Maly I Piotr, Eppler Elisabeth, Müller-Gerbl Magdalena
Musculoskeletal Research, Department of Biomedicine, University of Basel, Pestalozzistrasse 20, CH-4056 Basel, Switzerland.
Gen Comp Endocrinol. 2018 Mar 1;258:109-118. doi: 10.1016/j.ygcen.2017.05.008. Epub 2017 May 11.
Tissue-nonspecific alkaline phosphatase (TNAP) is playing a key role in bone calcification, as has been demonstrated in different mammalian species including human and rodents. However, to investigate age-related changes during life history, histochemical demonstration of TNAP is severely hampered, particularly in the elderly, by technical difficulties associated with sectioning calcified tissue. Sufficient fixation must precede decalcification since poorly fixed bone tissue is exposed to the deleterious effects of decalcification reagents. In order to find a method that would allow cryosectioning of bone without loss of TNAP activity, we assessed the efficacy of different fixation reagents regarding the effects on structural integrity and TNAP activity using liver and osseous tissue from younger and older horses. The results of this study reveal that glyoxal-based fixatives sufficiently preserved bone tissue for successful cryosectioning without compromising TNAP activity. The method described combines the demonstration of TNAP activity with optimal preservation of tissue morphology in osseous tissue of younger and even of older mammals. As a model species, we selected horse bones in light of potentially higher similarities to ageing history and lifelong locomotion in humans as compared to other, mostly smaller, experimental model species with a much shorter life span and artificial locomotive activity when kept in cages. This may serve as a basis for future studies addressing the impact of different life traits in iconic, domestic and companion animals, which are often patients in veterinary medicine, as well as for basic research on human physiology and pathologies of the musculoskeletal system.
组织非特异性碱性磷酸酶（TNAP）在骨钙化过程中起着关键作用，这一点已在包括人类和啮齿动物在内的不同哺乳动物物种中得到证实。然而，为了研究生命历程中与年龄相关的变化，TNAP的组织化学显示受到严重阻碍，尤其是在老年人中，这是由于钙化组织切片相关的技术困难。在脱钙之前必须进行充分固定，因为固定不佳的骨组织会受到脱钙试剂的有害影响。为了找到一种能够对骨进行冷冻切片而不损失TNAP活性的方法，我们使用年轻和年老马匹的肝脏和骨组织，评估了不同固定试剂对结构完整性和TNAP活性的影响效果。本研究结果表明，基于乙二醛的固定剂能够充分保存骨组织，以便成功进行冷冻切片，同时不影响TNAP活性。所描述的方法将TNAP活性的显示与年轻甚至年老哺乳动物骨组织中组织形态的最佳保存相结合。作为一个模型物种，我们选择马骨是考虑到与其他大多较小、寿命短且在笼养时具有人工运动活动的实验模型物种相比，马在衰老历程和人类终身运动方面可能具有更高的相似性。这可为未来研究不同生活特征对标志性、家养和伴侣动物（这些动物在兽医学中常为病患）的影响提供基础，也可为人类肌肉骨骼系统生理学和病理学的基础研究提供基础。
