Investigating cerebral neurovascular responses to hyperglycemia in a rat model of type 2 diabetes using multimodal assessment techniques

Figures

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  Figure 1. ECoG-LSCI setup and experimental protocol To assess ECoG signals and cerebral blood flow, an ECoG-LSCI neurovascular imaging system was utilized. Screw electrodes implanted in the S1FL were used to record neural activity via the ECoG. Electrical shocks were applied to the forepaws to assess changes in brain wave patterns in response to electrical stimulation. Concurrently, following craniotomy, an LSCI system was used to irradiate the cortex with a 660 nm laser. After images were taken with a CCD camera and uploaded to a computer, we used MATLAB for cerebral blood flow analysis. An ECoG recording from the S1FL (AP = +1.0 mm, ML = +4.0 mm), the reference electrode location, and the location where KCl was administered, along with the skull and the screw electrode placement, are shown in the picture.

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  Figure 2. Experimental schedule for the establishment of the HFD/STZ-induced T2DM animal model and ECoG-LSCI during peripheral stimulation (A) Rats were divided into two groups: the control and HFD/STZ groups. On day 21, the rats were administered STZ (40 mg/kg/d × 5 days, intraperitoneal injection) for 5 consecutive days, and blood glucose levels and body weights were monitored once a week until day 49. Diabetic rats (defined as those with blood glucose levels ≥ 288 mg/dL) were identified 28 days after STZ administration. Next, a glucose metabolism test was performed. After ECoG-LSCI, the rats were sacrificed, and the brains were removed for protein expression analysis. (B) One-half of the rat brain was dissected, and the location of the S1FL was determined. There were 3 holes in the brain tissue: 1 for the ECoG electrode, 1 for the reference electrode, and 1 for KCl delivery. Electrical signals in the S1FL were detected after 30 s of electrical stimulation at 6 mA. A combination of electrophysiology and LSCI was used to monitor electrical stimulation-evoked activity in the S1FL after 3 stimuli (30 s of electrical stimulation at 6 mA with 60-s intervals). ECoG and LSCI were used to…

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  Figure 3. Changes in body weight and blood glucose levels in HFD/STZ-induced diabetic model rats (A) Body weight changes in control rats and HFD/STZ-induced diabetic model rats were not significant. (B) After STZ injection, the blood glucose concentration in the HFD/STZ group was significantly greater than that in the control group on day 28. (C) Fasting blood glucose levels in the control group and HFD/STZ group. The fasting blood glucose level in the HFD/STZ group was significantly greater than that in the control group. (D) According to the insulin tolerance test, HFD/STZ model rats were not sensitive to insulin and showed insulin resistance. (E) According to the glucose tolerance test, blood glucose levels in the HFD/STZ group were greater than those in the control group. (F) The area under the curve (AUC) for the glucose tolerance test was significantly greater in the HFD/STZ group than in the control group. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 (n = 8 in each group). Data are represented as mean ± SD.

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  Figure 4. ECG recordings of rats in the control and HFD/STZ groups during electrical stimulation Three 30-s trains of electrical stimulation were applied to the S1FL. MATLAB was used to obtain calibrated evoked potentials and to analyze the average evoked potential plots. The baseline and average shock responses were determined from these plots. The electrical stimulation parameters included an amplitude of 6 mA, pulse width of 0.2 ms, frequency of 5 Hz, and duration of 30 s; the calibrated evoked potential was first determined, and then electrical stimulation was applied 10 s later for 30 s. (A) The mean evoked potential in the control group. The thumbnail represents the calibrated evoked potential. (B) Mean evoked potentials in the HFD/STZ group; the thumbnail shows the calibrated evoked potentials. (C) Analysis of electrophysiological results using MATLAB. Column analysis was performed using GraphPad Prism, and differences between the control and HFD/STZ groups were analyzed using an unpaired t test. Potential differences between the control and HFD/STZ groups; control vs. HFD/STZ, ∗∗∗p < 0.001. (D) Conduction latency; there was no significant difference between the control and HFD/STZ…

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  Figure 5. Effect of HFD/STZ-induced hyperglycemia and acute hyperglycemia on the number of CSD events and CSD velocity LSCI was used to detect alterations in cortical blood flow after KCl-induced CSD. CSD was induced by applying 4 M KCl for 40 min. The images on the left are centered on bregma, and P1 and P2 are marked. CSD velocity was determined by measuring blood flow in two circular regions of interest (ROIs) in the cerebral cortex separated by 3 mm. The velocity of the CSD was calculated by dividing the actual distance by the time difference between the peaks at that cortical location. The direction of the CSD is indicated by arrows. The degree of blood flow change over time was dependent on the distance between the two points and the time difference between the peaks at that cortical location. (A) An LSCI image of a control rat brain. (B) Dynamic changes in blood flow in the control group at 0, 125, 315, 480, 755, 1,025, 1,310, 1,550, 1,680, and 2,400 s. (C) LSCI images of the brains of rats in the HFD/STZ group. (D) Dynamic changes in blood flow in the HFD/STZ group at 0, 130, 270, 485, 690, 895, 1,170, 1,350, 1,645, and 2,400 s. (E) LSCI images of the brains of rats in the IPI…

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  Figure 6. Effects of HFD/STZ and acute hyperglycemia on cerebral neurovascular coupling Cerebral blood flow changes during KCl-induced CSD were recorded by LSCI, with 2 images captured per second (frame rate 2) for a total of 2,400 s. Using MATLAB, all the images were analyzed at a rate of 2 images/second, and the values were calculated to assess the CBF and determine the conditions under which CSD occurred. (A) From left to right, cerebral blood flow analysis in the control group, HFD/STZ group, and IPI group. (B) The duration of CSD in the control group, HFD/STZ group, and IPI group (from left to right). (C) After KCl-induced CSD was analyzed using MATLAB, the difference in the number of CSD events among the control group, HFD/STZ group, and IPI group was determined. (D) Differences in CSD velocity among the control group, HFD/STZ group, and IPI group. (E) Electrical stimulation-induced changes in blood flow in the control group, HFD/STZ group, and IPI group were compared. The data were recorded for a total of 60 s. Stimulation started at 10 s and continued for 40 s. (F) There was no difference between the groups before stimulation after normalization. There was a significant difference…

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  Figure 7. Expression levels of P-AKT473, P-GSK3β, BDNF, NeuN, and GFAP in the brains of HFD/STZ model rats (A) Representative western blot images of AKT473, GSK3β, BDNF, NeuN, GFAP, and β-actin. (B) Quantification of AKT473, GSK3β, BDNF, NeuN, GFAP, and β-actin levels. β-Actin was used as an internal control. In the western blot image, “-” indicates the control group, and “+” indicates the HFD/STZ group. Protein expression levels were quantified by densitometric analysis of the western blot bands and are presented as the means ± SEMs of eight rats per group. ∗p < 0.05, ∗∗∗p < 0.001 compared to control values. (n = 8 in each group). Data are represented as mean ± SD.

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  Figure 8. Summary of the effects of type 2 diabetes on brain signaling molecules HFD/STZ-induced diabetes results in hyperglycemia and impaired brain function. When glucose concentrations in the blood increase, tissue is damaged, resulting in a decrease in NeuN and BDNF expression and an increase in GFAP expression, causing synaptic dysfunction. Elevated glucose levels lead to decreased AKT and GSK3β levels. Dysfunction of physiological neurovascular coupling leads to decreased neuronal activity, imbalance of ion homeostasis, and abnormal blood flow regulation. NeuN, neuronal nuclear antigen; BDNF, brain-derived neurotrophic factor; AKT, AKT kinase serine; GSK3β, glycogen synthase kinase 3β.