Over the past few years, clinical and preclinical studies have reported that the threat of dementia and memory impairment is increasing due to type 2 diabetes (T2D), which affects more than 400 million patients worldwide (8, 37) and could affect 640 million by 2040 (45). Type 2 diabetes is a chronic metabolic disorder with a negative impact mainly on insulin-producing regions. In the brain, insulin resistance is extreme in areas that are responsible for cognition, namely the hippocampal region, which contains greater insulin receptor concentrations (5). Neuroimaging showed that an attribute of T2D is reduced hippocampal volume (31). The hippocampus, as the region carrying out such functions as thinking and memory, is the region where the impairment associated with sporadic Alzheimer’s disease (AD) has its cause. This impairment is in cognition and occurs (as does sporadic AD in general) most frequently in geriatric populations (7, 14), in whom T2D is also more common. Emerging studies have reported pre- or fully developed T2D in patients with Alzheimer’s (5), supporting the involvement of brain insulin resistance in sporadic AD pathophysiology. The dysregulation of the insulin-signalling pathway leads to compromised cognition (16).
Sevoflurane is a surgical anaesthetic inhalant (36) which caused cognitive dysfunction in various experiments on animals (41). Long-term exposure of rat models to a volatile anaesthetic resulted in symptoms similar to those present in autism spectrum disorders. Our research was conducted on senile rat models to determine the molecular mechanisms that induced neurotoxicity due to sevoflurane inhalation and whether modulating these mechanisms may limit neurotoxicity along with cognitive learning impediment and memory impairment in rat models. Several studies show that exposure to sevoflurane induces oxidative stress, increases neuronal apoptosis in the hippocampus, decreases learning and memory, and substantially attenuates brain-derived neurotrophic factor (BDNF) signals. As a result, they demonstrate that BDNF has a neuroprotective effect in senile rat models that have suffered neurotoxicity produced by sevoflurane and have impaired cognition.
Evidence of diminished expression of BDNF implied its significant contribution to the progression of different neurological disorders, including Parkinson’s disease and AD (28). This warrants the prescription of anti-diabetics to treat impaired cognition in patients with AD. BNDF serum levels in a mouse model increased significantly after the T2D drug metformin was administered in Parkinson’s studies (34). In addition, BDNF expression was significantly restored in a high-fat–diet rat model following treatment with metformin alone or in combination with another T2D drug, glimepiride, BDNF transcription nevertheless reducing in elderly C57BL/6J mice (2). The protective effect was manifested strongly after administration of vildagliptin, another anti-diabetic drug, with an increase in brain BDNF expression improving memory impairment in diabetic rats (17). Similarly, intragastric alogliptin administration once a day for three weeks has shown increases in BDNF levels in the brain cortex and hippocampal regions in C57BL/6J mice. No studies have yet been performed to assess the effects of glibenclamide (GBC) on neurotrophic expression or specifically on BDNF levels in older rat models with impaired cognition due to sevoflurane-induced dysfunction. Our research therefore aimed primarily at evaluating the impact of GBC on hippocampal learning, memory, and inflammation by assessing the two inflammatory cytokines tumour necrosis factor alpha (TNF-α) and interleukin 6 (IL-6). This experiment sought to recognise and understand the pathways involved in neurotoxicity due to enhanced neurotrophic signals induced by inhalation of sevoflurane, which contributed to increased oxidative stress and diminished cognition in neurons.
Experimental protocol
Effects of glibenclamide on memory in rats with sevoflurane-induced cognitive impairment as demonstrated by the Y maze test. Values are presented as (A) mean ± SEM (n = 15) of total arm entries and (B) percentage of spontaneous alternations: # – p < 0.05 and ## – p < 0.01 for significant difference
Effects of glibenclamide treatment on learning and memory in rats with sevoflurane-induced cognitive impairment in the Morris water maze test. Values are presented as mean ± SEM (n = 15) escape latency, time spent in the platform quadrant or the platform crossings. # – p < 0.05 and ## – p < 0.01 for significant difference
In comparison, the findings of a one-way variance study showed a significant disparity between the groups over the span of time spent in the quadrants of the platform (F (3, 44) = 16.198; P = 0.0087) and the crossings (F (3, 44) = 15.631; P = 0.0121). Figure 3 shows cognitive impairment similar to Alzheimer’s disease in the low-dose sevoflurane group with a significant decrease in platform quadrant time (P = 0.0087) and platform crossing (P = 0.0121) compared with the control group. In comparison, a significant difference was observed in the time spent in platform quadrants (P = 0.0077) and platform crossings (P = 0.013) between rats with low-dose sevoflurane–inflicted cognitive impairment which received GBC and rats which were administered the same low dose but no GBC. These results show that cognitive impairment in rats developed following inhalation of sevoflurane but that memory deficits improved after GBC administration to cognition-deficient rats.
Effects of glibenclamide treatment on interleukin 6 (IL-6) (A) and tumour necrosis factor alpha (TNF-α) (B) levels in the hippocampus of rats with sevoflurane-induced cognitive impairment. Values are presented as mean ± SEM (n = 15) of the concentrations. # –p < 0.05 for significant difference
Effects of glibenclamide treatment on attenuation of phosphoinositide 3-kinase (PI3K)/Akt signalling pathways in sevoflurane-induced cognitive impairment in the hippocampal regions of aged rats. Values are presented as mean ± SEM (n = 15) of the concentrations. # – p < 0.05 for significant difference versus the control group; * – p < 0.05 and ** - p < 0.01 for significant difference versus the low-dose sevoflurane group; GBC – glibenclamide; LDS – low-dose sevoflurane
Effects of glibenclamide treatment on BDNF expression in sevoflurane-induced cognitive impairment in rats. (A) Serum levels of BDNF and indicated oxidative stress markers in rats with or without effects of sevoflurane inhalation; (B) Expression of
Brain-derived neurotrophic factor (BDNF) expression in sevoflurane-induced cognitive impairment in rats. (A) Expression of BDNF protein in the control group; (B) BDNF expression in sevoflurane-induced cognitive impairment in the hippocampal regions of aged rats; (C) Expression of BDNF protein after treatment with glibenclamide in sevoflurane-induced cognitive impairment in the hippocampal regions of aged rats; (D) Relative quantification of BDNF expression in the control group, GBC-only group, low-dose sevoflurane group without GBC, and low-dose sevoflurane group with GBC (n = 5). Significant differences
The above effects of ROS generation resulting in oxidative stress further motivated us to evaluate serum rates of activity of MDA (F (3, 52) = 15.409; P = 0.0138), CAT (F (3, 52) = 19.47; P = 0.0003), GSH-Px (F (3, 52) = 48.84; P < 0.0001), and SOD (F (3, 52) = 20.41; P < 0.001). Sevoflurane stimulated marked serum level rises in each of these oxidative stress markers. In comparison, treatment with GBC greatly decreased serum rates of activity of MDA, CAT, GSH-Px, and SOD (Fig. 6) in the hippocampi of the rats with low-dose sevoflurane– mediated cognitive dysfunction relative to these rates in the vehicle-treated low-sevoflurane group.
Our findings showed that low-dose sevoflurane anaesthesia altered the production of BDNF in aged rat hippocampal neurons. A significant decline in the transcription of BDNF mRNA accompanied by simultaneous down-regulation of BDNF proteins was evident, as seen in Fig. 6. A substantial decline in serum BDNF levels in aged rats after inhalation of sevoflurane is clearly seen in the findings. In comparison, treatment with GBC following low-dose sevoflurane exposure caused a dramatic increase in serum BDNF levels as well as up-regulated BDNF protein production with a substantial increase in BDNF mRNA expression (F (3, 32) = 19.898; P = 0.0014) in rat hippocampi relative to the low-sevoflurane group administered no GBC.
The specific pathophysiological mechanisms underlying cognitive dysfunction and brain damage in diabetes mellitus are unknown; however, various factors such as hyperglycaemia, vascular illness, hypoglycaemia, and insulin resistance play considerable roles. Microvascular complications and poor glycaemic control (defined as elevated glycated haemoglobin) contribute to morphological and physiological modifications in the central nervous system seen in greater cortical atrophy and micro-structural malformations which lead to cognitive dysfunction linked with intellectual disability, deficient working and verbal memory, and involuntary movements. Hypoglycaemia and hyperglycaemia have both been shown as key contributors to cognitive impairment. Diabetes mellitus (type 2) accelerates the ageing process of the brain, causing cerebral atrophy; additionally, it appears to interfere with cerebral amyloidogenesis, resulting in tau hyperphosphorylation
Sulfonylureas and metformin, alone or in combination, were found to lower the risk of dementia in a previous study (21). There have been no preclinical or molecular studies on the possible mechanisms involved in these drugs’ regulation of cognitive function; however, two of the sulfonylureas, glyburide and glipizide, were shown to intervene as mammalian target of rapamycin (mTOR) inhibitors (23). Given the role of the mTOR pathway in memory and learning, as well as its suggested role in neurodegenerative disorders such as Alzheimer’s disease (9, 13), such drugs may be beneficial in the treatment of cognitive impairment in diabetic patients. A recent study showed that glimepiride protects neurons from amyloid-β induced synapse damage, and that reducing synapse damage may slow the onset of cognitive decline in Alzheimer’s disease (33). Finally, glibenclamide was found to be effective in restoring memory function and normalising blood– brain-barrier modifications in db/db mice, a mouse model of spontaneous diabetes with hyperinsulinaemia, but the effects of glibenclamide on cognitive improvement involving BDNF expression have not been studied.
BDNF is a ubiquitously expressed neurotrophin which is present in the hippocampal region and contributive to neuroplasticity. It is responsible for the proper functioning of the nervous system and for the development of the embryo. Learning and memory mechanisms are regulated by BDNF signals, as was demonstrated with an improvement in hippocampal-dependent cognitive tasks (25, 27). Anxiety and other emotions may arise with the involvement of the hippocampus and the level of BDNF in this region may be influential upon origination of these feelings (35). Hence reduced signalling by BDNF owing to the intake of sevoflurane induces clustered cognitive dysfunction. Boosting BDNF expression may contribute to mitigation of the impairment in cognitive abilities.
Studies have shown that BDNF and synaptotagmin-1 protein levels both decrease significantly after the intake of sevoflurane, which affects synapse transmission and memory development. It was also found that postsynaptic density protein 95 expression decreases cognition development with the ingestion of sevoflurane in the medial prefrontal cortex of the neonatal, adult or geriatric rat populations (43), although the precise role of these signalling pathways is uncertain.
The results showed that the pathology of impaired cognition featured decreased BDNF expression. Analysis of gene expression was not performed in our research, which is one of the drawbacks of our analysis. This study helps to explain the consequences of sevoflurane inhalation and the impact of BDNF on gene expression when GBC restores the neurotrophin to the normal level. While we did not determine if replenished BDNF could minimise or prevent hippocampal neuronal apoptosis due to sevoflurane exposure, our findings nevertheless suggest that apoptosis was inhibited by it; experimental verification is needed, however.
Higher concentrations of advanced glycation end products in the brain promote glycation in the plasma membrane and raise the number of receptors, leading to an alteration in the intracellular signalling pathway, an upregulation of gene expression, the release of pro-inflammatory molecules, and the accumulation of scavengers of reactive oxygen species, all of which could result in increased oxidative stress and more prevalent microvascular pathophysiological changes. All these processes have been linked to neural ageing and hippocampal atrophy (10).
In agreement with earlier published results, our analysis showed that impaired memory in older rats was due to the elevated levels of the hippocampal inflammatory cytokines IL-6 and TNF-α following sevoflurane inhalation (5, 10, 27, 30). We found that when administered with a lower dose of sevoflurane, glibenclamide was able to reduce certain hippocampal inflammatory cytokines in rats with cognitive disability. A study reported that the hippocampal TNF-α levels in ischaemia-reperfusion injury rats were decreased by GBC, which was also the orientation of our results (29).
Glibenclamide was also able to reduce sevoflurane-mediated memory impairment. A clinical study also reported that memory in diabetic patients had increased with GBC therapy. Similarly, to our results, a report showed better memory in ischaemic or traumatically brain-injured rats in the subjects treated with GBC (39). Another study observed a decline in cognitive dysfunction related to a high-fat diet in mice with obesity (32). In their Y-maze and MWT studies, Chen
The PI3K/Akt signalling mechanism was examined to further investigate the impact of GBC in rat models with impaired cognition due to low-dose sevoflurane inhalation. The association of neurotrophic factors with their receptors (6) resulted in the activation of the PI3K/Akt signalling pathway, contributing to morphological changes such as enhanced neuroplasticity and neuronal survival along with enhanced neuronal development, regeneration and differentiation. The PI3 kinase is a heterodimer consisting of p85 and p10 subunits which have respective regulatory and catalytic functions in the phospholipid kinase family. Glibenclamide stimulates PI3K, which transforms into phosphatidylinositol (4,5)-biphosphate and binds to Akt by an unclear mechanism. It induces Akt phosphorylation in the presence of 3-phosphoinositide-dependent protein kinase-1, leading to Akt activation (22). Phosphoinositide 3-kinase shows Akt as its essential downstream molecule, and upon activation, Akt may trigger the phosphorylation of other downstream molecules which regulate cell proliferation and existence, thus protecting the cells damaged by ischaemic injuries (44).
In histological analysis of the hippocampus, elevated levels of TUNEL-stained activated cells were found. In numerous other studies which reported apoptosis in the hippocampal region, such findings were also made, and activation of the pro-apoptotic protein signal was attributed to consistent sevoflurane ingestion. These studies also confirmed that anaesthetic agents induced pro-apoptotic effects, namely early neuronal deficiencies, which resulted in later-stage behavioural deficits (42) that were reduced when treated with GBC. Hence, the mechanism by which GBC reversed the effects of sevoflurane neuro-damage was the suppression of neuronal apoptosis.
Accumulated ROS could also result in enhanced apoptosis (26). Several reports have shown that anaesthetic agent–induced brain intoxication was characterised by elevated oxidative stress leading to escalated lipid peroxidation, cell destruction and neuronal death (24). Our results indicated increased MDA levels while sevoflurane decreased SOD and CAT levels. The fall in SOD and CAT levels owing to sevoflurane suggests a higher potential for the disruption of the pathological process. The decrease may be attributed to a particular route of absorption of sevoflurane which has a direct effect on the brain’s antioxidant process (1). In numerous animal experiments, it was also found that GBC minimised neuronal damage by modulating antioxidation and the concentration of free scavenger radicals inside the brain (18). At the same time, it was observed that GBC often decreases the oxidative stress induced by different pathways leading to renal injury or neutrophilic inflammation (46). We found that GBC decreased oxidative stress and restored the antioxidation mechanism in the hippocampus. This suggests that GBC greatly mitigated the effects of oxidative stress attributable to sustained sevoflurane exposure.
When assessed using the Morris water maze test, significantly augmented memory and spatial learning in sevoflurane-induced rats was noted following GBC (10 mg/kg b.w.) treatment. Interestingly, in the low-dose Sevo group we found longer escape latencies than in the control group. The results are shown in Fig. 6. We also observed that treatment with GBC (10 mg/kg b.w.) significantly reduced the time needed to identify the platform by rats with cognitive impairment induced by sevoflurane. Sevoflurane greatly reduced the number of times platform sites were crossed over when compared with the control group during the testing procedure. Furthermore, GBC was found to reverse the effects induced by sevoflurane. These findings suggested that GBC effected a marked improvement in memory and learning behaviour in sevoflurane-induced cognitively dysfunctional rats.
One limitation of the study is that it did not include the demonstration of how the administered GBC reaches the plasma, cerebrospinal fluid and hippocampus of the treated rats. Measuring the GBC concentration in these tissues in order to demonstrate that the observed effect is due to GBC would therefore be useful. Another limitation is that the effect of GBC treatment on blood glucose and/or insulin concentration was not analysed although it could have been to prove that the GBC application worked as an antidiabetic drug. The study also needs to be accompanied with the corresponding images of brain tissue/hippocampal histological analysis. The final possible shortcoming of the study is that it did not include immunostaining results for neuronal apoptosis.
Our study identified that the replenishment of BDNF due to the action of GBC inhibits oxidative stress and cognitive impairment mediated by sevoflurane. The PI3K/Akt signalling pathways were also triggered by GBC, resulting in decreased hippocampal neuronal death. Under GBC therapy at 10 mg/kg b.w., sevoflurane-induced neurotoxicity, spatial learning deficits, and cognitive loss were diminished as changes related to inactivation of IL-6 and TNF-α in the hippocampus. Our results indicate that GBC may be a novel drug for preventing sevoflurane inhalation– induced impaired learning in the hippocampus. Our study therefore suggests that BDNF may be a vital target for the development of potential therapies in cognitive deficits and neurodegeneration. Further research is still needed, however, for confirmation by molecular and genetic analyses.