Long-term functional outcomes and correlation with regional brain connectivity by MRI diffusion tractography metrics in a near-term rabbit model of intrauterine growth restriction

Abstract

Background: Intrauterine growth restriction (IUGR) affects 5-10% of all newborns and is associated with increased risk of memory, attention and anxiety problems in late childhood and adolescence. The neurostructural correlates of long-term abnormal neurodevelopment associated with IUGR are unknown. Thus, the aim of this study was to provide a comprehensive description of the long-term functional and neurostructural correlates of abnormal neurodevelopment associated with IUGR in a near-term rabbit model (delivered at 30 days of gestation) and evaluate the development of quantitative imaging biomarkers of abnormal neurodevelopment based on diffusion magnetic resonance imaging (MRI) parameters and connectivity.

Methodology: At +70 postnatal days, 10 cases and 11 controls were functionally evaluated with the Open Field Behavioral Test which evaluates anxiety and attention and the Object Recognition Task that evaluates short-term memory and attention. Subsequently, brains were collected, fixed and a high resolution MRI was performed. Differences in diffusion parameters were analyzed by means of voxel-based and connectivity analysis measuring the number of fibers reconstructed within anxiety, attention and short-term memory networks over the total fibers.

Principal findings: The results of the neurobehavioral and cognitive assessment showed a significant higher degree of anxiety, attention and memory problems in cases compared to controls in most of the variables explored. Voxel-based analysis (VBA) revealed significant differences between groups in multiple brain regions mainly in grey matter structures, whereas connectivity analysis demonstrated lower ratios of fibers within the networks in cases, reaching the statistical significance only in the left hemisphere for both networks. Finally, VBA and connectivity results were also correlated with functional outcome.

Conclusions: The rabbit model used reproduced long-term functional impairments and their neurostructural correlates of abnormal neurodevelopment associated with IUGR. The description of the pattern of microstructural changes underlying functional defects may help to develop biomarkers based in diffusion MRI and connectivity analysis.

Figure 1. Schematic and graphical representation of the study design and methods.

PANEL 1: (A) Illustrative image of unilateral ligation of 40–50% of uteroplacental vessels at 25 days of pregnancy, (B) Scheme of surgical procedures and study groups. PANEL 2: Illustrative pictures of neurobehavioral and cognitive evaluation in the Open Field Behavioral Test (A) and Object Recognition Task (B). PANEL 3: MRI acquisitions: (A) Fixed brains were scanned to obtain high resolution T1 weighted images and diffusion-weighted images. After masking brain volume, (B) global analysis was performed to obtain average DTI parameters (FA, linearity, planarity and sphericity coefficients). (C) Then voxel-based analysis of diffusion-related parameters was performed by elastic registration to a reference FA map. PANEL 4: (A) Illustrative image of tractography used for connectivity analysis. It was performed by measuring the ratio of fibers involved in anxiety and short-term memory networks over the total number of fibers reconstructed. (B) Manual delineation of brain regions involved in anxiety, attention and memory networks in coronal slices including prefrontal cortex (PrC), striatum (Ca + Pu), cingulate cortex (CiC), temporal cortex (TeC), thalamus (Th), amygdala (Am), hippocampus (Hp) and hippocampus formation (HpF).

Figure 2. Discriminatory index results and percentage of learning in study groups.

(A) Discriminatory index values of the study group (p = 0.013, adjusted for gender); (B) Percentage of controls and cases that reached the learning criteria (p = 0.03, adjusted for gender).

Figure 3. Fractional anisotropy, linearity, planarity and sphericity coefficients: regions showing statistically significant differences (p<0.01) between cases and controls.

Coronal slices of the 3D reference image displaying representative anatomical structures for specific coefficients. Slice locations are shown in the T1 weighted images at the top. PANEL 1: Representative anatomical regions showing a significant decrease in linearity (A) and planarity (B) coefficients and in fractional anisotropy (C) in cases compared to controls. PANEL 2: Representative anatomical regions showing a significant increase in planarity (A) and sphericity (B) coefficients in cases compared to controls.

Figure 4. Correlation maps between neurobehavioral and cognitive tests items and fractional anisotropy values.

Coronal slices (from anterior to posterior) of the 3D reference image are displayed. Colormap highlights the areas where the correlation coefficient is higher than 0.2. Spearman correlation p<0.001. PLANEL 1: (A) Latency of leaving the starting point, (B) Total squares crossed, (C) Total time exploring, (D) External squares crossed, (E) Time in external area, (F) Internal squares crossed, (G) Time in internal area, (H) Grooming, (I) Rearing. PLANEL 2: (A) Time exploring familiar object, (B) Time exploring novel object and (C) Discriminatory index.

Figure 5. Ratio of fibers involved in the anxiety or memory networks over the total number of fibers.

(A) Ratio of fibers adjusted for gender in anxiety network; in global analysis (p = 0.10), in left (p = 0.01) and right hemisphere (p = 0.59 †); (B) Ratio of fibers adjusted for gender in memory network; in global analysis (p = 0.08), in left (p = 0.03 †) and right hemisphere (p = 0.92 †). † Non-normal variables.

Figure 6. Reconstructed fibers in anxiety and memory networks in the experimental groups.

Coronal and axial views of anxiety and memory networks of one control (A) and one case (B). Reconstructed fibers are overlapped to the 3D reconstruction of T1 weighted images.