DOI:
10.1039/C6RA19715A
(Paper)
RSC Adv., 2016,
6, 93815-93825
Effects of cerebral glucose levels in infarct areas on stroke injury mediated by blood glucose changes†
Received
4th August 2016
, Accepted 19th September 2016
First published on 21st September 2016
Abstract
Admission hyperglycemia is considered to be related to poor outcomes of ischemic stroke. However, there is controversy regarding effects of attempts to lower blood glucose in stroke patients. This study aimed at determining the effects of blood glucose fluctuation on stroke injury by detection of cerebral glucose levels. A single intraperitoneal injection of glucose (0, 0.5, 1, 1.5 or 2 g kg−1) at 5 min before reperfusion caused blood glucose fluctuation (5–15 mmol L−1) lasting for 2 h after reperfusion. Blood glucose levels of 6–10 mmol L−1 decreased stroke injury after reperfusion for 24 h and 28 days compared with conditions of hyperglycemia (>10 mmol L−1) and hypoglycemia (<6 mmol L−1). High glucose concentration increased neuronal injury and death after oxygen–glucose deprivation. Under hyperglycemia and hypoglycemia, elevations in expression of MMP-2/-9 and decrease of tight junction proteins including occludin, claudin-5 and ZO-1 contributed to blood–brain barrier (BBB) dysfunction in infarct regions after ischemia–reperfusion injury, and reduction of hexokinase, pyruvate kinase and lactate dehydrogenase activities significantly inhibited the glucose metabolism in cortex and striatum after 24 h of reperfusion. BBB damage and reduced glucose metabolism caused accumulation of glucose in infarct areas. An obvious increase in cerebral glucose levels aggravated stroke injury.
1 Introduction
Hyperglycemia occurs in 30–40% of patients (without a history of diabetes) with acute ischemic stroke.1 Many studies have shown that admission hyperglycemia is associated with a worse prognosis and high mortality in ischemic disease, such as acute stroke and myocardial infarction. Evidence indicates that hyperglycemia, irrespective of diabetes mellitus status, may be a predictor of disease aggravation.2–6 However, no significant clinical benefit is observed by lowering blood glucose levels within 24 h of stroke onset using glucose–potassium–insulin treatment.7 Also, a meta-analysis indicated that controlling blood glucose level with insulin in the first hour of stroke does not obviously improve the functional outcome, but increases hypoglycemic episodes.8,9 A systematic review and meta-analysis of acute myocardial infarction also indicated that treatment of hyperglycemia did not stop the worsening progress and that admission hyperglycemia was not a good therapeutic target.2 Therefore, therapeutic attempts at lowering glucose are controversial in patients with ischemic stroke.
Most clinical and animal studies have focused mainly on the association between the hyperglycemia and stroke injury, with cerebral glucose changes generally being ignored.10 However, compared with blood glucose level, the cerebral glucose content is more closely relevant to the injury of ischemic stroke. How are cerebral glucose levels affected after stroke? How do changes in cerebral glucose affect the ischemic injury? Research into cerebral glucose levels may aid further explanation on the effects of blood glucose variance on stroke injury.
The cerebral glucose level is lower than the blood glucose level (cerebral glucose level is about 20% of that in blood).11 Under physiological conditions, the ratio of cerebral glucose to blood glucose significantly decreased following elevation of blood glucose level,12 indicating that the brain can control glucose entrance from the blood. The blood–brain barrier (BBB) and glucose transporter (GLUT) play crucial roles in regulating transport of glucose from blood to brain. The BBB selectively controls inward and outward transportation of materials via cell junctions and through cell surface transport systems and enzymes. Among the cell junctions at the BBB site, tight junctions are the most important controllers for restrictive functions of the BBB.13 The GLUTs also help to regulate glucose transport and maintain the cerebral glucose level.14 GLUT1 and GLUT3 isoforms are the primary transporters for glucose uptake in brain. Microvascular endothelial cells of BBB are highly enriched with GLUT1 (55 kDa).15 GLUT3 is found mostly in neurons, but is also found at low levels in neuropil and microvessel fractions.13,14,16 Therefore, BBB integrity and GLUTs function are crucial for regulation of glucose homeostasis in the brain.
This study aimed at identifying the effects of blood glucose fluctuation in acute reperfusion on ischemia/reperfusion (I/R) injury and recovery, with further analysis of cerebral glucose changes. Any cerebral glucose variance after I/R was investigated via BBB permeability, glucose transport and glucose metabolism in the brain.
2 Experimental procedures
2.1 Animals and middle cerebral artery occlusion (MCAO)
Male Sprague-Dawley rats (Experimental Animal Center of Chinese Academy of Medical Sciences, Beijing, China) were housed under standard conditions. The experimental protocols and animal care were performed according to the National Research Council's Guide for The Care and Use of Laboratory Animals, and were approved by the Institutional Animal Care and Use Committee of the Peking Union Medical College and Chinese Academy of Medical Sciences.
The rats (260–280 g) were fasted for 10 h to standardize glycemic state. Anesthesia was maintained with 2% isoflurane in nitrous oxide/oxygen (70
:
30). MCAO was performed as previously reported.17 Briefly, a nylon suture of 18–19 mm was inserted into the left internal carotid artery and gently advanced through the internal carotid artery to block the middle cerebral artery. The brain ischemia (at least 70% reduction of MCA flow compared with baseline) was confirmed by laser Doppler flowmetry (PeriFlux System 5000; Perimed, Stockholm, Sweden). Then, the rats were returned to their heated cages, with free access to water but not feed. After ischemia for 80 min, the success of the model was confirmed according to the neurological score,18 and rats with neurological score 1–4 were selected for further treatment. After ischemia for 90 min, the suture was removed to allow reperfusion. Food was given to the rats after determination of blood glucose.
2.2 Experimental design
The experimental schedule is shown in Fig. 1. To induce different levels of blood glucose in acute reperfusion, the animals were intraperitoneally injected with single dose of glucose (0, 0.5, 1, 1.5 or 2 g kg−1), or vehicle (distilled water, 5 mL kg−1) in the sham group, 5 min before reperfusion. Blood glucose level was measured using blood from the tail vessels with a commercial glucometer (Accu-Chek, Roche, Germany). Cerebral injury and functional recovery were assessed at 1 day and 28 days after reperfusion.
![image file: c6ra19715a-f1.tif](/image/article/2016/RA/c6ra19715a/c6ra19715a-f1.gif) |
| Fig. 1 Summary of the experimental schedule. I/R: ischemia and reperfusion; TTC: 2,3,5-triphenyltetrazolium chloride; EBST: elevated body swing test; OGD: oxygen–glucose deprivation; TUNEL: terminal deoxynucleotidyl transferase-mediated (dUTP) nick end labeling; HK: hexokinase; PK: pyruvate kinase; LDH: lactate dehydrogenase; MMP-2/-9: matrix metalloproteinases-2/-9; GLUT: glucose transporter. | |
2.3 TTC staining and behavior assessment
2,3,5-Triphenyltetrazolium chloride (TTC) staining was performed according to a previous method.17 The infarct area was analyzed using Image-Pro Plus 6.0 software: infarct area (%) = total infarct area/total section area × 100. At 24 hours after reperfusion, the neurological deficits of rats were assessed with the same scoring system.18
During the 4 weeks of observation, functional recovery was evaluated by the elevated body swing test (EBST), which was used to test asymmetric motor behavior and has been described in detail previously.19 The number of turns was recorded for 1 min, and the average scores for each testing (two sessions in each case) were calculated for each rat every week. The contralateral turns (%) = right turns/left turns × 100.
2.4 Magnetic resonance imaging (MRI)
MRI scans were performed after reperfusion for 1, 2, 3 and 4 weeks. The animals were anaesthetized with 1.5% isoflurane and fixed in a body restrainer with tooth-bar in an MRI spectrometer (PharmaScan 70/16, Bruker, Germany). Their brains were scanned using a rat head surface coil. T2-Weighted images were acquired with the following parameters: repetition time: 5 min; matrix size: 256 × 256 pixels; field of view: 30 × 25 mm; acquisition time: 10 min; slice thickness: 0.5 mm; TR = 5000 ms; TEeff = 33 ms. Thirty successive coronal images were acquired, from which the 3rd, 9th, 15th, 21st and 27th pictures were selected. Hyperintense infarct areas in T2-weighted images were assigned with a region of interest tool and analyzed using Image-Pro Plus 6.0 software: infarct area (%) = total infarct area/total section area × 100.
2.5 Glucose content and enzymatic activity detection in the brain
After anesthesia, the brains of rats were dissected out after transcardial perfusion with phosphate buffered saline (PBS). Prefrontal (1/7), middle (2/7–4/7, MCA-supported area) and posterior (5/7–7/7) cortex, striatum and hippocampus were freed from other parts.20 The tissues from different brain regions were homogenized with cold physiological saline (1 mg: 9 μL). The homogenate was centrifuged (5000 rpm for 15 min at 4 °C) to collect supernatants for determination of glucose content, hexokinase (HK), pyruvate kinase (PK) and lactate dehydrogenase (LDH) activity with commercial kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The total protein concentration was measured with a bicinchoninic acid (BCA) protein assay kit (PPLYGEN, China). All values were normalized by the total protein concentration.
2.6 Evans blue (EB) extravasation
EB (4% in saline, 2 mL kg−1, Sigma) was injected through the tail vein after reperfusion. After circulation for 30 min, EB was removed by intracardiac perfusion with PBS. Then the brains were quickly removed and placed on ice. Five sections (2 mm for each section) per rat were photographed using a digital camera. The ipsilateral hemisphere was weighed and homogenized with 2.5 mL of 50% trichloroacetic acid (TCA). The mixture was incubated overnight at 4 °C and then centrifuged at 4 °C and 12
000 rpm for 20 min. The EB concentration in diluted supernatant was measured with a spectrophotometer at 610 nm wavelength and compared against a standard curve. The amount of EB was normalized by the brain weight.
2.7 Organotypic brain slice cultures and oxygen–glucose deprivation/reoxygenation (OGD/R) injury
Brain slices were prepared according to the reported methods.21,22 Briefly, the Sprague-Dawley rats (7 days old) were sacrificed by decapitation and the cerebellum was immediately immersed in ice-cold artificial cerebral spinal fluid (ACSF) and bubbled with 5% CO2 and 95% O2 for 2 min. Coronal slices (300 μm) in the MCA-supported location were cut using a Leica vibrating-blade microtome (VT1200 S) with a speed setting of 0.5 mm s−1 and oscillation amplitude of 1.5 mm. The slices were in dissection medium (ACSF) for 0.5 h at 4 °C, and were transferred to inserts (pore size 0.4 μm, PICM03050, Millipore) in six-well plates (three slices/insert). The culture medium contained 50% MEM, 24% horse serum, 25% HBSS, 1% penicillin–streptomycin (all from Life Technologies) supplemented with 4 mM NaHCO3, 12.5 mM Hepes, and glucose to 25 mM (pH 7.2). After culture for 10–15 days at 37 °C with 5% CO2 in air, the brain slices were incubated with ACSF (without glucose, pH 7.2) in a hypoxic chamber (Billups-Rothenberg, USA) with the oxygen concentration (<0.2%) monitored by an oxygen analyzer (Nuvair, USA) at 37 °C for 2 h. Then, the slices were incubated with the culture medium containing 25, 50 or 75 mM glucose for 24 h.
Cellular uptake of the fluorescent dye propidium iodide (PI) was used to assess OGD/R-induced cell injury in organotypic slices. According to a reported method,23 PI was added to the culture medium (5 μg mL−1) and incubated for 1 h, then the red fluorescence-labeled slices were observed and imaged using an inverted fluorescence microscope (Nikon ECLIPSE Ti, Japan).
2.8 TUNEL assay
After reperfusion for 24 h, the brains of the rats in each group (n = 4) were removed after transcardia perfusion of PBS and fixed in 4% paraformaldehyde. Paraffin-embedded coronal sections (5 μm thick) were selected for terminal deoxynucleotidyl transferase-mediated (dUTP) nick end labeling (TUNEL) staining according to the kit's instruction (Roche Diagnostics, Germany). Briefly, the dewaxed sections were incubated with horseradish peroxidase (HRP)-labeled dUTP solution. After washing, converter-POD was added to the slides. Then the sections were counterstained with 3,3′-diaminobenzidine tetrahydrochloride (DAB). Images were obtained using an inverted digital imaging light microscope (Olympus BX51, Japan). The integrated optical density of photographs were analyzed using Image-Pro Plus 6.0 software.
2.9 Immunohistochemistry
According to a previous method,24 occludin (Santa Cruz Biotechnology, USA) stained sections were observed and imaged with a light microscope. The photographs were analyzed with Image-Pro Plus 6.0 software.
2.10 Western blotting assay
The brain tissues or collected cells were lyzed with RIPA buffer containing a protease inhibitor cocktail (Thermo Scientific, USA). Following the same procedure,24 protein expression of matrix metalloproteinases (MMP)-9, MMP-2, occludin, ZO-1 (Santa Cruz Biotechnology, USA) and claudin-5 (LifeSpan Biosciences, USA) was detected using chemical luminescence (PPLYGEN, China) and visualized with a luminescent image analyzer (ImageQuant LAS4000mini, Sweden). Densitometric analyses were performed using Gel-Pro Analyzer software (Media Cybernetics).
2.11 Statistical analysis
All data are presented as mean ± SD. Statistical analysis was done by one-way analysis of variance (ANOVA) followed by Bonferroni's multiple comparisons test, Dunnett's t-test or student's t-test as appropriate. Statistically significant differences between groups were defined as p < 0.05 and these are indicated in figure legends.
3 Results
3.1 Effects of blood glucose fluctuation on I/R injury
Blood glucose levels increased following elevation of glucose doses. Two hours after reperfusion, blood glucose concentrations in the glucose-treated groups got back to the level of the 0 g kg−1 glucose group (Fig. 2A). In the TTC stained sections, the white region represents the infarct area (Fig. 2C). Under the blood glucose fluctuation (5–15 mmol L−1) in acute reperfusion, I/R had varying influence on brain injury based on the results of TTC staining, neurological score and infarct area (Fig. 2B–D). I/R injury under blood glucose levels of 6–10 mmol L−1 (0.5 and 1 g kg−1 glucose groups) was more serious than in hypoglycemic (≈5 mmol L−1 in 0 g kg−1 glucose group) or hyperglycemic (>10 mmol L−1 in 1 and 2 g kg−1 glucose groups) conditions, wherein, glucose (1 g kg−1) treatment significantly decreased the neurological score and infarct area compared with the 0 g kg−1 glucose group (p < 0.05). However, the brain injury after 2 g kg−1 glucose exposure was more serious than in the glucose (0 g kg−1) group (p < 0.05).
![image file: c6ra19715a-f2.tif](/image/article/2016/RA/c6ra19715a/c6ra19715a-f2.gif) |
| Fig. 2 Effects of blood glucose fluctuation in acute reperfusion on ischemia–reperfusion injury after 24 h reperfusion. (A) Blood glucose changes after single intraperitoneal injection of glucose (0, 0.5, 1, 1.5 or 2 g kg−1) at 5 min before reperfusion. a p < 0.05, b p < 0.01, c p < 0.001 vs. 0 g kg−1 group, n = 10. (B) Neurological score of rats after reperfusion for 24 h. (C) Representative TTC stained brain sections. (D) Statistical analysis of infarct area in TTC stained slices. Data are expressed as mean ± SD. * p < 0.05, ** p < 0.01 vs. 0 g kg−1 group, n = 6. | |
During the 4 weeks of observation, the survival rate was 90% in the 1 g kg−1 glucose group, and 70% in the 0 or 2 g kg−1 glucose groups (Fig. 3A). The body weight of rats after 1 g kg−1 glucose treatment was higher than that of rats in the 0 and 2 g kg−1 glucose groups after 1 week' reperfusion (Fig. 3B). In the EBST test (Fig. 3C), sham-operated animals did not typically exhibit a side bias throughout the experiment, whereas rats with unilateral stroke displayed a strong tendency to turn toward the contralateral side. The group treated with 1 g kg−1 glucose showed a significant improvement compared with the 2 g kg−1 glucose group after injury for 4 weeks (p < 0.05). After reperfusion for 3 weeks, the infarct areas were markedly decreased in the 1 g kg−1 glucose group versus the 0 and 2 g kg−1 groups (Fig. 3D).
![image file: c6ra19715a-f3.tif](/image/article/2016/RA/c6ra19715a/c6ra19715a-f3.gif) |
| Fig. 3 Effects of blood glucose fluctuation in acute reperfusion on ischemia–reperfusion injury after reperfusion for 28 days. (A) Survival rate of rats. (B) Statistical analysis of body weight of rats. a1,b1,c1,a2,b2 p < 0.01; a3,b3,a4,b4 p < 0.05 vs. sham. (C) Results of elevated body swing test (EBST). (D) Representative coronal T2-weighted images of brain. The hyperintensity (white) corresponds to the ischemic area. Statistical analysis of infarct area in T2WI. Data are expressed as mean ± SD. * p < 0.05, ** p < 0.01 vs. 1 g kg−1 group, n = 7. | |
3.2 Changes in cerebral glucose levels after I/R treatment
Glucose content in different brain regions was determined (ESI Fig. 1,† Fig. 4A and B). Although blood glucose level was significantly elevated at 30 min after 1 or 2 g kg−1 glucose administration, the cerebral glucose levels remained at a stable level in the sham rats. However, I/R for 30 min caused elevation of glucose levels in different brain regions and a significant increase of glucose in the striatum in the 0 and 2 g kg−1 glucose groups compared with the sham group (p < 0.05). There was no obvious difference in cerebral glucose levels between the 1 g kg−1 glucose and 0 or 2 g kg−1 glucose groups after I/R 30 min for, but significant differences were seen after I/R for 1 day and 28 days. Data showed that cerebral glucose level was significantly increased in 0 g kg−1 glucose-treated rats (Mid-C, striatum) and 2 g kg−1-glucose treated rats (Pre-C, Pos-C, striatum) compared with the 1 g kg−1 glucose group (p < 0.05).
![image file: c6ra19715a-f4.tif](/image/article/2016/RA/c6ra19715a/c6ra19715a-f4.gif) |
| Fig. 4 Changes in cerebral glucose levels and effects on brain injury after reperfusion. Glucose levels were determined in different brain regions including prefrontal cortex (Pre-C), middle cortex (Mid-C), posterior cortex (Pos-C), striatum and hippocampus after blood reperfusion for 1 day (A) and 28 days (B). TUNEL-stained brain sections after reperfusion for 24 h and the statistical analysis, scale bar = 100 μm (C). Data are expressed as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. sham; # p < 0.05, ## p < 0.01 vs. 1 g kg−1 group, n = 6. | |
3.3 Cerebral glucose increase promotes cellular apoptosis, as detected by TUNEL
TUNEL-positive cells were brown after DAB staining (Fig. 4C). After reperfusion for 24 h, the apoptotic cells in all test groups increased versus the sham (p < 0.05). Compared with the 1 g kg−1 glucose group, TUNEL-positive cells were obviously increased in the 0 g kg−1 group and had a significant increase in 2 g kg−1 glucose group (p < 0.05). This indicates that cerebral glucose increase aggravated stroke injury.
3.4 Effects of different glucose levels on OGD/R injury in brain slices
PI-labeled cells (red) in brain slices indicated the injured cells caused by OGD/R exposure (p < 0.05, Fig. 5). Restoration of culture medium for 24 h caused significant increase of PI-positive cells in both cortex (Fig. 5A) and striatum (Fig. 5B) after OGD (p < 0.05). High glucose (50 and 75 mM) challenge markedly increased the cellular injury compared with the 25 mM glucose group (p < 0.05), indicating that high glucose exposure could aggravate OGD/R-induced injury of brain.
![image file: c6ra19715a-f5.tif](/image/article/2016/RA/c6ra19715a/c6ra19715a-f5.gif) |
| Fig. 5 Effects of glucose changes on brain injury after oxygen and glucose deprivation. Representative images of propidium iodide (PI) uptake in cortex (A) and striatum (B) in organotypic brain slices and statistical analysis of PI-positive cells. Data are expressed as mean ± SD. * p < 0.05, ** p < 0.01 vs. sham; # p < 0.05 vs. 1 g kg−1 group, n = 6. | |
3.5 BBB changes after I/R for 30 min
After I/R for 30 min, impairment of the BBB was assessed in infarct hemisphere. EB leakage mainly occurred at the initial part of MCA and the striatum (Fig. 6A). The EB content in the 1 g kg−1 glucose group was lower than in the non-glucose group, and was significantly decreased versus the 2 g kg−1 group (p < 0.05, Fig. 6B). Expression of MMP-2/9 and tight junction protein (TJP) was detected to determine the extent of BBB disruption. From Fig. 6C, expression of MMP-2/9 significantly increased in the 0 and 2 g kg−1 groups compared with the 1 g kg−1 group (p < 0.05). Immunohistochemical results showed strong expression of occludin in blood vessels in the sham group, with moderate expression 30 min after reperfusion in the 1 g kg−1 glucose treated group (Fig. 7A). Loss of occludin expression was observed in parts of blood vessels after 0 or 2 g kg−1 glucose exposure. As predicted, occludin expression significantly decreased after I/R injury (p < 0.05). Occludin expression in the striatum in the 2 g kg−1 group was much lower than in the 1 g kg−1 group (Fig. 7B, p < 0.05). Further studies indicated that ZO-1 expression in both the cortex and striatum, as well as claudin-5 expression in the cortex, was markedly decreased in the 0 and 2 g kg−1 groups versus the 1 g kg−1 group (Fig. 7C and D, p < 0.05). These results revealed that BBB permeability was subject to more serious damage under hypoglycemia or hyperglycemia in the early period of reperfusion.
![image file: c6ra19715a-f6.tif](/image/article/2016/RA/c6ra19715a/c6ra19715a-f6.gif) |
| Fig. 6 Effects of blood glucose fluctuation on blood–brain barrier permeability in ipsilateral hemisphere after reperfusion for 30 min. (A) Representative images of Evans blue-stained brain sections from rats subjected to 90 min middle cerebral artery occlusion (MCAO) followed by 30 min of reperfusion (n = 3). Evans blue was clearly observed in the ipsilateral hemisphere. (B) Quantitative analysis of Evans blue in brain (n = 4). (C) Protein expressions of MMP-2 and MMP-9 in the ipsilateral hemisphere (n = 3). Data are expressed as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. sham; # p < 0.05, ## p < 0.01, ### p < 0.001 vs. 1 g kg−1 group. | |
![image file: c6ra19715a-f7.tif](/image/article/2016/RA/c6ra19715a/c6ra19715a-f7.gif) |
| Fig. 7 Effects of blood glucose fluctuation on tight junction protein expression in ipsilateral hemisphere after reperfusion for 30 min. (A) Expression of occludin in vascular endothelial cell by immunohistochemical staining, scale bar = 100 μm. Protein expression of occludin (B), ZO-1 (C) and claudin-5 (D) in cortex and striatum of rats after reperfusion for 30 min. Data are expressed as mean ± SD. ** p < 0.01, *** p < 0.001 vs. sham; # p < 0.05, ## p < 0.01, ### p < 0.001 vs. 1 g kg−1 group, n = 3. | |
BBB injury promoted glucose translocation from blood to the brain. However, the cerebral glucose level was also controlled by the glucose metabolism conditions. High enzyme activities of HK, PK and LDH were observed after I/R for 30 min (ESI Fig. 2†), wherein, enzyme activities were markedly increased in the striatum in model groups compared with the sham. However, no obvious changes of enzyme activities were found between the 0 and 2 g kg−1 glucose and the 1 g kg−1 glucose groups.
3.6 Changes of BBB and enzyme (HK, PK, LDH) activities in brain after I/R for 24 h
I/R for 24 h caused obvious loss of tight junction proteins (Fig. 8A), wherein 2 g kg−1 glucose treatment significantly decreased expression of occludin, claudin-5 and ZO-1 compared with 1 g kg−1 glucose treatment (p < 0.05). Moreover, the claudin-5 and ZO-1 levels in the 0 g kg−1 group were lower than in the 1 g kg−1 group (Fig. 8B–D, p < 0.05). The HK, PK or LDH activities in Pre-C or striatum were much higher in the 1 g kg−1 glucose group than in the 0 and 2 g kg−1 groups (p < 0.05, Fig. 9). Therefore, more serious BBB damage and lower level of glucose metabolism contributed to cerebral glucose increase after 0 and 2 g kg−1 glucose treatment compared with 1 g kg−1 glucose treatment after I/R for 24 h.
![image file: c6ra19715a-f8.tif](/image/article/2016/RA/c6ra19715a/c6ra19715a-f8.gif) |
| Fig. 8 Effects of blood glucose fluctuation on expression of tight junction proteins in ischemic hemisphere after reperfusion for 24 h. (A) Representative images of occludin expression in vascular endothelial cell by immunohistochemical staining, scale bar = 100 μm. Protein expression of occludin (B), ZO-1 (C) and claudin-5 (D) in infarct region of brain. Data are expressed as mean ± SD. *** p < 0.001 vs. sham; # p < 0.05, ## p < 0.01 vs. 1 g kg−1 group, n = 3. | |
![image file: c6ra19715a-f9.tif](/image/article/2016/RA/c6ra19715a/c6ra19715a-f9.gif) |
| Fig. 9 Effects of blood glucose fluctuation on glucose metabolism in ischemic hemisphere after reperfusion for 24 h. (A) Hexokinase (HK), (B) pyruvate kinase (PK) and (C) lactate dehydrogenase (LDH) activities in prefrontal cortex (Pre-C), middle cortex (Mid-C), posterior cortex (Pos-C), striatum and hippocampus. Data are expressed as mean ± SD. * p < 0.05, ** p < 0.01 vs. sham; # p < 0.05, ## p < 0.01, ### p < 0.001 vs. 1 g kg−1 group, n = 6. | |
4 Discussion
The effects of blood glucose fluctuation on stroke injury were studied in the early period of reperfusion. First, blood glucose concentration changes were explored after single injection (i.p.) of glucose dosage (0, 0.5, 1, 1.5 or 2 g kg−1) in rats. Five minutes after glucose administration, blood glucose levels showed significant changes (ESI Fig. 3†). Therefore, intraperitoneal administration of glucose 5 min before reperfusion was adopted into the experimental design to study the effects of blood glucose fluctuation in early reperfusion on ischemia–reperfusion injury.
Although stress-induced hyperglycemia is to some extent adaptive and some regions of ischemic brain may require glucose supply,25 the glucose level in the infarct area was higher than in the normal region and was sufficient for the brain to use. Previous reports have documented that high glucose induces neuron injury, involving oxidative stress via NADPH-dependent generation of ROS and apoptosis.26,27 Consistent with these results, this study further clarified that high glucose exposure increases OGD/R-induced injury in brain slices. Hence, suggesting that hyperglycemia should be corrected in patients with acute stroke. Meanwhile, it is worth noting that hypoglycemic disaster can be caused by overcorrection of hyperglycemia.
Previous studies have indicated that ischemic stroke causes disruption of the BBB, which further disturbs the vascular permeability and promotes exacerbation of edema.28,29 Activation of protein kinase C (PKC)-β and consequent stimulation of oxidative stress are involved in the hyperglycemia-evoked BBB damage.30 In addition, hyperglycemia causes elevation of NADPH oxidase and MMP-2/-9 activities, and decrease TJP levels, which contribute to BBB dysfunction.30,31 Results from the present study suggest that high blood glucose levels elevate expression of MMPs and promote degradation of TJP. It was noteworthy that hypoglycemia had the same effects as hyperglycemia on BBB permeability, which may be associated with oxidative stress and inflammation.32,33 Additionally, endothelial dysfunction and loss of BBB integrity evoked by hypoglycemia may be mediated by Nrf2 suppression and inhibition of the protective effects of flow-induced shear stress.34,35 The molecular mechanisms underlying hyper- and hypoglycemia-elicited BBB endothelial dysfunction are not yet well understood and require further study.
Glucose deprivation enhances activity and expression of the SGLT-like glucose transporter, which is important in maintaining glucose concentration under hypoglycemic stress.36,37 In the current study, expressions of GLUT1 and GLUT3 were up-regulated in all test groups compared with the sham. However, their expressions showed no marked difference between the 1 g kg−1 glucose group and the 0 and 2 g kg−1 glucose groups after reperfusion for 30 min and 24 h (ESI Fig. 4†).
HK, PK and LDH, the key enzymes of glucose metabolism, were found to be significantly increased in the 0 and 2 g kg−1 glucose groups compared with the 1 g kg−1 group at the 1st day and 28th day but 30 min after reperfusion. In early reperfusion, hypermetabolism of glucose could provide more energy for ischemic and hypoperfused brain tissue.38 However, the BBB function disorder in acute reperfusion allowed glucose to enter the brain, and the increased glucose further damaged the neuronal cells and BBB following the reperfusion process. The mitochondrial respiratory chain appears to be susceptible to ischemia and reperfusion. Both ischemia and reperfusion damaged the complexes of the mitochondrial respiratory chain by production of free radicals.39 High glucose promotes production of superoxide by donating glucose-derived reducing equivalents to molecular oxygen.25 Despite the high activities of HK, PK and LDH, limitations on mitochondrial oxidation of pyruvate and secondary deterioration of mitochondrial function could markedly decrease glucose oxidation,40 and promote lactate production (ESI Fig. 5†). Moreover, the accumulated glucose cannot be used effectively because of cellular loss and injury in the later period of reperfusion. Therefore, glucose metabolism disruption resulted in accumulation of glucose, which mainly occurred in the later period of reperfusion.
5 Conclusion
In summary (Fig. 10), hyperglycemia (>10 mmol L−1) and hypoglycemia (<6 mmol L−1) in the early period of reperfusion can aggravate ischemic stroke injury because of marked cerebral glucose increase. Disruption of the BBB and decrease in glucose metabolism contributed to the elevation in cerebral glucose. Hence, it is important to regulate blood glucose level, especially before reperfusion, in attempts to decrease cerebral ischemia–reperfusion injury. The present study indirectly showed that fasting of animals before middle cerebral artery occlusion (MCAO) can provide a more stable model of stroke. Control of cerebral glucose levels provide a target for future study on therapy for stroke.
![image file: c6ra19715a-f10.tif](/image/article/2016/RA/c6ra19715a/c6ra19715a-f10.gif) |
| Fig. 10 Summary of the current study. I/R: ischemia and reperfusion, BBB: blood–brain barrier. | |
Acknowledgements
This work was supported by the National Natural Science Foundation of China (81274122, 81173578, 81202507, 81373997), the National Mega-project for Innovative Drugs (2012ZX09301002-004, 2012ZX09103101-006, 2012ZX09301002-001), the National High-Tech R&D Programme (863 Program) (2012AA020303), the Program for Changjiang Scholars and Innovative Research Team in University (PCSIRT) (IRT1007), the Specialized Research Fund for the Doctoral Program of Higher Education of China (20121106130001), Beijing Natural Science Foundation (7131013, 7142115), Beijing Key Laboratory of New Drug Mechanisms and Pharmacological Evaluation Study (BZ0150).
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Footnote |
† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra19715a |
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