Synopsis

Impaired brain glucose consumption is a possible trigger of Alzheimer’s disease (AD). Animal models can help characterize each contributor to the cascade independently. Here we perform a first-time longitudinal study of brain connectivity in the intracerebroventricular-streptozotocin rat model of AD. We report altered brain circuitry as early as two weeks in regions notoriously affected by AD (cingulate cortices, posterior parietal cortex and hippocampus), and widespread gradual breakdown of connectivity with time. The changes in brain connectivity induced by glucose metabolism disruption can bring further insight into the role of this mechanism in AD.

Introduction

Functional MRI (fMRI) studies of amnestic mild cognitive impairment and Alzheimer’s disease (AD) have revealed decreased resting-state activity of the default mode network which correlates strongly with clinical disease severity and progression^1,2.

However, the mechanism that triggers AD is not well-established, with amyloid plaques, neurofibrillary tangles of tau, microgliosis and glucose hypometabolism all likely involved in the cascade. One main advantage of animal models is the possibility to tease out the impact of each of these insults on brain connectivity.

Following an intracerebroventricular (icv) injection of streptozotocin (STZ), rats and monkeys develop impaired brain glucose metabolism (i.e. “diabetes of the brain”). Numerous studies have reported AD-like features in icv-STZ animals^3-5, but this model has never been characterized in terms of MRI-derived biomarkers beyond structural brain atrophy⁴.

Here, we report the longitudinal evolution of functional connectivity in icv-STZ rats compared to controls using resting-state fMRI.

Methods

All experiments were approved by the local Service for Veterinary Affairs. Male Wistar rats (N=9) (236±11 g) underwent a bilateral icv-injection of either streptozotocin (3 mg/kg, STZ group, N=5) or buffer (control group, N=4). Rats were scanned at four timepoints following surgery (Fig. 1), on a 14T Varian system. Briefly, rats were anesthetized using isoflurane for initial setup and promptly switched to medetomidine sedation (bolus: 0.1mg/kg, perfusion: 0.1mg/kg/h), which preserves neural activity and vascular response better than isoflurane. Resting-state fMRI data were acquired using a two-shot gradient-echo EPI sequence as follows: TE/TR=10/800ms; Matrix: 64x64; FOV: 23x23mm²; 8 1.12-mm slices; 370 repetitions (TA=10’). Two or three runs were acquired on each rat.

Data pre-processing included denoising⁶, distortion correction⁷, slice-timing correction and spatial smoothing⁸ (Gaussian kernel: 0.36x0.36x1 mm³). Independent component analysis (ICA) was performed on each individual fMRI run⁹ with high-pass temporal filtering (f > 0.01Hz) and 40 independent components (IC’s). Artefactual IC’s were manually identified and cleaned from the data¹⁰.

In parallel, fMRI images were registered to a rat brain atlas⁷ and 28 atlas-defined ROIs were automatically segmented. “Cleaned” fMRI data were used to compute individual functional connectivity (fc) matrices between these 28 ROIs, using global signal regression, which were further Fisher z-transformed (Fig. 2).

Mean fc matrices for each group and timepoint were calculated. Two-tailed t-test was used to assess between-group fc differences at each timepoint, while longitudinal changes were assessed separately for each group using one-way ANOVA.

Results

Figure 3 shows an example of anatomical images, matching fMRI images, and atlas-based anatomical labels registered into fMRI space.

Mean fc matrices at each timepoint revealed slightly reduced fc connectivity in the STZ group at 2 weeks (weaker positive and negative correlations), and partial recovery at 6 weeks prior to more significant and widespread changes from 13 weeks on (Fig. 3). ANOVA confirmed stable fc in the control group over the 2 – 27 week time range, contrasting with widespread and significant temporal changes in the STZ group (Fig. 4).

Discussion and Conclusions

This is the first study to characterize longitudinal fc changes caused by brain glucose metabolism disruption in the icv-STZ model. Group comparisons revealed most prominent differences in the posterior parietal cortex (PPC) and anterior cingulate at 2 weeks, retrosplenial cortex at 6 weeks, and PPC, hippocampus and subiculum at 13 weeks, which are all regions typically involved in AD. The reproducibility of fc matrices in the CTL group over time gives increased confidence in the detection of dramatic fc changes in the STZ group.

Our results are consistent with previous studies of the icv-STZ rat model using histopathology, namely with structural thinning of the PPC after one month, atrophy of the hippocampus at 6 weeks⁵ and intra-cellular accumulation of amyloid in the PPC and hippocampus after 3 months³. Finally, we report non-monotonic changes in fc, with partial recovery around 6 weeks, consistent with previously-established trends for structural thickness and memory performance³.

Future work will focus on acquiring a larger dataset, especially at the later timepoints. We will also characterize gray and white matter structural degeneration using diffusion MRI and correlate it to changes in fc.

Finally, the icv-STZ model displays functional changes reminiscent of AD, while the degeneration is induced primarily by impaired brain glucose metabolism. It therefore constitutes an excellent model of sporadic AD and should allow to further explore the hypothesis of AD being “type-3 diabetes”.

Acknowledgements

References

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