MRI confirms loss of blood-brain barrier integrity in a mouse model of disseminated candidiasis

Abstract

Disseminated candidiasis primarily targets the kidneys and brain in mice and humans. Damage to these critical organs leads to the high mortality associated with such infections, and invasion across the blood-brain barrier can result in fungal meningoencephalitis. Candida albicans can penetrate a brain endothelial cell barrier in vitro through transcellular migration, but this mechanism has not been confirmed in vivo. MRI using the extracellular vascular contrast agent gadolinium diethylenetriaminepentaacetic acid demonstrated that integrity of the blood-brain barrier is lost during C. albicans invasion. Intravital two-photon laser scanning microscopy was used to provide the first real-time demonstration of C. albicans colonizing the living brain, where both yeast and filamentous forms of the pathogen were found. Furthermore, we adapted a previously described method utilizing MRI to monitor inflammatory cell recruitment into infected tissues in mice. Macrophages and other phagocytes were visualized in kidney and brain by the administration of ultrasmall iron oxide particles. In addition to obtaining new insights into the passage of C. albicans across the brain microvasculature, these imaging methods provide useful tools to study further the pathogenesis of C. albicans infections, to define the roles of Candida virulence genes in kidney versus brain infection and to assess new therapeutic measures for drug development.

Keywords: Candida albicans; MRI; blood-brain barrier; extracellular vascular contrast agent Gd-DTPA; intravital two-photon microscopy; murine disseminated candidiasis; ultrasmall iron oxide particles.

Fig. 1

Axial images of representative (A) normal and (B,C and D) infected brain after Gd-DTPA infusion. Hyper-intense signals in infected representative brains (n=5) at day 0, 3 and 5 PI show non-localized regions with possible BBB damage in contrast to the normal brain. (E) Two selected ROIs shown in Y and X (10 pixel area) used to assess the dynamic contrast curve after Gd-DTPA infusion for normal mouse (F) and infected mouse brain (G). The normal mouse brain depicts the first pass effects of the contrast agent typical of a brain with intact BBB while the infected regions (G) show effects consistent with BBB breaching. (H) A pilot image showing selected regions of interest (ROI) in cortical areas. (I) The overall average intensity of all selected ROIs plus selected cortical ROIs of penultimate axial slices, showing increase in signal intensity in a quantitative longitudinal study corresponding to Gd-DTPA leakage due to BBB damage in infected mouse in comparison with the normal is summarized. The abbreviated units of 1F, 1G and I is arbitrary values of average signal intensities.

Fig. 2

Representative axial images of mouse brains after i.v. administration of Gd-DTPA in (A) control (B) day 1, (C) day 3 and (D) day 5 PI infected mice and their corresponding, fungal specific, GMS stained sections of the uninfected (E) and infected brains (F–H) with high magnification of selected areas (I–L). No visible changes are observed in the non-infected mouse brain (A) where Gd does not penetrate the BBB. Hyper-intense regions in C. albicans infected mice post-Gd infusion show visible differences in all tested time points, consistent with vascular BBB breaching. Furthermore, post Gd signal variation images for C. albicans infected mouse brains at three different time points (B–D, n=5) show varying degrees of Gd leakage (BBB breaching) with severity of infection indicated by hyper-intensity of the MRI images correlating well with the observed histology (E–L).

Fig. 3

(A) Average spin-spin relaxation times (T₂) and (B) apparent diffusion coefficients (ADC) for, multiple cortical ROIs (10 ROIs per region with pixel resolution of 0.0075 cm and 10 pixel area of 5.625×10⁻⁴ cm²) over 6 contiguous axial slices, of normal and infected brains. Structural changes in the micro-environment in the cortical areas due to the infiltration of Candida correlate with altered cellular integrity in the infected brains, thus leading to reduction in T₂. Perhaps due to the large vacuole present in Candida cells, ADC values were not significantly altered by infection. (C) Longitudinal changes in signal intensities of axial images of frontal and dorsal regions of infected brains after Gd-DTPA infusion. Increasing hyper-intense signal in infected brain cortex and hippocampus at days 1–5 PI (p< 0.05 0.001 and 0.001, respectively, n = 5) indicates increasing BBB damage.

Fig. 4

C. albicans colonization of the brain was visualized in real-time using intravital TPLSM. A representative maximal projection of a three-dimensional (3D) z-stack shows the distribution of GFP⁺ C. albicans (green) in the meninges and superficial brain parenchyma three days following infection (low panel). A similar projection captured from an uninfected mouse is shown as a negative control (upper panel). Both the filamentous (white arrows) and yeast (white arrowheads) forms of C. albicans were observed by TPLSM. Quantum dots (red) were injected intravenously just before imaging to visualize blood vessels. White scale bars = 50 microns. See associated Movie 1.

Fig. 5

Representative sections of transverse MR images of 8 week old BALB/c mice infected with or without C. albicans (n=5) and with or without USPIO 24 h post MRI (upper panels) and respective fungal specific GMS and H&E stained sections. (A) Infected mice without USPIO administration do not demonstrate T2* signal loss. Fungal specific GMS shows heavy fugal colonization and H&E section shows inflammatory cell infiltration. (B) Noninfected mice without USPIO administration do not demonstrate T2* signal loss. (C) noninfected mice with USPIO also show no T2* effects. The respective GMS and H&E stained sections in B and C show no colonization and no inflammatory cell infiltration. (D) Infected mouse with USPIO administration clearly demonstrates T2* signal loss. GMS staining shows heavy fugal colonization, and the H&E section shows inflammatory cell infiltration. 1 mm scale bar indicates magnification of the stained sections.

Fig. 6

T₂^* signal loss in transverse kidney and brain MR images of mice infected with C. albicans. Signal was quantified in the indicated regions of kidney and brain 24 hours after injection of USPIO agent and the indicated days post C. albicans inoculation. Control 8 week old BALB/c mice were uninfected but received USPIO. (A) Three arbitrarily demarcated medullary, corticomedullary and cortical regions of kidneys were assessed at the indicated times PI for signal intensities (*** p<0.001 compared with the control group, consistent with more USPIO-laden phagocytes). (B) T₂^* signal loss was quantified in frontal and dorsal cerebral brain regions at the indicated times post inoculation (*** p<0.001). (C) Quantitative analysis of brain infiltrating leukocytes in uninfected and day 3 C. albicans infected mice. Bar graphs show the frequency (upper panel) and absolute numbers (low panel) of neutrophils (CD45⁺ Thy1.2⁻ Ly6C⁺ Gr-1^hi) and monocytes/macrophages/dendritic cells (CD45⁺ Thy1.2⁻ Ly6C⁺ Gr-1^low). Data are presented as the mean±SD. Each group consisted of five mice.