Ouabain浮腫脳の電子顕微鏡的研究

ELECTRON MICROSCOPIC STUDY OF OUABAIN-INDUCED EDEMATOUS BRAIN

I．緒言

　脳損傷や脳腫瘍などに伴つて発生する脳浮腫においては，増加した液体の貯溜部位は皮質ではastrocyteであり，白質ではそれに加うるに細胞外腔であるということが明らかにされているが^{14）28）40）43）}，その発生機序はなお未解決のままである。従来，脳浮腫発生の要因として血管の透過性異常が重要視されてきたが，最近，脳浮腫の生化学的な研究が集積されるにいたり，その細胞腫脹の機序について新たな見地から検討が加えられている。すなわち，in vitroにおけるastrocyteの腫脹が嫌気的条件や，種々のエネルギー代謝阻害剤で増強されること^11）21）や，in vivoで作られた浮腫脳においてもエネルギー代謝障害，特にミトコンドリアの機能障害が存在する^{31）35）44）}ことから，脳浮腫にみられるastrocyteの腫脹する機序を，高エネルギーリン酸の減少にもとづく細胞膜の能動輸送（active transport）の障害で説明する人も多い^{31）32）35）44）}。

　一般に細胞内ではK^＋濃度が高く，細胞外ではNa^＋濃度の高いことが知られているが，このイオンの偏在性を保つているactive cation transportはエネルギー依存性であり^18），細胞の形態保持に重要な機構であるといわれている。Skou^36）がNa-K—ATPaseを発見して以来，細胞膜を介してのactive cation transportには，膜に存在するこの酵素がNa-K—pumpとして主役を演じていることや，active cation transportの阻害剤として知られていたouabain^34）が，この酵素を特異的に阻害することなどが，多くの研究によつて明らかにされてきた^{15）30）37）}。他方，このactive cation transportを阻害すると，細胞が腫脹することも確かめられておることから^41），脳浮腫の場合にもエネルギー産生能の低下が二次的に膜のactive cation transportを阻害し，細胞内にNa^＋の貯溜をまねき，細胞が腫脹することも推測できよう。

In the course of study on brain edema, ouabain, a specific inhibitor of Na-K-ATPase, was locally applied on the cat cerebral surface to ascertain whe-ther or not the primary disturbance of active cation transport across cell membrane was responsible for producing a state of brain edema.

A burr hole about 1 cm. in diameter was trephined on the parietal bone of cats, and after removing dura, the cerebral surface was directly immersed in 10^-3M or 10^-4M ouabain solution, or in control solutions. In the preliminary experiments electro-encephalograms were recorded in some immobilized animals under artificial respiration. The cortex im-mersed in 10^-3M ouabain solution in phosphate buffer showed a low voltage and slow wave activity in EEGabout 15 minutes after the immersion. No alteration in EEG appeared in the cortex immersed in 10^-4M ouabain or control solutions.

Biochemical analysis made in our laboratory of the materials immersed in 10^-3M ouabain solution revealed marked increase in sodium content in as-sociation with slight increase in water.

Based on these data, in vivo responses to ouabain of ultrastructure and Na-ion localization in cat cere-bral cortex were examined by electron microscope.

A) Ultrastructure

In light microscopy of osmium-fixed and epon-embedded materials, the cortex immersed in 10^-3M ouabain solution showed spongy state localized in upper two or three cortical layers.

Electron microscopic examinations revealed swell-ing of cell processes, mainly of neuronal elements, dendrite and presynaptic ending. In the mild lesions swelling was well confined to postsynaptic ending. Even in the severely affected lesions astrocytes and astrocytic processes as well as pericapillary end-feet showed no evidence of swelling in earlier stages within 60 minutes after the immersion. Astrocytic responses such as an increase of glycogen-like gra-nules and slight swelling of the processes were added to the changes of neuronal elements in 3 hours after the immersion.

B) Na-ion localization

In an attempt to observe the localization of Na-ion in the cerebral cortex by electron microscopy, the method of Komnick was applied with slight modifications. In normal cortex large amount of precipitate of NaSb (OH)₆ was present in astrocytes and astrocytic processes. Small amount of precipitate was recognizable in neurons and oligodendrocytes as well as in neuronal processes, mostly limited to endoplasmic reticulum, Golgi apparatus and nuclear envelope. Precipitate was hardly observed in the basement membrane nor in capillary endothelium in a striking contrast to pericapillary end-feet.

In cortex immersed in 10^-3M ouabain solution for 60 minutes, the swollen neuronal structures, particu-larly dendrites, had a high precipitate concentration.

From these results, it has been speculated that neuronal elements, rather than astrocytic, depend highly on active cation transport system, and that "cellular swelling " responsible for impairment of active cation transport is related to neuronal, rather than astrocytic, elements.