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Long-term effect of isoflurane anesthesia on functional connectivity detected by fMRI and local field potential measurements1A.I. Virtanen Institute for Molecular Sciences, Kuopio, Finland
Synopsis
Isoflurane, a commonly used anesthetic in preclinical studies, is known to alter functional connectivity during anesthesia. It has been found that isoflurane can induce brain plasticity and cause long-term changes on brain function. Therefore, we studied the connectivity changes caused by single isoflurane (3h, 1.8%) exposure after a one-month waiting period by resting state fMRI and local field potential (LFP) measurements. Treated rats exhibited significantly strengthened connectivity between hippocampus and somatosensory cortex both in fMRI and LFP, indicating long-term modulation of brain activity by single administration of isoflurane anesthesia compared to non-treated controls.
Introduction
In preclinical fMRI studies, anesthesia is typically required to minimize stress and movement of the animals during the measurements. Anesthesia can, however, dramatically alter brain function1 and/or change hemodynamic response2,3. Furthermore, it has been shown that the effects of anesthetics can extend beyond the duration of anesthesia. For example, isoflurane can influence plasticity and gene-expression several days or weeks after the initial exposure.4-12 Several experiments include, e.g., long surgical procedures13 under general anesthesia, that can possibly have a long-term effect on brain connectivity14, 15. However, no studies have been conducted to evaluate the long-term effects of isoflurane anesthesia on functional connectivity. Therefore, the aim of the study was to investigate the long-term effect of 1.8% isoflurane on brain function using fMRI and electrophysiological local field potential (LFP) measurements a month after the initial exposure.Subjects and methods
Male Wistar rats (n = 12) were exposed to 1.8%
isoflurane for 3 h and naïve rats (n=12) were used as controls. This protocol
was mimicking the anesthesia during a long invasive surgical procedure. After
one month, 6+6 rats were imaged with fMRI and another group of 6+6 rats were
used in LFP measurements. FMRI and LFP measurements were conducted with the
following isoflurane concentrations: 1.3%, 2.0%, 1.3%, and 3.0%, each 10min in
duration. FMRI was performed in a Bruker Pharmascan 7T magnet with single-shot
spin-echo echo planar imaging sequence with the following parameters: TR 2,000
ms, TE 45 ms, matrix size 64 × 64, field-of-view 2.5 × 2.5 cm, 11 slices of 1.5
mm thickness, and a bandwidth of 250 kHz. FMRI data was converted to NIfTI (http://aedes.uef.fi),
slice-timing corrected, motion-corrected, co-registered to a reference brain
(SPM8), and finally smoothed. Resting state functional connectivity (RSFC) (0.01 - 0.15 Hz) was calculated from either 4 or 12 regions of interest, covering the
LFP electrode locations or whole brain, respectively.
LFP was recorded with SciWork data acquisition
system (Datawave Technologies) with a 2049 Hz sampling rate. Cortical LFP was
recorded bilaterally from the somatosensory cortex (S1) left (S1L) and right
(S1R) (AP:-1, ML: +/- 3) using screw electrodes, and hippocampal LFP (50µm stainless steel wire electrodes) from the right dentate gyrus (DG)
(AP: -3.8, ML: + 1.6, DV: -4.3) and right cornu ammonis 1 (CA1) (AP: -3.8, ML:
+ 1.6, DV: -3.7). Data was analyzed with Matlab R2011a and Spike2, version 8. LFP
coherence was analyzed from averaging 30s long epochs during each isoflurane
concentration. Inter-channel correlation was measured from LFP power (amplitude
envelope) using either full band or band-pass filtered signal.16,17 Burst
suppression activity of burst frequency (Hz) and standard deviation (SD) of
suppression periods (s) was also analyzed.
Statistical testing for fMRI correlation, LFP coherence and correlation were conducted by two-tailed two-sample t-tests in Matlab. We used false discovery rate (FDR) to account for multiple comparisons for 12-ROI correlation and LFP coherence analyzes.
Results
Both fMRI and LFP measurements indicated that brain function was altered one month after the initial isoflurane exposure. In fMRI whole brain analysis, we found increased correlation in 11 thalamo-cortical and in 5 hippocampal-cortical connections in the isoflurane-exposed group compared to the control group (p<0.05, FDR adjusted) under 2.0% isoflurane (Figure 1). Also, increased CA1-S1R correlation was found in the 4-ROI fMRI analysis (p=0.025) under 2.0% isoflurane (Figure 2).
In LFP analysis, we found increased coherence between DG and S1R in delta band during the 2nd 1.3% isoflurane period (p=0.026, FDR adjusted) (Figure 3). Additionally, increased coherence was observed between DG and S1L in alpha band during the 1st 1.3% isoflurane period (p=0.034, FDR adjusted) and in delta band under the 2nd 1.3% isoflurane period (p=0.009, FDR adjusted). Correlation of LFP power was increased between S1R and DG, and S1R and CA1 (p=0.035 and 0.044 accordingly) in isoflurane exposed rats compared to controls under 1.3% isoflurane (Figure 2). Finally, no change in burst frequency or SD of suppression periods was found in either somatosensory cortex (p=0.470 and p=0.676, respectively) or in hippocampus (p=0.377 and p=0.676) (Figure 4).
Discussion and conclusion
These results suggest that a single prolonged isoflurane exposure has a persistent effect on brain function that lasts at least one month. Because no change was seen in burst frequency (established measures for the depth of anesthesia) or SD of suppression periods, the observed alterations in FC likely reflect neural network plasticity changes rather than a change in the depth of anesthesia. As a conclusion, in studies exploiting deep isoflurane anesthesia, extra caution should be taken while interpreting connectivity results, even if there is a long period after initial anesthesia.Acknowledgements
This work was supported by the Doctoral Program in Molecular Medicine of University of Eastern Finland. We thank MP for technical assistance in animal preparations. The funding sources had no further role in study design, or in the collection, analysis or interpretation of data.References
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