can also induce SE and lead to brain damage. Exposure to organophosphorus agents (OP) can cause signs of seizures such as myoclonic movements, respiratory distress, and death (Engel, 1993; McDonough & Shih, 1997). OP compounds inhibit the enzyme acetylcholinesterase that normally degrades the neurotransmitter acetylcholine. When acetylcholinesterase is inhibited, the result is a cholinergic hyperactivation in brain areas such as piriform cortex and the medial septal area leading to increased glutamatergic drive in the piriform, entorhinal, and perirhinal cortices and the hippocampus, causing the expression of motor seizures and SE (Myhrer, 2007). This excessive glutamatergic drive can cause neuroexcitotoxicity (Wasterlain and Shirasaka, 1994). The overactivation of N-methyl-D-aspartate (NMDA; a type of glutamatergic receptor) immediately induces an influx of Ca2+, leading to a series of molecular events that ultimately cause cell death (Delorenzo *et al*., 2005). As one of the results of brain damage caused by SE, certain brain areas display neuroplastic changes (like axonal sprouting) in neuronal circuitry. The axonal sprouting in the hippocampus is hypothesized in the literature as one of the causes of epilepsy (Mello *et al*., 1993; Okazaki *et al*., 1995).

sleep periods and a reduction of as little as 1.3 hrs may result in reductions in alertness (Bonnet and Arand, 1995). According to Newmark & Clayton, (1995), headaches and sleep problems are probably overlooked during medical evaluations during active duty. Sleep disturbances can be associated with depression (Vanbemmel, 1997) and interestingly, sleep deprivation can function as an antidepressant treatment in 40-60% of patients that suffer from depression (Hemmeter *et al*., 2010). Although the mechanisms are still unclear, this

Among all situations that cause alterations of brain oscillatory patterns, brain damage is the most critical, leading to seizures and sleep disturbances. Shouse, da Silva, & Sammaritano (1996) pinpointed that seizures and inter-ictal events have circadian distribution, indicating that some arousal and sleep states are seizure-prone, while others are seizure resistant, both modulating seizure occurrence. Kotagal & Yardi (2008) pointed out that seizures during the sleep state are reported in approximately one third of epileptic patients. Both normal sleep pattern and sleep deprivation modulate the frequency of epileptiform discharges observed

Brain damage can be caused mechanically, chemically or even influenced by genetic factors. Blast is currently the major cause of battlefield injuries and death. Blast overpressure waves affect organs such the brain, auditory system, the gastrointestinal tract, and predominantly the lungs (Wightman and Gladish, 2001; DePalma *et al*, 2005; Garner and Brett, 2007; and Long *et al*, 2009). Unfortunately, there are no currently approved neuroprotective agents for use in ischemic stroke or traumatic brain injury. Recently, Vespa and co-workers (2010) showed that TBI can lead to electrographic SE, a state in which prolonged and uninterrupted seizures occur without recovery, for a period of 30 min or more. The identification of SE is essential in avoiding the development of epilepsy. Seizures are clinical manifestations of hypersynchronous and hyperexcitatory neuronal activity in a given neuronal network and can lead to brain damage and further "rewiring" that causes a chronic epileptic state, characterized by SRS (Shorvon, 2000). It is known that patients that suffer TBI may, at some point, develop SRS and latency to SRS is dependent on the degree of damage (Salazar *et al*., 1995; Chen *et al*., 2009; Lowenstein, 2009). It is very important to distinguish EEG traces characteristic of each state from seizures and seizure-like events. The clear identification of electrographic SE is essential to interfere and attempt to avoid the

Exposure to certain compounds can also induce SE and lead to brain damage. Exposure to organophosphorus agents (OP) can cause signs of seizures such as myoclonic movements, respiratory distress, and death (Engel, 1993; McDonough & Shih, 1997). OP compounds inhibit the enzyme acetylcholinesterase that normally degrades the neurotransmitter acetylcholine. When acetylcholinesterase is inhibited, the result is a cholinergic hyperactivation in brain areas such as piriform cortex and the medial septal area leading to increased glutamatergic drive in the piriform, entorhinal, and perirhinal cortices and the hippocampus, causing the expression of motor seizures and SE (Myhrer, 2007). This excessive glutamatergic drive can cause neuroexcitotoxicity (Wasterlain and Shirasaka, 1994). The overactivation of N-methyl-D-aspartate (NMDA; a type of glutamatergic receptor) immediately induces an influx of Ca2+, leading to a series of molecular events that ultimately cause cell death (Delorenzo *et al*., 2005). As one of the results of brain damage caused by SE, certain brain areas display neuroplastic changes (like axonal sprouting) in neuronal circuitry. The axonal sprouting in the hippocampus is hypothesized in the

literature as one of the causes of epilepsy (Mello *et al*., 1993; Okazaki *et al*., 1995).

in the EEG and behavioral seizures do occur more frequently during NREM sleep.

phenomenon may help on the development of new antidepressants.

development of epilepsy.

Although prolonged seizures lasting 30 min or more are characterized as SE (Sloviter, 1999), recently, Chen and Wasterlain (2006) proposed the term ''impending status epilepticus'' for seizures that last at least 5 min, pointing out such seizures should be treated immediately.

The use of animal models of SE is an excellent tool to study SE and its consequences. Approaches such as telemetry have greatly reduced the number of animals used and greatly refined such studies. Models such as seizures induced by systemic and intra-hippocampal pilocarpine (Turski *et al*., 1983a; Cavalheiro *et al*., 1991; Furtado *et al*., 2002; Furtado *et al*., 2011; Castro *et al*., 2011), soman (McDonough *et al*., 1986; Carpentier *et al*., 1990; Petras, 1994; Shih and McDonough, 1997; Myhrer, 2007; de Araujo Furtado *et al*., 2010; Figueiredo *et al*., 2011), kainic acid (Ben-Ari *et al*., 1979; Williams *et al*., 2007) and electrical stimulation of the amygdala (Nissinen *et al*., 2000) have brought answers to fundamental questions about the mechanisms of seizures and treatment options. SRS was found in animals experiencing SE induced by pilocarpine (Leite *et al*., 1990; Mello *et al*., 1993) and kainic acid (Pisa *et al*., 1980; Cronin and Dudek, 1988; Hellier and Dudek, 1999) after a latent period. Also, self-sustaining SE and SRS can be provoked by uninterrupted electrical hippocampal stimulation (Lothman *et al*., 1989; 1990), perforant path stimulation (Mazarati *et al*., 1998; Mazarati *et al*., 2002) and electrical stimulation of the amygdala (Nissinen *et al*., 2000). Brain damage caused by OP (such as soman) can also lead to SRS. There are reports in the literature implying the occurrence of recurrent seizures in rats (McDonough *et al*., 1986b) and a full characterization of soman-induced SRS is described by de Araujo Furtado *et al*., (2010) using long-term EEG recording through telemetry.

Regardless of the fact that the discussion continues as to which brain changes lead to SRS, the occurrence of an initial insult may likely induce SRS (Sloviter, 1999). However, recent reports have shown that subjects that are challenged to a convulsive stimulus, but do not display SE, still have a probability of developing SRS much later (Navarro Mora *et al*., 2009; Pernot *et al*., 2009) suggesting that long-term video-EEG monitoring may be necessary in most studies in order to truly study epileptogenesis.

It is important to recognize that different patterns of seizures can be present after the brain receives a mechanical, electrical or chemical challenge. Although it is very complex, several seizure patterns have been found during the SE (Treiman *et al*., 1990). Fig. 2 shows characteristic EEG during SE and a summary of recurrent seizure patterns and SRS are presented in the next section.

Fig. 2. Representative cortical electrocorticograms showing electrical activity during different periods after soman exposure. (A) 13 min after exposure. (B) 33 min after exposure. SE can last for several hrs and, even after treatment, recurrent seizures may occur (see next section).

Use of Telemetric EEG in Brain Injury 209

there also appeared to be sustained oscillation in the alpha band (8.1 to 12 Hz) but with reduced power spectrum. Dominant frequencies of the delta band (0.1 to 4 Hz) also appeared mainly at the beginning of seizures, but were not sustained in the time (Fig. 4).

Fig. 4. Output of a representative SRS (60 sec.) wavelet transform spectral analysis. A – EEG (amplitude x time) of SRS; B – Frequency x time analyses of SRS; C- Intensity of frequencies

The use of telemetry to capture continuous recordings has the advantage of allowing the detection of SRS and long term changes in circadian brain oscillations. However, telemetry results in a large accumulation of data. A large volume of data can result in analysis delay, frustration and poor EEG interpretation. Unique tools capable of performing efficient spectral analyses (Rossetti *et al*., 2006; Romcy-Pereira *et al*., 2008; Lehmkuhle *et al*., 2009), seizure estimation, and spike detection (Saab and Gotman, 2005; White *et al*., 2006; Casson *et al*., 2007; Jacquin *et al*., 2007; Hopfengärtner *et al*., 2007) has been used in several studies on epilepsy. Artificial neural networks have proven to be the most reliable tool (Gabor *et al*., 1996; Gabor, 1998; Nigam and Graupe, 2004; Kiymik *et al*., 2004; Tzallas *et al*., 2007; Srinivasan *et al*., 2007; Patnaik and Manyam, 2008) but require tremendous computational power in order to be time effective when analyzing large data sets. Another alternative is the use of commercial software designed for seizure detection. However, most often, this type of software is "tuned" to specific parameters for human subjects, such as sleep stages and spike and wave activity. In some cases, these parameters must be changed between

**4. Assessment of the long-term EEG changes** 

analysis.

#### **3.2.1 Recurrent seizures**

After the termination of SE, there is normally a period without seizures that can last from minutes to hours. Subsequently, subjects may display recurrent seizures that can induce additional brain damage. These seizures come however in different patterns, the type 1 pattern (Fig. 3A) is characterized by low frequencies between 0.8 and 1.4 Hz (delta band), with high amplitude spikes. The type 2 pattern also oscillated in the delta band, but faster than type 1, between 1.4 and 3.7. This pattern is characterized by high and low amplitude spikes (Fig. 3B)*.* The type 3 pattern has frequencies oscillating in the theta band, between 4.8 and 5.4 Hz, with low spikes (Fig. 3C). The type 4 pattern is characterized by no spikes, but oscillates also in theta band, faster than type 3, between 5.5 and 6.5 (Fig. 3D). Long-term video-EEG monitoring may be necessary in most studies in order to detect epileptogenesis.

Fig. 3. Electrographic seizures patterns (10 sec) calculated and illustrated in wavelet transform analyses. A – Type 1 Pattern; B – Type 2 Pattern; C – Type 3 Pattern; D – Type 4 Pattern. The first fig (at the top) of each pattern shows the EEG (Amplitude x Time); the second fig (down left) of each pattern shows the frequencies in exact time (Frequency x Time); the third fig (down right) of each pattern shows the power of frequency (Intensity x Frequency).

#### **3.2.2 Spontaneous recurrent seizures**

Electrographic SRS are characterized by frequencies oscillating in the theta band (4.1 to 8 Hz) and are sustained during most of the duration of the seizure. From 25 sec up to 45 sec,

After the termination of SE, there is normally a period without seizures that can last from minutes to hours. Subsequently, subjects may display recurrent seizures that can induce additional brain damage. These seizures come however in different patterns, the type 1 pattern (Fig. 3A) is characterized by low frequencies between 0.8 and 1.4 Hz (delta band), with high amplitude spikes. The type 2 pattern also oscillated in the delta band, but faster than type 1, between 1.4 and 3.7. This pattern is characterized by high and low amplitude spikes (Fig. 3B)*.* The type 3 pattern has frequencies oscillating in the theta band, between 4.8 and 5.4 Hz, with low spikes (Fig. 3C). The type 4 pattern is characterized by no spikes, but oscillates also in theta band, faster than type 3, between 5.5 and 6.5 (Fig. 3D). Long-term video-EEG monitoring may be necessary in most studies in order to detect epileptogenesis.

Fig. 3. Electrographic seizures patterns (10 sec) calculated and illustrated in wavelet transform analyses. A – Type 1 Pattern; B – Type 2 Pattern; C – Type 3 Pattern; D – Type 4 Pattern. The first fig (at the top) of each pattern shows the EEG (Amplitude x Time); the second fig (down left) of each pattern shows the frequencies in exact time (Frequency x Time); the third fig (down right) of each pattern shows the power of frequency (Intensity x Frequency).

Electrographic SRS are characterized by frequencies oscillating in the theta band (4.1 to 8 Hz) and are sustained during most of the duration of the seizure. From 25 sec up to 45 sec,

**3.2.2 Spontaneous recurrent seizures** 

**3.2.1 Recurrent seizures** 

there also appeared to be sustained oscillation in the alpha band (8.1 to 12 Hz) but with reduced power spectrum. Dominant frequencies of the delta band (0.1 to 4 Hz) also appeared mainly at the beginning of seizures, but were not sustained in the time (Fig. 4).

Fig. 4. Output of a representative SRS (60 sec.) wavelet transform spectral analysis. A – EEG (amplitude x time) of SRS; B – Frequency x time analyses of SRS; C- Intensity of frequencies analysis.
