Characterizing region-specific glucose metabolic profile of the rodent brain using Seahorse XFe96 analyzer

Animals

Sprague Dawley male and female rats (3-month-old) were purchased from the Charles River Laboratories (Hollister, CA). C57BL/6J female mice (3-month-old) were purchased from the Jackson Laboratory (Bar Harbor, ME). All animals were kept in independent ventilating cages with access to laboratory chow and water ad libitum under a fixed 12:12 light-dark cycle. All animal procedures were reviewed and approved by the University of North Texas Health Science Center Institutional Animal Care and Use Committee and followed EU Directive 2010/63/EU Guide for the Care and Use of Laboratory Animals.

Preparation of acute brain slices

Animals were anesthetized using isoflurane inhalation and brains were immediately removed and placed in ice-cold oxygenated artificial cerebrospinal fluid (aCSF; in mmol/L: 120 NaCl, 3.5 KCl,1.3 CaCl₂, 1 MgCl₂, 0.4 KH₂PO₄, 5 HEPES, 10 D-glucose, pH 7.4). The aCSF was oxygenated by 95% O₂ with 5% CO₂ at least one-hour prior to brain dissection. Agarose (4%) was used to make gel block and brains were super-glued to the block and then fixed to the tissue holder of a vibratome (VT1000S, Leica, Nussloch, Germany) filled with oxygenated ice-cold aCSF. Coronal and sagittal sections (180 μm thickness) were obtained from cerebrum and cerebellum, respectively. The sections were transferred to a chamber containing aCSF with continuous oxygenation at room temperature.

Tissue punching and Seahorse XFe96 analysis

Brian sections were transferred to a 60 mm cell culture dish containing oxygenated aCSF at room temperature. Reusable biopsy punch (0.5 mm or 0.75 mm diameter, World Precision Instrument, FL, USA) was used to obtain tissue punches from different regions of the cerebral and cerebellar sections under a Zeiss surgical microscope (Carl Zeiss, Germany). The punch was gently pressed overtop the area of interest in the brain section and the obtained tissue punch was immediately injected into a poly-L-lysine (Sigma-Aldrich, USA) coated XFe96 Cell Culture Microplate (Agilent Technologies, CA) based on pre-defined layout. Each well contained 180 μl aCSF with 1.0 mM pyruvate solution as assay media. After loading the 96-well plate, punches were manually manipulated to position in the bottom center of each well. For each Seahorse assay, brain punches derived from the same animal were seeded in a 96-well plate with n = 12 to 16 for each group. The XFe96 Cell Culture Microplate was then transferred to a non-CO2 incubator at 37 °C for 30 min. During this period, 10X concentration of each drug for assay (prepared in aCSF) was loaded in each injection port (A, B, C, D) of the Seahorse XFe96 Assay sensor cartridge that had been hydrated overnight and exchanged for XF Calibrant solution 3 hours prior to assay initiation. The drug-filled sensor cartridge was inserted into the Seahorse XFe96 Analyzer (Agilent Technologies, CA) for calibration. Once the calibration finished, the calibration plate was replaced by the tissue-containing microplate to initiate the assay.

Assay protocol contained 4-cycle measurement of OCR/ECAR baseline. Four to twelve cycles measurement was made after injection of oligomycin (Sigma Aldrich, USA), gboxin, or genipin (Cayman Chemical, MI, USA). Four cycles measurement was made after injection of carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone (FCCP) supplied with pyruvate (Sigma Aldrich, USA). Four cycles measure were finally made after injection of rotenone (EMD Millipore, Germany)/antimycin A (Sigma Aldrich, USA). Each cycle consists of 2-min mix, 1-min wait and 3-min measure period. All the concentrations indicated are final concentration in the well after injection.

Immunohistochemistry and confocal microscopy

Young adult female rats were sacrificed by anesthesia and cardiac perfusion with ice-cold normal saline. The brains were harvested and fixed in 4% paraformaldehyde and processed for paraffin embedding. Coronal and sagittal brain sections (7-µm thick) were obtained from the cerebrum and cerebellum, respectively, for immunostaining. Sections were blocked with 0.2% Triton-X in Superblock Blocking Buffer™ (Thermo Scientific, USA) for 20 min at room temperature after de-paraffinization and antigen retrieval. Sections were then washed and incubated with primary antibodies (NeuN: Mouse mAb, 1:150 dilutions, catalog #MAB377, Miillipore; GFAP, Rabbit mAb, 1:250 dilutions, catalog #12389, Cell Signaling Technology) at 4 °C overnight followed by 60-min incubation of Alexa Fluor conjugated secondary antibodies (Thermo Fisher Scientific, USA). The sections were mounted with ProLong™ Gold antifade reagent with DAPI (Invitrogen, USA). Z-stack microscopic images were obtained by Zeiss LSM 510 META confocal microscope (Carl Zeiss, USA). Images were processed for maximum intensity Z-projection by ImageJ software (NIH, USA).

Western blots

Young adult mice were euthanized and perfused with ice-cold normal saline. The cerebral cortex, hippocampus, and cerebellum were dissected and protein extractions were obtained for Western blots carried out as previously detailed, ¹³ using following primary antibodies: β3-Tubulin (D71G9) (Rabbit mAb, 1:1000 dilutions, catalog #5568, Cell Signaling Technology); Aldehyde dehydrogenase 1 family, member L1 (ALDH1L1, Rabbit Ab, 1:1000 dilutions, catalog #ab87117, Abcam); GFAP (D1F4Q) (Rabbit mAb, 1:1000 dilutions, catalog #12389, Cell Signaling Technology); GAPDH (Mouse mAb,1:5000 dilutions, catalog #sc-3223, Santa Cruz Technology); Phospho-AMPKα (Thr172) (Rabbit mAb, 1:1000 dilutions, catalog #4188, Cell Signaling Technology); AMPKα (23A3) (Rabbit mAb, 1:1000 dilutions, catalog #2603, Cell Signaling Technology); Phospho-Acetyl-CoA Carboxylase (Ser79) (Rabbit Ab, 1:1000 dilutions, catalog #3661, Cell Signaling Technology); Acetyl-CoA Carboxylase (C83B10) (Rabbit Ab, 1:1000 dilutions, catalog #3676, Cell Signaling Technology); Phospho-LKB1 (Ser428) (C67A3) (Rabbit Ab, 1:1000 dilutions, catalog #3482, Cell Signaling Technology); LKB1 (D60C5F10) (Rabbit Ab, 1:1000 dilutions, catalog #13031, Cell Signaling Technology). The membrane was then incubated by HRP-conjugated secondary antibodies (The Jackson ImmunoReseach Laboratories). Chemiluminescence was detected with Biospectrum 500 UVP imaging system. Protein band density was quantified by ImageJ (NIH, USA) and normalized to GAPDH.

Data collection and statistical analysis

The animal data reporting of the current study has followed the ARRIVE 2.0 guidelines. ¹⁴ Raw data of OCR and ECAR curve for each well were complied with Agilent’s Wave 2.6.0 software. Wells with detached or broken tissue punch observed after assay were excluded from analysis. The basal respiration was measured as OCR in the absence of mitochondria inhibitors. The residual OCR values and OCR reduction after oligomycin or gboxin injection was used as proton leak-linked respiration and ATP-production coupled respiration, respectively. The OCR measurement after FCCP/pyruvate injection without oligomycin treatment was used as maximal respiration. The difference between maximal OCR and basal OCR was considered as spare respiration capacity. OCR measurement after rotenone/antimycin A (Rot/AA) injection represented non-mitochondria respiration (Figure 1(a)). ECAR was used for measure of glycolysis rate. The baseline ECAR was obtained at the starting assay conditions, while the stressed ECAR was obtained in the presence of stressor mix (oligomycin + FCCP). The metabolic phenotype showed the baseline OCR/ECAR of punches, as well as the maximal OCR (obtained after FCCP injection) and ECAR (after oligomycin/FCCP injection). In each figure (panel), data were derived from the brain punches from the same animal and seeded in the same plate. Each experiment was repeated at least 3 times under the same condition from different animals.

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Figure 1.

Metabolic analysis of acute tissue punches (0.5 mm diameter) from rodent brain sections (180 µm thick). (a) Schematic diagram depicts the step of brain harvesting, brain sectioning, tissue punching and seeding in 96-well XFe plate for Seahorse extracellular flux analysis (Created with BioRender.com). (b, c, d, e, f) Metabolic analysis of cerebral cortex (CX), basal ganglia (BG), and cerebellum (CE) punches from a 3-month-old female rat using Seahorse XF96 analyzer. (b) Continuous tracings of oxygen consumption rate (OCR) of CX, BG, and CE punches before and after inhibition of mitochondrial complex V (oligomycin, oligo), addition of mitochondrial oxidative phosphorylation uncoupler FCCP, and inhibition of mitochondrial complexes I/III (rotenone/antimycin A) (Rot/AA) in the same 96-well plate. Non-oligomycin groups (no oligo) were included to obtain the maximal OCR. (c) Scatter dot plots depict basal, maximal respiration, and non-mitochondria respiration (Non-MOC) of each brain region. (d) Continuous tracing of extracellular acidification rate (ECAR) of CX, BG, and CE punches before and after each treatment in the same 96-well plate. (e) Scatter dot plot depicts baseline and stressed ECAR of each brain region. (f) Metabolic profile of CX, BG, and CE from the same assay. Final concentrations: oligomycin 5 μM, FCCP 1 μM/pyruvate 0.75 mM, rotenone 1 μM/antimycin A 1 μM. Results of OCR/ECAR curve and scatter dot plots are shown as mean ± SD for normally distributed data, and median with interquartile range for non-normally distributed data. Metabolic profiles are shown as mean ± SEM. n = 4–17, *p < 0.05, **p < 0.01; ***p < 0.001; ****p < 0.0001 in ordinary one-way ANOVA followed by Tukey’s multiple comparison test or Kruskal-Wallis test followed by Dunn’s multiple comparison test.

Data adhering to normal distribution were depicted as mean ± SD and non-normally distributed data were presented as median and interquartile range (being the 25th and 75th percentile) unless otherwise stated. The Shapiro-Wilk test was used to test for normality. Two-tailed Student t-tests or Mann-Whitney U test were used to identify any significant difference when comparing two groups. One-way ANOVA followed by Tukey’s multiple comparison test or Kruskal-Wallis test followed by Dunn’s multiple comparison test were used for multiple groups comparison with one independent variable. The α level was set at 0.05 for all analyses, and p-value <0.05 was considered statistically significance. Statistical analyses were performed using Graph Pad Prism V.7.

Region-specific glucose metabolic profile of the young and old rat brain

To determine the glucose metabolism in the presence of native intracellular and extracellular environment in well-defined anatomical brain structure, we have optimized a method that enables dynamic metabolic function profiling by the Seahorse XFe96 analyzer using acute rat brain tissue biopsy punches modified from previously reports.^11,12 We obtained tissue punches from different brain regions at frontoparietal cortex, basal ganglia, hippocampal CA1, dentate gyrus, and cerebellar cortex for metabolic analysis using a Seahorse XFe96 analyzer (Figure 1(a)).

We compared different size cerebral cortical and cerebellar punches of 0.5 and 0.75 mm in diameter to obtain reliable OCR measurements. We found that the OCR readings of the 0.5 mm diameter cortical and cerebellar punches were within the 20–200 pmol/min range recommended by the manufacturer. In addition, the cerebellum demonstrated a lower basal OCR as compared with the cerebral cortex (Supplemental Figure 1).

We determined the glucose metabolism profile of cerebral cortical, basal ganglia, and cerebellar punches obtained from 3-month-old rats using Seahorse XF Cell Mito Stress Test. Inhibition of mitochondrial complex V by oligomycin has been found to attenuate the maximal OCR response to mitochondrial uncoupler FCCP. ¹⁵ Thus, non-oligomycin groups were included to obtain the maximal OCR rate. Consistently, we observed that cerebellar punches exhibited significantly lower basal OCR than cerebral cortical and basal ganglia punches. Data analysis further revealed that the cerebellum was significantly lower than cerebral cortex and basal ganglia in maximal respiration and non-mitochondria respiration (Figure 1(b) and (c)). Similarly, significantly lower basal and stressed ECAR were observed in the cerebellum as compared to basal ganglia and/or cerebral cortex (Figure 1(d) and (e)). Assessment of glucose metabolic phenotype indicated more energetic phenotypes for all groups in response to stress and that the cerebellum displays a more quiescent phenotype than cerebral cortex and basal ganglia evidenced by the lower glycolytic and aerobic activity at the cerebellum than the cerebral cortex and basal ganglia (Figure 1(f)).

We obtained tissue punches from hippocampal dentate gyrus, CA1, and cerebellum of 3-month-old rats for extracellular flux analysis in the same 96-well plate. Our results demonstrated that dentate gyrus exhibited basal OCR values similar to the cerebellum while the basal OCR of the CA1 was within the range of cerebral cortex and basal ganglia (Figure 2(a)). Data analysis further revealed that dentate gyrus and cerebellum were significantly lower than hippocampal CA1 in basal respiration and non-mitochondria respiration. No significant difference was observed in maximal respiration among CA1, dentate gyrus and cerebellum (Figure 2(b)). Dentate gyrus displayed similar basal ECAR as hippocampal CA1, while CA1 was significantly higher than the cerebellum and dentate gyrus in stressed ECAR (Figure 2(c) and (d)). Assessment of glucose metabolic phenotype indicated that the cerebellum had a quiescent phenotype as compared with hippocampal CA1 and dentate gyrus (Figure 2(e)).

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Figure 2.

Metabolic analysis of in hippocampal CA1, dentate gyrus (DG), and cerebellum (CE) punches from 3-month-old female rat using a Seahorse XFe96 analyzer. (a) Continuous tracings of OCR of CA1, DG, and CE punches before and after inhibition of mitochondrial complexes and addition of uncoupler in the same 96-well plate. Non-oligomycin groups (no oligo) were included to obtain the maximal OCR. (b) Scatter dot plots depict basal, maximal respiration, and non-mitochondria respiration (Non-MOC) of each brain region. (c) Continuous tracing of ECAR of CA1, DG, and CE punches before and after each treatment in the same 96-well plate. (d) Scatter dot plot depicts baseline and stressed ECAR of each brain region. (e) Metabolic profile of CA1, DG, and CE from same assay. Final concentrations: oligomycin 5 μM, FCCP1 μM/pyruvate 0.75 mM, rotenone (Rot) 1 μM/antimycin A (AA) 1 μM. Results of OCR/ECAR curve and scatter dot plots are shown as mean ± SD for normally distributed data, and median with interquartile range for non-normally distributed data. Metabolic profiles are shown as mean ± SEM. n = 4–19, *p < 0.05, **p < 0.01; ***p < 0.001; ****p < 0.0001 in ordinary one-way ANOVA followed by Tukey’s multiple comparison test or Kruskal-Wallis test followed by Dunn’s multiple comparison test.

We further determined metabolic phenotype in different brain regions of middle-aged rats. Tissue punches were obtained from cerebral cortex, basal ganglia, and cerebellum of 12-month-old rats, and glucose metabolism was assessed by Seahorse XF Cell Mito Stress test in the same 96-well plate. Consistently, the cerebellum displayed lower basal respiration and non-mitochondrial respiration than cerebral cortex and basal ganglia (Supplemental Figure 2a, b). The cerebellum also displayed lower baseline and stressed ECAR as compared with cerebral cortex and basal ganglia (Supplemental Figure 2c, d).

Region-specific glucose metabolic profile of the mouse brain

We conducted extracellular flux analysis in different brain regions from 3-month-old mice to explore the region-specific metabolic phenotype in young mouse brain. Consistently, the cerebellum displayed lower basal OCR and ECAR values compared to cerebral cortex and basal ganglia. The cerebellum was significantly lower than cerebral cortex and basal ganglia in basal respiration, maximal respiration, and non-mitochondrial respiration (Figure 3(a) and (b)). A significantly lower baseline and stressed ECAR was observed in the cerebellum as compared with cerebral cortex and basal ganglia (Figure 3(c) and (d)). Assessment of glucose metabolic phenotype indicated that the cerebellum displayed a more quiescent phenotype with lower glycolytic and aerobic activity than cerebral cortex and basal ganglia (Figure 3(e)).

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Figure 3.

Metabolic analysis of cerebral cortex (CX), basal ganglia (BG), and cerebellum (CE) punches from a 3-month-old female mouse using a Seahorse XFe96 extracellular flux analyzer. (a) Continuous tracings of OCR of CX, BG, and CE punches before and after inhibition of mitochondrial complexes and treatment of uncoupler in the same 96-well plate. Non-oligomycin groups (no oligo) were included to obtain the maximal OCR (not shown). (b) Scatter dot plots depict basal, maximal respiration, and non-mitochondria respiration (Non-MOC) of each brain region. (c) Continuous tracing of ECAR of CX, BG, and CE punches before and after each treatment in the same 96-well plate. (d) Scatter dot plot depict baseline and stressed ECAR of each brain region. (e) Metabolic profile of CX, BG, and CE from same assay. Final concentrations: oligomycin 5 μM FCCP 1 μM/pyruvate 0.75 mM, rotenone (Rot) 1 μM/antimycin A (AA) 1 μM. Results of OCR/ECAR curve and scatter dot plots are shown as mean ± SD for normally distributed data, and median with interquartile range for non-normally distributed data. Metabolic profiles are shown as mean ± SEM. n = 6–20, *p < 0.05, **p < 0.01; ***p < 0.001; ****p < 0.0001 in ordinary one-way ANOVA followed by Tukey’s multiple comparison test or Kruskal-Wallis test followed by Dunn’s multiple comparison test.

In separate analysis, dentate gyrus exhibited similar basal respiration, as the cerebellum. Data analysis suggested that the cerebellum was significantly lower than hippocampal CA1 in basal, maximal respiration, and non-mitochondrial respiration, as well as significantly lower than dentate gyrus in maximal respiration and non-mitochondrial respiration (Supplemental Figure 3). Glucose metabolic phenotype assessment implicated a more glycolytic and aerobic activity for hippocampal CA1 as compared to dentate gyrus and cerebellum that both maintained a more quiescent phenotype (Supplemental Figure 3e). Overall, the region-specific metabolic phenotype in cerebral cortex, basal ganglia, hippocampal CA1, dentate gyrus, and cerebellum displayed similar region-specific profile in rat and mouse.

Oxidative phosphorylation efficiency of mouse brain

We conducted titration experiments to select optimal concentrations of oligomycin on OCR. Oligomycin treatment induced a dose-dependent OCR reduction which reach plateau at 5 to 10 μM (Figure 4(a) and (b)). We further determined the ATP production and proton leak-linked respiration in the mouse brain using different concentrations of complex V inhibitors. Oligomycin and gboxin decreased OCR to similar levels in the cerebral cortex and cerebellum at 5 µM concentration. Increase of concentration of oligomycin and gboxin to 10 µM did not provide further inhibition of OCR (Figure 4(c) to (f)). Data analysis suggested that cerebral cortex has significant higher ATP production, proton leak-linked, and non-mitochondrial respiration than the cerebellum. Interestingly, ATP production-linked respiration is about 50% of the basal respiration in both cerebral cortex and cerebellum. Proton leak-linked respiration comprised up to 30% of basal respiration in the cerebral cortex and cerebellum (Figure 4(d) and (f)).

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Figure 4.

Optimization of mitochondrial complex V inhibition and measurement of non-mitochondria respiration, ATP production-linked respiration, and proton leak-linked respiration in the cerebral cortex and cerebellum. (a) Continuous tracings of OCR of CX punches from a 3-month-old female mouse before and after inhibition of complex V by different concentration of oligomycin (0, 1, 5, 10, 20, 50 μM). OCR was measured for 12 cycles of 85 minutes after addition of oligomycin. (b) Bar graph showing dose-dependent effect of oligomycin on OCR. Results are mean ± SD. n = 11–14. *p < 0.05, **p < 0.01 in ordinary one-way ANOVA followed by Tukey’s multiple comparison test. (c) Continuous tracings of OCR of CX and CE punches from a 3-month-old mouse before and after inhibition of complex V by oligomycin (5 or 10 µM) (oligo 5/10), addition of FCCP/pyruvate, and rotenone/antimycin A (Rot/AA). (d) Stacked bar graph showing non-mitochondria respiration, ATP production-linked respiration, and proton leak-linked respiration of CX and CE punches. (e) Continuous tracings of OCR of CX and CE punches from 3-month-old female mouse before and after inhibition of complex V by gboxin (5 or 10 µM) (gboxin 5/10), addition of FCCP/pyruvate, and rotenone/antimycin A. (f) Stacked bar graph showing non-mitochondria respiration, ATP production-linked respiration, and proton leak-linked respiration of CX and CE punches. Results are mean ± SD. n = 8-12. *p < 0.05, **p < 0.01; ^***p < 0.001 in either un-paired Student t-test for data or Mann-Whitney U test.

We determined the effect of uncoupling proteins (UCPs) inhibitor, genipin, on mitochondrial proton leak-linked respiration. Acute treatment of genipin at 50 and 100 μM significantly decreased proton leak coupled OCR with minimal effect on ATP production coupled OCR (Figure 5(a) to (c)). In addition, chronic treatment of genipin at 100 and 150 μM in aCSF supplied with pyruvate for 60 min displayed a trend to attenuate the basal OCR and significant reduction of proton leak coupled OCR without impact on ATP production coupled respiration (Figure 5(d) to (f)).

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Figure 5.

Inhibition of uncoupling proteins decreased proton leak coupled OCR in cerebral cortex punches. (a) Continuous tracings of OCR of the cerebral cortex punches from a 3-month-old female mouse before and after treatment of UCPs inhibitor (genipin 50, 100 μM), oligomycin 5 μM, FCCP 1 μM/pyruvate 0.75 mM, and rotenone (Rot) 1 μM/antimycin A (AA) 1 μM. (b, c) Bar graphs showing the effect of UCPs inhibition by genipin on ATP production coupled (b) and proton leak coupled (c) respiration. (d, e, f) Bar graphs show the effect of chronic treatment (60 minutes) of genipin on basal OCR (d), ATP production-linked OCR (e), and proton leak-linked OCR in the cerebral cortex punches from a 3-month-old female mouse. Results are mean ± SD. n = 12-15. ^*p < 0.05, ^**p < 0.01; ^***p < 0.001 in ordinary one-way ANOVA followed by Tukey’s multiple comparison test.

Region-specific metabolic signaling in the mouse brain

We determined the cellular components in the brain punches of different brain regions. Immunohistochemistry and confocal microscopy of the corresponding punch area demonstrated distinct brain structures with different cellular composites (Figure 6(a) to (c)). We further identified the neuronal and astrocytic markers and major metabolic signaling expression in different brain regions of 3-month-old mice. Representative Western blots and quantitative analysis indicated a significantly higher neuronal marker of β3-tubulin in cerebral cortex than hippocampus and cerebellum (Figure 7(a) and (d)). The expression of ALDH1L1, an astrocyte marker, was significantly higher in cerebral cortex than hippocampus (Figure 7(a) and (e)), while GFAP expression was higher in hippocampus and cerebellum than in cerebral cortex (Figure 7(a) and (f)). Activation of AMPK signaling was significantly higher in the cerebellum than in cerebral cortex and hippocampus evidenced by higher expression of phospho-AMPKα (Figure 7(b) and (g)), phospho-ACC (Figure 7(b) and (h)), and phospho-LKB1 (Figure 7(c) and (i)). These data indicated that cerebral cortex, hippocampus, and cerebellum displayed a diverse cellular composition and difference in activation of metabolic signaling.

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Figure 6.

Immunohistochemistry of GFAP, NeuN, and DAPI depict different cellular components of different brain regions. (a) Coronal section of the cerebrum depicts the location of tissue punches at cerebral cortex and basal ganglia and representative confocal images show neuron and astrocyte components of cerebral cortical and basal ganglia punches. (b) Coronal section of hippocampus depicts the location of tissue punches at hippocampal CA1 and dentate gyrus and representative confocal images show neuron and astrocyte components of CA1 and dentate gyrus punches. (c) Sagittal section of the cerebellum depicts the location of the tissue punches at cerebellar cortex and representative confocal images show neuron and astrocyte components of cerebellar cortex punches. Brain sections were stained by GFAP (green), NeuN (red), and nucleus counterstained by DAPI (blue).

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Figure 7.

Cellular composition and metabolic signaling in different mouse brain regions. (a) Western blots of β3Tubulin, GFAP, ALDH1L1, GAPDH at the cerebral cortex (CX), hippocampus (HP), and cerebellum (CE) of 3-month-old mouse. (b) Western blots of AMPKα, pAMPKα, ACC, pACC, GAPDH at CX, HP, CE of 3-month-old mouse. (c) Western blots of LKB1, pLAKB1, GAPDH at CX, HP, CE of 3-month-old mouse. (d-i). Quantitative analysis of β3-Tubulin, ALDH1, GFAP, pAMPKα, pACC, and pLKB1 expression at CX, HP, and CE. Results are mean ± SD (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001 in repeated measure for one-way ANOVA followed by Tukey’s multiple comparison.