A METHOD OF STAINING THE MEDULLATED FIBRES “ E K BLOC”; AND A MODIFICATION OF THE MARCHI METHOD.

By DAVID ORR, M.B., C.M. (Edin.), Pathologist, Coicnty Asylzim, Prestwich.

(PLATE XVI.)

AT the March meeting of the Manchester Pathological Society I read a iiote of a new method of staining the medullated fibres of the brain and spinal cord, the paper being illustrated by a series of lantern slides. At that time a short extract of the paper appeared in the Society’s Transactions.‘

Further experience of the method, and the introduction of some slight but important modifications, have enabled me to obtain much better results, and the pictures obtained after a more extended trial have given me confidence to publish the details of the method as I now use it.

Although the methods of staining the Inyeline sheath are many and varied, there are few by means of which one can obtain accurate and reliable histological appearances. Some fail to give a sufficiently black reaction, others leave the very fine fibres in the grey matter of the brain unstained ; in fact, out of the many methods in use a t the present time, three alone seem worthy of recommendation, namely, Weigert’s, Heller’s, and Azoulay’s.

I n the first-as most are aware-the staining reagent used is hcematoxylin ; in the other two osmic acid is employed. Since Weigert published an account of his method in 1884 i t has been universally used in the tracing of lesions in the spinal cord and brain, yielding admirable results ; but for the study of the fine fibres of the brain and the neurokeratin network of peripheral nerves, osmic acid is, in my opinion, infinitely preferable to hiematoxylin.

On comparing Heller’s and Azoulay’s methods, one finds that the former yields sharper and more constant results, but during the process of differentiation subsequent to the reduction of the osmic acid by pyrogallic acid, in the Heller method, it occasionally happens that the finest medullated fibres fail to retain the stain, a result which might lead one to erroneously suspect their absence.

l Brit. Med. Joum., London, March 25, 1899.

388 DAVID ORR.

It was this slight defect in the sections prepared according to Heller’s formula which led me to try to devise a method which could be relied upon to give constant results, osmic acid being chosen as the reagent on account of its giving the blackest reaction with myeline.

Hitherto thin sections of Miiller- or formalin-hardened tissues have been treated with osmic acid, the black reaction then being obtained by reduction of the acid to metallic osmium upon the niyeline sheaths and intervening tissues, these latter yielding up the stain during the process of differentiation with potassium, permanganate, and oxalic acid.

Certainly, attempts have been made to bring about the penetration of formalin-hardened and fresh blocks of nervous tissue with osmic acid, but these have failed on account of the tendency of osmic acid to become reduced around the periphery of the block of tissue, and thus to prevent the central parts from taking part in the reaction. In this way a condition of “ case-hardening ” is brought about, the peripheral parts alone being blackened, the centre giving an absolutely negative result. It is evident, then, that some substance must be introduced along with the osmic acid which will retard its reduction, and a t the same time act as a vehicle to carry it into the very centre of the piece of tissue.

With the view of finding such a substance as would further the penetration and retard the reduction of the osmic acid, various experi- ments were undertaken, all with the object of obtaining a black reaction for the medullatecl fibres in formalin-hardened blocks of nervous tissue.

Combinations of osiiiic acid with ammonia, nitric acid, peroxide of hydrogen, and formalin were tried, the inevitable result being ‘( case-hardening ” a t the margin-where the fibres were sometimes well stained-and a white centre free from any reaction whatsoever.

Evidently, then, the above substances possessed no power either of aiding penetration or of retarding reduction of the acid, and not until a piece of formalin-hardened cord was immersed in Hermann’s solution was the difficulty overcome. ’ Hermann’s solution, which consists of platinic-chloride, osmic, and acetic acids, seemed to be able to penetrate formalin-hardened tissues with ease, a fairly good reaction being obtainable in three days ; a t the end of which time one could still readily detect the characteristic odour of osmic acid.in the mixture.

From this result one naturally argued that acetic acid played a very important rale in the penetrating power possessed by Hermann’s mixture, and the result obtained from the immersion of a formalin- hardened piece of cord in a mixture of osmic and acetic acid amply confirmed one’s surmises. The piece of tissue in three or four days was found to be thoroughly blackened throughout, and there existed not the slightest trace of (‘ case-hardening.”

ST.4INING MEDULLATED N E R V E FIBRES ‘‘ EN BLOC.” 389

By using pieces of tissue of various thicknesses it was found that very thin pieces were blackened throughout in twenty-four hours, and that naturally, as the thickness of the piece of tissue was increased, longer time was required to ;~llow of thorough and equal penetration, until a point was reached at which the process stopped, owing to the osmic acid in the solution becoming completely reduced by the formalin in the tissues.

It seems highly probable that formalin owes its reducing properties to the free formic acid, which is always unavoidably present in all preparations of commercial formalin. This fact is emphasised by Kronthal’ in a recent paper, in which he describes a new method of obtaining a black reaction for nerve cells by first introducing lead formiate into the fresh blocks of tissue, and the subsequent reduction of the lend by immersing the tissue in equal parts of sulphuretted hydrogen water (freshly prepared) and 10 per cent. formalin.

According to this author, the free formic acid in the formalin used, if not the actual reducing agent, seems to aid reduction very considerably.

Having obtained the knowledge that formalin-hardened tissue could be penetrated and blackened by the acetic-osmic acid mixture, several pieces of brain and cord were immersed for four days in a solution consisting of osmic acid 1 per cent. 4 c.c., and glacial acetic acid 1 C.C. On the fourth day the solution was darkened, and the tissues being penetrated further addition of osmic acid seemed unnecessary.

The thickness of the pieces of tissue did not exceed & in., and in every case penetration was complete and equal throughout. After embedding in pmtffin, sections were cut; and having been freed from paraffin were differ- entiated in potassium permanganate 2 per cent., and then transferred to oxalic acid 1 per cent., as in Heller’s method.

In a certain number of instances it was found that the differentiation process went too far, evidently on account of the strength of the potassium permanganate, which was then reduced to $ or <z per cent., rendering the process much slower and avoiding over-differentiation.

Some very good preparations were obtained of the neurokeratin network of peripheral nerves, its structure coming out with great distinctness, although the nerves remained in the solution for twenty-four hours only. Here one had to be careful to use very dilute potassium permanganate solution, and that only for a short space of time.

Not being entirely satisfied with the reaction obtained for the very fine fibres of the cerebral cortex, fresh nervous tissue was immersed in the mixture, with the result that one found the exceedingly fine fibres of the subpial layer of the cerebral cortex brought out with much greater certainty and distinctness. Moreover, this modification makes the method much more rapid, and for some months I have entirely discarded formalin-hardened pieces of brain, being convinced that the staining of fresh pieces of brain is much to be preferred.

’ h’eurol. Ccnlralbl., Leipzig, 1899, No. 6.

390 DAVID ORR.

Glacial acetic acid was added to the osmic acid in the case of formalin-hardened blocks of tissue, but I find that acetic acid in 1 per cent. solution answers equally well, and has not the same tendency to damage fresh nervous structures.

A small piece of fresh tissue from the cerebral cortex or cord, not exceed- ing $ in. in thickness, is placed in osmic acid 2 per cent. 8 c.c., added to which is acetic acid 1 per cent. 2 c.c.; the osmic acid being increased in strength to 2 per cent. in order to hasten fixation, and because it was found that the stronger acid intensified the black reaction. Should the mixture be darkened at the end of twenty-four hours then it ought to be renewed. The tissue is thoroughly permeated with the osmic acid in forty-eight houm, and is now placed in formalin 10 per cent. for three days, in order to complete reduc- tion and hardening. At the present moment I would recommend paraffin embedding for brain tissue and celloidin for cord.

As before, the sections must be passed through permanganate and oxalic acid until differentiation is complete. I n a few instances, instead of placing the osmic acid fixed blocks of tissue in formalin, they were immersed for three days in Weigert’s chrome-alum-copper fluid, to which had been added a small percentage of formalin. This modification seemed to yield no blacker reaction, and did not give the tissue the excellent cutting consistence which formalin-hardened tissues possess.

I have had an opportunity of studying numerous sections from the brains of the insane, and have obtained excellent and constant results with the method, its chief advantage being the distinctness and certainty with which it brings out the fine medullated fibres coursing in the grey matter. In the case of portions of cord treated by the above method some excellent results have been obtained, the lesion found in the posterior columns in a case of ataxic general paralysis being very well demonstrated, as seen from the accompanying illus- tration.

MODIFICATION OF MARCHI’S METHOD.

It is then embedded in paraffin or celloidin.

Being satisfied that acetic acid greatly increased the penetrating power of osmic acid, one was tempted to see with what success degenerated fibres could be demonstrated in bichromate-hardened nervous structures, for admirably adapted though Marchi’s fluid may be for the study of degeneration in the central and peripheral nervous system, yet its well-known limited power of penetration often leads one to suspect that the appearances shown by it do not represent in their entirety the amount of pathological change present.

Briefly Marchi’s method is as follows :-Small pieces of cord or brain are fixed in Miiller’s fluid for from fourteen days to one month. They are then placed in a mixture of Miiller’s fluid and osmic acid 1 per cent. in equal proportion for ten days. They are then washed in running water for twenty- four hours, subsequently being hardened in alcohol, embedded in celloidin, cut and mounted.

S T A I N I N G M E D U L L A T E D NERVE FIBRES “ E N BLOC.” 391

The normal myeline sheaths appear yellow, the degenerated ones black, or they may only be represented by black niyeline droplets. The piece of tissue must not he thicker than 2 mm., and one must carefully renew the fluid should the smell of osmic acid disappear.

Frequent agitation of the fluid is also recommended, in order to facilitate its equal penetration. Knowing that acetic acid added to osmic acid greatly increased its penetrating property, I immersed bichromate-hardened pieces of brain and cord in the acetic-osmic acid mixture in order to see if a reaction for degenerated fibres could be obtained.

The pieces of brain tissue varied in thickness from one-eighth to a quarter- inch, and were selected from the brains of the insane ; the pieces of cord being taken from cases of general paralysis. I n order to check the results, pieces of tissue from the same cases were placed in Marchi’s fluid. In both cases the tissues were allowed to lie in their respective fluids for ten days.

A careful comparison of the two results obtained showed con- clusively that the addition of acetic acid was evidently of great importance. Microscopically, one found that the Marchi fluid brought out very few fibres indeed in the brain, and in some instances the centre of the piece of tissue failed to show any reaction whatsoever. On the other hand, in the case of the pieces of brain placed in the acetic-osmic acid mixture, one was really surprised to find so many of the medullated fibres affected to a greater or less extent by degeneration. I n every instance the block of tissue was thoroughly penetrated, the centre showing the Marchi reaction equally as well as the periphery. The pieces of tissue were selected from cases of acute mania and melancholia, chronic insanity, secondary dementia, and general paralysis ; and in all the types of insanity, except in one or two cases of acute melancholia of recent origin, one obtained the char- acteristic reaction for degenerated myeline in a considerable number of the medullated fibres.

As to the frequency with which one meets with such degenerative processes in the brains of the insane I am not as yet in a position to say, as one would prefer to examine many more cases before recording any results obtained from the research of so wide and difficult a subject.

For some time past, in the initial stages of fixation and hardening, I have been using a mixture of 2 per cent. of bichromate and formalin ; the proportion of formalin not exceeding 5 per cent. of the mixture. Nervous tissues are placed in this solution for twenty-four hours, and thereafter preserved in 2 per cent. of bichromate alone. The addition of formalin hastens fixation, and if the bichromate is changed occasionally any formaliii left in the tissues becomes absolutely destroyed. Should any formalin be left, then a mixed result is obtained, isolated degenerated fibres being . seen a t the periphery of the section, while in the centre normal fibres were partly

392 DAVID ORR.

or entirely stained. Having selected the tissue to be stained, a t the end of ten days penetration is completed, and the tissue, without washing in water, may now be embedded in paraffin or celloidin. The paraffin process seems to me to be preferable, as one can thereby obtain thinner sections-a matter of great iniportance if counterstain- ing of the axis-cylinder ifi desired.

The axis cylinder stain which I have used is Van Gieson’tl, according to the formula given in Pollack‘s recent book of methods.’

With this stain the axis cylinders are brought out red, the normal sheaths coming out in yellow, and thus a fairly good contrast is obtained with the blackened morbid fibres. Pollack fails to state the length of time which one ought to allow for obtaining clear pictures of the axis cylinders. Sections of 1 p require from ten to fifteen minutes in the acid-fuchsine and picric-acid mixture, after having been previously stained in Delafield‘s hzmatoxylin for three minutes.

I n a recent number of the Rev. neurol., reference is made to a paper by Bouch,:L in which he advocates the addition of sodium iodate to osmic acid, claiming that by this addition penetration is greatly facilitated. I have tried the addition of both sodium and potassium iodate in five cases, and as yet have not found either of any advantage ; in fact, it seems doubtful if Bouch’s modification possesses any advantage over the Marchi mixture.

Besides the reaction for degenerated myeline, which is always brought out, the axis cylinders seem to stain under certain conditions which are to me a t present inexplicable. Recently, while examining an area of atrophic softening, removed from the baeal ganglia of a case of chronic insanity, I came upon a morbid change in the axis cylinder, an illustration of which accompanies this paper.

Scattered throughout the degenerated area-which was composed of morbid medullated fibres, myeline droplets, pigment, and broken- down nerve cells-there were numerous axis cylinders stained jet black, and entirely in some instances denuded of niyeline covering. I have never met with this appearance before or since, but to me it is uuggestive of fatty change, and in the accompanying illustration one of those axis cylinders is shown, in which a large droplet s e e m to have formed in the course of the fibre. I n thiu instance the myeline covering is unstained, and the illustration shows how varicosity of the medullary sheath may be caused sometimes-though not always, as is held by some writers-by local swellings upon the axis cylinder.

Before concluding, I should like to take this opportunity of thanking Dr. W. F. Robertson for many valuable hints while con- ducting my earlier experiments in the laboratory of the Scottish asylums.

Die “Farbetechnik des Nervensystems,” Berlin, 1898, Aufl. 2. 2Rev. ? m w o l . , Paris, 1899, No. 2.

JOCRSAI, OF PATHOI.OGY.-Yoi.. V l . PLATE XVI.

DESCRIPTIOK OF PLATE XVI.

FIG. 1.--To show the neurokeratin network of n peripheral nerve. Zeiss, Obj. A,

FIG, 2.-To show the fine niedullated fibres of the outermost layer of the cortex.

oc. 3.

Zeiss, Obj. A, Oc. 3.

FIG. 3.-To show the lesion of the posterior columns of the cord in a case of ataxic general paralysis. Zeiss, Obj. A', Oc. 3.

FIG. 4.-To show the degenerated tracts brought out by the modified Marchi'a fluid. Zeiss, Obj. A*, Oc. 3.

FIG. 5.-To show the blackening of the morbid axis-cylinder with varicosity. Zeiss, Apoch. Obj. 2 mm. Apert. 1'30, Compens. Oc. 8.