We thank Jonas and colleagues
1 for their interest in our work. In their immunohistochemical study,
2 six morphological stages were described to characterize the change of microglia from a resting to an activated state with reference to the soma size, process number, process diameter, branches number, and process length/soma diameter. We fully agree with Jonas that a longitudinal, in vivo study would be pertinent to validate the histologic observation. While serial morphological changes of the microglia can be visualized in the CX3CR1
GFP/+ transgenic mice using the in vivo confocal scanning laser ophthalmoscopy (CSLO) imaging system,
3 we believe that some modifications in the instrumentation and the study design would be required to distinguish the six different stages as described.
One shortcoming of the CSLO compared with a confocal scanning laser microscope is a weaker resolving power. While individual processes and soma of the microglia can be distinctively visualized with in vivo CSLO imaging, it may not be possible to measure the diameters of the processes and soma reliably in the CSLO images for a full classification. The application of in vivo confocal or multiphoton microscopy with adaptive optics may provide the required resolution for the measurement. Optic nerve injury models adopted in our previous study including optic nerve crush and acute elevation of IOP induce generalized activation of microglia.
3 Models eliciting localized damage to the retina (e.g., a spot laser damage) is necessary to capture both the “A” (activation) and “R” (deactivation) stages. It is hopeful that the advancement of in vivo imaging techniques can provide better insights into the roles of microglial activation in retinal and optic nerve diseases.