Background & Aims

Clinical studies have shown that women tend to have greater pain sensitivity, which can increase the risk of pain becoming chronic (1). Therefore, gaining insights into gender-based differences is crucial for effective pain management. While preclinical animal studies have explored gender differences, they have rarely focused on differences related to mechanical stimulation. In our recent research, we employed fMRI to uncover heightened activation in female mice compared to males when subjected to mechanical stimulation (2). However, since Blood-oxygen-level-dependent (BOLD)- fMRI reflects indirect neuronal activity, our current study combines fMRI with direct calcium recordings during hind paw stimulation to provide a more comprehensive understanding of these gender-based distinctions.

Methods

Male and female C57BL/6J mice, aged 8 to 10 weeks, underwent stereotactic surgery for the delivery of GCamP-expressing AAV specifically targeted to excitatory neurons (CamKII) in the S1 hind limb (S1HL) and cingulate cortex (CG) structures. After a 4-week period, fibers were implanted in both structures to facilitate calcium recordings at a rate of 2 kHz during scanning. These recordings were performed within a 9.4T MRI scanner (Bruker), utilizing a surface coil, while the animals were under combined isoflurane/medetomidine anesthesia (3). Electrodes were inserted into the hind paw to administer electrical stimulation at 1 (low, innocuous) or 3 (high, noxious) mA with frequencies of 3 or 9 Hz. The fMRI measurements were conducted using a gradient EPI sequence (TR/TE 1000/18 ms, 18 slices, 0.5 mm thickness, matrix 70×60, FOV 16×16 mm). Stimulation followed a block paradigm consisting of 10 repetitions of 10 seconds ON and 50 seconds OFF (4). The data analysis was performed using MATLAB.

Results

Stable expression of GCamP was detected in both the S1HL and CG regions. In BOLD images, we observed signal voids primarily localized to the site of fiber implantation, and detected a strong correlation between BOLD signals and calcium recordings. Next, we analysed calcium transients in S1HL and CG at both stimulus intensities. In both sexes, we observed increased amplitudes with high vs. low stimulus intensity in the S1HL (p=0.03). In contrast, there was no change in the CG (p=0.8). However, there was an observed increase in recurrent activity in CG following stimulation. Therefore, we investigated the area under the curve (AUC) at different intervals (1-10, 11-20, 21-30 seconds) following stimulus onset and conducted a principal component analysis (PCA) involving all factors. Interestingly, a sex-related effect was evident solely in CG (p=0.03), driven by an elevated AUC in female mice, indicating enhanced recurrent network processing in response to the stimulus.

Conclusions

By integrating optical calcium recording in CG and S1HL with BOLD fMRI (ofMRI) in mice, we identified elevated calcium signal amplitudes in response to noxious stimulus intensities, likely reflecting the sensory-discriminative processing in S1HL. This phenomenon remained consistent across sexes. However, in female mice, we observed heightened recurrent activity in CG, marked by an extended AUC that persisted beyond the stimulation phase, suggesting an augmentation in higher-order processing.

References

1.Mogil JS. Qualitative sex differences in pain processing: emerging evidence of a biased literature. Nat Rev Neurosci. 2020;21(7):353-365. doi:10.1038/s41583-020-0310-6
2.Chen H fen, Lambers H, Nagelmann N, et al. Generation of a whole-brain hemodynamic response function and sex-specific differences in cerebral processing of mechano-sensation in mice detected by BOLD fMRI. 2023;(August):1-14. doi:10.3389/fnins.2023.1187328
3.Pradier B, Wachsmuth L, Nagelmann N, et al. Combined resting state-fMRI and calcium recordings show stable brain states for task-induced fMRI in mice under combined ISO/MED anesthesia. Neuroimage. 2021;245(September):118626. doi:10.1016/j.neuroimage.2021.118626
4.Amirmohseni S, Segelcke D, Reichl S, et al. Characterization of incisional and inflammatory pain in rats using functional tools of MRI. Neuroimage. 2016;127:110-122. doi:10.1016/j.neuroimage.2015.11.052

Presenting Author

Bruno Pradier

Poster Authors

Bruno Pradier

PhD

University of Muenster

Lead Author

E-mail

Anna Szinyei

Lead Author

E-mail

Lydia Wachsmuth

Lead Author

E-mail

Huifen Chen

Lead Author

E-mail

Daniel Segelcke

University Hospital Muenster

Lead Author

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Cornelius Faber

Lead Author

E-mail

Esther Pogatzki-Zahn

University Hospital Muenster

Lead Author

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Topics

  • Gender/Sex Differences