Synopsis

fMRI in rodents has become a widely used technique with various applications in preclinical research. In this context, most studies are performed with anesthetized animals. Medetomidine evolves as a frequently used sedative but long term stability is limited -prolonged stability can be achieved by addition of Isoflurane. Several recent fMRI studies have used this combination. We evaluated the effects of this combination of anesthetics using a multimodal setup of BOLD fMRI and Ca²⁺ recordings. Even small addition of Isoflurane resulted in a significant decrease of neural (Ca²⁺) and hemodynamic (BOLD) responses to a distinct electrical stimulus.

Purpose

Methods

BOLD

fMRI 20 rats (10 female Fischer, 5 male SD and 5 female SD) were investigated by fMRI, 3 rats additionally obtained simultaneous Ca²⁺ recordings. Animals were initially anesthetized using 5% Isoflurane. Electrodes were inserted into one forepaw and animals were transferred to a 9.4 T small animal MRI. Medetomidine infusion was started (0.04 mg/kg bolus, followed by infusion of 0.05 mg/kg/h) and Isoflurane was discontinued. Measurements began 40 min after Medetomidine infusion was started. The forepaw was electrically stimulated (5s stimulation (9Hz, 1mA), 25 s rest), repeatedly for 600 s. fMRI BOLD was performed using a single-shot GE-EPI (TR=1 s, TE= 18 ms, 600 repetitions, resolution 0.32 x 0.35 mm², 9 slices, slice thickness 1.2 mm). Stimulation experiments were performed under stepwise increased and later decreased Isoflurane concentrations (seven steps: 0.0%, 0.2%, 0.4%, 0.7%, 1.0%, 1.25%, 1.5%). fMRI was repeated for each step, using the same stimulation paradigm, and allowing 10 minutes waiting time after concentration had been adjusted.

Ca²⁺ + BOLD

AAVs encoding GCaMP were injected into the somatosensory cortex of the forelimb (S1FL). After at least 4 weeks rest, animals were anesthetized with ~2% Isoflurane. A glass fiber (diameter 200 µm) was implanted in S1FL and was connected to a blue laser (488 nm) delivering excitation light. Using the same fiber, fluorescent light was detected and sampled with 2 kHz (fig. 1).¹ Medetomidine infusion was started and Isoflurane was discontinued. The animal was transferred to the MRI for simultaneous BOLD + Ca²⁺ recordings. Anesthesia, stimulation and fMRI stayed the same. We additionally recorded Ca²⁺ responses to 3 Hz stimulation (1mA, 5 s stim.+ 25 s rest) for 300 s for each Isoflurane concentration. Animals subject to Ca²⁺ recordings were perfused transcardially and brains were removed for histological validation of GCaMP expression (fig. 4). fMRI analysis was performed with SPM 8. Using custom-written MATLAB scripts, a ROI (3x3 Voxel) was defined in the activated area and the amplitude of the BOLD timecourse was extracted. The amplitude the Ca²⁺ signal on stimulation was also analyzed and was reported with reference to baseline fluorescence (Δf/f). Onset and full width at half maximum was also analyzed.

Results

BOLD

Increasing Isoflurane concentrations gradually reduced the amplitude of the BOLD response. Addition of 0.2% Isoflurane reduced the amplitude by 27% (p=0.014), 0.7% Isoflurane resulted in a reduction of 62% (p<0.001). (fig. 3a)

Ca²⁺

Increasing Isoflurane concentrations gradually reduced the amplitude of detected calcium signal. Addition of 0.2% Isoflurane reduced the amplitude by 39% (p=0.038), 0.7% Isoflurane by 63% (p<0.001). Onset and full width at half maximum did not change significantly. For stimulation with 3 Hz, primary responses upon each stimulus were observed, regardless of the Isoflurane concentration (fig. 2a,d,g). For stimulation with 9 Hz, at high Isoflurane concentrations responses were observed only for the first pulses of the stimulus train (fig. 2b,e,h).

No significant differences between the results of test series with increasing (first half of experiment) vs decreasing (second half of experiment) Isoflurane concentrations were observed, nor between male vs female, nor between Fischer vs SD rats.

Discussion

Previous studies already stated that adding Isoflurane to Medetomidine sedation provides stable physiological conditions for prolonged fMRI studies. However, adding Isoflurane comes at the expense of losing signal in both fMRI (hemodynamic + neural signal) and Ca²⁺ recordings (neural signal).

The combination of both anesthetics reduces the MAC of Isoflurane² meaning that already little admixture of Isoflurane has significant effects - the amplitude of the BOLD response is decreased significantly. Isoflurane induces vasodilation of cerebral vessels³ In contrast, Medetomidine causes vasoconstriction of both arteries and veins⁴, and attenuates cerebrovascular dilation when combined with Isoflurane⁵. One could explain the effects observed in BOLD with hemodynamic effects alone. Ca²⁺ recordings however indicate that neural activity changes, too. This effect was not an effect of time because while decreasing the Isoflurane concentration stepwise, both BOLD and Ca²⁺ amplitude recovered.

Conclusion

Acknowledgements

References

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