Histological outcome evaluation of selected brain preparation protocols for white fiber dissection

Background:White fiber dissection is essential for studying brain connections. However, preparation protocols have not been validated.Methods:We microstructurally analyzed Klingler’s brain preparation method and freezing process and assessed changes under two protocols:freeze-only and freeze-thaw. The microstructure changes of these protocols were evaluated by measuring the ratio of the total gap area to the white matter area and determining the mean eccentricity value to assess the degree of anisotropy.Results:Sixty hemispheres were allocated to ten different freezing protocols. In the freeze-only protocols, the total gap area ratio was significantly higher compared to that of specimens fixed with only formaldehyde, particularly after continuous freezing for 3-4 weeks;however, the difference in eccentricity was not significant. In the freeze-thaw protocols, both the area ratio and eccentricity were significantly higher compared to the freeze-only. The optimum degree of fiber separation in the freeze-thaw protocols reached its peak with four cycles of 1-week freezing periods interrupted by six hours of thawing.Conclusion:The Klingler method assists in the separation of the white fibers through the gaps formed by ice crystals, but an appropriate degree of anisotropy is reached when the freezing protocol is interrupted by at least four thawing cycles.