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Cortical Dysplasia in Rats Provokes Neurovascular Alterations, GLUT1 Dysfunction, and Metabolic Disturbances That Are Sustained Post-Seizure Induction

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Abstract

Focal cortical dysplasia (FCD) is associated with blood-brain barrier (BBB) dysfunction in patients with difficult-to-treat epilepsy. However, the underlying cellular and molecular factors in cortical dysplasia (CD) associated with progressive neurovascular challenges during the pro-epileptic phase, post-seizure, and during epileptogenesis remain unclear. We studied the BBB function in a rat model of congenital (in utero radiation-induced, first hit) CD and longitudinally examined the cortical brain tissues at baseline and the progressive neurovascular alterations, glucose transporter-1 (GLUT1) expression, and glucose metabolic activity at 2, 15, and 30 days following a second hit using pentylenetetrazole-induced seizure. Our study revealed through immunoblotting, immunohistochemistry, and biochemical analysis that (1) altered vascular density and prolongation of BBB albumin leakages in CD rats continued through 30 days post-seizure; (2) CD brain tissues showed elevated matrix metalloproteinase-9 levels at 2 days post-seizure and microglial overactivation through 30 days post-seizure; (3) BBB tight junction protein and GLUT1 levels were decreased and neuronal monocarboxylate transporter-2 (MCT2) and mammalian target of rapamycin (mTOR) levels were increased in the CD rat brain: (4) ATPase activity is elevated and a low glucose/high lactate imbalance exists in CD rats; and (5) the mTOR pathway is activated and MCT2 levels are elevated in the presence of high lactate during glucose starvation in vitro. Together, this study suggests that BBB dysfunction, including decreased GLUT1 expression and metabolic disturbance, may contribute to epileptogenesis in this CD rat model through multiple mechanisms that could be translated to FCD therapy in medically refractory epilepsy.

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Data Availability

The data that support the findings of this study are available from the corresponding author upon reasonable request. Some data may not be made available because of privacy or ethical restrictions.

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Funding

This work is supported in part by the National Institute of Neurological Disorders and Stroke/National Institutes of Health grant R01NS095825 awarded to Chaitali Ghosh.

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Authors and Affiliations

  1. Cerebrovascular Research, Department of Biomedical Engineering, Lerner Research Institute, Cleveland Clinic, 9500 Euclid Avenue, Cleveland, OH, 44195, USA

    Chaitali Ghosh, Rosemary Myers, Sherice Williams & Mohammed Hossain

  2. Department of Biomedical Engineering and Molecular Medicine, Cleveland Clinic Lerner College of Medicine of Case Western Reserve University, Cleveland, OH, USA

    Chaitali Ghosh

  3. Charles Shor Epilepsy Center, Neurological Institute, Cleveland Clinic, Cleveland, OH, USA

    Christina O’Connor, Michael Nemeth & Imad M. Najm

  4. Department of Quantitative Health Sciences, Lerner Research Institute, Cleveland Clinic, Cleveland, OH, USA

    Xuefeng Liu

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  1. Chaitali Ghosh
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Contributions

CG designed the experiments and wrote the manuscript. RM performed the immunohistochemistry (DAB) staining, immunohistochemistry analyses, quantification, and cell culture experiments. CO, SW, and MN cared for and treated the animals for the experiments and obtained the cortical brain tissue specimens used for analysis with the assistance of CG and IN. In addition, SW performed initial histological and immunohistochemistry staining (fluorescent) and analysis. MH performed the western blot and biochemical assays. XL performed the statistical analysis of the data. All the authors contributed to editing the manuscript.

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Correspondence to Chaitali Ghosh.

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All animal procedures were approved by the Animal Research Committee of Cleveland Clinic Foundation. All studies were performed in accordance with the approved guidelines by the Institutional Animal Care and Use Committee and the Institutional Biosafety Committee of Cleveland Clinic. All efforts were to minimize animal suffering.

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IN serves on the Speaker Bureau for Eisai, Inc., and as a member of ad hoc advisory board for Eisai, Inc. and LivaNova. None of the other authors has any potential conflict of interest to disclose.

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Supplementary Information

Supplemental Fig. 1

General experimental design and rat groups. NL, XRT (irradiated in utero), PTZ (seizure-induced/SE) and XRT+PTZ and the overall experimental outline is depicted (PNG 2013 kb)

High resolution image (TIF 3681 kb)

Supplemental Fig. 2

Long-lasting BBB disruption post-seizure induction in rats with CD. FITC-albumin leakage as a surrogate of BBB disruption was followed with and without seizure induction in CD rats. The representative images of the brain cortex after 2 days post-PTZ (PND47), 15 days post-PTZ (PND60) and 30 days post-PTZ (PND75) show increased FITC-albumin extravasation in XRT, PTZ and XRT+PTZ cortical tissues compared to normal (NL). Quantification of BBB leakage in these areas was performed using Image J software. Increased BBB albumin leakage to the brain parenchyma was consistently elevated in the XRT+PTZ cortex, which continued until 30 days post-PTZ compared to NL. The result was expressed as mean ± SEM by two-way ANOVA, ***p<0.001. Scale bar = 200 μm (PNG 1105 kb)

High resolution image (TIF 7965 kb)

Supplemental Fig. 3

GFAP expression post-seizure induction in rats with cortical malformation. GFAP (red) immunostaining showed slightly upregulated glial cell activation (reactive gliosis) in XRT+PTZ cortex regions with FITC-albumin (green) leakages in rats. Activated astrocytes were not observed in the PTZ group at 30 days post-PTZ (PND75). Scale bar = 200 μm (PND47) and 50 μm (PND75) (PNG 4015 kb)

High resolution image (TIF 16871 kb)

Supplemental Fig. 4

Full western blots (a-e) for all targets analyzed. The representative blots from western blot analysis of MMP-9, Claudin-5, Claudin-1, GLUT1, mTOR, the mTOR pathway panel and MCT2 are provided with their corresponding β-actins (PNG 2037 kb)

High resolution image (TIF 569 kb)

Supplemental Fig. 5

Glucose and lactate absolute levels. Cortical glucose and lactate levels were measured and plotted in mmol/L in normal (NL), XRT, PTZ and XRT+PTZ rats. The glucose levels were significantly lower in XRT and XRT+PTZ compared to NL at PND47, PND60 and PND75. The PTZ group’s glucose levels were significantly increased at PND47 but returned to NL by PND60. Simultaneously increased lactate levels were seen in XRT and XRT+PTZ rats at PND47 but returned back to normal by PND75 (PNG 380 kb)

High resolution image (TIF 1715 kb)

Supplemental Fig. 6

Exogenous high lactate treatment effect on mTOR pathway and lactate transporter in vitro. Increased protein expression of phospho-mTOR (Ser2448), phospho-S6K (Ser371) and MCT2 is found in human embryonic kidney (HEK) cells after lactate (20 mM) treatment during glucose starvation vs. normal glucose media (0 mM lactate) control, examined by western blot. β-actin is used for normalization. The results are expressed as mean ± SEM by two-sample t-test, **p<0.01, ***p<0.001 (PNG 381 kb)

High resolution image (TIF 1716 kb)

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Ghosh, C., Myers, R., O’Connor, C. et al. Cortical Dysplasia in Rats Provokes Neurovascular Alterations, GLUT1 Dysfunction, and Metabolic Disturbances That Are Sustained Post-Seizure Induction. Mol Neurobiol 59, 2389–2406 (2022). https://doi.org/10.1007/s12035-021-02624-2

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