The application of mechanical force on the brain initiates a complex series of interacting (sometimes non-monotonic) biochemical cascades, which, along with the initial impact, characterize TBI pathophysiology. Disruption of the cell membrane can lead to an ionic disturbance of Na+, K+, Ca2+, Mg2+, and Zn2+ (Vink et al., 1988; Katayama et al., 1990; Soares et al., 1992; Nilsson et al., 1993; Smith et al., 1993). The high concentration of K+ and Ca2+ in the extracellular space triggers release of neurotransmitters (e.g., glutamate), which can further exacerbate the ionic disturbance creating a vicious cycle (Faden et al., 1989; Katayama et al., 1990; Nilsson et al., 1990; Lyeth et al., 1993; Reeves et al., 1997; Shin and Dixon, 2015). This wave of depolarization can lead to excitotoxic cell death beyond what is observed in the injury core and surrounding penumbra (Sullivan et al., 1976; Dixon et al., 1987; Lowenstein et al., 1992; Hicks et al., 1993; Yamaguchi et al., 1996; Leonard et al., 1997; Yakovlev et al., 1997; Floyd et al., 2002; Witgen et al., 2005; Fedor et al., 2010). Moreover, there is considerable evidence that the pathophysiological release of neurotransmitters can alter the function of glutamatergic (Faden et al., 1989; Miller et al., 1990; Smith et al., 1993; Schwarzbach et al., 2006), cholinergic (Robinson et al., 1990; Yamamoto et al., 1993; Jiang et al., 1994; Lyeth et al., 1994; Delahunty et al., 1995; Shin and Dixon, 2015), GABAergic (Reeves et al., 1997; Witgen et al., 2005; Bonislawski et al., 2007; Gupta et al., 2012), and dopaminergic receptor systems (Donnemiller et al., 2000; Massucci et al., 2004; Shin et al., 2011), resulting in potential long-term cellular and circuit dysfunction independent of cell death.

One specific change related to excessive activation of the nervous system following TBI is the accumulation of intracellular calcium and the subsequent activation of calcium dependent catalytic enzymes such as calpain (Kampfl et al., 1997; Khorchid and Ikura, 2002). While hyperactivation of calpains is commonly associated with apoptosis (Patel et al., 1996), calpains also advance cytoskeletal and plasma membrane breakdown as well as disruption of Na+ channel function (Hicks et al., 1995; Folkerts et al., 1998; Saatman et al., 1998; Johnson et al., 2013). Changes in the cytoskeleton and membrane can trigger further ionic imbalance and specifically lead to high intraaxonal Ca2+ levels, which further challenge the already damaged axons resulting from the primary injury (Graham et al., 2000; Kita et al., 2000; Baker et al., 2002; Johnson et al., 2013; Li et al., 2014).  In addition, TBI results in deficits impacting certain forms of behavioral plasticity (Ip et al., 2002; Griesbach et al., 2004) and formation of long term memories (Rimel et al., 1981; Leininger et al., 1990; Fedor et al., 2010; Gurkoff et al., 2013; Zhang et al., 2015). Disruption to cognition and plasticity following TBI is of particular relevance to our research interests and will be the focus of this review. Specific emphasis will be placed on how oscillatory activity contributes to information processing and how modifying injury- perturbed EEG could be relevant to reversing deficits in the clinical population. To this end we will first elaborate on what local field oscillations are and how they are generated in the brain.

#### DEFINING AN OSCILLATION

There is both growing evidence and excitement that neuromodulation, and specifically invasive electrical neurostimulation, can be used to improve function in patients with neurological disorders (Lozano and Lipsman, 2013; Suthana and Fried, 2014; Tekriwal and Baltuch, 2015). In the case of TBI it is clear that functional consequences can be severe and persist for many years after the insult (Jennett et al., 1981; Whiteneck et al., 2004; DeKosky et al., 2010; Ponsford et al., 2014). And at least some of these cognitive and behavioral deficits could be mitigated with neurostimulation (Buzsáki and Watson, 2012; Lozano and Lipsman, 2013; Shin et al., 2014). Although the precise mechanism is still being delineated, it is hypothesized that driving specific neural circuits can entrain physiological circuit activity ultimately improving behavioral outcomes. Stemming from this, based on our recent findings we hypothesize that TBI-induced alterations in neural connectivity result in altered oscillations, as observed in the EEG. Further, we hypothesize that stimulating the injured nervous system to restore or substitute these oscillations will improve outcome. However, essential to the implementation and assessment of any intervention is the knowledge of the underlying mechanisms involved. Therefore, the next sections will summarize the basic neurophysiology associated with brain oscillations observed in the EEG and how these oscillations contribute to neural function. Critically this overview will introduce concepts from the perspective of developing research strategies to determine whether electrical neurostimulation can be used to improve cognitive outcome in TBI patients.

EEG is the measurement of change in the extracellular field potential recorded from the scalp that is generated by the sum of ionic movements across synapses, dendrites, soma, axons and the electroconductive cerebral spinal fluid. Similar activity measured from intracranial electrodes is commonly referred to as intracranial EEG (iEEG) or electrocorticography (ECoG). For simplicity, we will refer to all recordings, scalp and intracranial, as EEG for the remainder of this manuscript. The average of ionic movements within the immediate surrounding volume of an implanted electrode is referred to as the local field potential (LFP). There are many cellular actions that sum together to contribute to the total change in the ionic balance measured by a depth electrode, such as synaptic activity, Ca2+ fluctuations, intrinsic currents and resonances, spike after-hyperpolarization, gap junctions and glial interaction (Berridge and Rapp, 1979; Buzsáki et al., 2012). The magnitude of the electric field detected in the EEG is related to the alignment of the electrode relative to the processes of cells in any given region (Buzsáki et al., 1986; Montgomery et al., 2009). Specifically, an electrode placed parallel to the dipole created by ionic movements will result in the highest amplitude recording (Kringelbach et al., 2007).

A synchronized and reoccurring change in ionic movements results in an oscillation that can be observed in the EEG. Oscillations may arise due to a variety of mechanisms, such alternating excitation-inhibition (or excitation-excitation or inhibition-inhibition) of neurons, pacemaker cells, resonance or subthreshold membrane oscillation (James et al., 1977; Buzsáki et al., 1983; McCormick and Bal, 1997; Marshall et al., 2002; Klausberger et al., 2003; Wang, 2010). There are multiple discrete oscillatory bands ranging from 0.05 to 500 Hz that have been operationally defined based on functional states of the brain (Klausberger et al., 2003; Penttonen and Buzsáki, 2003; Buzsáki and Watson, 2012). While the general structure of many oscillations is similar (e.g., alternating excitation-inhibition, pacemaker cell), granularly each rhythm is quite distinct from one other. How, when, and where an oscillation is generated defines its operation and contribution to information processing, and in the case of a brain injury, the pathophysiology of a disorder. Therefore, in order to understand how TBI might affect the generation or maintenance of oscillations, and how to develop and assess potential strategies to restore oscillations, it is critical to consider how mechanistically an oscillation is generated.

## UNDERSTANDING HOW OSCILLATIONS ARE GENERATED

One of the earliest and most studied examples of oscillations observed in the EEG is from studies of sleep progression. For example, a defining characteristic of early non-REM (NREM) sleep is the presence of spindle waves, which are 1–3 s bursts of activity in the 7–14 Hz range every 3–10 s (Brown et al., 2012). To describe spindle generation it is important to consider both which brain regions as well as which specific cellular mechanisms are responsible for generating rhythmicity. Spindles arise due to the thalamic reticular nucleus (TRN) hyperpolarizing thalamocortical neurons with a rhythmic burst of inhibitory synaptic potentials (IPSPs; Avanzini et al., 1989; Bal et al., 1995a,b). This hyperpolarization leads to the activation of low-threshold T-type Ca2+ channels (IT), which even at low, negative membrane potentials can generate an action potential. Subsequently thalamocortical neurons generate a burst of excitatory synaptic potentials (EPSPs) that activate the TRN as well as corticothalamic neurons giving rise to a spindle (Crunelli et al., 1989; Bal et al., 1995a,b). Convergence of excitatory input onto TRN activates low-threshold Ca2+ channels, which send prolonged IPSPs back to thalamocortical neurons starting the oscillatory cycle anew (Steriade and Deschenes, 1984; Avanzini et al., 1989; McCormick and Bal, 1997). Thus, the time to go through one full cycle prescribes the observed frequency of a spindle (Bal et al., 1995a).

Thalamocortical bursting activity gives rise to another dominant NREM sleep oscillation in the delta frequency band (0.5–4 Hz; McCormick and Bal, 1997; Brown et al., 2012). Unlike the spindle waves, delta oscillations are generated in a single cell by the interplay between ionic currents (Steriade et al., 1993b). Low-threshold Ca2+ bursting in thalamocortical neurons is followed by a hyperpolarizing overshoot. This de-inactivates IT and opens the hyperpolarization-activated cation channel causing an h-current (Ih). Ih slowly depolarizes the cell towards the threshold for a Ca2+ spike by activating IT. Depolarization past −65 mV and subsequently −35 mV inactivates IT and deactivates Ih, respectively, and leads to an action potential (Crunelli et al., 1989; McCormick and Bal, 1997). Repolarization overshoots start the cycle again. However, it should be noted that other mechanisms have been proposed to account for the thalamocortical delta oscillation (Ball et al., 1977; Steriade et al., 1993a).

Specific to our understanding of oscillations during sleep it is easy to imagine how the precise activity of a series of receptor systems and the related interaction of ionic currents would be sensitive to the large ionic imbalance that follows TBI (Vink et al., 1988; Katayama et al., 1990; Soares et al., 1992; Nilsson et al., 1993; Smith et al., 1993). Consistent with this assertion, TBI is associated with sleep disturbances (Mathias and Alvaro, 2012) and specifically a decrease in delta power during NREM sleep for at least 12 weeks post injury (Parsons et al., 1997). Therefore, when considering how TBI alters oscillations and the potential for neurostimulation one has to determine not only which circuits and specific mechanisms are affected, but also when one needs to stimulate.

Our primary interest related to TBI and EEG is how injury may alter hippocampal oscillations and cognitive function. This interest is driven by a rich history in TBI-induced spatial learning deficits, deficits that we now know are concurrent with altered hippocampal oscillations (Fedor et al., 2010; Lee et al., 2013, 2015). Unlike the previously described oscillations, hippocampal theta (3–12 Hz), and specifically in CA1 is generated and maintained by the interaction of multiple rhythm generators as well as intrinsic membrane properties of hippocampal neurons that contribute to the detected rhythmic slow wave (Green and Arduini, 1954; Vanderwolf, 1969; Buzsáki et al., 1986; Kirk, 1998; Kocsis et al., 1999; Mormann et al., 2008; Montgomery et al., 2009; Colgin, 2013; Watrous et al., 2013). In the hippocampal CA1 subfield there are two well characterized dipoles of theta: the distal dendrites and soma (**Figure 1**). The first dipole, measured strongest near the hippocampal fissure, is attributed to layer 3 entorhinal cortex (EC) and CA3 subfield rhythmic excitation of distal dendrites of CA1 (Bland, 1986; Alonso and García-Austt, 1987; Konopacki et al., 1987; Kamondi et al., 1998; Kocsis et al., 1999). This dendritic depolarization co-occurs

connections. Projections from the MSN are left out for clarity and are represented by halos (circular on interneurons, linear on CA1 pyramidal cell).

with somatic hyperpolarization, which reflects inputs from the medial septum nucleus (MSN; Green and Arduini, 1954; Petsche et al., 1962; James et al., 1977; Bland, 1986; Vertes et al., 2004). The MSN consists of three types of neurons: GABAergic, cholinergic and glutamatergic. In fact, afferents from each of these neuronal subtypes play a role in the generation of the second dipole. Specifically, the interplay of phasic GABAergic inhibition, tonic cholinergic and glutamatergic excitation of hippocampal interneurons results in CA1 theta (Cole and Nicoll, 1984; Smythe et al., 1992; Tóth et al., 1997; Apartis et al., 1998; Wang, 2002; Hajszan et al., 2004; Colom et al., 2005; Vandecasteele et al., 2014; Fuhrmann et al., 2015). The septal GABAergic cells act as pacemakers of theta generation in CA1 pyramidal cells through disinhibiting hippocampal interneurons (Freund and Antal, 1988; Ylinen et al., 1995; Wang, 2002). In addition, MSN cholinergic and glutamatergic neurons directly modulate excitability in CA1 pyramidal cells, which in turn excite back projecting hippocamposeptal interneurons completing the septohippocampal loop (**Figure 1**; Gaykema et al., 1991; Tóth and Freund, 1992; Tóth et al., 1993; Manseau et al., 2008; Mattis et al., 2014; Sun et al., 2014). This interplay between septohippocampal interneurons has the added effect of disinhibiting and inhibiting the soma of CA1 pyramidal neurons at the theta frequency, which can be measured at or dorsal to the pyramidal layer. Interestingly, hippocampal interneurons are vulnerable to cell death after TBI (Tóth et al., 1997b; Almeida-Suhett et al., 2015; Huusko et al., 2015). In addition there is evidence that injury can alter function in these neurons (O'Dell et al., 2000; Ross and Soltesz, 2000; Mtchedlishvili et al., 2010; Gupta et al., 2012; Almeida-Suhett et al., 2015; Drexel et al., 2015). Any change in interneuronal number or function could contribute to changes seen in the theta band post injury. While it is well accepted that TBI can result in cell death and dysfunction in interneurons in general, in order to get a better understanding of the hippocampal pathophysiology it will be important for future studies to examine which specific classes of interneurons (**Figure 1**, e.g., O-LM, PV basket, axo-axonic) that contribute to CA1 theta generation are also affected by TBI (Klausberger et al., 2003).

These theta rhythm generators also work in concert with the intrinsic properties of hippocampal neurons. Specifically, there are intrinsic resonant and subthreshold membrane oscillating events which contribute to the hippocampal oscillations. For example, CA1 pyramidal cells have resonance (preferred frequency for maximal response) at theta frequency due to the interplay between voltage gated ionic currents. Depolarizations activate IM (K+ current), which has the effect of hyperpolarizing the cell. Hyperpolarization activates Ih (mix Na+/K+ current), which brings the potential closer to spike threshold. With the addition of a persistent Na+ current (INAP) these currents oppose each other resulting in a membrane resonance in the theta frequency (Pike et al., 2000; Hu et al., 2002). This CA1 rhythmicity is further amplified with subthreshold membrane oscillations via persistent Na+ and Ca2+ currents (Leung and Yim, 1991; García-Muñoz et al., 1993; Fransén et al., 2004). In a similar vein, both EC cells projecting to the hippocampus (Alonso and Llinas, 1989; Alonso and Klink, 1993; Klink and Alonso, 1993; Dickson et al., 2000; Quilichini et al., 2010) and hippocampal inhibitory interneurons (Maccaferri and McBain, 1996; Chapman and Lacaille, 1999; Pike et al., 2000) have a natural resonance and subthreshold membrane oscillation in the theta frequency range due to a mix of voltage-sensitive Na+ and K + currents. Furthermore, the MSN displays intrinsic bursting in the theta range (Vinogradova et al., 1980; Zhadina and Vinogradova, 1983). Thus, the magnitude of synaptically driven theta from the generators in the EC and septum is boosted by multiple intrinsic resonances from cells within the hippocampus as well as extrinsic to the hippocampal formation (Goutagny et al., 2009).

In summary, there are bands of oscillations starting as low as <0.1 Hz and ranging to as high as 600 Hz. Over the years we have operationally defined discrete ranges of oscillations (e.g., theta and gamma) based on specific cellular mechanisms as they relate to observed behavioral relationships. Research into individual oscillatory bands has revealed that mechanisms for the generation and maintenance of oscillations are complex and varied, with interactions of synaptic and intrinsic generators summing together to provide a single detected change in the LFP. However, it remains an open question to what extent TBI alters any, or perhaps all, of the specific mechanisms involved in the generation of individual oscillatory bands and ultimately the neural network underlying cognition.

#### HOW OSCILLATIONS INTERACT AND CONTRIBUTE TO INFORMATION PROCESSING

While each electrode yields a single LFP measure, that LFP is made up of several components. In fact, the combination

of synaptic and intrinsic membrane events frequently leads to power in multiple oscillatory bands. Specifically, when one decomposes an individual LFP one can see that each oscillation occurs simultaneously (**Figure 2**). **Figure 2** illustrates sinusoidal waves whose frequency corresponds to the slow, delta, theta and gamma oscillatory bands. A more exhaustive list of oscillatory frequencies was described by Penttonen and Buzsáki (2003). In general slow wave oscillations, and relevant to the current discussion those in the theta frequency range, are thought to synchronize distal regions of the brain promoting plasticity, while faster gamma oscillations are hypothesized to link and/or activate local neuronal ensembles (Bragin et al., 1995; Penttonen and Buzsáki, 2003; Buzsáki and Draguhn, 2004). There are several published reviews relating to a broader analysis of EEG and their role in plasticity and learning (Bas¸ar et al., 2001; Buzsáki, 2005; Lakatos et al., 2008; Knyazev, 2012; De Gennaro and Ferrara, 2003). However, based on the current level of understanding of these oscillations as they pertain to TBI is limited and therefore an in depth description of these findings is beyond the scope of this review. But, if we want to understand the extent of the effect of TBI on oscillations, it is important to not only consider one specific frequency band at a single electrode, for example hippocampal theta, but instead consider how multiple frequency bands are related at a single recording site (i.e., cross frequency coupling), and also how similar frequency bands are related between distal electrodes (i.e., phase coherence). Thus, in order to better understand the effects of brain injury on EEG it will be necessary to sample from multiple regions within a circuit as well as to investigate a range of frequency interactions in addition to a power analysis.

Interactions that take place across different frequencies at a single recording site are referred to as cross frequency coupling. The interplay between two frequencies could take place across several different domains (**Figure 3**). For example, crossfrequency power-power coupling (amplitude-amplitude) occurs when the power of the low frequency oscillation dictates the power of the high frequency oscillation; cross-frequency phasephase coupling (n:m phase locking) refers to a fixed number of high frequency oscillations nested in each slower cycle; crossfrequency phase-frequency and phase-power coupling indicates that the frequency and power of the faster wave is modulated by the phase of the slower oscillation, respectively (Jensen and Colgin, 2007; Belluscio et al., 2012). For a more thorough review on the significance of each of these interactions as they relate to cognition see (Axmacher et al., 2006; Lisman and Buzsáki, 2008; Colgin, 2013; Lisman and Jensen, 2013).

Phase coherence is the relationship of two oscillations of the same frequency across different electrodes. For example, there can be phase-phase coupling of oscillations such that the phases of each ongoing oscillation are in sync (i.e., the peak of one oscillation always occurs in the same phase of a second oscillation measured at a second site). Likewise, two similar frequencies can correlate in their power, independent of the phase. Specifically, as the power of an oscillation increases at one recording site, a similar increase in power is observed at a second electrode. These types of specific interactions suggest that oscillations are not simply a local phenomenon but instead have a role in network activity.

In addition to phase coherence, oscillations can organize the firing of individual neurons by summing together subthreshold excitatory inputs or organizing the firing procession of assemblies of neurons (O'Keefe and Recce, 1993; Skaggs et al., 1996; Tukker et al., 2007). While there are different implications for the specific type of interaction observed, in general interaction between rhythms supports neural communication, plasticity, formation of functional ensembles and consolidation of long-term memories (Buzsáki and Draguhn, 2004; Fell and Axmacher, 2011; Belluscio et al., 2012). The common variable is that oscillatory patterns contribute to higher order information processing including the formation of neuronal ensembles.

Ensemble formation, or the linking of a group of cells, is at the heart of information processing (Fries, 2005; Buzsáki, 2010). Oscillations are capable of promoting ensembles partly through temporally precise segregation and boosting of

Elsevier (Jensen and Colgin, 2007).

communication between some groups of neurons. Theta-gamma phase modulation (cross frequency phase-amplitude coupling) can explain how one region of the hippocampus, say CA1, can be involved in multiple networks virtually simultaneously. For instance, CA3-CA1 shows the greatest coherence and phase locking of single unit firing in the slow gamma band (gammaS: 25–50 Hz), which peaks in the early descending phase of the CA1 theta. On the other hand, medial EC (MEC)-CA1 has high coherence at mid gamma frequency (gammaM 50–90 Hz) which is strongest at the peak of theta (Colgin et al., 2009; Belluscio et al., 2012; Schomburg et al., 2014). Thus, CA1 inputs are segregated across the phase of theta cycle and therefore individual pyramidal neurons have the potential to temporally align with multiple ensembles within a phase of theta (for detailed discussion, see Buzsáki and Schomburg, 2015). Alternating back and forth between functional networks could be important, as an example, for shifting between encoding new and retrieval of previous information (Montgomery and Buzsáki, 2007; Colgin et al., 2009), assembling discrete stimuli into a single representation (Gray et al., 1989; Engel et al., 1991), selective attention/gain modulation (Fries et al., 2001) and associative binding (Headley and Paré, 2013). Critically, in many cases theta provides the temporal structure for local gamma, while simultaneously coupling cell assemblies between regions and ''chunking'' (Buzsáki, 2010) information into discrete processing units (Senior et al., 2008; Buzsáki and Moser, 2013; Colgin, 2013). Given that theta is altered post-TBI (Fedor et al., 2010; Lee et al., 2013, 2015), it will be important for future studies to examine how TBI may affect these tightly coupled interactions and whether changes in these interactions might underlie injury-induced behavioral deficits.

Theta modulation can also strengthen synaptic connections and organize information flow. For instance, hippocampal theta phase locks local cortical gamma activity across multiple regions and the firing of individual cortical neurons (Sirota et al., 2003; Hyman et al., 2005; Jones and Wilson, 2005; Siapas et al., 2005; Fujisawa and Buzsáki, 2011). Convergence of cortical inputs onto the hippocampus coincides with a time (i.e., theta phase) that is optimized to support hippocampal synaptic plasticity (Berry and Thompson, 1978; Huerta and Lisman, 1995; Seager et al., 2002). In turn, local hippocampal plasticity is shaped by a difference in gamma phase-phase synchronization between subfields. During tonic REM sleep CA3-CA1 gamma coherence is decreased, while dentate gyrus-CA3 gamma is increased. This releases CA1 from CA3 recurrent collateral control and allows the dentate gyrus to modify CA3 synaptic activity. However, during brief interspersed periods of phasic REM theta and gamma coherence across all three subfields is increased and so is CA1 firing (Montgomery et al., 2008). Thus, it seems that CA1 is excluded until hippocampal information is transmitted back to the cortex (Buzsáki, 1989; Wilson and McNaughton, 1994; Ji and Wilson, 2007). Shifting between local cell assemblies ensures accurate transmission of information, discrete synaptic modifications free of interference and a receptive receiver to form an ensemble. Unfortunately, it is yet to be determined if these interactions are affected by TBI and if they are correlated with cognitive and behavioral deficits.

While to this point we have focused on theta, ensemble formation can be modulated at other frequencies. For example, during NREM sleep cortical slow oscillations (0.02–0.8 Hz) drive the cortex to alternate between a depolarized and a hyperpolarized (up/down) state (Steriade et al., 1993a; Cowan and Wilson, 1994; Timofeev et al., 2001). This slow oscillation also propagates to the thalamus and the hippocampus. A depolarized cortical state is associated with thalamacortical spindles which can bias high frequency CA1 burst activity, commonly referred to as sharp wave-ripples (Buzsáki et al., 1992; Battaglia et al., 2004; Mölle et al., 2006; Buzsáki and Silva, 2012). These ripples are synchronized to a particular phase of the spindles and drive the activation of specific cortical ensembles (Siapas and Wilson, 1998; Sirota et al., 2003; Isomura et al., 2006; Wierzynski et al., 2009). This hippocampocortical interplay may bind hippocampal output with coactive cortical ensembles. Together, these interactions have the added effect of associating two different networks in the spirit of coordinating information storage and promoting formation of long term memories through reciprocal excitation of ensembles. Ultimately, depending on the effect of TBI these ensembles could be as important of targets as those organized by theta oscillations.

It is important to note that the very functions ascribed to oscillations are perturbed in TBI patients. On a variety of neuropsychological exams patients score worse on tests of attention, concentration, working memory, reaction time, judgement and measures of effort (Rimel et al., 1981; Levin et al., 1988a; McDowell et al., 1997; Bales et al., 2009). These failures of information processing consolidate into deficiencies in verbal and visual memory, episodic memory, multitasking, executive function and cognition (Levin et al., 1988b; Hanks et al., 1999; Millis et al., 2001; Alvarez et al., 2003; Bales et al., 2009; Bootes and Chapparo, 2010; McCauley et al., 2014; Mäki-Marttunen et al., 2015). Deficits in higher order information processing are compounded by sleep-wake disturbances (Kempf et al., 2010; Shay et al., 2014; Skopin et al., 2015). TBI patients report a range of sleep-related disorders including difficulty falling asleep, more nighttime awakenings and daytime naps, increased fatigue, and prolonged sleep (Parcell et al., 2006; Kempf et al., 2010; Mathias and Alvaro, 2012). These sleep deficits are accompanied by altered REM and NREM sleep as detected by nighttime scalp recordings, as well as changes in nocturnal hormone secretion (Parsons et al., 1997; Frieboes et al., 1999). Given the overlap between functions associated with oscillations and observed deficits in TBI patients, additional research is critical to understand whether dysfunction can be ascribed to an alteration in oscillations. Moreover, if there is a relationship between oscillations and outcome in brain injured patients treatments aimed at modulating EEG are seemingly an appropriate starting point.

#### ALTERED EEG AFTER TBI

There are data that clearly indicate that TBI alters oscillations both in pre-clinical models as well as in patients (for detailed Pevzner et al. Making Waves in the Brain

table on altered EEG, see Thatcher et al., 1989; Wallace et al., 2001; Rapp et al., 2015). In rodent models, there is a pronounced decrease in alpha, beta, delta and theta power following mechanical injury (Dixon et al., 1987; Ishige et al., 1987; McIntosh et al., 1987; Paterno et al., 2016). While most of these reductions return to baseline levels within minutes to hours after the injury (Sullivan et al., 1976; Dixon et al., 1988; McIntosh et al., 1989), some, like diminished theta, can persists for as long as 8–10 weeks after the insult (Fedor et al., 2010). A prolonged decrease in theta power is accompanied by other neurophysiological irregularities, even in brain regions spared from significant cell death. One such area, the CA1 subfield, exhibits altered excitation and inhibition, reduced LTP and pathological spine anatomy days to weeks after injury (Reeves et al., 1997; Sick et al., 1998; Sanders et al., 2000; Schwarzbach et al., 2006).

Analysis of human TBI patients resembles the reported prolonged recovery of EEG in experimental models of TBI. While modifications in brain activity can be seen as early as 24 h even after a subconcussive head trauma (Johnson et al., 2014), altered EEG following TBI can last for years after injury (Thatcher et al., 1989; Alvarez et al., 2008; Kempf et al., 2010; Slobounov et al., 2012). The changes in the EEG are not confined to a single oscillatory band, as they have been reported for the alpha, beta, delta, theta and gamma range (Alvarez et al., 2008; Tomkins et al., 2011; Rapp et al., 2015). Alterations of EEG activity are not state dependent, as changes are observed when a patient is at rest (Virji-Babul et al., 2014; Borich et al., 2015), actively moving (Slobounov et al., 2012) and during sleep (Parsons et al., 1997; Frieboes et al., 1999). In fact, abnormalities in scalp EEG are so consistent in patients they have been used to differentiate between injured and non-injured subjects, classify the severity of the injury, and some suggest, predict long term outcome after TBI (Thatcher et al., 1989, 1991, 2001; Alvarez et al., 2003; Arciniegas, 2011). For example, one of the criteria used to diagnose mild TBI many months after injury is an increase in coherence and a decrease in phase offset between frontal and temporal lobes, along with a decrease in power between frontal and posterior cortical regions (Thatcher et al., 1989). Furthermore, reversal of pathological EEG power ratio with administration of a neurotrophic peptide correlated with improvement in attention and working memory (Alvarez et al., 2008). The persistence of an abnormal EEG after a head injury suggests a potential link to prolonged psychological symptoms.

As is clear from the previous sections, it is not rigorous enough to determine that EEG is altered following injury. Unfortunately for the patient, it is also unlikely that there is a single mechanism that can easily explain why the EEG has changed. Therefore, it is critical to identify a starting point for research. While many neural systems and processes may be affected by a head injury, of particular interest (in our laboratory) is hippocampal dysfunction and the generation/maintenance of theta. TBI alters hippocampal neurotransmitter systems involved in the generation of theta, including acetylcholine, glutamate and GABA (Saija et al., 1988; Faden et al., 1989; Katayama et al., 1990; Robinson et al., 1990; Marshall et al., 2002). Rapid and prolonged increases in neurotransmitter levels act on local receptors causing long-lasting adaptation (Miller et al., 1990; Delahunty, 1992; Jiang et al., 1994; Delahunty et al., 1995; Schwarzbach et al., 2006). Thus, even after the injury-induced alteration of extracellular concentration of neurotransmitters returns to basal levels, modified receptors may have an ectopic response to subsequent activation, potentially affecting the timing and strength of receptor coupled processes essential to rhythm generation (Lyeth et al., 1992; Fineman et al., 1993; Kato et al., 2007; Marcoux et al., 2008). Another consequence of an intense glutamate discharge is excitotoxicity (Choi, 1988). This cell loss is readily observed in CA3 and dentate gyrus (Hicks et al., 1993; Floyd et al., 2002; Witgen et al., 2005), both of which are contributing nodes to CA1 theta (Bland, 1986; Kocsis et al., 1999; Marshall et al., 2002). Within the dentate gyrus, GABAergic interneurons in the hilus seem to be especially vulnerable to excitotoxicity, due to an increased excitatory drive onto their glutamatergic receptors (Tóth et al., 1997b; Hunt et al., 2011). Consequently, GABAergic cell death profoundly alters the excitability of not only the dentate gyrus, but the hippocampus as a whole leading to deficits in LTP and theta generation (Reeves et al., 1995, 1997; van den Pol et al., 1996; Witgen et al., 2005; Mtchedlishvili et al., 2010; Dinocourt et al., 2011). This hippocampal pathological process results in delayed degeneration in the MSN, a critical pacemaker for theta generation (Leonard et al., 1997). More specifically, cholinergic neurons within the septum show a marked susceptibility to cell death days to weeks following mild/moderate fluid percussion (Leonard et al., 1994; Schmidt and Grady, 1995) and a controlled cortical impact injury (Dixon et al., 1997). Such neuronal atrophy leads to enlarged ventricles and a proliferation of astrocytes, detected up to a year after TBI (Smith et al., 1997). Furthermore, downstream structures to the MSN, such as the hippocampus, show changes consistent with cholinergic hypofunction. In order to compensate for a decrease in evoked cholinergic neurotransmission (Dixon et al., 1996), there is an increase in the protein responsible for packing acetylcholine into presynaptic vesicles, downregulation of inhibitory autoreceptors and a hypersensitivity hippocampal cholinergic receptors and subsequent response of 2nd messengers (Jiang et al., 1994; Delahunty et al., 1995; Ciallella et al., 1998). MSN function could be further encumbered by post-traumatic epilepsy (Santhakumar et al., 2001; Frey, 2003; Pitkänen and McIntosh, 2006). Chronic seizure activity is related to a decrease (Garrido Sanabria et al., 2006) and altered firing of putative theta generating GABAergic cells in the septum (Colom et al., 2006). These observations have fueled the hypothesis that at least temporal lobe epilepsy in part arises due to an imbalance in septohippocampal theta and that theta stimulation could potentially be used as antiepileptic (Kitchigina et al., 2013; Fisher, 2015).

Degeneration and white matter damage also likely interfere with normal patterns of brain oscillations. Axonal abnormalities may arise from the initial shearing forces from the impact and an ionic imbalance in the extracellular space (Hicks et al., 1995; Graham et al., 2000; Li et al., 2014). Loss of ionic equilibrium leads to axonal increase of Ca2+ permeability, calpain activation, mitochondrial dysfunction and eventually breakdown of the cytoskeleton (Maxwell et al., 1997; Johnson et al., 2013). These changes may culminate in deafferentation/denervation and inappropriate synaptic plasticity (Povlishock and Katz, 2005; Hunt et al., 2011). Many of these axonal changes can be detected weeks after the insult and correlate with behavioral abnormalities (Kempf et al., 2010; Spain et al., 2010). It is not surprising then that compromised axonal structure and function results in irregular oscillatory interactions, even years after the injury. These structural deformities along with neurochemical aberrations contribute significantly to the observed deficits in the propagation of functionally relevant hippocampal theta (Fedor et al., 2010), and subsequently brain function after TBI (Hanks et al., 1999; Millis et al., 2001). These data clearly indicate that multiple TBI-induced mechanisms can play a role in altered brain oscillations and their interactions thus contributing to long-term impaired cognition.

## THETA DBS

Pathologies associated with TBI are wide-ranging, occurring at the molecular, physiological and structural level. These alterations in turn may lead to changes in network activity, affecting neural communication and plasticity. Abnormal rhythm generation could potentially hinder and prolong recovery after a TBI insult (Thatcher et al., 1991; Tomkins et al., 2011). Furthermore, once a patient has progressed out of the acute post-injury phase of the disease, neuroprotection is no longer a viable therapeutic option. Therefore, there is an urgency to develop treatment strategies for TBI patients who have chronic disability. DBS represents a potential intervention that can drive neural networks, improve neurophysiology and ultimately behavioral outcome in a subset of brain-injured patients. The advantage of neurostimulation, say over pharmacology, is its ability to target specific regions, inherent higher temporal resolution and ability to generate specific patterns of electrical input, all of which are critical factors in the generation and interaction of oscillations. Furthermore, neurostimulation has shown promise in alleviating symptoms in motor, cognitive, behavioral and psychiatric conditions (Brunoni et al., 2011; Lozano and Lipsman, 2013; Suthana and Fried, 2014). The relative success of DBS in each specific situation is determined by a growing body of parameters, beyond the scope of this article to survey (Kuncel and Grill, 2004; Butson and McIntyre, 2007; Birdno and Grill, 2008). Therefore, given the focus of this review we will highlight the potential use of one stimulation strategy, the use of low frequency stimulation within the theta range.

Exogenous induction of theta in structures like the hippocampus can improve cognitive processes in experimental animals. Hippocampal theta can be achieved either with direct hippocampal stimulation or by targeting afferent structures such as the fornix or MSN. Using rodent models, pre-training stimulation of MSN decreased the time to acquire discriminatory learning (Deupree et al., 1982) while post-training stimulation facilitated memory consolidation (Landfield, 1977; Wetzel et al., 1977). These results mirror the positive correlations observed between endogenous theta and enhanced acquisition and retention (Landfield et al., 1972; Berry and Thompson, 1978; Seager et al., 2002; Mandile et al., 2003; Mitchell et al., 2008). Importantly, the uniqueness of these findings lies not in the MSN per se, but in the theta oscillation. The critical role of theta oscillations specifically was revealed in studies were theta stimulation of the fornix was able to drive hippocampal theta oscillations following chemical inactivation of the MSN (Green and Arduini, 1954; Petsche et al., 1962; Vertes et al., 2004). Not only did stimulation drive theta, but also rescued the behavioral impairment (McNaughton et al., 2006). Further substantiating the selectivity of theta range, high frequency MSN stimulation does not facilitate mnemonic processes (Landfield, 1977; Wetzel et al., 1977). Therefore hippocampal theta, generated endogenously or extrinsically, plays a critical role in neural computations supporting animal cognition.

The beneficial effects of stimulation in the theta range are not limited to cognitive processes. For example, low frequency stimulation has been shown to be beneficial after an acute spinal cord contusion where an 8 Hz stimulation of the raphe nucleus improved motor coordination and sensory processing, increased white matter integrity and reduced astrocytosis (Hentall and Burns, 2009; Hentall and Gonzalez, 2012). Therefore it is possible that stimulation of theta following TBI might also have indirect effects that could improve the hippocampal milieu postinjury facilitating anatomical as well as physiological recovery. Epilepsy treatment is also closely associated with low frequency stimulation. Effective reduction of kindling from a 60 Hz induced seizure is achieved with 3–5 Hz stimulation (Gaito et al., 1980; Kile et al., 2010; Koubeissi et al., 2013). Building on this framework, Fisher recently proposed a novel hypothesis that MSN stimulation in the theta range may benefit patients with epilepsy (Fisher, 2015). Therefore stimulation following severe TBI might also have the added benefit of reducing or preventing post-traumatic epilepsy.

# THETA DBS IN TBI MODELS

Recently, several studies have described the restorative effect of theta stimulation after TBI injury. Lee et al. (2013, 2015) stimulated the MSN at 7.7 Hz and recorded an increase in hippocampal theta power along with better spatial performance in the Barnes maze following a moderate lateral fluid percussion injury (**Figure 4)**. Several stimulation controls bolstered the hypothesis that theta band stimulation was specifically augmenting the septohippocampal system in TBI rats. In particular, successful MSN stimulation in the theta range was intensity specific, there was no effect on overall motor output (i.e., distance traveled) and MSN stimulation at 100 Hz did not rescue the deficit in spatial performance (i.e., spatial search strategy) in the maze (Lee et al., 2015). Moreover, the authors concluded the effect was restorative and not simply enhancing function as similarly stimulated sham animals experienced no improvement in spatial learning. Likewise, the Hentall group observed positive effects on spatial memory in the watermaze and forelimb reaching movements when, following lateral fluid percussion, they stimulated the raphe nucleus, part of the ascending system that generates theta, at 8 Hz (Vertes et al., 2004; Carballosa Gonzalez et al., 2013).

In conjunction with behavioral outcomes, stimulation reversed cortical cell loss, white matter degeneration and decreases in cortical and hippocampal levels of cAMP, an intracellular second messenger (Carballosa Gonzalez et al., 2013). These proof of principle studies illustrate the potential of theta stimulation to augment physiological and behavioral outcome following TBI.

Importantly, theta stimulation has not been the only successful stimulation paradigm observed in experimental models of TBI. In a model of mild TBI, theta burst stimulation (TBS) was able to rescue working memory in the T-maze delayed non-match to sample task. Rather than using a continuous single pulse (7.7 or 8 Hz) fixed stimulation (Sweet et al., 2014) used a TBS protocol to stimulate the fornix, specifically with five 50 ms long bursts of high frequency (200 Hz) pulses per second. The hypothetical advantage of TBS as compared to continuous theta stimulation is that the 200 Hz gamma stimulation partially recapitulates endogenous patterned firing at a physiologically relevant theta frequency. In fact TBS has been demonstrated to induce long lasting LTP (Rose and Dunwiddie, 1986; Staubli and Lynch, 1987; Diamond et al., 1988; Kirkwood et al., 1993). Accordingly, Sweet et al. (2014) reported that TBS, but not low (5 Hz) or high (130 Hz) frequency stimulation of the fornix, improved performance of TBI rats in the T-maze. Spatial memory in the water maze was also improved with TBS; however, 5 Hz stimulation was not tested.

There are several key takeaways from these two successful stimulation paradigms. The first is that task might matter. There is evidence that, depending on task, there is a shift in the frequency of the theta oscillation (Kramis et al., 1975; Watrous et al., 2013). Therefore, the specific frequency within the theta range may be critical to improving outcome and the target may be different for different behaviors. Following that reasoning, while 5 Hz stimulation may not have improved T-maze performance, it is possible that 7.7 (or some other frequency) may have. In fact, in a study by McNaughton et al. (2006), it was observed that to optimally restore behavior in the watermaze after chemical inactivation of the MSN, it was best to stimulate the fornix with an endogenous EEG pattern recorded from the supramamillary nucleus as compared to fixed 7.7 Hz or an irregular theta stimulation pattern (with an average frequency of 7.7 Hz). These data indicate that not all ''theta'' is the same, and that the specific frequency within the theta band may be highly relevant.

The fact that these initial reports of DBS in TBI did not report completely homogenous results is worth noting. The data highlights the need for additional research to investigate the large parametric space available for potential stimulation parameters. There are many additional variables to be considered: intermittent vs. constant stimulation; endogenous stimulation (where theta is recorded from an uninjured site and played back in the injured hippocampus as in McNaughton et al., 2006) vs. exogenous fixed frequency; theta burst vs. single pulse; variants in voltage, pulse width and square as compared to sinusoidal; which regions, nuclei or subfields to target; at which point during the task/behavior to stimulate or whether to stimulate offline during sleep or to stimulate relative to an endogenous oscillation independent of the behavior. A closed loop system where stimulation was based on the recorded EEG (from a different region) would subsequently be amenable to biofeedback (Wallace et al., 2001; Rosin et al., 2011; de Hemptinne et al., 2015).

Different stimulation parameters will not only influence the efficacy of the treatment, but also most likely the extent of unintended effects. The most commonly reported adverse events are related to the implant rather than stimulation and include inflammation, headache, pain at the implant site, and mild paresthesia surrounding the implant (Kenney et al., 2007; Fisher et al., 2010; Salanova et al., 2015). However, in studies of stimulation for treatment of epilepsy there are reports of cognitive dysfunction, depression and suicide in a small number of patients (Bergey et al., 2015; Salanova et al., 2015). Therefore it will be important to monitor which symptoms TBI patients report receiving low frequency stimulation of the septohippocampal system. While complications with the surgery and device itself are minimal, as argued by Fisher (2015) in his proposal to stimulate the MSN in epileptic patients, there are however potential risks of eliciting seizures or promoting addiction to constant stimulation (for discussion on long-term safety of DBS, see Kenney et al., 2007). These potential risks (e.g., prior epileptic activity, predisposition to addiction, mood/affect disorders) should be taken into consideration when enrolling patients so as to minimize potential harm. The inclusion criterion could be further refined based on the mechanism of action of neurostimulation in the theta range. If DBS is masking an enduring effect or if it is restorative will potentially influence the type of therapy one gets, such as; when should DBS be administered relative to the injury and in response to what type of injury? Will immediate intervention interfere with the healing process or will waiting too long make the system unamenable? How long will the benefits of DBS persist, if

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stimulation is discontinued? Should the treatment continue indefinitely or should there be a clinical marker/threshold to stop or augment the stimulation? Thus, there is a clear need for considerable pre-clinical animal work and potentially computational modeling to better understand and explore the complex parameter space that is DBS and the mechanisms behind it, if we are to optimize the potential of neurostimulation for clinical translation.

#### CONCLUSION

After years of research, there are few proven interventions that reduce injury-induced cellular cascades and ultimately, cell death and dysfunction following TBI. While the latest census estimates over 5.3 million patients live with chronic disability, it is clear that that number has grown and continues to grow. Therefore, there is a clear need for pre-clinical research expressly focused on the injured nervous system in the chronic stages of the disease. Oscillations are known to play a key role in physiological circuit function, whether it is the progression of oscillations through the sleep cycle or theta oscillations in the hippocampus. Initial evidence suggests that injury-induced disruption of these oscillations has a profound impact on neural connectivity and behavior. In fact, changes in EEG can be used as a biomarker to confirm mild and moderate TBI. Additionally, limited studies of DBS in brain injured rats demonstrate that the injured brain can be modulated by entraining or replacing oscillations, with improved outcomes. Future preclinical studies are needed to explore a very large parametric space that spans not only multiple stimulation targets and paradigms but also different injury mechanisms as well as a range of cognitive behavioral tasks and dependent measures, extending beyond spatial navigation. The potential for DBS is clear. We believe that further research into electrical neuromodulation of the injured brain will result in an exciting avenue to promote behavioral, cognitive and neurophysiological recovery following TBI.

#### AUTHOR CONTRIBUTIONS

AP, KS, and GGG contributed to the conception, writing and editing of the manuscript. AI, DJL contributed significantly to the conception and editing of the manuscript.

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

Copyright © 2016 Pevzner, Izadi, Lee, Shahlaie and Gurkoff. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution and reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Hippocampal Neurophysiologic Changes after Mild Traumatic Brain Injury and Potential Neuromodulation Treatment Approaches

#### Fady Girgis, Jonathan Pace, Jennifer Sweet and Jonathan P. Miller\*

Department of Neurosurgery, University Hospitals Case Medical Center, Case Western Reserve University, Cleveland, OH, USA

Traumatic brain injury (TBI) is the leading cause of death and disability in individuals below age 45, and five million Americans live with chronic disability as a result. Mild TBI (mTBI), defined as TBI in the absence of major imaging or histopathological defects, is responsible for a majority of cases. Despite the lack of overt morphological defects, victims of mTBI frequently suffer lasting cognitive deficits, memory difficulties, and behavioral disturbances. There is increasing evidence that cognitive and memory dysfunction is related to subtle physiological changes that occur in the hippocampus, and these impact both the phenotype of deficits observed and subsequent recovery. Therapeutic modulation of physiological activity by means of medications commonly used for other indications or brain stimulation may represent novel treatment approaches. This review summarizes the present body of knowledge regarding neurophysiologic changes that occur in the hippocampus after mTBI, as well as potential targets for therapeutic modulation of neurologic activity.

#### Edited by:

Akiva Cohen, University of Pennsylvania & Research Institute of Children's Hospital of Philadelphia, USA

#### Reviewed by:

Mazyar Fallah, York University, Canada Trent Anderson, University of Arizona, USA

\*Correspondence:

Jonathan P. Miller<EMAIL_ADDRESS>
Received: 18 November 2015 Accepted: 25 January 2016 Published: 09 February 2016

#### Citation:

Girgis F, Pace J, Sweet J and Miller JP (2016) Hippocampal Neurophysiologic Changes after Mild Traumatic Brain Injury and Potential Neuromodulation Treatment Approaches. Front. Syst. Neurosci. 10:8. doi: 10.3389/fnsys.2016.00008 Keywords: hippocampus, mild traumatic brain injury, neurophysiology, neuromodulation, cognitive dysfunction, drug repositioning

# INTRODUCTION

Every year, one out of every 200 people worldwide will suffer a traumatic brain injury (TBI), including 1.7 million people in the United States alone, with an annual associated cost of approximately \$76.5 billion (Faul et al., 2010). It is estimated that 5.3 million Americans live with long-term disability as a result of their injury (Centers for Disease Control and Prevention [CDC], 2013). Young men and the elderly are particularly vulnerable because of an increased incidence of trauma and falls, respectively (Susman et al., 2002). TBI is also prevalent in the military, with approximately 20% of all returning deployed American troops suffering some form of brain injury from a blast exposure (Terrio et al., 2009), the vast majority of these being mild injuries.

Mild TBI (mTBI), sometimes termed concussion, accounts for 75% of all head injuries (Faul et al., 2010). Symptoms include headache, sleep impairment, memory deficit, difficulty concentrating (Levin et al., 1987), and an increased incidence of affective disorders such as depression and anxiety (Park et al., 2008). These clinical problems are persistent and severe in 10% of cases, lasting for months to years, sometimes indefinitely (Greig et al., 2014). Due to the striking prevalence and disruptive clinical manifestations of mTBI, research has focused on elucidating the pathophysiological mechanisms underlying primary and secondary brain injury in order to develop therapies that may alter or prevent these processes. More recently, changes in neural circuitry underlying many of the clinical manifestations of mTBI, especially memory and affective disorders, has been studied in an attempt to understand mechanisms of disruption and how modulation of these networks may be used to therapeutic advantage. In order to determine how novel neuromodulation treatments might be able to improve TBI-associated deficits, it is important to establish how individual neurons and subpopulations of neurons contribute to network activity and how these networks respond to injury. This review will discuss neurophysiological changes in the hippocampus associated with mTBI, and ways in which therapeutic neuromodulation might be used to reverse these deficits.

#### BIOCHEMICAL AND GENETIC CHANGES IN THE HIPPOCAMPUS FOLLOWING TBI

Neurological injury that occurs after a traumatic insult can result from two different mechanisms: primary and secondary injury. Primary injury refers to the mechanical forces of shearing and compression at the time of impact, while secondary injury involves the subsequent cascade of pathological events that occurs minutes to days after the insult (Greve and Zink, 2009). Such secondary processes include hypoxia/ischemia, edema, raised intracranial pressure, excitotoxity through glutamate release, calcium dysregulation, cytoskeletal proteolysis, free radical oxidative damage, altered synaptic physiology, cytokine release causing inflammation, and ultimately neuronal cell death (Choi et al., 1987; Raghupathi, 2004; Maas et al., 2008; Barkhoudarian et al., 2011; Greig et al., 2014; Merlo et al., 2014). These secondary injury mechanisms are especially important for therapeutic consideration as they can theoretically be altered or inhibited with the aim of preventing injury or protecting brain tissue that has the potential to recover.

There are several commonalities between the biochemical cascades involved in secondary mTBI injury mechanisms and those found in degenerative central nervous system (CNS) diseases such as Parkinson's disease (PD; Eakin and Miller, 2012; van Bregt et al., 2012), Alzheimer's disease (AD), Huntington's disease (HD), and amyotrophic lateral sclerosis (Daneshvar et al., 2011). As a result, pathways that have been extensively characterized in these diseases can be extrapolated and applied to analogous pathways involved in TBI in order to better understand the mechanisms of injury and potential novel therapeutic approaches. This has important implications for diagnosis: a complex mathematical model of semantic predications was recently used to search almost 100,000 citations of neural injury networks and produced a list of 17 potential biomarkers that may indicate mTBI, with glutamate, glucose, and lactate being the most common (Cairelli et al., 2015). In combination with other compounds, such as the apolipoprotein E-4 allele which adversely influences recovery following brain injury (Lawrence et al., 2015), these biomarkers may aid in the early diagnosis of mTBI, allowing the prompt administration of neuroprotective drugs or neuromodulatory therapies.

Glutamate is particularly important, as this excitatory neurotransmitter is known to play a strong role in secondary injury mechanisms throughout the brain. Microdialysis studies, in both rodents (Folkersma et al., 2011) and humans (Chamoun et al., 2010), have shown that glutamate concentrations increase in the acute phase following injury, whereas magnetic resonance spectroscopy (MRS) studies have shown a persistent decrease in glutamate concentrations following injury. The reason for this discrepancy is that microdialysis measures extracellular glutamate, while MRS measures both intra and extracellular glutamate (Guerriero et al., 2015).

Specifically in the hippocampus, glutamate decreases following moderate to severe temporal cortex injury, as measured by in vivo proton MRS in a direct cortical injury model in rodents (Harris et al., 2012). Although the hippocampus was considered perilesional tissue in this experiment, the temporal neocortex, i.e., the lesional tissue, also showed a decrease in glutamate in the acute phase. Human MRS studies also in the acute phase following concussion, however, showed no changes in hippocampal glutamate concentrations, despite finding decreased glutamate levels in the motor cortex (Henry et al., 2010). A reason for this discrepancy may be that the human study was in patients with mTBI, whereas the rodent study looked at moderate to severe TBI.

Another important compound altered in the hippocampus after TBI is the neurotransmitter gamma-aminobutyric acid (GABA). The expression of various GABA receptor subunits is changed, causing reductions of inhibitory postsynaptic currents and increases in excitatory post-synaptic currents after TBI in rodents (Hunt et al., 2011; Almeida-Suhett et al., 2015; Drexel et al., 2015). As a result, deficits occur in long-term potentiation, which can translate into cognitive impairment. Similarly, up and down-regulation of other proteins can lead to impairments in hippocampal synaptic transmission, including those involved in the extracellular signal-regulated kinase (ERK) pathways (Atkins et al., 2009), and the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex (Carlson et al., 2016).

The regulation of gene transcription affects not only the creation of proteins involved in neurotransmission after injury, but also the synthesis of neurons themselves. Because TBI is associated with hippocampal neuronal damage and death, it is important to develop therapeutic strategies to either protect immature neurons or enhance neurogenesis in the hippocampus after injury. Methods shown to drive neurogenesis post injury include forced overexpression of insulin-like growth factor 1 (IGF-1; Carlson et al., 2014), and the transplantation of synthetic human progenitor cells into the hippocampus after TBI (Blaya et al., 2015). Interestingly, TBI severity has been found to correlate with the degree of post-traumatic neurogenesis in rat hippocampus. Whereas mTBI had no effect on neurogenesis, moderate TBI promoted neural stem cell proliferation without increasing neurogenesis, and severe TBI increased neurogenesis (Wang et al., 2015a). A summary of the biochemical and genetic changes following TBI in rodent hippocampus can be found in **Table 1**.

#### TABLE 1 | Biochemical changes in rodent hippocampus following TBI, with effects categorized by subsection of the hippocampal complex.

CA, cornu ammonis; GABA, gamma-aminobutyric acid; GCL, granule cell layer; RNA, ribonucleic acid.

#### HIPPOCAMPUS BIOCHEMICAL PATHWAYS AND NEUROPROTECTIVE AGENTS IN mTBI

Since mechanisms of secondary injury after mTBI are similar to other diseases, it is likely that drugs that are already in use for other indications may have efficacy for mTBI as well. One example of such a mechanism is the glucagon-like peptide-1 (GLP-1) signaling pathway. GLP-1 was initially described based on its role in the pancreatic regulation of insulin, but receptors are also found in the CNS and changes in this pathway have been implicated in a number of neurodegenerative diseases (Li et al., 2009, 2010, 2012; Martin et al., 2009; Salcedo et al., 2012). Through coupling to a cAMP second messenger pathway, activation of GLP-1 receptors elicits neurotrophic actions such as neuritic growth while protecting neurons against various insults (Perry et al., 2002). As a result, there have been a number of investigations into modulation of this pathway to treat neurological disease.

Exendin-4 is a synthetic GLP-1 receptor agonist currently approved as a drug for the treatment of hyperglycemia in type-2 diabetes that crosses the blood brain barrier (Kastin and Akerstrom, 2003). When administered after mTBI, this neuroprotective agent has been shown to prevent changes in rodent hippocampal gene expression following mTBI. Many of these genes protected by Exendin-4 are also associated with memory loss in AD (which has been associated with TBI), and administration ameliorated memory deficits after injury (Tweedie et al., 2013). Additional rodent studies using markers of cell death have shown that Exedin-4 improved hydrogen peroxide mediated oxidative stress and glutamate toxicity in neuronal cultures in vitro. In vivo, using markers of cognitive function, the drug ameliorated mTBI-induced deficits in novel object recognition and maze testing, both when administered prior to and after the insult (Eakin et al., 2013; Rachmany et al., 2013). Therefore, data is emerging to support a shift in indication for the use of Exedin-4 from an antihyperglycemic medication to a potential agent to minimize secondary injury after mTBI.

Another agent with potential efficacy for secondary injury after mTBI is N-Acetyl-cystein (NAC), which is currently used as a mucolytic agent and an antidote to acetaminophen overdose. NAC has been shown to have antioxidant and neurovascularprotective effects after mTBI (Ellis et al., 1991; Hicdonmez et al., 2006; Chen et al., 2008) and has been demonstrated to reverse behavioral deficits in mTBI rodents when administered 30–60 min after injury (Eakin et al., 2014). Specifically in the hippocampus, NAC was found to decrease levels of cytosolicfree Ca2+ and reactive oxygen species, reduce apoptosis, and lower caspase-3 and -9 neuronal activities, following TBI in rats (Nazirog˘lu et al., 2014). In humans, NAC has been shown in a randomized double-blind trial to improve auditory, vestibular, and cognitive functional sequelae after blast-induced mTBI in military personnel (Hoffer et al., 2013). Although the mechanism of action in the trauma setting has yet to be elucidated, the 40 year safety profile and ease of administration of the medication makes it an attractive option for widespread use.

Numerous other compounds have been found to exert effects in the hippocampus following injury. As summarized in **Table 2**, different substances affect different subsections of the hippocampal complex. Although all these investigations were performed in rodents, several of these biochemical compounds may prove to have beneficial effects in humans suffering from cognitive and memory impairments following TBI.

## HIPPOCAMPAL CIRCUITRY AND NEUROPHYSIOLOGIC CHANGES AFTER mTBI

In addition to biochemical changes after injury, changes in electrical neural activity also play a role in the sequelae of TBI. The high prevalence of memory deficits after injury point to a relative vulnerability of the hippocampus and mesial temporal structures as compared to the rest of the brain (Hamm et al., 1993; Finset et al., 1999; Comper et al., 2005), and electrophysiologic studies have shown that changes occur in hippocampal circuit excitability after TBI (Witgen et al., 2005; Ortiz and Gutiérrez, 2015). In addition, moderate and severe TBI is associated with neuronal cell loss in several hippocampal regions (Lowenstein et al., 1992) as well as alterations in cellular homeostasis (D'Ambrosio et al., 1999) and dysregulated synaptic transmission (Toth et al., 1997; D'Ambrosio et al., 1998) in this region. On the other hand, mTBI has been associated with more subtle changes in firing patterns that are present even in the absence of morphological changes.

Studies comparing control rats to those who had undergone lateral fluid percussion induced mTBI found changes in working memory in the absence of differences in histology or neuronal loss (Eakin and Miller, 2012). Single unit recordings from cornu ammonis 1 (CA1) and CA3 subfields during a working memory task showed no differences in firing rates or spike characteristics between the two groups, but rats exposed to mTBI were found to have significantly fewer cells with spatiotemporal activity, and this correlated with task performance. Moreover, in a similar experiment looking at the burst characteristics of hippocampal cells during an object exploration task, memory deficits were found to be associated with decreased burst activity of certain subsets of neurons within the pyramidal cell layer (Munyon et al., 2014). Therefore, functional impairment may stem from an alteration in the activity of certain subpopulations of cells within the hippocampus.

These changes in neuronal firing patterns within the hippocampus can persist after TBI and correlate with changes in cognitive function (Witgen et al., 2005). In the analysis of local field potentials after injury, hippocampal broadband power decreased (Paterno et al., 2015), and specifically disruption of theta rhythm (4–7 Hz) has been implicated in the pathophysiology of memory loss (Winson, 1978; Fedor et al., 2010). It has been theorized that using electrical stimulation to artificially counteract and reverse these disrupted firing patterns within the hippocampus and other structures may regulate cellular and network processes and restore functional memory circuits (Diamond et al., 1988; Nakao et al., 2003; Shirvalkar et al., 2010; Tabansky et al., 2014; Hanell et al., 2015). Accordingly, theta burst stimulation (200 Hz in 50 ms trains, five trains per second; 60 mA biphasic pulses) delivered to the fornix of rats with mTBI improved deficits in learning and memory, whereas non-oscillatory low or high frequency stimulation did not (Sweet et al., 2014). In a related manner, theta band stimulation of the medial septal nucleus of mTBI rats resulted in a transient increase in hippocampal theta activity and improved spatial working memory (Lee et al., 2013), whereas 100 Hz gamma stimulation did not (Lee et al., 2015).

In humans, theta burst stimulation of the fornix has been studied in patients undergoing stereo-electroencephalography evaluation for drug-resistant epilepsy, and was shown to produce evoked potentials in the ipsilateral hippocampus. This resulted in significant and reversible improvements in immediate and delayed performance on a visual-spatial memory task (Miller et al., 2015). Temporally patterned stimulation paradigms may therefore present novel neuromodulatory strategies in the treatment of post-traumatic memory loss and cognitive deficits. Potential targets include the hippocampus, its associated white matter tracts, as well as the medial prefrontal cortex. The dysfunction of the latter is thought to play a role in the failure of emotion regulation after mTBI through top down amygdalar regulation (van der Horn et al., 2016), a mechanism that may result in persistent post-concussive symptoms.

CA, cornu ammonis; GABA, gamma-aminobutyric acid; Trk, tyrosine kinase; NMDA, N-methyl-D-aspartate; mTOR, mammalian Target Of Rapamycin; IL-1β, Interleukin-1 beta.

# IMAGING IN mTBI

Recent advances in imaging have allowed for in-depth structural analysis of brain regions both acutely after concussive injury and in the chronic phase. This is important because the ability to identify abnormalities that may previously have been missed is the first step in developing modalities to treat these phenomena, or in the least decide which patients require treatment. Although no single imaging modality has yet proven to be sufficient for all patients with TBI due to the heterogeneity of the condition (Amyot et al., 2015), each method offers its own advantages.

Conventional magnetic resonance imaging (MRI) and computed tomography (CT) are typically negative in mTBI patients with persistent symptoms of post-concussive syndrome. However, compared to controls, mTBI patients were found to have lower cortical thickness of the right temporal lobe and left insula, and increasing number of mTBIs was associated with lower cortical thickness in more brain areas, including bilateral insula, right middle temporal gyrus, and right entorhinal area (List et al., 2015). These findings are worrisome because if further deterioration of these structures occur as part of the aging process, subtle cognitive symptoms may progress to clinical dementia earlier than in age-matched controls. Diffusion tensor imaging is another technique that has recently emerged as a powerful adjunct to conventional MRI. It enables the measurement of fractional anisotropy, a value that indicates the degree of restriction experienced by water molecules diffusing along white matter fibers. Such a tool offers a noninvasive glimpse into the neurophysiology of these tracts and may allow detection of demyelination and axonal damage that would otherwise be missed on a conventional MRI. Indeed, fractional anisotropy was significantly reduced in the right superior longitudinal fasciculus of patients with mTBI and normal MRI as compared with controls, showing that diffusion tensor imaging is more sensitive than standard imaging in this population (Adam et al., 2015). Another study showed similar but subtler findings, as only those with mTBI who had abnormalities on conventional MRI had white matter changes on diffusion tensor imaging (Panenka et al., 2015).

Although increasing abnormality on post-injury CT and MRI correlate with lower neuropsychological performance (Bigler et al., 2015), diffusion tensor imaging has also shown promise in predicting neuropsychological outcomes following mTBI in patients with normal CT and MRI. Immediately following injury and 6 months post-trauma, patients with mTBI showed significant differences in fractional anisotropy and radial diffusivity as compared to controls, specifically in the corona radiata, anterior limb of internal capsule, cingulum, superior longitudinal fasciculus, optic radiation, and genu of corpus callosum, changes which demonstrated associations with neuropsychological outcomes across both time points (Veeramuthu et al., 2015). Another study has shown significantly reduced fractional anisotropy in the internal capsule of both TBI and depression patients (Maller et al., 2010), further underscoring the potential role for diffusion tensor imaging as a noninvasive biomarker useful in the prognostication of post-concussive symptoms.

MRS is another noninvasive technique that measures metabolic changes within brain tissue. In combination with vascular imaging techniques such as transcranial Doppler ultrasound, these modalities have begun to shed light on metabolic processes and the control and regulation of cerebral blood flow following mTBI (Len and Neary, 2011). In the hippocampus, N-acetyl aspartate to choline and N-acetyl aspartate to creatine ratios were decreased in patients with mTBI in comparison to control subjects (Hetherington et al., 2014). Similar changes were found following mTBI in the thalamus, with a reduction of N-acetyl aspartate to creatine and choline to creatine ratios, findings that are classically associated with ischemia (Sours et al., 2015). Of interest, these results correlated with self-reported sensory symptoms, leading the authors to speculate that novel interventions could target these changes in the hopes of treating patients with persistent post-concussive symptoms, as well as use the absence of such imaging features as an indicator to help predict safe return to work.

Magnetoencephalography is an imaging modality largely restricted to the study of epilepsy, but may prove useful as a sensitive detector of local field potentials in brain areas affected by mTBI. A recent innovative study looked at the tracking of eye movements as a marker for sustained attention over time and internal anticipatory control, measurements that are impaired in chronic mTBI while the patients were undergoing magnetoencephalography (Diwakar et al., 2015). Compared to controls, mTBI patients demonstrated impaired eye tracking that was concurrent with abnormal beta activity, which was suppressed in the right parietal cortex and enhanced in the left caudate and frontal-temporal areas. The clinical significance of these findings is yet to be seen, but this is a strong example of novel imaging methods being used in new and original ways to better understand the neurophysiology of injured neural circuits, methods that may alter the manner in which mTBI is diagnosed and treated in the future.

#### FUTURE DIRECTIONS

Direct modulation of neural activity via deep brain stimulation (DBS) currently does not play a direct role in the setting mTBI, but it does carry promise in the treatment of post-concussive affective disorders, trauma-induced tremor, and neurodegenerative disorders. DBS is an effective treatment for certain patients with severe major depression (Lozano et al., 2012) and obsessive-compulsive disorder (Kisely et al., 2014), but has yet to be investigated in the treatment of these disorders following brain injury. Likewise, PD often responds well to DBS of the subthalamic nucleus or globus pallidus internus (Okun, 2012), but data is lacking in the setting of trauma-induced Parkinson's. However, tremor following severe brain injury has been shown in small studies to respond to both thalamic and pallidal DBS (Issar et al., 2013; Carvalho et al., 2014).

If neurostimulation augments improvements in cognitive deficits after brain injury, memory disorders may present other potential applications for DBS (Shin et al., 2014). Forniceal DBS in a mouse model of Rett syndrome restored in vivo hippocampal long-term potentiation and neurogenesis, and rescued contextual fear memory and spatial learning (Hao et al., 2015). In humans, the fornix and nucleus basalis of Meynert are being investigated as possible DBS targets for improvement of memory in the treatment of AD (Sankar et al., 2014; Suthana and Fried, 2014). Although these strategies seem promising, they will likely be tested for efficacy and safety in the setting of severe TBI, prior to being applied in patients with mTBI.

Less invasive neuromodulation options for the treatment of mTBI include transcranial magnetic stimulation (TMS) and transcranial direct current stimulation (TDCS). TMS uses a surface coil to create a low frequency electric current that is delivered to the surface of the brain, while TDCS uses electrodes placed directly on the scalp. There is evidence in severe TBI that TMS may aid in motor recovery, aphasia, visuospatial neglect (Castel-Lacanal et al., 2014), and cognitive function (Polanowska and Seniów, 2010), but high frequencies are contraindicated due to the risk of seizures, especially in the setting of trauma as the injured brain is already predisposed to epileptiform activity. TDCS uses lower currents and therefore has a lower risk of seizures, but subsequently may also have less clinical effect, as current studies show mixed results in the treatment of post-concussive symptoms (Kang et al., 2012; Le´sniak et al., 2014; Ulam et al., 2015). TMS is currently the most extensively studied brain stimulation modality in TBI, but most interventions have been non-targeted and focused on the chronic phase of recovery after the injury (Li et al., 2015). As can be expected, better clinical outcomes occur when these neuromodulatory approaches are coupled with cognitive therapy and neurorehabilitation (Chantsoulis et al., 2015; Page et al., 2015).

Finally, nanotechnology and brain-computer interfaces may present new opportunities. By using a computer to bridge the gap between the brain and peripheral nerves in the setting of an injured spinal cord, brain-computer interfaces are ''a form of extended embodiment that become integrated into the individual's conception of himself as an autonomous agent'' (Glannon, 2014). Such systems are still in the experimental stage, but offer hope to those patients with severe motor deficits. Similarly, carbon nanotubes are being used to synthesize nanoelectrode arrays that permit real time monitoring and modulation of electrochemical events at the neural and synaptic level (Andrews, 2007), and may offer a glimpse into the future of the field of neuromodulation. As with DBS, however, these modalities will likely be reserved for patients with severe TBI until efficacy, safety, and economic feasibility can be established.

#### CONCLUSION

Despite the high incidence and prevalence of mTBI, only recently have the pathophysiologic mechanisms begun to emerge. Understanding the biochemical signaling pathways involved in the propagation of secondary injury mechanisms has led to the discovery of novel uses for medications

#### REFERENCES

currently used for other indications. Biochemical markers and advances in imaging are allowing for earlier diagnosis of concussions, and developments in the area of DBS for the treatment of memory and affective disorders are creating new avenues for the management of post-concussive syndrome. Ultimately, these advances in neuromodulation strive to improve outcomes in mTBI and substantially impact the lives of patients and families suffering from this condition.

#### AUTHOR CONTRIBUTIONS

FG, JP, JS and JPM wrote and edited the manuscript.

attachment protein receptor complex formation and alters synaptic vesicle distribution in the hippocampus. J. Neurotrauma 33, 113–121. doi: 10. 1089/neu.2014.3839

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

Copyright © 2016 Girgis, Pace, Sweet and Miller. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution and reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Facilitating Mitochondrial Calcium Uptake Improves Activation-Induced Cerebral Blood Flow and Behavior after mTBI

#### Madhuvika Murugan1 , Vijayalakshmi Santhakumar 2 and Sridhar S. Kannurpatti 1 \*

*1 Department of Radiology, Rutgers New Jersey Medical School, Newark, NJ, USA, 2 Department of Pharmacology, Physiology and Neuroscience, Rutgers New Jersey Medical School, Newark, NJ, USA*

Mild to moderate traumatic brain injury (mTBI) leads to secondary neuronal loss via excitotoxic mechanisms, including mitochondrial Ca2+ overload. However, in the surviving cellular population, mitochondrial Ca2+ influx, and oxidative metabolism are diminished leading to suboptimal neuronal circuit activity and poor prognosis. Hence we tested the impact of boosting neuronal electrical activity and oxidative metabolism by facilitating mitochondrial Ca2+ uptake in a rat model of mTBI. In developing rats (P25-P26) sustaining an mTBI, we demonstrate post-traumatic changes in cerebral blood flow (CBF) in the sensorimotor cortex in response to whisker stimulation compared to sham using functional Laser Doppler Imaging (fLDI) at adulthood (P67-P73). Compared to sham, whisker stimulation-evoked positive CBF responses decreased while negative CBF responses increased in the mTBI animals. The spatiotemporal CBF changes representing underlying neuronal activity suggested profound changes to neurovascular activity after mTBI. Behavioral assessment of the same cohort of animals prior to fLDI showed that mTBI resulted in persistent contralateral sensorimotor behavioral deficit along with ipsilateral neuronal loss compared to sham. Treating mTBI rats with Kaempferol, a dietary flavonol compound that enhanced mitochondrial Ca2+ uptake, eliminated the inter-hemispheric asymmetry in the whisker stimulation-induced positive CBF responses and the ipsilateral negative CBF responses otherwise observed in the untreated and vehicle-treated mTBI animals in adulthood. Kaempferol also improved somatosensory behavioral measures compared to untreated and vehicle treated mTBI animals without augmenting post-injury neuronal loss. The results indicate that reduced mitochondrial Ca2+ uptake in the surviving populations affect post-traumatic neural activation leading to persistent behavioral deficits. Improvement in sensorimotor behavior and spatiotemporal neurovascular activity following kaempferol treatment suggests that facilitation of mitochondrial Ca2+ uptake in the early window after injury may sustain optimal neural activity and metabolism and contribute to improved function of the surviving cellular populations after mTBI.

Keywords: TBI, mitochondria, CBF, dietary, calcium uniporter, kaempferol, whisker barrel, oxidative metabolism

#### Edited by:

*Akiva Cohen, University of Pennsylvania/Children's Hospital of Philadelphia, USA*

#### Reviewed by:

*Leslie S. Satin, University of Michigan Medical School, USA Han-Chiao Isaac Chen, University of Pennsylvania, USA*

> \*Correspondence: *Sridhar S. Kannurpatti<EMAIL_ADDRESS>
Received: *24 July 2015* Accepted: *19 February 2016* Published: *08 March 2016*

#### Citation:

*Murugan M, Santhakumar V and Kannurpatti SS (2016) Facilitating Mitochondrial Calcium Uptake Improves Activation-Induced Cerebral Blood Flow and Behavior after mTBI. Front. Syst. Neurosci. 10:19. doi: 10.3389/fnsys.2016.00019*

# INTRODUCTION

Preclinical systems level studies of TBI during development can be approached using neuroimaging combined with behavioral assessment from the same subjects as currently done in younger TBI patients. As the number of children and young adults seeking care for TBI continues to increase, developmental aspects of mild to moderate traumatic brain injury (mTBI) which lead to disruption of neuronal circuits and formation of maladaptive circuits with aberrant behavioral responses (Pitkanen et al., 2009; McNamara et al., 2010; Li et al., 2015) need to be characterized. As brain activity related to behavioral tasks such as cognition, sleep, sensory processing, and motor responses result from spatially segregated neuronal assemblies (Harris, 2005), spatiotemporal mapping of stimulus-induced hemodynamic representatives of neural activity are significant for systems-level evaluation of neural plasticity (Logothetis et al., 2001). Hence functional Magnetic Resonance Imaging (fMRI) methods that enable spatiotemporal mapping of brain activity through changes in cerebral blood oxygenation, water diffusion or cerebral blood flow (CBF) are increasingly used to assess TBI patients (Kim et al., 2010; Slobounov et al., 2010; Johnson et al., 2012). Functional Laser Doppler Imaging (fLDI) is a preclinical neuroimaging method enabling high spatial resolution (100µm) mapping of stimulus inducedneural activity represented by changes in microvascular CBF (Kannurpatti and Biswal, 2004, 2006). Accurate spatiotemporal mapping of neural events is limited by the sensitivity to microvascular signals compared to the contribution from large vessels. This ability to selectively discriminate signal change from arterioles and capillary compartments (closest in range to neural activity) is defined as functional resolution (Truong and Song, 2009). As shown by our previous studies, fLDI provides excellent functional resolution (Kannurpatti and Biswal, 2011) and complements preclinical fMRI for brain spatiotemporal mapping (Sanganahalli et al., 2013a). In this study, we used fLDI in a similar configuration that provided high microvascular specificity to detect activity close to neural ensembles (Kannurpatti and Biswal, 2011). Neural activity change in response to whisker stimulation between sham and mTBI animals was evaluated by assessing the spatiotemporal change in microvascular CBF in the somatosensory barrel field (S1BF), a region well characterized by electrophysiological and optical mapping studies of neuroplasticity (Welker, 1976; Diamond et al., 1993; Dowling et al., 1996; Kannurpatti and Biswal, 2011).

Secondary neuronal damage due to excitotoxic mechanisms and mitochondrial Ca2+ overload by increased Ca2+ influx is inevitable after TBI (Yokobori et al., 2014). However, the developing brain is relatively resistant to excitotoxic damage including relatively lesser excitotoxic Ca2+ dependent mitochondrial oxidative stress when compared to the adult (Kannurpatti et al., 2004). Hence the surviving neurons and their relatively Ca2+ resistant mitochondria may influence TBI prognosis during the developmental years. Human mitochondrial energy metabolism and Ca2+ uptake capacities diminish after TBI (Verweij et al., 2000), making the potentially surviving mitochondrial populations a critical therapeutic target (Giorgi et al., 2012). Slowing of mitochondrial Ca2+ uptake reduces neuronal activity in vivo (Mathiesen et al., 2011; Fluegge et al., 2012; Sanganahalli et al., 2013a,b) along with decreased oxidative energy metabolism (Kann et al., 2003; Mathiesen et al., 2011; Sanganahalli et al., 2013a,b). Hence we tested the impact of facilitating Ca2+ influx, known to boost neuronal electrical activity, and oxidative metabolism (Sanganahalli et al., 2013a), on long-term outcome after mTBI in developing rats. Kaempferol, a dietary flavonol compound and a known enhancer of the mitochondrial Ca2+ uniporter channel (mCU) was used to facilitate mitochondrial Ca2+ influx after mTBI. Kaempferol treatment improved post-injury behavioral outcomes and reduced alterations in neural circuit activity without augmenting mTBI-induced neuronal loss. The results indicate that facilitation of mitochondrial Ca2+ influx, known to boost ongoing neuronal electrical activity, and metabolism, may be a primary mechanism sustaining normal neural circuit functions in the surviving neuronal population leading to improved outcomes after mTBI.

# METHODS

# Animals

Male Sprague-Dawley rats (23–24 days old; weighing 60–80 g) were procured from Charles River Laboratories, Wilmington, MA, USA and used in this study. Rats were housed in pairs under controlled conditions. All procedures were carried out in accordance with the institutional guidelines and approved by the Institutional Animal Care and Use Committee of Rutgers-New Jersey Medical School and ARRIVE guidelines.

# Lateral Fluid-Percussion Injury

Lateral fluid percussion injury (FPI) is a mixed injury model (both focal and diffuse) leading to axonal, somal, and microvascular swelling. This leads to tissue distortion and axonal shearing both proximal and distal to the injury location (Lindgren and Rinder, 1965; Dixon et al., 1987; McIntosh et al., 1989). In comparison to a more focal cortical contusion injury (CCI), lateral FPI induces significant cell death in distant regions even as far as the corresponding contralateral cortex (Peterson et al., 2015), and neurophysiological changes in deeper regions such as the hippocampus (Gupta et al., 2012; Li et al., 2015; Pang et al., 2015). In the current mTBI model, lateral FPI was performed in rats when they reached an age of 25–26 days according to the procedures established previously in our laboratory (Gupta et al., 2012; Li et al., 2015). The injury age (P25- P26) in the current rat model corresponded to the developmental time range of approximately a year in humans (Sengupta, 2013) and can be classified as a developmental mTBI model.

In brief, rats were anesthetized with ketamine (80 mg/kg i.p) xylazine (10 mg/kg i.p) and positioned on a stereotaxic frame. Ketamine provided rapid surgical plane anesthesia followed by quick recovery for the survival surgery procedure. A 3 mm craniotomy was performed on the left side of the skull −5 mm posterior to the bregma and 3 mm lateral to the sagittal suture keeping the dura intact. A Luer-Lock syringe hub was glued surrounding the exposed dura using a cyanoacrylate adhesive. After 24 h, injury was induced by attaching the Luer-Lock hub of an isoflurane-anesthetized rat to the FPI device (Virginia Commonwealth University, VA, USA). A pendulum drop delivered a brief 20 ms impact on the intact dura. The impact pressure was measured by an extra-cranial transducer and controlled between 2.0 and 2.2 atm. For sham group (n = 5), the rats were anesthetized and attached to the FPI device without the pendulum drop. Righting reflex recovery time was recorded after injury in all animals and was in the range of 5.352 ± 2.97 min. Sham and injured animals were monitored within their cage environment on a daily basis.

## Drug Treatments

A subset of mTBI rats (n = 5) were administered 3 doses of kaemferol (1 mg/Kg i.p) diluted in saline containing 10% dimethyl sulfoxide (DMSO). The first dose was delivered 1 h following the FPI and the other doses on consecutive days. Another subset of mTBI-rats (mTBI + vehicle, n = 4) was administered 3 doses of vehicle (saline with 10% DMSO). The kaempferol dose level used (1 mg/Kg i.p) was significantly lower than the dose level (20 mg/Kg i.p) shown to have significant antioxidative effects in vivo (Lagoa et al., 2009). Intraperitoneal administration was adopted due to several benefits such as gradual absorption and broader time-peaks of plasma concentrations (Barve et al., 2009). Oral administration was not preferred due to concerns of intestinal microflora-related metabolism of the compound (Vissiennon et al., 2012). As the average half life of intravenous delivered kaempferol was 6 h and average lifetime 24 h (Barve et al., 2009), we designed the dosing regimen in 24 h intervals. 1 mg/Kg intravenous infusion of kaempferol can produce an estimated peak plasma concentration of ∼250µM/L, which is known to boost neural activity and oxidative metabolism in vivo (Sanganahalli et al., 2013a,b). Hence the intraperitoneal route of 1 mg/Kg kaempferol used would lead to plasma concentrations comparable to that achieved by human dietary intake of flavonol rich foods (DuPont et al., 2004).

#### Behavioral Testing

Following mTBI, all animals exhibited neurological deficits as assessed by a forelimb flexion test (data not shown). In order to access whisking-related sensorimotor impairment, a specific behavioral test namely the whisker stimulation-induced motor response (WSIMR) was performed at 1, 3, 7, 14, and 21 days following mTBI. We developed the WSIMR as a modification of the whisker-stimulated forelimb placement test (Schallert et al., 2000), since the forelimb placement responses in younger rats (23–30 days range) were not fully mature. During the WSIMR test, the animal was placed in a test cage and the whiskers on one side were gently stroked multiple times (10 stroke maximum) at a frequency ∼5 Hz in a caudo-rostral direction with an applicator stick. The animal did not have any initial visual cue to the approaching stroke from the experimenter. Ten trials with an inter-trial gap of 30 s were performed and repeated 5 times on each side of the animal with a repetition gap of 2 min. Whisker deflections during the behavioral experiments were performed by a single experimenter to maintain consistency of the force, frequency, and angle of stimulation. Motor responses such as avoidance of stick and the proficiency of reaction to each stroke was noted and scored on a scale of 0–3: Active avoidance by running away = 3, quick head movement away from the stroking stick = 2, slower head movement away from the stroking stick = 1 and no reaction to stroking = 0. A cumulative score was obtained after averaging all measurements from each side of whisker stroking.

# Functional Laser Doppler Imaging (fLDI)

We performed initial pilot experiments in a different set of animals (not included in this study) adopting the mTBI experimental approach to enable fLDI measurements. Removal of the luer-lock cap from the skull was piloted in three animals post-injury which led to increased thickness of the skull around and adjoining the capped area. During the preparation of the thinned skull, the texture of the skull area was very flakey with presence of extra intracranial blood vessels which resulted in excessive bleeding and comparably different quality of ipsilateral-contralateral window preparations. Hence cap removal immediately after injury was discontinued. In the next pilot, we retained the cap after injury using normal cyanoacrylate adhesive strength. However, cap removal in the adult animal prior to fLDI measurements became difficult. Hence a gradual reduction of the cyanoacrylate adhesive strength was tested and an appropriate concentration enabling easy cap removal prior to the thinned skull window preparation was used in all animals included within the study.

Rats after 42–48 days following mTBI (P67-P73) and agematched shams were anesthetized with urethane 1.2 g/kg i.p and placed on a warm pad for fLDI measurements. Animal core temperature was monitored and maintained at 37 ± 0.5◦C with a rectal probe and homeothermic blankets (Baxter K-MOD100, Gaymar Industries, USA). After endotracheal intubation and mechanical ventilation, end-tidal CO2 (ETCO2) and arterial oxygen saturation (SPO2) were continuously monitored throughout the experimental duration (8100 Poet-Plus Vital Signs Monitor, Criticare, WI, USA). ETCO2 levels were maintained between 32 and 35 mmHg through appropriate ventilator adjustments. Animals received a bolus of gallamine triethiodide (250 mg/kg, i.p) after the start of ventilation to achieve muscle relaxation. To enable fLDI measurements animals were positioned on a stereotaxic frame with the scalp retracted from the frontoparietal cortex by a dorsal midline incision to expose the cranium. The Luer-Lock syringe hub implanted for the induction of mTBI was gently dislodged and removed from the skull using a pair of scissors. Inflammation was evaluated visually by ensuring that the fur growth was normal with no scalp wounds around the cap. After the midline scalp incision and cap removal for fLDI measurement, the exposed skull area beneath the cap was examined to ensure that the prior craniotomy area was dry and closed by skull and tissue growth. On inspection in almost all the rats imaged, the skull beneath the cap region had completely grown and closed at 42–48 days post-injury. There was no inflammation in any of the cap-retained animals during observations prior to the fLDI measurements. Furthermore, no inflammation was observed in two animals from which the cap fell off during the duration of housing for the experiments. The temporalis muscle was disconnected from the cranium and a window of skull area measuring 5 × 5 mm centered at −2 mm and +5 mm lateral to the bregma enclosing the somatosensory barrel field (S1BF) on either hemisphere was thinned to translucency using an aircooled dental drill. Functional imaging using fLDI was carried out using a Moor LDI scanner (Moor Instruments, Sussex, UK) as described previously (Kannurpatti and Biswal, 2011). Briefly, a beam of laser from a low power (2 mW ± 20%, 632.8 nm) He-Ne source scanned the thinned skull window in a raster pattern and the scattered Doppler shifted beam was collected and focused onto a photo-detector. The laser Doppler flux signal which was proportional to the tissue perfusion/CBF at each measurement point was calculated and stored in the resulting image pixels. Distance between the scanner head and the thinned skull was 23 cm leading to an in-plane resolution of about 120 × 120 µm2 with fLDI images of 42 × 54 pixel matrix obtained covering a field of view of 5 × 6.5 mm2 . Functional images were obtained from the somatosensory field of view on both hemispheres at a rate of 16 sec/image and a dead time of 4 s between each image. The current configuration was optimized for maximum microvascular specificity to detect activity close to neural ensembles (Kannurpatti and Biswal, 2011). Urethane was used due to its minimal cardiovascular effects, respiratory, spinal reflexes and confounds on neurovascular coupling (Masamoto and Kanno, 2012). Urethane also provided stable anesthesia over longer periods (several hours) (Sanganahalli et al., 2013a) and was best suited for the imaging procedure using fLDI.

## Whisker Stimulation during fLDI Acquisition

Whiskers from B-E rows and the stradlers α, β, γ, and δ were glued together using a masking tape of rectangular area of 0.5 × 0.6 cm2 to include up to 5 arcs. Whisker deflections were carried out in the caudo-rostral direction using air puffs of 15 ms duration from a pressurized air source delivering a pressure of 60 psi through a solenoid (James Long Company, Caroga Lake, NY) controlled by a A310 pulse generator (WPI instruments, Sarasota, FL). Air puffs were delivered through a short plastic tubing of length 50 cm connected to a standard 1 ml pipette tip with sufficient velocity within the duration of 15 ms that deflected the whiskers by ∼40◦ in the rostro-caudal direction as characterized previously in our laboratory (Kannurpatti and Biswal, 2011). Deflected whiskers returned back to their original position before the delivery of the next air puff for the stimulus frequency of 5 Hz used in the current experiments. Whisker stimulation was performed on both sides of the animal in a sequential manner with contralateral whisker stimulation matched by ipsilateral fLDI measurement.

# Analysis and Statistical Parametric Mapping of fLDI Image Data Sets

The Moore LDI scanner acquisition program processed the blood flow related signal derived from the Doppler spectrum via an analog to digital (A/D) converter. The light intensity and the blood flux signals from the A/D converter was compensated for noise effects and color-coded in arbitrary flux units (relative perfusion units; rpu) before image reconstruction. Further data processing and statistical analysis was performed on the reconstructed image data using custom made programs developed in our laboratory (MATLAB, Natik, MA). Each experimental run contained 3 periods of whisker stimulation interspersed with 4 periods of rest representing an "ON/OFF" cycle along a block of 25 images. Experimental runs were repeated 5 times on each cortical field of view and averaged. An idealized box-car function representing the "ON/OFF" cycle of whisker stimulation was used as the reference waveform. The stimulus reference function was statistically cross correlated with the fLDI flux signal on a pixel-wise basis to determine activation. Cross correlation estimates the degree to which any two time-dependent signals are correlated. The distribution of the cross correlation coefficient throughout the image when no stimulus was presented (null distribution) was approximately a normal distribution and used to determine the threshold cross correlation coefficient value. A threshold of 1.96 times the standard deviation for the null distribution corresponding to an error probability of P < 0.05 was used for determining the activation threshold that corresponded to a correlation coefficient value of 0.22. Activated pixels in the functional maps were further controlled for false positives through a family-wise error control using a cluster size threshold of 0.04 mm2 . Functional images were color coded according to the correlation coefficient values and overlaid on the gray scale baseline CBF images obtained by averaging all resting period images. The functional images (color overlay) and the baseline CBF images have identical spatial resolution.

#### Histology

After the terminal fLDI experiments, rats were perfused with 4% paraformaldehyde in PBS, pH 7.4. Brains were removed and postfixed for 24 h in the same paraformaldehyde solution. Following post-fixation, brains were cryoprotected in 30% sucrose in PBS for 24–48 h. Coronal sections (40µm-thickness) were cut at approximately three horizontal planes with respect to the bregma (−0.1, −2.0, and −4.0 mm) using a Leica cryostat and mounted on gelatin-coated glass slides for histological analyses. The sections were stained with cresyl violet to mark the neurons. Brain tissue sections from both the injured-ipsilateral hemisphere and the uninjured-contralateral hemisphere were used for histological evaluations. To assess neuronal cell survival, six random counting frames/animal (three in each hemisphere), were considered in three distinct regions depending upon their lateral distance and depth from the injury site. (1) layer-IV of the S1BF region (−0.1 mm to bregma), (2) Layer-II and III of the S1BF region (−2 mm to bregma), and (3) hippocampal hilus (−4 mm to bregma). Neuronal cell bodies were counted from each frame obtained at a magnification of 40x using an oil immersion objective on an inverted optical microscope.

#### Statistics

Data are presented as mean ± SEM for the behavioral results and mean ± SD for all others. WSIMR test on different time points were analyzed using ANOVA with post-hoc pair-wise comparisons to assess differences in performances between ipsilateral and contralateral-side. For fLDI and histology measurements, average from each group was calculated and the significant differences between the sham and treatment groups were analyzed using ANOVA followed by post-hoc HSD test or a paired t-test. A P < 0.05 was considered to be statistically significant.

# RESULTS

Using the lateral fluid percussion rat model of TBI (Gupta et al., 2012), mTBI was performed on the hippocampal plane on the left hemisphere at −5 mm to the bregma (**Figures 1A,B**). This model, producing both focal and diffuse injuries, is a good representative of mild to moderate injuries in humans and well characterized in its functional impact on hippocampal (Gupta et al., 2012) and somatosensory neural circuits in rats (Lifshitz and Lisembee, 2012).

Sensorimotor behavior was assessed using the WSIMR test in order to correlate the S1BF circuit activity mapped using fLDI. Scores were assigned depending upon the vigor of face withdrawal when the whiskers on each side were stroked. In sham animals, a progressive improvement was observed in the WSIMR scores as rats reached a young-adult age of 1.5 months (**Figure 2A**). In mTBI animals, there was a significant ipsilateral-contralateral asymmetry in addition to a time-dependent improvement in WSIMR scores (**Figure 2B**). Ipsilateral-contralateral asymmetry in behavioral performance was significant at all time points up to 21 days post-injury (**Figure 2B**) without any change in contralateral performance when compared to sham (**Figures 2A,B**; ANOVA). As WSIMR testes higher order cortical functional integration requiring the engagement of sensorimotor circuits to elicit behavioral responses, the progressive decrease in the ipsilateral-contralateral asymmetry during the 21 day period after injury indicates that recovery in higher order brain functions may continue through the late stage (>21 days post-injury). In two additional groups of mTBI animals, one was treated with the mitochondrial Ca2+ uniporter mCU channel enhancer, kaempferol, during the early window (0–3 days post injury) while the other received vehicle treatment. Kaempferol (1 mg/Kg, i.p) or vehicle (saline with 10% DMSO) treatments were performed at 1 h, 24 h, and 48 h after injury. Behavioral assessment revealed that kaempferol treatment significantly improved WSIMR scores after and abolished the ipsilateral-contralateral asymmetry (**Figure 2D**; ANOVA) compared to vehicle treated rats which showed persistent statistically significant ipsilateralcontralateral asymmetry (**Figure 2C**; ANOVA, P < 0.01; Post-hoc Tukey's HSD test). The brief 3 day post-injury kaempferol treatment was able to completely normalize the ipsilateral-contralateral asymmetry around 14 days post-injury and thereafter (**Figure 2D**).

After completion of behavioral monitoring, each animal underwent functional imaging of CBF using fLDI 42–48 days post-injury (P67-P73). fLDI spatiotemporal mapping was performed during the adult stage as the immature somatosensory cortical barrel representation continues to develop exhibiting dynamic changes in metabolism, electrical activity, neural cell

of view on either hemisphere covering the somatosensory whisker barrel representation. (C) Description of the integrated experimental design to assess mTBI prognosis after mitochondrial treatment through behavioral, fLDI, and histological markers.

types, and transmitter systems which are complete and stable after 2 months of age. As detailed in the methods section, FPI models produce neuronal damage spread across a wider distance in the brain with cell death occurring even in the contralateral cortex (Pang et al., 2015; Peterson et al., 2015). The somatosensory barrel field (S1BF) chosen for functional imaging of brain circuit activity to correlate with whisker deflection-induced sensorimotor behavior in the intact animal model was distal to the site of impact. fLDI imaging field of view was placed on the S1BF region centered −2.5 mm caudal, 5 mm lateral to the Bregma and was ∼3 mm away from the site of fluid percussion impact (**Figure 1**). fLDI flux value in a pixel represents the product of the number of red blood cells and their velocity. Hence larger vessels with relatively higher CBF show larger fLDI flux values (>500 rpu) relative to smaller vessels such as arterioles and venuoles (<500 rpu), which can be distinguished in the baseline CBF images (**Figures 3A,D**). CBF change induced by underlying neural activity and oxygen metabolism forms the basis of spatiotemporal functional mapping of brain activity (Logothetis et al., 2001). Brain activation in response to a stimulus is determined by statistical parametric mapping of the hemodynamic response on a pixel-by-pixel basis. We performed statistical parametric mapping (Methods Section Analysis and Statistical Parametric Mapping of fLDI Image Data Sets) to detect whisker stimulation-induced CBF changes within the somatosensory cortex. Increases in CBF response yielded positive correlation coefficients whereas CBF decreases yielded negative correlation coefficients. Pixels with correlation coefficients ≥0.22 (positive CBF activation) or ≤ −0.22 (negative CBF activation) corresponding to a significance threshold of P < 0.05 were considered activated, representing the stimulusinduced cortical activation (color overlay in **Figures 3B,E**). Spatiotemporal mapping of hemodynamics in response to evoked stimuli can be positive or negative in direction. However, the negative hemodynamic responses cannot always be attributed to deactivation as CBF decreases can occur during various situations such as neuronal deactivation (Shmuel et al., 2002), blood diversion to an adjacent active cortical region (Harel et al., 2002; Kannurpatti and Biswal, 2004), or neurovascular uncoupling due to pathology (Schridde et al., 2008). Hence the positive and negative spatiotemporal CBF changes can be more appropriately defined as "activation" with respect to the resting state. Positive CBF responses contralateral to the injury, was similar to that observed in sham animals, but qualitatively appeared to be less focal than sham (**Figures 3A–C**). Small clusters of negative CBF changes in response to evoked stimulus accompanied the positive CBF responses (**Figure 3B**). The sparse negative CBF responses, usually in small clusters, originated from higher baseline flux pixels (over large vessels >500 rpu), and consistent with our previous fLDI mapping studies on normal rats. These small clusters of negative CBF change can

be attributed to a passive upstream decrease in CBF in certain large vessels diverting blood to adjacent cortical areas (Harel et al., 2002; Kannurpatti and Biswal, 2004). Activation area was estimated as the product of pixel area and the number of activated pixels in response to whisker stimulation. In the mTBI animals, fLDI activation was asymmetric where the injured ipsilateral hemisphere's positively activated area decreased by about 50% (**Figures 3D–F**). A prominent negative CBF activation with large clusters of activated pixels predominantly over microvasculature with low baseline fLDI flux values (<500 rpu) was observed in the mTBI animals (**Figure 3E**). These larger clusters of negative CBF responses from the microvascular pixels arise due to vasoconstriction originating from suppressed neuronal activity (Devor et al., 2007) and are distinct from the small negative CBF clusters observed mostly over large vessels in sham animals. The large cluster negative CBF responses from the microvascular pixels indicated substantially reduced excitability within the S1BF region ipsilateral to the injury in the mTBI animals. Such an alteration of stimulus-induced ipsilateral CBF response in mTBI animals may have multiple origins including impaired excitatory neuronal electrical activity, enhanced neuronal inhibition (Johnson et al., 2014), impaired neurovascular coupling and/or impaired blood perfusion in the injured and adjoining regions (Hayward et al., 2010). Overall, the results highlight mTBI induced neurodegeneration with sustained behavioral deficit and altered stimulus-induced spatiotemporal CBF responses indicative of increased neuronal inhibition and/or neurovascular uncoupling.

In separate mTBI animal groups the impact of mitochondrial Ca2+ uptake facilitation was assessed using fLDI after kaempferol or vehicle treatments. fLDI responses in vehicle treated mTBI animals (**Figures 4A–C**) was similar to that observed in the untreated mTBI animals where the stimulus-induced positive CBF response was significantly asymmetric (**Figures 4A–C**). Stimulus-induced positive CBF response not only improved in the kaempferol-treated animal group but the asymmetry in the ipsilateral-contralateral neurovascular activity was also significantly reduced (**Figures 4D–F**) compared to vehicle-treated mTBI animals (**Figures 4A–C**). No prominent microvascular-negative CBF responses were observed in the ipsilateral hemisphere after kaempferol treatment.

As brain perfusion is diminished chronically in TBI patients matching spatially with regions of neural functional deficits (Kim et al., 2010), we assessed the impact of mTBI on the baseline CBF in the developmental mTBI model used in this study. Baseline CBF was averaged over all pixels within the image field of view during the resting condition (in the absence of whisker stimulation). Baseline CBF significantly decreased across the ipsilateral cortex compared to contralateral (**Figures 5A,B**) accompanied by notable vascular rearrangements

within the ipsilateral cortical hemisphere (**Figure S1**). These results are consistent with earlier MRI findings of reduced CBF accompanied by increased vascular density in the peri-lesional cortex months after the injury (Hayward et al., 2010). Kaempferol treatment, however, did not lead to any improvement in the baseline CBF decreases observed after mTBI (**Figure 5D**).

To assess neural survival after injury, coronal sections of fixed brain tissue were stained with cresyl violet to evaluate neuronal density in the distal regions such as layer IV of S1BF and hippocampal hilus and S1BF layers II-III stereotaxically closer to the injury impact site. **Figures 6A–C** show representative cresyl violet stained section of a typical mTBI brain across both hemispheres from the distal and proximal regions relative to the site of impact Average neuronal counts obtained from multiple animals indicated a significant neuronal loss within the ipsilateral hippocampal hilus (**Figure 6F)**, typical to this injury model (Gupta et al., 2012; Li et al., 2015). Cortical layers of the S1BF region distal and proximal to the injury site also indicated significant neuronal loss in the ipsilateral hemisphere compared to contralateral (**Figures 6D,E**). No significant difference in neuronal cell density was observed between the ipsilateral and contralateral hemispheres of sham animals. However, neuronal density in the ipsilateral cortex in all injured groups significantly reduced compared to shams (**Figure 6**; paired t-test). Kaempferol and vehicle treatments did not induce further changes in neuronal density in injured cortex either proximal or distal to the injury site (**Figure 6**; ANOVA).

#### DISCUSSION

Using a systems approach, the current study assessed mTBI outcomes in developing rats with clinically relevant neuroimaging markers. As mitochondrial Ca2+ uptake capacity is known to decrease after TBI along with impaired oxidative metabolism, neural loss, and neurovascular changes (Verweij et al., 2000; Pandya et al., 2007; Hayward et al., 2010; Lifshitz and Lisembee, 2012), we tested if mitochondrial Ca2+ influx facilitation, known to boost neuronal electrical activity and metabolism (Sanganahalli et al., 2013a), will impact mTBI prognosis.

Kaempferol, a dietary flavonoid compound and a specific enhancer of the mitochondrial Ca2+ uniporter channel (mCU) (Montero et al., 2004), capable of crossing the blood brain barrier and diffusing freely into cells and organelles (Liu et al., 2006), was administered in a dose relevant to dietary intake. Dietary therapy is recognized to be advantageous to patients recovering from mild TBI without necessitating hospitalization and intravenous access for drug administration and considered important in preclinical treatment studies of TBI (Elkind et al., 2015). Based on previous dose-dependent studies determining that

to the site of injury. (A) Sham, (B) mTBI, (C) mTBI animals treated with vehicle, and (D) mTBI animals treated with kaempferol. Data represent group level Mean ± SD, sham group; *n* = 3 and mTBI group; *n* = 6 vehicle group; *n* = 3, kaempferol group; *n* = 6. Significantly different, one-way ANOVA, *Post-hoc* Tukey's HSD test. mTBI (Ipsi) vs. (Con); *P* < 0.004 and *F* = 11.88. mTBI + vehicle (Ipsi) vs. (Con); *P* < 0.04 and *F* = 11.84. mTBI + kaempferol (Ipsi) vs. (Con); *P* < 0.04 and *F* = 6.57. No significant differences were observed in the baseline CBF levels within the same hemispheres when compared between the mTBI + vehicle and mTBI + kaempferol groups.

1–3 mg/Kg kaempferol delivered intravenous could enhance the baseline spontaneous/evoked neural activity and CBF responses (Sanganahalli et al., 2013a,b), a similar dose range was used with daily intraperitoneal injections of 1 mg/Kg. The current treatment can be expected to produce plasma concentrations significantly lower than 250µM/L and comparable to ranges of kaempferol concentrations produced by human dietary intake of flavonol rich foods (DuPont et al., 2004). All animals receiving kaempferol survived with no adverse effects during the treatment which was 1000 times lower than the suggested safety limits of 1000 mg/Kg (Shih et al., 2013).

States of impaired mitochondrial Ca2+ uptake capacity has the potential to disrupt normal neural signaling and shown to diminish both spontaneous and activation-induced CBF and fMRI-BOLD functional responses in the working brain in vivo (Kannurpatti and Biswal, 2008; Sanganahalli et al., 2013a). Early post-injury brain functions are suboptimal and may coincide with the window of neuronal death/survival events (Lifshitz and Lisembee, 2012). Eelectroencephalography (EEG) and fMRI studies of humans in the early stages after mTBI indicate diminished neural circuit activity (Gosselin et al., 2011). The early-stage evolution of brain events may critically depend on mitochondrial functions within the surviving neuronal populations (Vagnozzi et al., 2007; Calderon-Cortes et al., 2008; Lifshitz and Lisembee, 2012; Sauerbeck et al., 2012; Bartnik-Olson et al., 2014). The current in vivo results reflect robust changes in cortical excitation where mTBI-induced brain functional activation showed decreased stimulus-evoked positive CBF responses and increased stimulus-evoked negative CBF responses (**Figures 3D–F**). Stimulus-evoked negative CBF responses were prominent on the injured hemisphere indicating a post-traumatic alteration in the underlying neural circuit activity or their neurovascular coupling, which may not be a desirable outcome in TBI patients. The prominent negative CBF responses in mTBI animals (large clusters of activated area) could be driven by suppressed neuronal activity within the somatosensory cortex (Shmuel et al., 2002) or neurovascular uncoupling due to pathology (Schridde et al., 2008). This is distinct from the smaller negative CBF response clusters observed in sham animals which likely reflects a passive reduction in CBF in upstream vessels of larger caliber (Kannurpatti and Biswal, 2004).

Diminished oxidative metabolism measured by oxygen utilization (indicative of mitochondrial dysfunction) has been observed in pediatric TBI patients (Ragan et al., 2013). As demonstrated by our earlier studies, diminished oxidative metabolic state achieved by inhibiting mitochondrial Ca2+ uptake after treatment with the mCU inhibitor Ru360 attenuated stimulus-evoked somatosensory neuronal activity and CBF responses (Kannurpatti and Biswal, 2008; Sanganahalli et al., 2013a). In contrast to Ru360, kaempferol treatment was shown to have the opposite neurophysiological effects of enhanced oxidative metabolism and neuronal activity (Sanganahalli et al., 2013a). In the current study where mTBI rats were treated with kaempferol, the brief and early mCU enhancement resulted in sustained beneficial effects eliminating inter-hemispheric asymmetry in the stimulus-evoked CBF response into adulthood. Additionally, the treatment reduced changes in neurovascular activity and activation-induced negative CBF responses across the injured ipsilateral hemisphere (**Figures 4D–F**). Kaempferol treatment improved somatosensory behavioral performance as early as 7 days after mTBI and the effects were sustained during the entire duration of testing up to 21 days after mTBI (**Figure 2D**). Hence, mitochondrial Ca2+ uptake impairment may be an early event within the mitochondrial populations after developmental mTBI and a potential early treatment target. Since inter-hemispheric asymmetry in somatosensory behavior was eliminated after kaempferol treatment by 7 days after mTBI and was sustained at 21 days post injury, no further behavioral testing was conducted beyond 21 days. Development of inter-hemispheric asymmetry in the spatiotemporal CBF responses measured by fLDI and restoration of inter-hemispheric symmetry by kaempferol treatment observed over 48 days after injury parallels the mTBI-induced behavioral impairment and its mitigation by kaempferol treatment 21 days after injury.

No significant neuronal survival differences were observed between kaempferol and vehicle treatments (**Figure 6**; ANOVA), indicating that mitochondrial Ca2+ uptake facilitation effects of the drug did not trigger adverse mitochondrial Ca2+ deregulation events including mitochondrial permeability transition. As the

FIGURE 6 | Cresyl violet histological staining of coronal sections of post-mortem brains after terminal fLDI imaging. (A–C) Representative sections over the S1BF and hippocampal hilar regions in different magnifications. (D–F) Number of stain positive neuronal cell bodies estimated per frame from different layers of the S1BF region and hippocampal hilus. Data represent group level Mean ± SD, sham group; *n* = 3 and mTBI group; *n* = 6 mTBI + vehicle group; *n* = 3, mTBI + kaempferol group; *n* = 6. Paired *t*-test comparisons between ipsilateral and contralateral neuronal counts indicated significant neuronal loss in the ipsilateral cortex and no significant protective effect of kaempferol treatment. Ipsilateral neuronal counts tested across all groups with a one-way ANOVA followed by a *post-hoc* Tukey's HSD test indicated significant differences between sham and all injured groups whereas no significant difference was observed in the contralateral neuronal counts. Kaempferol treatment after mTBI did not change neuronal survival in the adult. (S1BF layer II-II; ANOVA; *F* = 9.82; Sham vs. mTBI; *P* < 0.01; Sham vs. mTBI + vehicle; *P* < 0.01; Sham vs. mTBI + kaempferol; ns; mTBI vs. mTBI + vehicle; ns; mTBI vs. mTBI + kaempferol; *P* < 0.05, mTBI + vehicle vs. mTBI + kaempferol; ns. S1BF layer IV; ANOVA; *F* = 7.01; Sham vs. mTBI; *P* < 0.01; Sham vs. mTBI + vehicle; *P* < 0.01; Sham vs. mTBI + kaempferol; *P* < 0.01; mTBI vs. mTBI + vehicle; ns; mTBI vs. mTBI + kaempferol; ns, mTBI + vehicle vs. mTBI + kaempferol; ns. Hippocampal hilus; ANOVA; *F* = 20.18; Sham vs. mTBI; *P* < 0.01; Sham vs. mTBI + vehicle; *P* < 0.01; Sham vs. mTBI + kaempferol; *P* < 0.01; mTBI vs. mTBI + vehicle; ns; mTBI vs. mTBI + kaempferol; *P* < 0.05, mTBI + vehicle vs. mTBI + kaempferol; ns).

developing brain is relatively resistant to excitotoxic Ca2+ dependent mitochondrial oxidative stress and mitochondrial Ca 2+ overload compared to the adult (Kannurpatti et al., 2004; Robertson et al., 2004), mitochondrial Ca2+ uptake facilitation treatments may have beneficial effects of improved neuronal activity with minimal impact on detrimental mitochondrial Ca2+ dependent events. Hence, mitochondrial Ca2+ influx facilitation in the immature brain may prove to be beneficial as it maintained optimal neuronal activity in the surviving neurons with no exacerbation of secondary neuronal death. Mitochondrial Ca2+ uptake enhancement also did not improve the baseline CBF decreases observed after mTBI (**Figure 5D**). This indicated that pharmacological facilitation of mitochondrial Ca2+ uptake activity might not have a significant effect on cerebrovascular cellular compartments and their functions. However, possible effects on vascular cell survival and proliferation may need further investigation as increased vascularization along with decreased CBF has been observed as a long term response in the peri-lesional cortex after mTBI (Hayward et al., 2010) and reproduced in the adulthood stage in the current studies (**Figure 5** and **Figure S1**).

In vivo Ca2+ fluxes in the working brain in different compartments are difficult to demonstrate directly but its functional consequences can be measured in vivo. Impact of enhanced mitochondrial Ca2+ influx leads to increased mitochondrial oxidative metabolism and increased neuronal electrical activity which has been demonstrated in the intact animal model by our previous studies (Sanganahalli et al., 2013a). Hence enhanced mitochondrial Ca2+ uptake is the most likely event leading to altered neural functional states affected by kaempferol. Kaempferol as a dietary flavonol possesses antioxidative and anti-inflammatory effects (Middleton et al., 2000; Hamalainen et al., 2007) and may possibly impact the observed changes in brain circuit activity after treatment. Independent in vivo studies suggest that dose levels in the range 20 mg/Kg i.p and above are required for any significant antioxidative effects from kaempferol treatments (Lagoa et al., 2009). Although the 1 mg/Kg i.p dose of kaempferol used in this study may be suboptimal for any antioxidative effects as no neuroprotective impact was apparent in our results, non-specific antioxidative, and anti-inflammatory effects of kaempferol treatment in the developmental mTBI model cannot be excluded and needs further investigation. The current study in vivo tested a single compound targeting mCU activity. In order to quantify the complete physiological impact of mitochondrial Ca2+ uptake after developmental mTBI, similar systems level evaluation with several other mCU-targeting compounds may be required. Furthermore, stimulus-induced CBF response alterations after mTBI can have a neuronal and/or neurovascular origin. Hence future single subject electrophysiological and functional imaging correlations may be needed to determine the exact origins of the mTBI-induced functional activity changes within the brain.