This is the first molecular comparison of the potential mechanisms underlying the neuroprotective effects of MT and CB, and of the molecular targets and associated cell signaling pathways that mediate their protective effects on hippocampal neurons after TBI. Previous reports have shown that, in addition to their nootropic effects and common gene targets, both MT and CB reduce neuronal injury both in vitro
and in vivo
. These studies served as the primary rationale for the choice of these two structurally different compounds for our comparative pathway analysis. However, neither drug has previously been evaluated in a fluid-percussion TBI model 
The complex pathogenesis of TBI is associated with injury-induced changes in multiple molecular pathways 
, making it difficult to evaluate novel therapeutic agents with multiple postulated mechanisms of action in pre-clinical animal models. Recent studies have shown that distinct molecular networks that underlie core biological processes can be used as biosensors or biomarkers for human diseases 
, and distinct genetic programs (coordinated activation and/or repression of genes in known cell signaling pathways) are associated with neurodegenerative diseases 
. Considering these facts, we hypothesized that distinct alterations in a specific set of biological pathways associated with cell survival or cell death could serve as biomarkers of TBI pathology. Therefore, we reasoned that effective therapeutic drugs would alter expression of common genes in these specific injury-induced cell survival and/or cell death pathways. Moreover, in order to effectively alter physiological responses that regulate neuronal fate after TBI, effective neuroprotective agents will likely influence multiple components of a coordinated response. In this study, we used a systems biology approach to investigate drug-induced changes in a broad spectrum of biological pathways affected by TBI, albeit with a special focus on pathways associated with cell survival or cell death since our objective was to gain insight into the neuroprotective mechanisms of these drugs 
. Recent reports using this approach have demonstrated that neurodegenerative diseases influence broad neuronal networks 
, and that distinct disease-modifying pathways can be associated with neurodegeneration 
. This has led to insightful discoveries such as cessation of organ growth in adult rats being mediated by a defined genetic program that coordinates downregulation of multiple growth-promoting genes 
The main finding of our study is that a distinctive common molecular signature characterizes the effects of MT and CB, thus suggesting an avenue for investigation of the pathophysiology of TBI, as well as evaluation of potential treatment strategies for TBI. Our data show that the cumulative genomic response to these drugs appears to be a homeostatic improvement or normalization in injury-induced signaling. Specifically, MT and CB attenuate injury-induced gene expression associated with neurodegeneration and cell death, and, contrary to our expectation that these drugs would increase mobilization of protective genes, both drugs attenuate injury-induced gene expression associated with cell survival.
Another important outcome of our study is that we show, for the first time, that MT and CB reduce the number of degenerating neurons after TBI in the hippocampus, a brain region critical for learning and memory. These data are concordant with previous studies showing that these drugs prevented ischemia-induced synaptic dysfunction in the hippocampus 
Because the goal of this study was to understand drug-induced neuroprotection, it was essential to examine gene expression in only neurons that survived TBI, without the contaminating data deriving from dying neurons. In an earlier study, LCM made it possible for us to selectively capture only surviving, FJ-negative pyramidal neurons from the CA3 subfield of the rat hippocampus at 24 h post-TBI 
. In this previous study, by comparing changes in gene expression in surviving cells (FJ-negative) to those in non-surviving cells (FJ-positive) after TBI, we identified specific cell signaling networks known to play an essential role in injury response and survival. These data, followed by examination of the combined expression profiles of the pathways in which many (6 or more) co-expressed or functionally linked genes were altered by drug treatment, provided the most useful insight into the common mechanisms of these neuroprotective drugs. Our observation that both drugs similarly affected a distinct set of common target genes that were induced after TBI in these canonical pathways, suggests that altered expression of these specific genes was associated with their protective effects.
While it is impossible to predict whether the surviving neurons in untreated TBI rats would have died or survived at a later time point, we know that, at 24 h post TBI, the neurons analyzed in our study were not undergoing neurodegeneration as determined by FJ staining. However, even surviving neurons are expected to show a transcriptional profile reflecting their exposure to TBI in the absence of drug treatment, and surviving neurons from treated animals would show gene expression profiles influenced by drug treatment.
Whether or not the final outcome is death or survival, hippocampal neurons mount a protective molecular response after TBI. In the case of the dying neurons, the response is inadequate. This suggests that neuronal survival or death is regulated by a cell survival rheostat that is determined based on a ratio of cell survival to cell death genes that, in turn, depends on the stochastic expression of these genes in hippocampal neurons before TBI 
. This stochastic component might mask small, not necessarily statistically significant albeit biologically significant, changes in expression induced by drug treatment. In many of these cell survival or cell death associated pathways, the common gene targets of both drugs only became evident when the 2-fold change cut-off was not applied and used to screen out certain pathways for further study. Indeed, by first selecting pathways based on biological relevance and then examining the alterations in gene components, we gained an insight into the neuroprotective mechanisms of MT and CB that we would have missed totally by only studying statistically significant pathways. Interestingly, the rheostat-like effects of small regulatory non-coding microRNAs on target mRNAs are often less than 2-fold, within the boundaries of random variations, but still biologically significant 
. Our data support the concept that a cell survival rheostat could be influenced by drugs that induce subtle changes in individual gene expression, within co-expression networks, resulting in coordinated and biologically significant changes in cell signaling pathways. In many cases, the fold changes in gene expression induced by drug treatment were less than two-fold. However, the key observation we made after IPA analysis is that both CB and MT produced consistent and similar changes in co-expressed genes in dozens of canonical pathways associated with cell survival/plasticity/regeneration or cell death/stress/degeneration. Consequently, a coordinated pattern of significant gene expression changes only became apparent when the effect of these drugs was evaluated on entire cell signaling pathways rather than on individual genes with significant p-values.
Pathway analysis revealed several biologically relevant events. Specifically, acute and long-term neurodegeneration is the result of TBI-induced dysregulation of genes in multiple cell signaling networks, and neurons regulate synaptic plasticity by regulating the expression of activity-induced gene expression 
.Thus, our data suggest that CB and MT exert their neuroprotective effects via homeostatic normalization of injury-induced gene expression to pre-injury levels. Moreover, the similar transcriptional profiles induced by CB and MT support our hypothesis that neuroprotective drugs work through a common set of molecular targets associated with cell survival or cell death.
The attenuation or normalization of injury-induced gene expression is one of the defining molecular signatures in our study, suggesting that, paradoxically, neuroprotection is associated with an overall lack of mobilization of protective genes. Although we did observe upregulation of some survival-associated genes in the NFAT and PI3K pathways, the attenuating effects of both drugs on several pathways associated with cell death and stress signaling are also consistent with previous studies showing that improved levels of neuroprotection were obtained with therapeutic agents that targeted multiple pathways involved in cell death 
Both CB and MT display inhibitory effects on 11βHSD1, and, hence, glucocorticoid production, resulting in inhibition of glucocorticoid-regulated targets such as neuropeptide Y and tyrosine hydroxylase 
. However, pathway analysis confirmed our original hypothesis that the neuroprotective effects of MT and CB are mediated, in part, by common molecular targets other than 11βHSD1. In fact, many of the TBI-induced gene expression changes observed in our study have not been previously associated with glucocorticoid treatment.
Extensive in silico
validation of genes affected by both compounds (GEO accession GSE31357, References S1
) showed that many of the genes present in pathways affected by MT and CB treatment are associated with regenerative and/or pro-survival phenotypes, while others are associated with cell death signaling. A key feature of recovery after injury is the reactivation of cellular programs characteristic of embryological development 
. The CREB, NFAT, PKA and PI3K/AKT signaling pathways are associated with cell survival, but are also well known to be essential in development and regeneration. Expression of genes associated with tumorigenesis was down-regulated or normalized by both drugs (see References S1
). Tumor cells often have the ability to invade, resist cell death-inducing signals, and metastasize to other regions of the body by activating developmental regulatory programs 
. Therefore, a limited repertoire of cell survival signals appears to be common to both tumorigenesis and neuronal survival after TBI. Several genes in the amyloid processing pathway were also affected by MT and CB. This is particularly relevant because much evidence links early onset of Alzheimer's disease (AD) in TBI survivors to accumulation of amyloid-β (Aβ), due to a TBI-induced imbalance in Aβ genesis and catabolism 
Because TBI produces significant oxidative stress in the rat hippocampus 
, we also examined the effects of MT and CB on canonical pathways associated with generation of reactive oxygen species. ROS are byproducts of oxidative phosphorylation 
and nitric oxide signaling 
, primarily in the mitochondria. The effects of both drugs on mitochondrial gene expression provided provocative insights, as previous studies have shown that the effects of glucocorticoids on mitochondrial function follow a U shaped curve (low doses are protective while higher doses are associated with suppression of Bcl-2 levels and decreases in neuronal survival) 
. CB, but not MT, has been shown to uncouple mitochondrial OxPhos 
. Here we showed, for the first time, that the overall effect of both drugs on genes involved in OxPhos would predict a reduction of ROS consistent with a mild uncoupling of OxPhos. Previous studies have shown that increased expression of uncoupling proteins decreases production of mitochondrial ROS 
. Moreover, deletion of uncoupling protein 2 (UCP-2) in mice exacerbated ischemic brain injury, and was associated with suppression of cell repair genes and antioxidants 
Interestingly, compounds that partially inhibit OxPhos can increase tolerance to subsequent hypoxia 
. Because compounds that reduce mitochondrial oxidant stress, such as the L-type calcium channel blockers, nicardipine and nimopidine, also have been shown to have neuroprotective effects, they have been proposed as treatments for neurodegenerative disorders 
. Nonsteroidal, anti-inflammatory drugs also uncouple OxPhos 
, which suggests an additional mechanism for their neuroprotective effects in neurodegenerative disorders such as AD 
Finally, one proposed neuroprotective strategy involves reduction of oxidative stress using therapeutic agents such as resveratrol 
or interventions such as caloric restriction 
. Resveratrol has been shown to exert its neuroprotective effects by a cell signaling pathway involving UCP2 
, suggesting that, in general, other uncouplers of OxPhos have therapeutic potential for TBI. In line with this, a recent study suggested that novel protective drugs could be discovered by their ability to shift energy metabolism from mitochondrial respiration to glycolysis, which is known to suppress oxidative stress and cell death after ischemia 
Although the undesirable side-effect profiles of these drugs preclude long-term use in TBI patients, the demonstration that, following a single treatment, MT or CB reduces neurodegeneration, indicates that acute, short-term treatment after TBI might safely mitigate some of the deleterious effects of the initial injury. These two drugs associated with neuroprotection and cognitive enhancement induce a distinctive molecular profile associated with a reduction in oxidative stress and cell death. Additionally, many drugs in clinical use for other purposes may exhibit similar effects. Thus, the screening of such drugs for efficacy in TBI patients is warranted. Drugs identified using these criteria may also be beneficial for other neurodegenerative disorders in which similar cell survival pathways are affected.
Microarray data is MIAMI compliant and has been deposited in the Gene Expression Omnibus under the accession number GSE31357.