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Histological Analyses Methods of Meniscus In the experimental orthopaedic studies, it has been suggested to support biochemical, biomechanics analyses with histological analyses in the evaluation of cartilage healing models. In the histological evaluation of tissues, generally qualitative analyses have been applied, whereas quantitative analyses have been done on a limited scale or have never been done. Providing a scientific validity for the study is directly related to the choice of analytic methods in accordance with the aim, quantitation the parameters evaluated and their interpretations. From this point of view in this review, it has been attempted to present the methodology that can be applied in the histological preparation and analysis of meniscus tissue., Türkçe Özet: Deneysel ortopedik çalışmalarda, kıkırdak iyileşme modellerinin değerlendirilmesinde, biyokimyasal, biyomekanik incelemelerin histolojik incelemeler ile desteklenmesi önerilmektedir. Dokuların histolojik değerlendirilmesinde genellikle kalitatif incelemeler yapılırken kantitatif incelemeler sınırlı miktarlarda yapılmakta ya da hiç yapılmamaktadır. Çalışmanın bilimsel geçerliliğinin sağlanması, amaca uygun analitik metodların seçilmesi, değerlendirilecek parametrelerin kantite edilmesi ve bunların yorumlanmasıyla doğrudan ilişkilidir. Bu noktadan yola çıkarak bu derlemede, menisküs dokusunun histolojik preparasyonu ve incelemesinde kullanılabilecek metodoloji ortaya konulmaya çalışılmıştır.

1. Ulusal Sinir Bilimleri Kongresi’nde (16-20 Mart 2002, Eskişehir) poster olarak sunulmuştur. Sinir dokusunun ışık mikroskobik fiksasyonunda ve birçok nöroanatomik boyama metotlarında mikrodalga kullanımı, kaliteyi arttırmak, standardizasyonu sağlamak, zaman ve kullanılan kimyasallar açısından ekonomik bir çalışma gerçekleştirmek amacıyla önerilmektedir. Bu çalışmada amaç; santral sinir sisteminde myelinin ışık mikroskobik incelenmesinde fiksasyon ve boyanma aşamalarında mikrodalga ışınımını kullanarak eşit kalitede preparat elde etmek, preparasyon süresini kısaltarak patoloji laboratuvarlarında arşiv parafin bloklarında uygulanabilecek, demyelinizan hastalıkların tanısında kullanılabilecek kısa süreli boyama prosedürünü gerçekleştirmektir. Sıçan beyin dokusu myelinli lifleri, normal myelin boyanmasında kullanılan Weil ve Kluver-Barrera metotları uygulanarak boyandı.Konvansiyonel şartlarda iki gün süren fiksasyon, mikrodalga grubunda 16.5-18.5 dakikada gerçekleşti. Weil metodunda boyama süresi 2 dakikaya, Kluver-Barrera metodunda 15 dakikaya düşürüldü. Süre kısalmalarının yanısıra morfolojik yapıda bozulmaya rastlanılmadı. Çalışmanın sonunda demyelinizan hastalıkların saptanmasında, myelinin ışık mikroskobik fiksasyonunda ve normal myelinin boyanmasında zamanın kısıtlı olduğu durumlarda hem kısa sürede, hem de kaliteli boyanmayı elde etmek amacıyla mikrodalga ışınımının kullanılabileceği düşünüldü. Microwave application is recommended for the fixation of nerve tissue in light microscopic analyses and various neuroanatomical staining methods to increase the quality of the preparation and standardization as well as achieve an economical study concerning the consumed time and chemicals. The purpose of this study is to obtain qualitatively equal preparations by using microwave irradiation in fixation and staining steps of the light microscopic analyses of the myelin in the central nervous system and, by shorthening the preparation time, to achive a concise staining procedure in order to apply on archieve paraffin blocks and to use for the diagnosis of demyelinating diseases in pathology laboratories. The myelinated fibers of rat brain tissue were stained by Weil and Kluver-Barrera methods which are routinely used for myelin staining. The fixation was obtained in 16.5-18.5 minutes in the microwave-group while it takes 2 days in conventional conditions. In the Weil’s method the fixation time was shortened to 2 minutes and in Kluver-Barrera’s method to 15 minutes. No morphological disturbance was detected due to the time reductions. As the result of the study, it is suggested that, in order to obtain qualified staining in a short time, microwave applications can be used during light microscopic fixation of myelin in the diagnosis of demyelinating diseases as well as for normal myelin staining when the time is limited.

Corrosion casting is an important tool for the study of microvascular structure, but unlike tissue preparation for light and electron microscopy, few controlled studies of the methods for preparation of microvascular casts for scanning electron microscopy have been reported. Most vascular casting begins with rinsing cells from the lumen of the blood vessels, but the necessity of this has not been shown. Because of a report of successful casting without rinsing and light microscopic evidence of edema with complete rinsing prior to casting, this preparation procedure was examined. Casts of lung vasculature that were thoroughly rinsed were compared to those that were not. Of the six rats that were not lavaged, only one gave a completely filled cast, but of the six animals that were well rinsed, only one did not fill. Except for the filling and mild periarterial edema, no significant difference in cast quality or features was detected. Rinsing improves the frequency of obtaining completely filled casts. The edema does not greatly interfere with the capillary image and its location gives insight into this process in the lung.

To determine the importance of the pulmonary or bronchial arteries as the site of injection to cast the lung vasculature of rats, we retrospectively compared a group of normal Sprague-Dawley rats that were cast through either the caudal vena cava and right ventricle leading to the pulmonary arteries or through the aorta and bronchial arteries. There was no difference in the appearance of the whole casts or cast-to-lung weight ratios between the two groups, although an incompletely cast lung that was injected through the aorta showed preferential filling of the pleural and peribronchial regions. Nuclear impressions, a sign of good filling, were more frequent and the grade of the images was higher in the group filled through the vena cava. The microvascular density index was not different between the two groups, and a multivariate analysis of variance showed the injection site was not important in determining vascular density. The pulmonary vessels fill nearly as well when injected from the bronchial circulation indicating that the bronchial circulation can immediately perfuse the lung if the pulmonary arteries are obstructed, and casting can be carried out from either side with nearly the same results.

Scanning electron microscopy of corrosion casts is an important tool for the study of microvascular structure but few systematic studies on methods of specimen preparation have been reported. This study sought to determine the relative importance of factors involved in corrosion. It compared potassium hydroxide, sodium hydroxide and water as major corrosive agents. It tested the size of the tissue sample, and the use of prealkali autolysis, detergent, and proteolytic enzymes in a Latin square designed study. The main findings were that sodium and potassium hydroxide were better than water (P < 0.0001) and the longer the corrosion time the better corroded the samples were (P < 0.0001 ). Although not a controlled factor, higher room temperature was also associated with better corrosion. The use of proteolytic enzymes, detergent, and warm tap water alone before the alkali treatment did not significantly improve the corrosion in this study, although this does not preclude an effect with another experimental design.