After TBI, individual neurochemical changes can be used to elucidate the specific cellular mechanisms involved in TBI. Additionally, MRS can indicate the intracellular regions of interest directly. Thus, this approach can provide information complementary to that obtained by using microdialysis probes, and does so non-invasively. Using 1H-MRS, T2-weighted images and brain water-content analysis in this study, we attempted to determine whether the previously noted protective effect of GCP-II-KO against TBI may be caused by neurometabolic alterations [13]. We therefore investigated Glu and NAA changes in the hippocampus after CCI, using high-field 1H-MRS in vivo. Both Glu and NAA levels in the ipsilateral hippocampus were significantly lower at 24-h post-TBI in GCP II-KO mice than in WT mice (p < 0.05). However, the reduction in Glu and NAA levels was less marked in GCP II-KO mice than in WT mice (p < 0.05). We also showed that the extent of both cortical edema and brain swelling was lower in KO + CCI than in WT + CCI mice, as was the brain water-content.
In the CNS of mammals, Glu is the most abundant excitatory neurotransmitter. Rapid neural depolarizing and release of the excitatory neurotransmitter Glu induced by CCI can lead to overstimulation of Glu receptors, which initiates neuronal damage. Therefore, we considered that 1H-MRS measurements of Glu may provide an in-depth understanding about excitotoxicity after CCI.
At the very early stage of TBI, changes in the concentration of Glu may indicate an imbalance in excitatory and inhibitory activities in the hippocampal region. Our previous studies had shown that the GCP-II inhibitors can, to a certain extent, reduce the release of Glu into the extracellular fluid, with an increase in NAAG levels in animal models that have undergone moderate TBI [8, 9]. Previous 1H-MRS studies have suggested a decrease in the Glu/Cr ratio in the hippocampal region of rats at 2 h and 4 h after CCI, which was deemed to be associated with the glutamate–glutamine cycle and the increased energy requirement of the hippocampal neurons after trauma [14, 15]. We found a decrease in the Glu/Cr ratio at 24 h after CCI in GCP II-KO and WT mice, which indicates that the CCI-induced decrease in Glu levels may contribute to neurological dysfunction in the long run. Concurrently, the less marked reduction in Glu levels in the ipsilateral side of GCP-II-KO CCI mice than in that of WT CCI mice demonstrated that TBI had a less marked effect on mice lacking the GCP-II gene. We hypothesized that NAAG persisted in the synaptic septum after TBI in mice lacking the GCP II gene, inhibiting further release of Glu from the presynaptic membrane, and slowing the deterioration of hippocampal neurons after TBI. Our finding of a decline in Glu levels by 24 h after CCI is consistent with those of a similar study [15].
In the adult brain, NAA is the second-most abundant amino acid. Little is known about the function and metabolism of NAA, even though it is one of the key organic constituents of the brain [16]. Because NAA is found almost exclusively in neurons [17], neuronal injury and death in various diseases and injuries are reflected by the reduction of NAA [18, 19]. The noteworthy decline in the NAA/Cr ratio by 24 h after CCI is in accordance with the findings of other studies that have used similar approaches [15]. In addition, after TBI, the prolonged decline in NAA can lead to diffuse axonal injury or neurodegeneration [20]. We found that the marked decrease in NAA by 24 h after injury in the ipsilateral hippocampus region of KO CCI mice was statistically significantly less than that observed in WT mice. Although the function of NAA is not yet fully understood, it is considered to be a biomarker reflecting neuronal mitochondrial status [20]; consequently, the decrease may be due to impaired NAA synthesis by the mitochondria [7, 15, 21]. Several studies have provided evidence to support the view that a reduction in NAA reflects irreversible neuronal/axonal loss, and is caused by defective NAA synthesis in the mitochondria [22, 23]. Moreover, it has been found that the decrease in NAA in diffuse TBI is partly accompanied by an ATP decline, and recovers only with the restoration of ATP levels [24].
Immediately after CCI, a depolarizing cascade rapidly and simultaneously releases excitatory neurotransmitters, including Glu and NAAG, into the extracellular fluid. At the mGluR3, the escape of abundant excitotoxic Glu is partially inhibited by NAAG, which acts as a potent agonist of the receptor. However, the released NAAG is rapidly inactivated by GCP-II. Previous studies have reported that inhibition of GCP-II plays an endogenous protective role by increasing extracellular levels of NAAG, and that inhibiting GCP-II may be an important approach to increasing extracellular NAAG. Our recent study showed that GCP-II-KO exerts a neuroprotective effect against TBI by preserving mitochondrial integrity in the ipsilateral cortex [25]. It is possible that GCP-II-KO mitigates CCI-induced ipsilateral hippocampal mitochondrial dysfunction, which would exacerbate the consumption of Glu and inhibit the production of NAA due to a lack of ATP.
We found relatively mild brain edema in the ipsilateral hemisphere of GCP-II-KO mice based on T2 images and the measurement of the brain-water content (Fig. 4). After TBI, the disrupted blood–brain barrier allows water to move to the extracellular compartment (vasogenic edema) from the vessels, which is followed by cellular water uptake (cytotoxic edema/cell tumefaction/swelling). Cytotoxic edema particularly affects astrocytes, as these cells are 10 times more numerous than neurons. The mechanisms underlying the tumefaction remain unclear. It has been commonly believed that direct cell damage contributes to the consumption of ATP. To maintain volume homeostasis, brain cells exude organic osmolytes, including Glu [24]. This requires energy consumption and changes in the transmembrane ion gradients, leading to neuronal depolarization, which induces an increased influx of Na+ and Ca2+ into the cell. The concomitant energy failure injures the Na+/K+ ATPase pump system, and the Na+ overload cannot be compensated. Consequently, water enters into the cell and the cell volume increases [26].
Our research had some limitations. The injured cortical areas were not included in the study because of unstable detection results. The relationship between additional metabolites and TBI remains to be studied in future. Despite this, 1H-MRS is becoming increasingly valuable for examining human survivors of TBI and for improving novel therapies. Nevertheless, further studies are necessary to elucidate how the neurochemical status observed by 1H-MRS relates to the pathological mechanisms associated with TBI.
