Hyperglycemia (both peak glucose and persistent hyperglycemia) in TBI patients is associated with injury severity and clinical outcomes[7, 23, 24]. Previous studies have shown that primary trauma evokes a cascade of changes that result in secondary axonal injury. The role of blood glucose in the secondary mechanisms of neuronal damage after DAI has not yet been clarified. In this study, we found that abnormal histopathological changes were exacerbated when DAI rats suffered hyperglycemia. Meanwhile, the expression of markers of axonal damage, including β-APP, NF-L, and NF-H, and the number of TUNEL-positive cells were increased significantly in DAI rats with hyperglycemia, indicating that hyperglycemia led to more serious secondary axonal damage after DAI. Similarly, the link between hyperglycemia and a poor prognosis is also observed in ischemic stroke, subarachnoid hemorrhage, intracerebral hemorrhage and other neurodegenerative diseases.
Next, the mechanisms underlying hyperglycemia leading to secondary damage after DAI were studied. Persistent hyperglycemia results in acidosis, electrolyte disturbances, inflammation, vessel disorders, BBB rupture, and hyperpermeability. The BBB is a highly specialized arrangement of the vasculature that has evolved to provide protection from potentially harmful pathogens that enter the bloodstream[26, 27]. In many CNS diseases, such as stroke and subarachnoid hemorrhage, loss of BBB tight junction integrity leads to increased paracellular permeability[26, 27]. Vascular endothelial cells are a significant target of hyperglycemic damage, but the mechanisms underlying such damage to the cerebral microvasculature are not fully understood[28, 29]. In this study, a possible role of hyperglycemia in the regulation of BBB integrity following DAI was investigated. Hyperglycemia aggravated DAI-induced significant brain edema and Evans blue leakage in vivo, and OGD induced a decrease in TEER and increased HRP flux in vitro. The damage to BBB permeability caused by hyperglycemia contributed to a decrease in tight junction proteins, including occludin-1, ZO-1, and claudin-5. Meanwhile, previous studies have also reported that hyperglycemia leads to endothelial dysfunction and cerebrovascular changes during both ischemia and reperfusion[30, 31]. However, the role of hyperglycemia in the secondary mechanisms of BBB damage after DAI has not yet been clarified.
Neuroinflammation is an important common determinant of increased BBB permeability. PPARγ is a subunit of PPAR and is a ligand-activated nuclear transcription factor. PPARγ can be activated by its ligand. PPARγ ligands attenuate degenerative processes in the brain through the control of anti-inflammatory mechanisms, oxidative stress, neuronal death, neurogenesis, differentiation, and angiogenesis. Once activated, PPARγ can combine with specific DNA response elements to regulate the transcription and expression of genes, and neuroinflammation is markedly suppressed in many cerebral injuries. Previous studies have demonstrated that PPAR-γ activation can exert neuroprotective effects on many animal models of acute brain insults and can reduce brain edema. For example, AQP4 deletion protects BBB integrity by reducing inflammatory responses due to upregulation of PPAR-γ expression and attenuation of proinflammatory cytokine release after acute severe hypoglycemia. Our findings indicate that PPARγ activation may alleviate axonal injury by reducing BBB disruption. In this study, PPARγ expression was decreased significantly after DAI and OGD. In the hyperglycemia-treated groups, PPARγ expression was further decreased, indicating that the secondary damage to the BBB induced by hypoglycemia was associated with the reduction in PPARγ expression.
Our previous study also showed that the PPARγ agonist RSG can protect BBB integrity by decreasing the levels of inflammatory mediators through a caveolin-1-dependent pathway. Upregulation of caveolin-1 by RSG has been demonstrated to require superoxide formation and activation of Src, EGFR, and the Mek1-Erk1/2 and p38 MAP kinase pathways[34, 35]. Caveolin-1 is a plasmalemmal anchoring protein and modulator of vascular function and glucose homeostasis. A chronic hyperglycemic condition directly decreased caveolin-1 expression in the brain neurons of diabetic rats, and the downregulation of caveolin-1 induced by chronic hyperglycemic conditions is independent of mTOR signaling[36, 37]. In this study, hyperglycemia significantly reduced caveolin-1 expression after DAI and OGD. After RSG treatment, caveolin-1 expression was increased, but all protective effects of RSG were abrogated when caveolin-1 expression was downregulated. These results indicate that RSG protected BBB integrity by upregulating the expression of TJ proteins through the caveolin-1 pathway after DAI combined with hyperglycemia. In contrast, one study found that high glucose could lead to the hyperpermeability of monolayer endothelial cells through the VEGF/KDR pathway and caveolin-1 overexpression. The role of caveolin-1 in hyperglycemia-induced pathological changes requires further study.
Experimental studies have shown that a hyperglycemic condition activates the production of IL-1, IL-6, and TNF-𝛼[39–41]. All these inflammatory factors have been reported to be related to secondary axonal injury. IL-1𝛽 and TNF-α overexpression was strongly related to axonal injury, while IL-6 mRNA and protein expression levels were also positive at sites where axonal injury was observed. Moreover, brain injury itself stimulates systemic inflammation, leading to increased permeability of the BBB, which is exacerbated by secondary brain injury. High glucose has been demonstrated to exacerbate neuroinflammation and apoptosis in the intermediate stage post-TBI by inhibiting the MEK5/ERK5 pathway. In this study, the levels of inflammatory mediators, including TNF-α, IL-1β, and IL-6, were increased after DAI and OGD. Furthermore, hyperglycemia significantly increased TLR4 expression and the levels of TNF-α, IL-1β, and IL-6. After RSG treatment, TLR4 expression and TNF-α, IL-1β, and IL-6 levelswere decreased. Inhibition of caveolin-1 by Cav-1 siRNA abrogated the protective effect of RSG. Thus, RSG protected the BBB model by increasing the expression of TJ proteins and decreasing the levels of inflammatory cytokines through the caveolin-1/TLR4 pathway after DAI combined with hyperglycemia. Our previous findings indicated that TLR4 inhibition at 1 d after DAI effectively alleviated pathological changes, including apoptosis, neuronal and axonal injury, and glial responses, with decreased inflammatory factor levels. Moreover, caveolin-1 could negatively regulate TLR4 activation[43, 44]. Reduced caveolin-1 expression in monocytes could aggravate the TLR4-mediated inflammatory cascade. Studies have also found that cav-1 binds to TLR4 and inhibits lipopolysaccharide-induced proinflammatory cytokine (TNF-α and IL-6) production in murine macrophages. Mutation analysis revealed a caveolin-1 binding motif in TLR4, which is essential for this interaction and for the attenuation of proinflammatory signaling.
In conclusion, our findings indicate that hyperglycemia exacerbated axonal injury, cell apoptosis, and glial activation and destroyed BBB integrity by downregulating the expression of TJ proteins and accelerating the release of inflammatory mediators, accompanied by inhibition of PPARγ and activation of the caveolin-1/TLR4 signaling pathways after DAI. Our findings provide evidence that hyperglycemia exerts marked deleterious cerebral effects after DAI and suggest that PPARγ agonists hold considerable promise with respect to new DAI accompanied by hyperglycemia treatment.