Investigation of the structural and molecular alteration of the astrocytes by thermal stimulation using a PVC-Gel heater-based chamber

Introduction: : Temperature fluctuations critically affect brain homeostasis, and hyperthermia exacerbates neurological dysfunction and inflammation. Astrocytes, as the predominant glial cells in the CNS, are key regulators of neuro-inflammation, ionic balance, and blood-brain barrier integrity. Despite their sensitivity to temperature changes, real-time observation of astrocytic responses under thermal stress remains limited due to challenges in controlling temperature precisely while maintaining optical access. To address this, we developed a PVC-Gel heater-based chamber optimized for astrocyte culture and live imaging. The platform provides spatially uniform heating within physiological ranges (37℃ to 42℃ ±1℃), and >90% optical transmittance across the visible spectrum. Utilizing a PVC-Gel heater, we aim to observe the astrocyte changes according to temperature, thereby providing basic data for temperature sensitivity and neuro-inflammation research.

Materials and

Methods: : The PVC-gel heater was fabricated by mixing dibutyl adipate, tetrahydrofuran, and polyvinyl chloride, casting it into a thin film, and sandwiching it between ITO-coated PET sheets. Primary astrocytes were isolated from the cerebral cortex of 1-2 day-old mice and cultured in DMEM. Cells cultured in the chamber were exposed to a 100 Hz AC electric field to generate heat for temperature stimulation. Morphological changes in astrocytes were observed by imaging the changes in the phase-contrast images in real time during the 15-minute heating session. Cell viability was assessed using a live-dead assay. Immunofluorescence staining for DAPI and GFAP was performed to examine the changes in the nuclei and structure of astrocytes after thermal stimulation. qPCR analysis was performed to investigate temperature-sensitive and inflammation-related gene expression.

Results, Conclusions, and Discussions:: Results and

Discussion: Astrocytes exposed to the hyperthermic temperature(40 ℃, 42 ℃) exhibited progressive cell detachment, as revealed by real-time changes in the phase contrast images. Compared to the 37 ℃ control, thermally stimulated cells showed an increased bright area surrounding the cells, indicating detachment from the bottom. The intensity in the DAPI signal decreased from the astrocytes stimulated by the hyperthermic temperature, indicating the DNA damage caused by the thermal stimulation. The GFAP-stained region from the astrocyte with thermal stimulation showed a decreased area and form factor, revealing morphological simplification. qPCR analysis demonstrated that temperature-sensing markers such as HSP70, TRPV1, and inflammatory markers such as IL-1β, Stat3 showed elevated expression at higher temperatures. These changes indicated that astrocytes actively sense thermal stimuli and initiate molecular programs for protection and immune regulation.

Conclusions: Our results demonstrated that the PVC-Gel heater enables precise thermal stimulation and real-time observation of astrocytic response. Using this platform, we could confirm changes in the structural and molecular dynamics of astrocyte thermosensitivity and inflammation. Therefore, this research has the potential to advance temperature-related pathophysiology and develop therapeutic strategies.

Acknowledgements and/or References (Optional):: This work was funded by Basic Research Funds from the Department of Mechanical Engineering at KAIST (A0601003029).