Diffusion tensor imaging correlates with cytopathology in a rat model of neonatal hydrocephalus

Abstract

Background: Diffusion tensor imaging (DTI) is a non-invasive MRI technique that has been used to quantify CNS abnormalities in various pathologic conditions. This study was designed to quantify the anisotropic diffusion properties in the brain of neonatal rats with hydrocephalus (HCP) and to investigate association between DTI measurements and cytopathology.

Methods: DTI data were acquired between postnatal day 7 (P7) and P12 in 12 rats with HCP induced at P2 and in 15 age-matched controls. Animals were euthanized at P11 or P22/P23 and brains were processed with immunohistochemistry for glial fibrillary acidic protein (GFAP), ionized calcium-binding adaptor molecule (Iba-1), and luxol fast blue (LFB) to assess astrocytosis, microglial reactivity and degree of myelination, respectively.

Results: Hydrocephalic rats were consistently found to have an abnormally low (at corrected p-level of <0.05) fractional anisotropy (FA) value and an abnormally high mean diffusivity (MD) value in the cerebral cortex (CX), the corpus callosum (CC), and the internal capsule (IC). Immunohistochemical analysis demonstrated trends of increasing astrocyte and microglial reactivity in HCP rats at P11 that reached statistical significance at P22/P23. A trend toward reduced myelination in the HCP rats was also found at P22/P23. Correlation analysis at P11 for the CC demonstrated statistically significant correlations (or trends) between the DTI measurement (the decreased FA and increased MD values) and the GFAP or Iba-1 rankings. The immunohistochemical rankings in the IC at P22/P23 were also significantly correlated or demonstrated a trend with both FA and MD values.

Conclusions: This study demonstrates the feasibility of employing DTI on the brain in experimental hydrocephalus in neonatal rats and reveals impairments in multiple regions of interest in both grey and white matter. A strong correlation was found between the immunohistochemical results and the changes in anisotropic diffusion properties.

Figure 1

In vivo diffusion tensor imaging of a control rat at postnatal day 8. In the fractional anisotropy-based color coded axis maps (A and B), the colors are used to indicate the preferred diffusion direction in a coronal plane. Red, green, and blue represent transverse, dorso-ventral and rostro-caudal directions, respectively. The brightness is coded by the FA value. On this map, both white matter (corpus callosum, internal capsule, and external capsule) and grey matter (cortex, caudate and putamen) can be identified. C and D are mean diffusivity maps showing the same structures.

Figure 2

Mean diffusivity maps of hydrocephalic rats at postnatal day 11. The mid-sagittal image (A) shows the location of the five coronal slices (B-F) arranged from rostral to caudal. The expansion of the lateral ventricle and the posterior recess of the cerebral aqueduct (CA) can also be seen. Abbreviations: CC, corpus callosum; CPu, caudate-putamen; CX, cortex; EC, external capsule; FX, fornix; IC, internal capsule; HC, hippocampus; LV, lateral ventricle; PVWM, periventricular white matter.

Figure 3

Box-plots comparing the fractional anisotropy (FA) and mean diffusivity (MD, 10^-3mm²/s) values in corpus callosum in hydrocephalic rats and the control rats. All ages combined, n = 12 for hydrocephalic and n = 15 for control rats. The Wilcoxon two-sample test showed a significant difference (p < 0.05, corrected for multiple comparison).

Figure 4

Representative photomicrographs of GFAP for astrocytes (A-H) and Iba-1 for microglia (I-P) immunohistochemistry from the external capsule, internal capsule, corpus callosum, and fornix in P11 saline control animals (A, C, E, G for GFAP; I, K, M, O for Iba-1) and hydrocephalic animals (B, D, F, H for GFAP; J, L, N, P for Iba-1). For GFAP, despite the immaturity of the tissue, the hydrocephalic animals exhibit the same severe glial reaction observed in the P22/23 animals as evidenced by large intensely stained cell bodies and thick processes (B, straight arrows). For Iba-1, note the round intensely stained profiles that represent highly phagocytic microglial somata in the white matter of control animals. These profiles are increased in hydrocephalic animals at this age, but are not found in any 22 or 23-day old animals. Scale bar = 25 μm and cresyl violet counterstain for all panels.

Figure 5

Representative photomicrographs of GFAP (A-F), Iba-1 (G-L), and LFB (M-R) immunohistochemistry of the external capsule, internal capsule, and the fornix of P22/23 control (A, C, & E for GFAP; G, I, and K for Iba-1; M, O, & Q for LFB) and hydrocephalic animals (B, D, & F for GFAP; H, J, & L for Iba-1; N, P, & R for LFB). GFAP staining in the hydrocephalic animals reveals severe reactive astrocytosis when compared to the saline control animals. Panel A shows typical characteristics of resting astrocytes that includes lightly stained cell bodies with thin processes. Panel B demonstrates distinctive characteristics of reactive astrocytes including a darkly stained cell body with thick processes (arrow). For Iba-1, control animals (G, I, & K) exhibit resting microglial cells which are characterized by a small unremarkable cell body with thin processes. The hydrocephalic animals (H, J, & L) display reactive microglia cells which are typified by a darkly stained, enlarged, distorted cell body with thick processes (arrows, J and L). At the end stages of reactivity they exhibit a "balled up" appearance known to represent a phagocytic state (arrow, L). LFB staining is dark and uniform in the saline controls which indicates the preservation of myelin (M, O, & Q). Whereas, the hydrocephalic animals exhibit a more diffuse and variable staining pattern, providing evidence of demyelination most likely resulting from the mechanical stress that accompanies HCP (N, P, & R). Scale bar = 25 μm for all panels except O and P, scale bar = 50 μm.