Determination of the most accurate technique among various histological methods for evaluation of the efficacy of neuroprotective agents in animal model of multiple sclerosis

Here we aimed to comprise several histological methods to determine the most accurate technique for evaluation of efficacy of neuroprotective agents in animal model of multiple sclerosis (MS).

Male C57BL/6 mice were treated for 6 weeks in control, control + Benzoraic acid (BA, as neuroprotective agent) 20, 40, 80 mg/kg during last 2 weeks, Cuprizone (0.2%) as demyelinating agent, and Cuprizone (0.2%) + BA (20, 40, 80 mg/kg during last 2 weeks) groups. At the end of experiment, histological examinations including immunohistochemistry (IHC), Hematoxylin & Eosin (H & E) staining, Luxol Fast Blue (LFB) staining, and Transmission Electron Microscopy (TEM) techniques were performed for evaluation of myelin damages.

Significant myelin damage was detected by all of the used histological methods in Cuprizone group (p < 0.001 compared to control group). LFB technique could not show improvement in damages by all doses of BA. The H&E, IHC and TEM methods showed significant protective effects of the high BA dose compared to Cuprizone group. Only the TEM method could show significant (p < 0.05) protective effect of the medium BA dose.

TEM was the most and LFB was least sensitive methods. Present study suggests simultaneous using of methods such as H & E for better assessment of neuroinflammation and IHC for confirmation of TEM results. Moreover, in the case of restriction in methods, TEM and IHC are the most sensitive methods and could provide reliable results related to efficacy of neuroprotective agents in this model.