Synopsis

Keywords: Brain Connectivity, fMRI (resting state), Light sedation

Post traumatic brain injury (TBI) neuroinflammation has been linked to many long-term outcomes of TBI. To better understand the interrelationship of the neuroinflammation and changes of functional connectivity, we followed rats after TBI in a series of functional magnetic resonance imaging (fMRI) and positron emission tomography (PET) experiments. We observed hypoconnectivity in the corticothalamic connections, which was laterally altered at the acute time point and correlated with the observed level of neuroinflammation in the lateroposterior thalamic nuclei. This sheds light on the potential role of focal post-traumatic neuroinflammation shaping large scale functional connectivity in the post-traumatic brain.

Introduction

Neuroinflammation induced by traumatic brain injury (TBI) has been associated with many long-term outcomes of TBI, such as epilepsy¹ and cognitive impairment². While the initial injury disrupts the cortical areas, neuroinflammation is often observed in thalamic regions³. Moreover, the thalamus is closely interconnected with the cerebral cortex and other subcortical structures^4,5, making it a vulnerable node of circuit dysfunction after TBI⁶. While both changes in the functional connectivity (FC) and the neuroinflammation of the thalamus after the injury have been widely studied, the combined effect of both is yet underexplored⁷. In this study, we wanted to gain a better understanding of the consequences of the TBI on corticothalamic FC and its interrelationship with thalamic inflammation. To achieve this, we have followed the rats after TBI in a series of functional magnetic resonance imaging (fMRI) and positron emission tomography (PET) experiments.

Methods

Traumatic brain injury was induced by lateral fluid-percussion injury (LFPI)⁸ in 19 animals and a control group of 6 animals was sham-operated. The rats were habituated and measured under a light 0.5% isoflurane sedation as described before⁹ at 10 days, 2 months, and 6 months after the induction of TBI. The baseline fMRI data were acquired 1 week before the injury. The daily habituation time was gradually increased and the overall habituation period was 3 days for the baseline and 2 days for the consequent measurements. The fMRI was measured with gradient-echo echo-planar imaging sequence (TR = 1 s, 17 slices) for 25 minutes (1500 repetitions). In addition to the traditional preprocessing pipeline (slice timing correction, motion correction, and registration to a reference brain), we implemented a motion scrubbing and Independent component analysis-based approach for motion removal, as described before⁹. The region of interest (ROIs) were drawn on the reference brain according to an anatomical atlas¹⁰. The ROIs included cingulate cortex (Cg), retrosplenial cortex (Rs), and lateroposterior thalamic nuclei ipsilateral (LPi) and contralateral (LPc) to the injury. The ROI selection was done based on PET to represent thalamic inflammation areas (LPi, LPc) and their cortical connections (Cg, Rs). The ROI analysis of FC was performed on motion-free parts of the signal as described previously⁹.
The animals underwent PET imaging using a TSPO radioligand [¹⁸F]-FEPPA at 2 weeks post-injury induction. The ROIs were drawn in the myocardium and bilaterally in the lateroposterior thalamic nuclei. To remove the effect of the injected dose, the correlation of the uptake from the myocardium to the uptake of thalamic areas was modeled by fitting a linear model in the sham-operated animal, and then subtracting the uptake from the thalamic areas in all datasets. For correlation analysis between the uptake of the tracer and the FC, the effect of an injected dose was further removed.

Results

The PET data showed increased uptake of the TSPO-tracer in TBI animals in the perilesional thalamus, cortex, and hippocampus (Figure 1), which points to neuroinflammation in these areas. Specifically, in the thalamic area, we observed increased uptake in the lateroposterior and posterior thalamic nuclei. The sham-operated animals did not show any increased uptake of the tracer (Figure 1). The FC analysis of MRI data showed decreased connectivity in the TBI animals after the injury (Figure 2). The hypoconnectivity is mostly pronounced in the LPi at 10 days post-injury, while the connectivity of LPc decreased only slightly at this time point. Laterally altered connectivity of LP is still apparent at 2 months past the injury, while it diminishes by the 6 months time point (Figure 2). In the acute time point, we also detected decreased connectivity in the cortical connections in sham-operated animals, which is likely associated with the effect of craniectomy over the left parietal cortex. Additionally, the difference in the correlation values of the RS to the LPi and LPc in TBI animals correlated to the corrected FEPPA uptake in LPi in the acute time point (ρ = -0.5, p < 0.05). While the correlation was not significant at the chronic time points, the interrelation is still apparent in the 2 months post-injury (Figure 3).

Discussion

Our study is one of the first longitudinal studies following the interrelationship of neuroinflammation and changes in functional connectivity after TBI in rats. Since the corticothalamic circuits are the focus of this study, the light sedation protocol in habituated rats was used to reduce the effect of anesthesia on these connections. For a better understanding of the large-scale circuits and networks, additional cortical areas should be included. However, since variable-size cortical lesions, craniectomy, and bleeds create artefacts on the fMRI images, more advanced preprocessing and ROI selection criteria are needed. Nevertheless, as in the LFPI animal model, thalamic neuroinflammation is commonly found in the lateroposterior thalamic nuclei, which is interconnected with the cingulate and retrosplenial cortex, which are not included in the primary lesion area, our analysis still provides a good insight into the post-TBI pathology.

Conclusion

We have observed hypoconnectivity in the corticothalamic connections, which was laterally altered at the acute time point and correlated with the observed level of neuroinflammation in the LPi. This sheds light on the potential role of focal post-traumatic neuroinflammation in shaping large-scale functional connectivity in the post-traumatic brain.

Acknowledgements

This project is co-funded by the Horizon 2020 Framework Programme of the European Union (Marie Skłodowska Curie grant agreement No 740264) and the Academy of Finland.

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