Abstract/References

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.

1. De Benedictis A, Duffau H.　Brain hodotopy: from esoteric concept to practical surgical applications.　Neurosurgery, 68(6):1709-1723, 2011.　discussion 23.

2. Wysiadecki G, Clarke E, Polguj M, Haładaj R, Żytkowski A, Topol M.　Klingler’s method of brain dissection:review of the technique including its usefulness in practical neuroanatomy teaching, neurosurgery and neuroimaging.　Folia Morphol (Warsz), 78(3):455-466, 2019.

3. Shah A, Jhawar S, Goel A, Goel A.　Corpus Callosum and Its Connections:A Fiber Dissection Study.　World Neurosurg, 151:e1024-e1035, 2021.

4. Komaitis S, Stranjalis G, Kalamatianos T, Drosos E, Kalyvas AV, Skandalakis GP, et al.　A stepwise laboratory manual for the dissection and illustration of limbic and paralimbic structures:lessons learned from the Klingler’s technique.　Surg Radiol Anat, 44(7):1045-1061, 2022.

5. Catani M, Mesulam MM, Jakobsen E, Malik F, Martersteck A, Wieneke C, et al.　A novel frontal pathway underlies verbal fluency in primary progressive aphasia.　Brain, 136(Pt 8):2619-2628, 2013.

6. Kronfeld-Duenias V, Amir O, Ezrati-Vinacour R, Civier O, Ben-Shachar M.　The frontal aslant tract underlies speech fluency in persistent developmental stuttering.　Brain Struct Funct, 221(1): 365-381, 2016.

7. Wang X, Pathak S, Stefaneanu L, Yeh FC, Li S, Fernandez-Miranda JC.　Subcomponents and connectivity of the superior longitudinal fasciculus in the human brain.　Brain Struct Funct, 221(4):2075-2092, 2016.

8. Yagmurlu K, Middlebrooks EH, Tanriover N, Rhoton AL, Jr.　Fiber tracts of the dorsal language stream in the human brain.　J Neurosurg, 124(5): 1396-1405, 2016.

9. Komaitis S, Skandalakis GP, Kalyvas AV, Drosos E, Lani E, Emelifeonwu J, et al.　Dorsal component of the superior longitudinal fasciculus revisited: novel insights from a focused fiber dissection study.　J Neurosurg, 132(4):1265-1278, 2019.

10. Ludwig E KJ.　Atlas cerebri humani:The inner structure of the brain demonstrated on the basis of macroscopical preparations.　Journal of the American Medical Association, 162(17):1580, 1956.

11. Dziedzic TA, Balasa A, Jeżewski MP, Michałowski Ł, Marchel A.　White matter dissection with the Klingler technique:a literature review.　Brain Structure and Function, 2020.

12. Zemmoura I, Blanchard E, Raynal PI, Rousselot-Denis C, Destrieux C, Velut S.　How Klingler’s dissection permits exploration of brain structural connectivity?　An electron microscopy study of human white matter.　Brain Struct Funct, 221(5): 2477-2486, 2016.

13. Hartel RW.　Crystallization in foods.　Gaithersburg, Md.:Aspen Publishers;2001. x, 325 p. p.

14. McCabe WL, Smith JC, Harriott P.　Unit operations of chemical engineering. 7th ed.　Boston: McGraw-Hill;2005. xxv, 1140 p. p.

15. Cook KLK, Hartel RW.　Mechanisms of Ice Crystallization in Ice Cream Production.　Comprehensive Reviews in Food Science and Food Safety, 9(2):213-222, 2010.

16. Tavare NS.　Simulation of Ostwald ripening in a reactive batch crystallizer, 33(1):152-156, 1987.

17. Wang X, Watanabe M, Suzuki T.　Factors Affecting the Changes of Ice Crystal Form in Ice Cream Quantitative Evaluation of the Shape Change of Ice Crystals by Using Fractal Analysis.　Transactions of the Japan Society of Refrigerating and Air Conditioning Engineers, 25(1):29-33, 2008.

18. Bevilacqua A, Zaritzky NE, Calvelo A.　Histological measurements of ice in frozen beef.　International Journal of Food Science & Technology, 14(3):237-251, 1979.

19. Bomben JL, King CJ.　Heat and mass transport in the freezing of apple tissue.　International Journal of Food Science & Technology, 17(5):615-632, 1982.

20. Miyawaki O.　Analysis and Control of Ice Crystal Structure in Frozen Food and Their Application to Food Processing.　Food Science and Technology Research, 7(1):1-7, 2001.

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