Synopsis

Keywords: Stroke, Ischemia, coma, spreading depression

We have demonstrated spreading depression as a key event contributing to coma induction following the ET-1 injection to the brainstem of rats. Multi-modal fMRI enables the whole brain functional mapping to present the spatial dynamic changes during coma induction, in particular, paired with the incidence of SD events.

INTRODUCTION

Previously, our lab developed a robust coma rat model by introducing the ET-induced ischemic injury in the dorsomedial brainstem1. Here, we optimized the injection protocol of endothelin-1 (ET-1) into the tegmentum, enabling simultaneous fMRI and fiber photometry-based Ca²⁺/Glu recording with real-time micro-injection of ET-1. This method enables measuring brain dynamic changes during the onset of coma and the comatose state progression in the acute 12-hour time window. Interestingly, the spreading depression (SD) event has been reliably detected during the first 10-15 min following the onset of coma induction. Whole brain fMRI presented oxygenation changes and Glu signals indicated the initiation of the iso-electric state, as well as the SD. This work revealed the critical contribution of SD during coma induction and highlights the critical usage of multi-modal fMRI to specify the underlying brain dynamic changes

METHODS

Animal Model
Male Long Evans male rats (~250g) were induced with 5% and maintained at 1%-2% isoflurane. The rats’ rectal temperature was maintained at around 37^oC. The genetically encoded reporter iGluSnFR or GCaMP6f was expressed by the AAV virus in the forepaw somatosensory cortex. Optical fiber (200 µm) was inserted into the cortex (e.g. somatosensory cortex) expressing the fluorescent biosensors for recording. Isoflurane is withdrawn after the coma induction. Animals were incubated during scanning.
MR Techniques
All images were acquired with a 14 T/ 13 mm horizontal bore magnet (Magnex), interfaced to a Bruker AV-Neo console (Bruker), and equipped with a 6 cm gradient set, capable of providing 1.2 T/m (Resonance Research). A transceiver surface coil was used to acquire fMRI images. fMRI scans with block design were performed using 3D Echo planar imaging sequence: TR=1.5s, TE=7.5ms, FOV=2.4X2.4X2.0 cm³, 96X96X40 matrix.

RESULTS

We have implemented microinjection of ET-1 with peek tubing into the brainstem tegmentum through the ventral surface of the brain (Fig 1A). As shown in Fig 1B, after the coma induction the apnea was detected based on a change in shape and amplitude of the ventilatory pressure waveform (spontaneous breathing vs. apnea insert). The cortical and brainstem activity, as measured by local field potentials (LFP), is abruptly vanished in 3-5 min after the ET-1 injection and withdrawal of the anesthetic. We combined fiber photometry for glutamate (Glu) signal recording using the genetically encoded sensors( iGluSnFR) and whole-brain MR imaging during the coma induction. The extracellular glutamate recording showed spontaneous spikes decreased after the ET-1 injection(Fig 2), which was accompanied by a positive Glu spike, i.e. SD event. During ET-1 microinjection, the BOLD fMRI responses can trace the vasoconstriction in the brainstem injection site (negative BOLD, blue dot). In 8-10 min, we observed broad vasoconstriction across the brainstem (blue patch), and positive BOLD of higher-level regions, which may indicate flow compensation. The SD occurred 12 min after ET-1 injection, coinciding with a largely spread negative BOLD signal in the brain, which could be caused by suppression of neuronal activity and also the diffusion of ET-1 (Fig 3). A control group, of which the ET-1 is directly injected into the 4th ventricles, will be used to elucidate the cause of the spreading of negative BOLD signals following SD. Fig 4 shows two representative SD-related fMRI maps following coma induction, presenting highly reproducible global negative fMRI responses. However, it should be noted that detailed brain functional changes coupled with SD need to be verified from the global negative BOLD signals in our future study.

CONCLUSIONS

We optimized a brainstem coma induction model with real-time monitoring of the LFP, whole-brain fMRI, and fiber photometry-based Glu recording during coma induction and acute coma recovery. We observed that spreading depression events occurred during coma induction, contributing to neuronal activity suppression (as evidenced by iso-electric lines).

Acknowledgements

This research was funded by NIH funding (RF1NS113278, RF1NS124778, R01NS122904, R01NS120594), NSF grant 2123971, and the S10 instrument grant (S10 MH124733–01) to Martino’s Center.

References

1 Pais-Roldan, P. et al. Multimodal assessment of recovery from coma in a rat model of diffuse brainstem tegmentum injury. Neuroimage 189, 615-630, doi:10.1016/j.neuroimage.2019.01.060 (2019).