The occurrence of hypoglycemia is common in the daily glucose management of diabetic patients, and a growing number of studies have suggested that hypoglycemia plays an important role in the pathogenesis of cognitive dysfunction in diabetic patients [19–21], but the underlying molecular mechanism remains unclear. In the present study, we demonstrated that severe hypoglycemia has a significant deleterious effect on cognitive function in diabetic mice. More importantly, we demonstrate for the first time that this effect may be associated with pericyte dysfunction and BBB disruption.
Glucose is the main source of human energy and cannot be synthesized and stored in the brain, so a continuous glucose supply to the brain is essential for maintaining cognitive function. Severe hypoglycemia can cause permanent neuronal damage and further structural damage to the brain, leading to altered cognitive function [9]. Previously, we found that mitochondrial homeostasis in hippocampal tissue was imbalanced after diabetic mice experienced repeated non-severe hypoglycemia, thereby causing cognitive impairment [22, 23]. In the present study, our group continued our previous model of severe hypoglycemia in diabetic mice [18], in which a single injection of STZ caused absolute destruction of pancreatic islet β cells and clinical manifestations of type 1 diabetes mellitus with obvious “excessive drinking, polyphagia, polyuria, and weight loss”. To eliminate the influence of confounders such as physical strength and emotion of the mice after severe hypoglycemia on cognitive function testing, the mice that underwent the behavioral tests performed the grip test after a full 1-week rest. The results showed similar abilities in grip strength and swimming speed among the groups, with no measurable differences in sensorimotor coordination and strength between the groups, suggesting that the absence of gross motor impairment may affect the cognitive function of the mice. The Morris water maze subsequently demonstrated that severe hypoglycemia in the diabetic state can cause significant deficits in memory consolidation and working memory capacity in mice.
The BBB is a highly selective, semi-permeable border that separates circulating blood from extracellular fluid in the brain and central nervous system, and is formed by EC in the capillary wall, astrocyte ends wrapped around the capillaries, and pericytes embedded in the BM of the capillaries. Pericytes, a BBB component, share a BM with EC, and in regions lacking a BM, the intersection of pericytes and EC membranes, termed a peg–rivet contact, forms a direct connection that controls molecular exchange between pericytes and EC [24]. Under physiological conditions, pericytes play an important role in BBB formation and maintenance, neurovascular system regulation, inflammatory cell transport, and toxic metabolite removal from the brain [25]. In recent years, the relationship between BBB function and AD has become a topic of interest. A study [26] showed that AD patients had greater BBB leakage compared to the healthy population, and the investigator concluded that BBB disruption is an early biomarker of cognitive dysfunction in humans. It has also been argued [27] that BBB integrity is lost prior to cognitive decline. Pericyte deficiency has been observed in animal models of AD and in postmortem histological studies [28]. In another study [29], capillaries in the brain tissue of AD patients and in mice bred to develop AD pathology were extruded by the pericytes and showed significant dysfunction. The authors calculated that this capillary constriction was sufficiently severe to halve blood flow, which is comparable to the reduction in blood flow to the part of the brain affected by AD, and they proposed that restoring perivascular cell function in the brain holds promise for treating AD. In conclusion, pericytes may play an important role in the pathogenesis of cognitive dysfunction. Therefore, is hypoglycemia-induced cognitive dysfunction associated with pericyte/BBB disruption? Studies have found that [25] hypoglycemia potentially damages BBB integrity and function, and hypoglycemia exposure severely affected the expression of BBB oxidation and inflammatory stress markers. However, there are no studies on BBB disruption in diabetic mice after severe hypoglycemia. In the present study, diabetic mice with severe hypoglycemia showed increased Evans blue infiltration and significant brain edema, suggesting severe BBB disruption. BBB/neurovascular unit impairment is now considered one of the key factors contributing to the development of diabetic encephalopathy [30], and diabetic mice exhibit enhanced BBB permeability after severe hypoglycemia, which may be associated with the loss of perivascular cells in the brain. To clarify the mechanism of BBB disruption, we examined the indicators related to pericyte function and distribution, and show that pericyte-specific expression of the proteins PDGFR-β and α-SMA was reduced in the brains of diabetic mice after severe hypoglycemia, suggesting that the pericytes were lost. The transmission electron microscopic findings also suggest that diabetic mice have BBB disruption and pericyte damage after severe hypoglycemia. The preliminary results of the present study suggest that the mechanism of cognitive dysfunction after severe hypoglycemia in diabetic mice may be related to BBB disruption, which may result from pericyte dysfunction and loss. Therefore, how might pericytes in hypoglycemia affect BBB function?
The presence of oxidative stress during hypoglycemia has been well demonstrated, with increased reactive oxygen species (ROS) production in mitochondria isolated from the hippocampus of diabetic rats exposed to recurrent hypoglycemia [31]. In addition, elevated levels of inflammatory factors such as TNF-α and IL-6 have been observed in hypoglycemia, causing an acute inflammatory state in the organism [32]. MMPs are zinc-dependent proteases that degrade many structural components of the extracellular matrix and non-extracellular matrix proteins [13], and include MMP9. Oxidative stress and an inflammatory environment contribute to MMP9 activation and secretion by pericytes, which decreases the expression of TJ proteins and leads to BBB destruction [33]. MMP9 inhibitors reduce pericyte-associated BBB leakage [34]. In the present study, MMP9 expression was increased in diabetic mice and severely hypoglycemic mice, and the levels of occludin and claudin-5, closely related to BBB function, were further decreased; ultramicroscopy of mouse brain hippocampal tissue also showed significant TJ disruption. It is suggested that severe hypoglycemia in the high-glucose state can cause increased MMP9 expression in brain pericytes, which further causes degradation of the TJ proteins and leads to BBB disruption.
Notably, in the present study, although MMP9 levels were increased and TJ proteins were decreased in the diabetic state, no further BBB disruption or reduced pericyte numbers by hyperglycemia was observed, and the water maze behavior also suggested no significant cognitive dysfunction in the diabetic mice alone. Although diabetes is associated with increased risk of neurodegeneration and dementia [35], the specific effects of hyperglycemia on pericyte/BBB function remain controversial. Several studies have suggested [36] that the mechanism of pericyte dysfunction and consequent BBB leakage in diabetic patients is related to enhanced ROS and reduced ATP production due to oxidative stress caused by prolonged hyperglycemia. In contrast, others have found that BBB function remained unchanged in diabetes [15]. Yet, our results show that hyperglycemia alone caused a slight impairment in pericyte function and in BBB morphology and function compared to that in normal mice. However, it was insufficient to cause significant BBB disruption and subsequent cognitive dysfunction as compared to the severe hypoglycemia group. We conjecture that the possible reason for the high glucose in this model is the short duration of the study (only 5 days from sampling and 12 days from the water maze test), and the glycosylation products and oxidative stress of the high glucose were insufficient to cause serious BBB disruption and thereby lead to cognitive dysfunction. However, according to the trend of our results, it is reasonable to think that long-term hyperglycemia will destroy pericyte/BBB function. To explore the effect of hyperglycemia on pericyte/BBB function, further investigations can extend the duration of hyperglycemia in diabetic mice or use diabetic genotype mice.
It is well known that neuronal damage is a key factor in cognitive dysfunction [37]. The loss of pericytes may disrupt the BBB through the leakage and deposition of vascular and neurotoxic macromolecules from peripheral circulation sources. The associated microvascular degeneration and edema may cause chronic hypoxia by further increasing the entry of neurotoxic substances and causing reduced blood flow, resulting in structural and functional changes in neurons, thereby affecting interneuronal interconnections and ultimately exacerbating the development of primary neurodegenerative disease [38]. Pericyte-deficient mice have increased neuronal apoptosis, reduced neuronal numbers, and behavioral deficits in the cortex and hippocampus [39]. In the present study, HE staining revealed significant neuronal destruction in the severely hypoglycemic mice, and we speculate that the cause may be the leakage and deposition of vascular and neurotoxic macromolecules after pericyte/BBB dysfunction that further exacerbate neuronal damage, thereby causing cognitive dysfunction. The specific molecular mechanism requires further research.
This study has some limitations. First, this study used STZ injection to create a diabetic mice model, and it was reported that STZ injection could directly cause brain damage [40]. However, in the present study, mice in the group that experienced severe hypoglycemia had significantly more impairment in cognitive function and BBB compared with mice in the diabetic group that received STZ injection alone, suggesting an independent or additive effect of hypoglycemia on these impairments. Second, the effect of severe hypoglycemia on pericytes/the BBB has not been further explored at the cellular level, and the specific mechanisms have not been explored in depth. Our study shows, for the first time, that the severe hypoglycemia-induced cognitive dysfunction in diabetic mice is associated with pericyte/BBB dysfunction.