The influence of exogenous recombinant HSP 70 on the alteration of membrane stiffness in hippocampal neurons following the modeling of neonatal hypoxic-ischemic injury in mice

Abstract

Introduction: The use of atomic force microscopy (AFM) to investigate membrane stiffness in neurons provides valuable insights into cellular mechanisms and their alterations in response to various pathophysiological conditions. Heat shock protein HSP 70, a component of the cellular stress response system, plays a role in stabilizing the protein structures of cellular organelles. However, studies examining changes in the stiffness of hippocampal neuronal membranes in its presence, particularly following cerebral circulation disturbances, have not been conducted yet.

Materials and Methods: The study was performed on a mixed culture of hippocampal neurons derived from 9-day-old male CD-1 mice, obtained 24 hours after modeling neonatal hypoxia-ischemia. The following groups were formed: Intact culture; HI culture; HI + rhHSP70 10^{-6 M}; HI + rhHSP70 10^{-8 M}; HI + rhHSP70 10^{-9 M}; HI + rhHSP70 10^{-12 M}, with the substance added in dilutions from an initial dose of 0.1 µg/g. The Young's modulus was measured using force spectroscopy, and maps of local stiffness of various surface areas were generated.

Results and Discussion: The neonatal hypoxia-ischemia model resulted in an 18% increase in the stiffness of the neuronal cell surface compared to the control group (p<0.001). The addition of rhHSP70 at concentrations of 10^{-6 M} and 10^{-8 M} to the HI culture led to an increase in membrane stiffness by 20% (p<0.001) and 3% (p<0.0034), respectively, while dilutions of rhHSP70 at 10^{-9 M} and 10^{-12 M} resulted in a decrease in membrane stiffness by 35% (p<0.001) and 22% (p<0.001) compared to the intact group, respectively. In comparison to such in the neuronal culture group after neonatal hypoxia-ischemia modeling, membrane stiffness with the addition of rhHSP70 at 10^{-8 M}, 10^{-9 M}, and 10^-12 M decreased by 17% (p<0.0004), 65% (p<0.001), and 49% (p<0.001), respectively.

Conclusion: Thus, the addition of rhHSP 70 results in a reduction in membrane stiffness in the mixed culture of hippocampal neurons in mice, compared to the intact culture obtained after neonatal hypoxia-ischemia. The AFM method allows for the assessment of how various molecules, such as heat shock proteins (e.g., rhHSP70), influence the mechanical properties of membranes, which may be critically important for the development of new therapeutic agents.

Graphical Abstract