MODE OF ACTION OF CERTAIN MYELIN STAINS

BARBARA SMITH AND R. A. DUNBAR Department of Pathology, St. Bartholomew's Hospital, London

PLATES XXVTI AND XXVIII

HISTOCHEMICAL methods for specific lipids are scanty, and their inter- pretation is controversial. It is known that if brain is stored in formalin, phospholipids, particularly phosphatidyl serine and phosphatidyl ethanolamine are lost early from the tissue (Weil, 1929; Brante, 1949). The latter author showed that after about a year in fixative the phos- phatidyl choline and sphingomyelin also begin to fall, so that after some years there may be very little phospholipid left. According to Heslinga and Deierkauf (1962) the amounts of cholesterol, cerebrosides, sulphatides, phospho-inositides and sphingomyelin are unaffected by storage in unbuffered formalin for up to 24 yr. Phosphatidyl choline, phosphatidyl ethanolamine and phosphatidyl serine are broken down during prolonged storage. It follows therefore that material fixed for a long period in formalin should have lost much of its stainable phos- pholipid, and methods for lipids that are negative in this material, but positive in material fixed for only a short time, can be considered specific for phospholipids, as the other myelin constituents are the same in both cases.

MATERIALS AND METHODS

Ten human spinal cords that had been stored in unneutralised formol-saline for periods between 10 and 15 yr were compared with 2 that had been fixed for 6 8 mth and 2 fixed for 2-3 wk. There was no significance in the staining properties of the last 2 groups, and they will be referred to as short-fixed. The pH of the formalin in which the long-fixed cords were stored was between pH 4.0 andpH 4.7. The staining methods used were sudan black B in 70 per cent. alcohol, periodic acid-Schiff (Culling, 1963, p. 229), performic acid-Schiff (Pearse, 1951), lux01 fast blue (Kluver and Barrera, 1953), Okamoto's mercury diphenylcarbazone method (Pearse, 1960, p. 852) and the nile blue method (Cain, 1947).

Sections from short-fixed material were blocked by bromination (Pearse, 1960, p. 847) and deamination (Pearse, 1960, p. 800) before staining by the methods given above, and some sections were methylated (Pearse, 1960, p. 801) and then stained with lux01 fast blue. Serial sections from the same material were extracted with cold acetone, ether at 37" C and methanol-chloroform (1 :1) at 60" C, all for 1 hr and then stained with sudan black B. These sections were compared with unextracted sections and also with unfixed cryostat sections of spinal cord treated in the same way. Portions of long- and short-fixed spinal cord were washed overnight, homogenised in an Ultra Turrax and extracted with methanol-chloroform at 37" C for 1 hr. The amount of iodine with which the extract would combine was measured with Dam's reagent. In each case a long- and short-fixed cord were processed together and the results compared.

3. PATH. BACT.-VOL. 91 (1966) 117 H 2

118 BARBARA SMITH AND R. A. DLINBAR

Long-fixed

RESULTS The difference between the staining reactions of myelin in the long-

and short-fixed cords is shown in the table and in figs. 1-3, together with the effects of deamination and bromination on the short-fixed material. Methylation increases the lux01 fast blue reaction enormously.

With unfixed sections exposed to lipid solvents and stained with sudan black B, no stainable lipid remains after methanol-chloroform extraction, only a trace after warm ether and a markedly reduced amount after cold acetone (fig. 4). After increasing periods of formalin fixation of the same cord from 5 to 30 days, more and more stainable

Short-fixed I Short-foted with prior with prior

bromination 1 deamination

TABLE

The staining reactions of long- and short-fixed spinal cords

Method S hort-fixed I

~~

Sudan black B . . I Periodic acid-Schiff . . - Performic acid-Schiff . ‘ f Nile blue . . . , Blue Okamotoreaction . . 1- Lux01 fast blue . . ‘ f I 1- I - -

lipid remains after extraction (fig. 5). Even after 5 days’ fixation methanol-chloroform does not remove all the sudanophilic material and after 30 days considerable amounts remain. Cold acetone, particularly, becomes less and less effective in removing lipid and after 30 days the extracted section can only just be distinguished from the normal.

Extracts of 7 of the long-fixed cords took up 20 per cent. less iodine than the short-fixed cords.

DISCUSSION Baker (1944-45) pointed out that part of the action of formalin

on lipids was one of true fixation and that much less lipid was extract- able by lipid solvents from fixed than from fresh material in vitro. This is demonstrated particularly well by the extraction and staining of sections and shows that extraction methods are not valid on sections of fixed material. Even immersion in hot methanol-chloroform overnight does not extract all the sudanophilic material. For this reason it was decided to compare short- and long-fixed material, rather than use fresh material as a control.

There is no difference between the long- and short-fixed material in their staining intensity with sudan black B. This is to be expected., as a fat-soluble dye will stain as long as any lipid is left. The results

M YELIN STAINS I19

of the Schiff reactions are difficult to assess because formalin-fixed myelin is Schiff-positive without any prior oxidation. This is not true of unfixed myelin, and fixed material is rendered negative by pre- treatment with 5 per cent. aqueous phenylhydrazine. Wolman and Grew (1952) suggested that formaldehyde combines with the ethylene groups of the unsaturated fatty acids producing carbonyl groups which would be Schiff-positive. Prior treatment of normal material with periodic acid increases the colour very considerably, owing to oxidation of glycol groups in the cerebrosides. As expected, there is no change in this intensity after long fixation. There is also some increase in the colour developed if performic acid is used as the oxidiser. Lillie (1952) has suggested that performic acid oxidises ethylene groups to aldehydes. If Wolman and Greco are right, this presumably involves the ethylene groups not already broken by the formaldehyde. The loss of staining in the long-fixed material could be due either to the reduction of the number of double-bonds present by the continued action of formalin or to the loss of unsaturated compounds into the fixative. The latter is more likely, as the long-fixed sections stained by performic acid- Schiff are paler than short-fixed sections stained with Schiff‘s reagent without any prior oxidation.

Okamoto’s method is dependent on attaching mercury to the lipid molecule and staining the mercury with diphenylcarbazone. It is not known for certain where the mercury molecule is attached, but again a likely site is the ethylene linkage. This is made more probable by the fact that sections pretreated with bromine, which attaches itself to the carbon double-bond, cannot be stained. The nile blue method is said by Cain (1947) to stain neutral lipids red and fatty acids and phosphatides, particularly lecithin, blue. In the short-fixed material the myelin is dark blue, without any red in it, after the sections have been mounted about an hour. The long-fixed myelin is very definitely a reddish-purple. This finding would be compatible with the loss of phosphatides from the sections. The red-staining material is probably present in the short-fixed material, but is obscured by the very stronz blue.

The loss of staining in the long-fixed material is more marked with luxol fast blue than any other method. In the short-fixed material, the colour is unaffected by prior bromination, and reduced by prior deamination: it is also greatly increased by methylation, which blocks the carboxyl groups and makes more amino groups available to com- bine with the dye. Copper phthalocyanin itself is insoluble and is rendered soluble by its attached radicles which are also responsible for its staining affinities. As far as we know, in the case of luxol fast blue, these radicles are sulphonyl groups and an unknown base. Lux01 fast blue behaves like an acid dye and is in many ways similar to eosin. Ischaemic or anoxic neurones stain strongly with both eosin and lwol fast blue, probably owing to the fall in pH of the cell. This reduces the number of carboxyl groups and increases the number of

120 BARBARA SMITH A N D R. A . DUNBAR

amino groups available for dye-binding. Pearse (1960, p. 318) has suggested that it stains lipids only when dissolved in a solvent that can penetrate them. When luxol fast blue is dissolved in water instead of alcohol it does colour the myelin, but is removed so easily by lithium carbonate that it seems likely that it has not penetrated the tissue. Using spot tests Pearse (1955) found that lecithin, sphingomyelin and phosphoryl choline were positive, but rather surprisingly cephalin was negative.

The distribution of saturated and unsaturated fatty acids among the various lipids is not known exactly, but the phosphatides certainly contain a far larger proportion of unsaturated fatty acids than do cerebrosides, and in the normal spinal cord the cholesterol is not esterified. Stains for unsaturated fatty acids will therefore colour mainly, but not exclusively, phosphatides. It is known that long-fixed brains contain little or no phospholipid (Brante, 1949) and the fall in the iodine value of the cord homogenates after long fixation suggests that some of the unsaturated fatty acids have been lost.

i t is suggested that the performic acid-Schiff reaction and the Okamoto reaction for phospholipids are mainly if not entirely dependent on the presence of unsaturated fatty acids. They are therefore specific for phospholipid only when no other compounds containing unsaturated fatty acids are present, which is not true of myelin. Part of the blue colour given by nile blue is due to phospholipid and part to fatty acid, so it cannot be used as a specific stain for either. The periodic acid- Schiff reaction stains hexose-containing lipids, but it is not useful histochemically in formalin-fixed myelin because this is Schiff-positive without prior oxidation. Luxol fast blue in alcohol remains the only stain studied that is apparently specific for phospholipids; possibly they are the only compounds that contain enough available basic groups to react with it.

SUMMARY Spinal cords fixed in formalin for 10-15 yr lose most of their

phospholipid. Sudan black 3 and the periodic acid-Schiff reaction stain long- and

short-fixed material equally well. The performic acid-Schiff reaction, the Okamoto reaction for phos-

pholipid and the blue of the nile blue reaction are reduced in the long- fixed myelin and the luxol fast blue reaction is almost absent.

It is suggested that the performic acid-Schiff and the Okamoto reaction stain unsaturated fatty acids, which occur predominantly but not exclusively in phospholipids.

Luxol fast blue may react with the amino groups of the phos- phatides and is therefore, in myelin, relatively specific for phospholipids.

We are grateful to Mr P. Crocker for the photographs. The long-fixed cords were kindly given us by Dr D. R. Oppenheimer of the Radcliffe Infirmary, Oxford, and Dr Sabina Strich of the Institute of Psychiatry.

SMITH AND DUNBAR

MYELIN STAINS

PLATE XXVII

FIG. 1.-Spinal cord fixed in formalin for (a) 3 wk and (6) 14 yr. Lux01 fast blue.

FIG. 2.-As fig. 1 . Performic acid-Schiff.

FIG. 3.-As fig. 1. Okamoto’s method.

SMITH AND DUNBAR PLATE XXVIII

MYELIN STAINS

FIG. 4.-Serial fresh cryostat sections of spinal cord stained with sudan black B. (a) Unextracted. (b) Extracted in cold acetone for one hour. (c) Extracted in ether at 37" C for 1 hr. (d) Extracted in methanol-chloroform at 60" C for 1 hr.

FIG. %-As fig. 4, but fixed in formalin for 7 mth.

MYELIN STAINS 121

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