The spatiotemporal assessment of tissue dynamics after the introduction of disruptive factors is crucial for evaluating their impact and for developing effective countermeasures. Here, we report a 4D-spatiotemporal imaging approach using second harmonic generation (SHG) imaging microscopy, enabling an advanced time-resolved analysis of three-dimensional tissue features. This is of particular interest as topical administration of drugs during spinal surgeries is a standard practice for preventing and treating postoperative complications like infections. Local drug concentrations on tissue are high in these scenarios, and given the dura's role as a protective barrier for the brain and spinal cord, potential drug-induced damage should be evaluated critically. By employing 4D-SHG imaging, we gained detailed insights into changes in dimensional properties of thin section samples, namely, width, height, and volume, as well as into alterations within the hierarchic structure of collagen. The latter thereby allowed us to postulate a mode of action, which we attributed for the herein investigated samples to the pH of the formulation.