Prolonged systemic hyperglycemia does not cause pericyte loss and permeability at the mouse blood-brain barrier

Abstract

Diabetes mellitus is associated with cognitive impairment and various central nervous system pathologies such as stroke, vascular dementia, or Alzheimer's disease. The exact pathophysiology of these conditions is poorly understood. Recent reports suggest that hyperglycemia causes cerebral microcirculation pathology and blood-brain barrier (BBB) dysfunction and leakage. The majority of these reports, however, are based on methods including in vitro BBB modeling or streptozotocin-induced diabetes in rodents, opening questions regarding the translation of the in vitro findings to the in vivo situation, and possible direct effects of streptozotocin on the brain vasculature. Here we used a genetic mouse model of hyperglycemia (Ins2^AKITA) to address whether prolonged systemic hyperglycemia induces BBB dysfunction and leakage. We applied a variety of methodologies to carefully evaluate BBB function and cellular integrity in vivo, including the quantification and visualization of specific tracers and evaluation of transcriptional and morphological changes in the BBB and its supporting cellular components. These experiments did neither reveal altered BBB permeability nor morphological changes of the brain vasculature in hyperglycemic mice. We conclude that prolonged hyperglycemia does not lead to BBB dysfunction, and thus the cognitive impairment observed in diabetes may have other causes.

Figure 1

(a) Fasting blood glucose levels (mmol/l) and (b) body weight (g) of male Ins2^AKITA and WT littermate controls. Data is presented as mean ± SEM. n = 2 to 15 mice.

Figure 2

Blood-brain barrier permeability measurements in male Ins2^AKITA and WT littermate controls. (a) Representative stereomicroscope fluorescence images of brains showing 1 kDa Alexa Fluor 555 cadaverine permeability in Ins2^AKITA and WT after 2 h of dye circulation (n = 2). (b) Representative confocal images of coronal sections of 1 kDa Alexa Fluor 555 cadaverine injected mouse brains. ANPEP positive mural cells, green; PECAM1 positive vasculature, white. No Alexa Fluor 555 cadaverine leakage into brain parenchyma was observed either in Ins2^AKITA or WT mice (n = 2, scale bar 30 μm). (c) Quantification of 1 kDa Alexa Fluor 555 cadaverine permeability in 26.5–32 week-old Ins2^AKITA and WT mice after 2 h circulation (n = > 8, 3 independent experiments). y-axis shows the fold change in relative fluorescence units (RFU) per gram of brain tissue in relation to WT. (d) Quantification of 1 kDa Alexa Fluor 488 cadaverine permeability in 38 week-old Ins2^AKITA and WT mice after 2 h circulation (n = 3). y-axis shows the fold change in relative fluorescence units (RFU) per gram of brain tissue in relation to WT. (e) Evans Blue dye permeability in 30 week-old Ins2^AKITA and WT littermate control mice after overnight circulation (n = > 2). y-axis shows optical density (OD) at 620 nm per gram of tissue. Pdgfb^Ret/Ret served as positive control for tracer leakage into the brain parenchyma. n.s., not significant, student’s t test. Data is presented as mean ± SEM.

Figure 3

Characterization of pericyte coverage and pericyte-specific gene and protein expression in 26.5–32 week-old Ins2^AKITA and WT littermate controls. (a,b) Representative images of pericyte-specific protein expression in WT and Ins2^AKITA mice, (a) Aminopeptidase N (ANPEP, green) and Vitronectin (VTN, red); (b) Laminin alpha 2 (LAMA2, green) and desmin (DES, red). Endothelium visualized with PECAM1, cyan. n = 2, scale bar 30 μm. (c) The skeletal length of PECAM1 positive capillaries and ANPEP positive pericytes in Ins2^AKITA and WT was measured and plotted as the percentage of the pericyte length over vessel length (n = 6, *p = 0.0125, student’s t test). (d,e) qPCR analysis on isolated brain microvasculature fragments for pericyte-specific gene Pdgfrb (d) and Rgs5 (e). WT controls are set as 1 and Ins2^AKITA results are presented as fold change over WT (n = 5). n.s. = not significant, student’s t test. Data is presented as mean ± SEM.

Figure 4

Representative image of immunostaining corresponding to the glucose transporter protein SLC2A1 (GLUT1) in 30 week-old Ins2^AKITA and WT cerebral cortex. Mural cells (ANPEP, green), glucose transporter 1 (GLUT1, red), endothelium (PECAM1, cyan). n = 2, scale bar 50 µm.

Figure 5

Characterization of reactive astrocytes in 26.5–32-week-old Ins2^AKITA and WT littermate controls. (a) Representative image of reactive astrocytes in Ins2^AKITA and WT cerebral cortex, astrocytes (GFAP, green), vasculature (PECAM1, white) (n = 5, scale bar 50 μm). (b) Total cerebral cortex Gfap mRNA expression in Ins2^AKITA (n = 6) and WT (n = 4) mice presented as fold change over Hprt reference gene expression. (c) Gfap mRNA expression from isolated cerebral microvascular fragments of Ins2^AKITA and WT mice presented as fold change over Gapdh reference gene (n = 7). n.s. = not significant, Student’s t test.

Figure 6

Characterization of microglia in 26.5–32 week-old Ins2^AKITA and WT littermate controls. (a) Representative images of microglia (AIF1, red) in Ins2^AKITA and WT cerebral cortex. Vasculature is stained with anti-PECAM1 antibody. Notice the fewer microglial processes in Ins2^AKITA cerebral cortex when compared to WT, scale bars 10 μm (n = 6). (b) Quantification of the number of AIF1 + microglia per field in WT and Ins2^AKITA cerebral cortex (n = 6), n.s. = not significant, student’s t test. (c) Quantification of the microglial filament length per field (μm) (n = 6), **p = 0.0012, student’s t test.