Comparison of the Brain and Medulla Spinalis Ultrastructural Evaluation Using Five Different Fixatives

Abstract

Objectives: Ultrastructural examination of nervous tissues is essential for neuroscientific study. Nervous tissue is sensitive to ischemia and hypoxia. Thus, the fixation and process of this tissue is critical for evaluating ultrastructure of nervous tissue. It is difficult to distinguish whether the findings are due to the fixative used in the experimental research or the experimental agent. In this study, we aimed to investigate the effects of the different five fixatives on the brain and medulla spinalis, which are the central nervous system organs, by evaluating the ultrastructural changes. Materials and Methods: In this study, five male Wistar Albino rats (200-250 gr) were anesthetized. The perfusion fixation was performed by 4% paraformaldehyde (PFA). The extracted brain and medulla spinalis pieces were immersed into five different fixation solutions as (I) Trump’s solution 4% PFA and 1% glutaraldehyde (GA), (II) 2% PFA and 2.5% GA with 2.5 mM CaCl, (III) 2% PFA and 2.5% GA, (IV) 2.5% GA, (V) Trump’s solution 4% PFA and 1% GA (1% OsO4 containing potassium ferrocyanate). After routine processes, all ultrathin tissue sections were investigated under the transmission electron microscopy. Results: In Group 4, the cell nucleus, perinuclear space, mitochondrion, rough endoplasmic reticulum and myelin sheet, endothelium and basal lamina of nervous tissues of brain and medulla spinalis were evaluated as normal ultrastructure compared to the other groups. Conclusion: In present study, we showed that 2.5% GA solution prepared with cacodylate buffer was convenient and effective for nerve tissues of electron microscopic routine process.

Keywords

• Transmission Electron Microscopy (TEM)
• Fixatives
• Trump’s Solution
• Phosphate Buffer
• Cacodylate Buffer

İnce Yapı İncelemelerinde Beyin ve Medulla Spinalisin Beş Farklı Tespit Solüsyonuyla Korunmasının Karşılaştırılması

Abstract

Amaç: Sinir dokularının ince yapı düzeyinde incelenmesi nörobilimsel çalışmalar için gereklidir. Sinir dokusu hipoksi ve iskemiye hassastır. Bu nedenle, bu dokunun tespit ve takip edilmesi sinir dokusunun ince yapı düzeyinde değerlendirilmesi açısından kritiktir. Bulguların deneysel araştırmada kullanılan tespit solüsyonundan mı yoksa deneysel etkenden mi kaynaklandığını ayırt etmek zordur. Bu çalışmada, beş farklı tespit solüsyonunun merkezi sinir sistemi organları olan beyin ve medulla spinalis üzerine etkilerini, hücre ve organellerin ince yapı düzeyinde gösterilmesini ve karşılaştırılmasını amaçladık. Gereç ve Yöntem: Çalışmamızda beş erkek Wistar albino sıçana (200-250 gr) anestezi uygulandı. Perfüzyon tespiti %4 paraformaldehit (PFA) ile gerçekleştirildi. Çıkarılan beyin ve medulla spinalis parçaları; (I) Trump’ın solüsyonu %4 PFA ve %1 glutaraldehit (GA), (II) %2 PFA ve %2,5 GA (2,5 mM CaCl içinde), (III) %2 PFA ve %2,5 GA, (IV) %2,5 G, (V) Trump’ın çözeltisi %4 PFA ve %1 GA (potasyum ferrosiyanat içeren %1 OsO4) olmak üzere beş farklı tespit solüsyonuna daldırıldı. Rutin takip işlemlerinden sonra, tüm ince doku kesitleri geçirimli elektron mikroskobu altında incelendi. Bulgular: Grup 4’te beyin ve medulla spinaliste gözlemlenen hücre çekirdeği, perinükleer boşluk, mitokondri, granüllü endoplazma retikulumu, miyelin tabakası, endotel ve bazal laminasının diğer gruplara göre normal bir yapıda olduğu elektron mikroskobu altında izlendi. Sonuç: Bu çalışmada, beyin ve medulla spinalisin elektron mikroskobu altında incelenmesi için tespit işleminde kakodilat tamponu ile hazırlanan %2,5 GA çözeltisinin uygun ve etkili olduğunu gösterdik.

Keywords

• Geçirimli Elektron Mikroskobu (TEM)
• Tespit Solüsyonu
• Trump Solüsyonu
• Fosfat Tamponu
• Kakodilat Tamponu

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