Effect of cyanide intoxication on the metachromatic material found in the central nervous system.

In the course of studies on the rat brain, tissue was fixed in formalin-alcohol.Frozen sections stained with toluidine blue revealed numerous y-meta- chromatic bodies (MB), mainly in relation to myelin.These bodies were felt to be a product of fixation (artifact) probably caused by the solution of some myelin component and its reprecipitation mainly within the white matter.Similar bodies have been described previously but there is no uniformity of opinion as to their origin, chemical nature, or significance (Ferraro, 1928; Grinker and Stevens, 1929; Smith, 1949; Wislocki and Singer, 1950; Wolman, 1960).The close relationship to, and probable derivation from, myelin suggested that analysis of the chemical structure of the meta- chromatic bodies in normal rat brain might increase our understanding of myelin.Also a close similarity appeared to exist between them and the material found inside the 'mucocytes' described by Grinker and Stevens (1929) and possibly the mucoid sub- stance in the cytoplasm of swollen oligodendrocytes.Lastly, we thought that any early pathological change in myelin might be reflected and perhaps magnified in the metachromatic bodies.With this in mind, we studied the metachromatic bodies in the white matter lesions of cyanide-poisoned rats. MATERIALS AND METHODSThirty-four predominantly young adult Lewis rats of both sexes with a minimum body weight of about 220 g. were used.These were divided into two groups.(1) Seven rats were not subjected to any experimental procedure and were used as normal controls.Four of these were anaesthetized with chloroform and killed by exsanguination.The fifth was killed rapidly with ether, the sixth was kept anaesthetized with ether for five minutes, when it was killed also by the ether, and the last was anaesthetized with intraperitoneal sodium nembutal (2-5 mg./100 g. 'National Research Council postgraduate resident research associate supported by the National Aeronautics and Space Administration.Supported by U.S. Public Health Service grant no.MH-1 1698-01.body weight) for 30 minutes and then killed by an over- dosage of nembutal.These animals were included in the study to assess whether narcosis or the mode of killing had any effect on the appearance or distribution of the metachromatic bodies.No such effect was found.(2) Twenty-seven rats were exposed for 20 to 30 minutes to HCN gas according to the 'static method' of Levine (1960).These were killed after varying intervals of time ranging from directly after exposure (usually by over- dosage of HCN at the end of 30 min.) up to 80 days.The brains of animals killed directly after exposure and up to one week after exposure are described in this communication.The rest of the brains were fixed in Cajal's formalin ammonium bromide (2% ammonium bromidein 15 % formalin (F.A.B.)) and used for a separate neuroglial study.Most of the animals were anaesthetized with ether and killed by exsanguination and their brains promptly removed and processed for the demonstration of neuro- glia (Ibrahim, Briscoe, Bayliss, and Adams, 1963) and myelin lipids (Adams, 1965).Some whole brains and some slices from most other brains were fixed in 5 % formalin in 80% ethyl alcohol for a minimum of two days before they were cut on the freezing microtome and examined.The cytological methods were the same as described previously (Ibrahim et al., 1963).Only tissues fixed in 80% ethanol and tissues fixed for a long time in 10% formalin and formol-calcium (1% calcium acetate in 10% formalin) contained a product of fixation (artifact) resembling that found in formalin-alcohol-fixed blocks.The two artifacts were compared histochemically.The histochemical methods employed were mainly taken from the schemata given by Adams (1965).They will be referred to under Results.However, the methods employed for examining metachromic phenomena will be mentioned here since there is still disagreement con- cerning the methodology and its interpretations (see Pearse, 1960, for a discussion of the problem).Formalin-alcohol-fixed blocks were washed well in water, and frozen sections 20 ,u thick were cut and collected in water.These were washed for about 30 minutes, since too large a trace of fixative hampers staining.The sections were placed on slides previously dipped in warm chrome- gelatin (0 05 % chromium potassium sulphate in 2 % gelatin) and dried at room temperature.The sections 545 guest.