This study showed that severe hypoglycemia in a T1D mouse model can lead to BBB leakage, pericyte dysfunction, and neuronal damage, causing the onset of cognitive dysfunction, which is consistent with the results of our previous study . Further findings in this study revealed that the above mechanism of injury may be related to the excessive activation of oxidative stress and mitochondrial dysfunction due to glucose reperfusion after hypoglycemia. Interventions using the mitochondria-targeted antioxidant Mito-TEMPO in both in vivo and in vitro models have revealed that it may improve cognitive function in mice by resisting mitochondrial oxidative stress, reducing pericyte loss and apoptosis, attenuating BBB leakage, and neuronal damage.
Severe hypoglycaemia is a common and serious complication of insulin therapy in patients with T1DM . The close association between T1DM and cognitive dysfunction, the cause of which may be related to recurrent episodes of hypoglycemia [1, 2]. The brain is most vulnerable to hypoglycaemia because it is highly dependent on glucose as its primary fuel and has little storage capacity for glucose . Studies in rodents have shown that severe hypoglycemia leads to significant brain damage through a variety of mechanisms, including ROS production, mitochondrial permeability shifts, and oxidative DNA damage . Among these, glycemic recovery after hypoglycemia (i.e., glucose reperfusion injury) is thought to be one of the direct causes of exacerbated brain damage in patients with diabetes . In this study, we constructed a T1D mouse model of severe hypoglycemia to observe the effects of severe hypoglycemia on cognitive function in mice experiencing a rapid rise in blood glucose to a high glucose state. Our results revealed that glucose reperfusion after hypoglycemia could cause increased levels of oxidative stress and mitochondrial dysfunction in the brain. We found significant damage to hippocampal and cortical neurons in histology, and impaired cognitive function was observed in mice. Intervention with the mitochondria-targeted antioxidant Mito-TEMPO significantly reduced neuronal death and improved cognitive function in mice while reducing mitochondrial ROS production and improving mitochondrial morphology and function. Therefore, the role of oxidative stress in cognitive dysfunction due to hypoglycemia remains unclear.
Oxidative stress is a severe imbalance between ROS and reactive nitrogen species (RNS) production and antioxidant defenses . Mitochondria are a major source of intracellular ROS  and are considered one of the major targets of ischemia-reperfusion injury and a key regulator of neuronal cell life and death . Hypoglycemia, a source of oxidative stress, may lead to neuronal damage in the CA1 region of the hippocampus and accelerate cognitive decline by exacerbating hyperglycemia-induced oxidative stress and inflammation . Among these, glucose reperfusion following severe hypoglycemia is considered a period of marked ROS production and oxidative stress. The scavenging capacity of SOD, an effective free radical scavenger, plays a key role in maintaining the dynamic balance between ROS production and mitochondrial integrity . MDA is a product of the reaction of lipids with oxygen-free radicals, and its content represents the degree of lipid peroxidation. SOD and MDA are important indicators for evaluating oxidative stress in terms of antioxidant and oxidative capacity, respectively , and are often used together in the field of research related to oxidative stress. In this study, increased brain tissue ROS and MDA levels were found in the DH group of mice, accompanied by a decrease in SOD activity, confirming that severe hypoglycemia can enhance oxidative stress in the brains of T1D mice.
Several studies have shown the beneficial effects of natural mitochondrial antioxidants such as Okamoto maple  and melatonin  in neurodegenerative diseases. During induced acute hypoglycemia, vitamin C infusion (as an antioxidant) may reduce oxidative stress and inflammation in patients with T1DM . Mito-TEMPO is a specific scavenger of the mitochondrial superoxide . Mito-TEMPO was reported to cross the blood-brain barrier and prevent nicotine-induced ischemic brain injury , and also improved cognitive function by reducing the accumulation of tau oligomers in the cortical neurons of mice . Therefore, Mito-TEMPO may have therapeutic potential in hypoglycemia-induced brain injury. In this study, intervention with Mito-TEMPO, a mitochondria-targeted antioxidant, reduced ROS and MDA expression, increased SOD activity in mouse brain tissue, and improved neuronal death and cognitive dysfunction in the cortical and hippocampal CA1 regions of mice caused by severe hypoglycemia, a mechanism of action that may be mediated by the targeted scavenging of mitochondrial ROS and attenuation of oxidative stress. Therefore, we suggest that by enhancing antioxidant defenses, for example, through the administration of antioxidants, it may be possible to reduce oxidative stress-induced damage and improve synaptic dysfunction and neuronal damage caused by hypoglycemia and glucose reperfusion.
Brain endothelial cells interact with astrocyte endfeet, pericytes, and basement membranes to form neurovascular units (NVUs), which are essential components of the BBB . The BBB controls the composition of the neuronal internal environment and is essential for normal neuronal and synaptic functions . Degeneration of the BBB and dysregulation of blood vessels can be detected in the early stages of patients with Alzheimer's disease (AD) . Furthermore, damage to the BBB is now considered to be one of the key mechanisms leading to diabetic encephalopathy . Hypoxia/glucose deprivation can go so far as to lead to mitochondrial dysfunction and a subsequent reduction in ATP production, leading to destruction of the BBB and exacerbating brain damage . Our results show that severe hypoglycemia could cause a decrease in ATP content in the brain of mice, and treatment with Mito-TEMPO resulted in a significant increase in ATP content, suggesting an improvement in mitochondrial function. A previous study by our team  found that severe hypoglycemia can cause TJ protein deficiency and increased BBB leakage in diabetic mice; however, the exact mechanism has not been clarified. It has been found that TJ can be disrupted by oxidative stress, and changes in TJ protein levels and/or in the cellular localization/transport of TJ proteins are among the factors contributing to BBB disruption . In this study, mice experiencing severe hypoglycemia and glucose reperfusion were found to have reduced TJ protein expression and increased blood-brain barrier leakage, and electron microscopic findings also showed disruption of the BBB and loss of TJ. This damage was reversed by the Mito-TEMPO intervention. Based on the above, we hypothesized that BBB leakage and TJ loss are related to the oxidative stress caused by severe hypoglycemia. Therefore, there are two main questions that must be addressed: at which target does oxidative stress generated by hypoglycemia primarily cause BBB leakage? What cellular functions need to be focused on?
There is evidence that BBB disruption in patients with AD is associated with pericyte dysfunction and that pericyte loss occurs early in the AD disease process, at the stage of mild cognitive impairment . Pericytes are highly sensitive to oxidative stress and in many diseases such as diabetes and AD, pericytes are found to be the first NUV cells to die . BBB disruption can be prevented by reducing oxidative stress and protecting pericyte function . Matrix metalloproteinases (MMPs) are protein-degrading enzymes that degrade extracellular matrix proteins and are common culprits of oxidative stress-induced BBB damage. MMP-9 is the major MMP that is most closely associated with barrier permeability following oxidative injury . It has been reported that oxidative stress can contribute to the secretion and activation of MMP-9 by pericytes , which in turn leads to TJ disruption and increased BBB leakage. Based on the importance of pericytes in the maintenance of BBB structure and function, we focused on pericytes as the cause of BBB leakage due to hypoglycemia. In our previous study, we found that hypoglycemia could induce cell loss and increase MMP-9 expression in the perivascular brain of diabetic mice . The present study further confirmed these results and constructed an in vitro hypoglycemic model, and found that glucose deprivation followed by re-hyperglucose (simulating glucose reperfusion after severe hypoglycemia in the diabetic state of T1DM) on top of high glucose cultures could cause increased mitochondrial ROS production in HBVP cells, mitochondrial disruption, and decreased mitochondrial membrane potential, ultimately leading to apoptosis of HBVP cells. Furthermore, an increase in pericyte numbers and an improvement in pericyte function after intervention with Mito-TEMPO were observed in both in vivo and in vitro trials. Therefore, we suggest that mitochondrial oxidative stress induced by hypoglycemia and/or glucose reperfusion leads to pericyte dysfunction and reduced numbers, causing BBB leakage and, ultimately, cognitive impairment.
Chronic hyperglycemia plays an important role in BBB function and cognitive dysfunction in the brain in diabetes , and may contribute to neuronal damage by increasing polyol pathway fluxes, late glycosylation end-product formation, and oxidative stress . In this study, increased brain ROS production was observed in the T1D group. In vitro, high glucose decreased HBVP cell viability, increased cellular ROS production, mitochondrial disruption, and decreased mitochondrial membrane potential. However, no further effects of hyperglycemia on reduced pericyte numbers, BBB leakage, neuronal damage, or cognitive dysfunction were observed. We hypothesize that this is due to the short duration of hyperglycemia in the mouse model used in this study (3 days of hyperglycemia followed by execution for histological testing and 10 days of hyperglycemia followed by cognitive behavioral testing). The acute short duration of hyperglycemia was insufficient to cause damage to the BBB and neurons in mice, leading to significant cognitive impairment, which is not contradictory to the conclusion of the current study that hyperglycemia is an important risk factor for cognitive impairment. Our studies have demonstrated that acute short-term hyperglycemia can cause a rapid increase in oxidative stress (e.g., 3 days of hyperglycemia in mouse experiments resulted in a significant increase in ROS in the mouse brain, and in cellular assays, 40 h of hyperglycemia incubation resulted in a significant increase in oxidative stress indicators in HBVP cells).
This study had some limitations. The model of insulin-induced severe hypoglycemia used in this study is less accurate than the use of the glucose clamp in terms of simulating the duration and degree of hypoglycemia. However, the insulin-induced hypoglycemia model has the advantages of being simple, easy to perform, and equally efficient, and is now widely used as a test method, second only to the glucose clamp, in studies related to acute and chronic complications associated with hypoglycemia.