337
Studies of the Impregnation of Nervous Tissue Elements
II. The Nature of the Compounds Responsible for the Impregnation of
Axons; Practical Considerations
By M. WOLMAN
{From the Department of Pathology, The Hebrew University, Hadassah Medical School,
Jerusalem, Israel)
SUMMARY
Axoplasm and axolemma differ in their chemical and physical characteristics. The
impregnation of axons is mainly due to a compound which is soluble in hot organic
solvents and which appears to be situated in the axoplasm.
Ethylenic linkages and hydroxyl groups play no role in the impregnation of axons.
Sulphydryl and carbonyl groups appear to be partly responsible. These groups are
situated mainly in the axoplasm, although sulphydryls are present also in the axolemma. The impregnation of axolemma depends to a great extent on chemical groups
which could not be identified.
The fact that solutions of Ag diammine are more suitable for impregnating axons
than those of AgNO 3 might be due to one of the following factors: (i) that the reducing
groups of the axons are more easily oxidized at high pH levels; (2) that the complex
diammine ion might exert a higher oxidizing activity when reacting with some tissue
molecules; (3) that the rate of reaction of the reducing groups of the tissue with the
Ag diammine ion might be faster than that with the silver ion.
The Bielschowsky procedure may be carried out successfully on sections of tissue
embedded either in paraffin wax or carbowax. With the former procedure care should
be taken to minimize contact with mixtures of hot solvents. A further intensification
of impregnation of paraffin sections is obtained by treating sections for 24 hours in
4% formaldehyde prior to the Bielschowsky procedure.
INTRODUCTION
I
N the previous article (Wolman, 1955) evidence was given that the metallic
impregnation of axons depends on the reducing capacity of their constituents. Such a capacity depends on the presence of molecular groups having
suitable redox potentials.
The present paper deals mainly with the nature of these groups and with
the solubility characteristics of their parent molecules. Some inconsistencies,
real and apparent, between current opinions and the data emerging from these
experiments are also discussed. Finally, some practical aspects of the study for
the staining of axons are indicated.
The techniques used in this study are basically similar to those reported in
the previous article.
SOLUBILITY CHARACTERISTICS OF THE COMPOUNDS RESPONSIBLE FOR THE
IMPREGNATION
In formalin-fixed, carbowax-embedded sections kept for 24 hours in absolute ethanol, ether, or ethanol-ether mixture (1:1), axons could be stained
[Quarterly Journal of Microscopical Science, Vol. 96, part 3, pp. 337-341, 1955.]
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Wolman—Studies of the Impregnation of
by the Bielschowsky procedure as well as by Cajal's gold-sublimate method.
In some cases (e.g. after absolute alcohol extraction) the impregnation was
stronger and sharper than in the controls, probably because of the defatting
of the sections which allowed better penetration (cf. Liesegang, 1911; Foot,
I932)Extractions at room temperature with pyridine, ethanol-xylol (1:1), methanol-chloroform (2:1), ethanol-chloroform (1:1), or ethanol (24 hours) followed by xylol (24 hours) resulted in a somewhat decreased impregnation.
This decrease was mainly at the expense of the interior of the axons (axoplasm), while the axolemma was much less affected. Extraction by hot
methanol-chloroform and hot pyridine for 3-4 hours at 560 markedly reduced
the intensity of the impregnation of axons, and especially of the axoplasm.
The staining of paraffin-embedded sections by Bielschowsky's procedure is
known to give erratic, often highly unsatisfactory results. It has been found
that results could be greatly improved if: (a) the blocks were passed directly
from the clearing fluid into paraffin without a xylol-paraffin or benzol-paraffin
hot bath; (b) the block was sufficiently thin to require only a short immersion
in the clearing agent-paraffin mixture at a high temperature; and (c) the block
of tissue was dried on filter paper before being put into the paraffin oven.
Conclusion
The impregnation of axons is due in part to a lipid constituent which is
soluble in hot solvents and is located mainly in the axoplasm.
ATTEMPTS TO CHARACTERIZE THE ACTIVE GROUPS RESPONSIBLE FOR THE
IMPREGNATION OF AXONS
The effect of the following reactions on the subsequent impregnation of
axons by the Bielschowsky procedure was studied.
(a) Effect of reagents which react mainly with ethylenic linkages. Immersion
of the sections in 1% OsO4 for 1-24 hours resulted in a marked staining of
the myelin sheaths, but not of the axons. This treatment did not interfere with
the subsequent impregnation of axons.
Bromination for 1-4 hours in bromine vapours or in a 10% solution of
bromine in CC14 (Lillie, 1954) markedly reduced the impregnation of myelin,
but did not affect the impregnation of axons to the same extent.
Staining by the performic acid-Schiff reaction (Lillie, 1952) did not render
axons visible.
(b) Effect of reagents which react mainly with hydroxylic groups. Although
acetylation by acetic-anhydride-pyridine mixture for 24 hours at room temperature, and by pure acetic anhydride under the same conditions resulted in
a decreased impregnation of axons, the failure of treatment by NaOH, both
in aqueous (McManus and Cason, 1950) and in alcoholic solutions, to reverse
this effect, proved that the decreased impregnation was due to the dissolving
capacity of the reagents. Benzoylation by a 10% solution of benzoyl-chloride
in dry pyridine for 24 hours (Pearse, 1954) did not influence the impregnation
Nervous Tissue Elements (II)
33^
of axons either, except for the dissolving effect of pyridine. Similar results
were obtained with phenyl-isocyanate. Sections which were dried in the air
and then kept for 3 days in a 0-5% solution of the reagent in xylol, followed
by thorough washing in xylol, and then by impregnation, did not differ
markedly from sections which were kept in xylol only and then impregnated.
Diazotization of phenolic rings by tetrazotized orthodianisidine following
the technique used by Seligman (cf. Ashbel and Seligman, 1949) or by diazotized sulphanilic acid (Lison, 1953, p. 417) did not affect the impregnation
of axons.
(c) Effect of reagents which react mainly with sulphydryl groups. Treatment
by o-i M iodoacetic acid for 24 hours at room temperature and by Lugol's
solution for 2 hours greatly reduced the intensity of impregnation of the
axons. This reduction was at the expense of both axolemma and axoplasm.
It has already been noted that treatment by ammonium sulphide increased
the intensity of the impregnation of axons.
Attempts at staining of axons by Seligman's DDD reagent resulted in a very
faint or no staining of axons. (The reagent was kindly supplied by Dr. A. M.
Seligman of the Beth Israel Hospital, Boston, Mass.) Tetrazolium salts at
high pH levels stained axons (see Wolman, 1955).
(d) Effect of reagents which react mainly with carbonylic groups. Blocking of
carbonyl groups by aniline chloride and phenylhydrazine (Lillie, 1952),
hydroxylamine and sulphanilic acid (Pearse, 1954) decreased the intensity of
impregnation of the axons. The decrease appeared to involve only the axoplasm. Schiff's leucofuchsin did not stain axons.
(e) Effect of oxidizing agents. Oxidation by periodic acid (1 % for 2 hours),
by chromic acid (4% CrO 3 for 1 hour), and by potassium dichromate (3% for
1 and 5 days), followed by thorough rinsing and then by the Bielschowsky
procedure, resulted in some increase in the impregnation of axons.
(/) Effect of reducing agents. Treatment of the sections by 10% formalin for
24 hours resulted in an intensified impregnation of axons. This intensification
was especially apparent in sections embedded in paraffin. A similar but less
striking effect was obtained by immersing the slides for 5-10 minutes in a 5%
solution of (NH4)2S.
DISCUSSION
(a) Characteristics of the compound responsible for the impregnation. The
experiments reported indicate that the reducing groups responsible for the
reduction of silver ions are not ethylenic links or hydroxyls. Two types of
groupings, viz. carbonyls and sulphydryls seem to be important for the
impregnation of axons. Romanes (1950), who conducted a limited number of
tests along the same lines, could not find evidence that sulphydryl groups
were responsible for the impregnation of axons, and believed that aldehydic
groups were of major importance.
The results of the extractions by lipid solvents and of the blocking of some
reactive groups yielded further information. The axolemma and the axoplasm
34°
Wolman—Studies of the Impregnation of
appear to differ in their chemical constitution. The axoplasm contains a
compound which can be dissolved by hot lipid solvents. Its staining in carbowax embedded and in frozen sections appears to be due largely to its content of carbonyl and sulphydryl groups. The fact that techniques which are
considered specific for these groups do not visualize axons satisfactorily might
be due to their low sensitivity.
The impregnation of the axolemma, on the other hand, appears to depend
only in part on the presence of sulphydryl groups. Other chemical groups
which cause the reduction of silver ion in the axolemma remain unknown.
Periodic acid oxidation (Lhotka, Myhre, and Combs, 1953; Lhotka and
Myhre, 1953) and chromate oxidation (Golgi's method, cf. Kallins, 1926) are
known to increase the impregnability of tissue constituents. The means by
which these oxidative agents increase the intensity of axon impregnations can
be deduced from known facts. Chromate and periodate oxidation may result
in the formation of new carbonyls, and this effect may compensate for a
possible loss of other reducing groups which have been oxidized.
The effect of the reducing agents tested might be due to reduction of labile
oxidation-reduction systems (such as sulphydryl or polyphenol groups), or in
the case of formaldehyde to the formation of reducing groups by the effect of
formaldehyde on some lipids (cf. Wolman and Greco, 1952).
(b) The oxidizing activity of silver diammine solutions. The factors regulating
the oxidizing potential of silver diammine solutions have been discussed by
Nauta and Gygax (1951) and by Garven and Gairns (1952). These authors
suggested that the oxidizing potential of the solutions is due to dissociation of
the complex Ag-diammine ion into Ag+ and ammonium ions. The oxidizing
potential was supposed to depend on the minimal concentrations of silver ions
present in the solution. In support of their thesis Nauta and Gygax reported
that silver diammine solutions which contain more Na+ ions (up to a given
optimum) impregnate sections more strongly than pure Ag-diammine solutions (prepared by adding NH 4 OH to AgNO3 solutions). These data could
not be substantiated, and, furthermore, the theory does not account for the
fact that Ag-diammine solutions, in which the concentration of Ag+ ions is
considerably lower, impregnate structures more strongly than AgNO3 solutions.
In the preceding paper it was implied that Ag-diammine solutions act as
stronger oxidants than AgNO3 solutions, but this assumption appears contrary
to the known data of physical chemistry, which assign a higher oxidizing
potential to AgNO3.
These difficulties cannot be explained in a final manner, although three
possibilities may be suggested.
(a) It is possible that axons are higher on the oxidation-reduction scale, that
is: can be oxidized by stronger oxidants only than myelin in the neutral and
nearly neutral pH range, while in the alkaline range they are more easily
oxidized than myelin. Such behaviour is known in hexoses which are much
more easily oxidized in an alkaline pH.
Nervous Tissue Elements (II)
341
(b) The complex diammine ion may behave abnormally when reacting with
complex tissue molecules because of participation of electrons not belonging
to the external orbit. (A suggestion made to the author by Dr. A. Katchalsky
of the Department of Physical Chemistry.)
(c) Ag-diammine, although of a lower oxidation potential than AgNO3,
might be more reactive than AgNO3 towards the reducing groups of the
axons. In other words, while both solutions have a high enough potential to
oxidize axons, the reaction-rate with Ag-diammine would be faster.
PRACTICAL APPLICATIONS OF THE STUDY
Axons can be impregnated by the standard Bielschowsky procedure in
carbowax embedded material after fixation in formalin. The advantages of
carbowax embedding over frozen sections include the ease of handling and
cutting the sections and the uniformity of results, as the whole section is
immersed and air bubbles are eliminated.
The results may be somewhat improved by extracting the sections with
alcohol for 24 hours, or with 20% pyridine in alcohol for 10-30 minutes.
Axons can be demonstrated in paraffin embedded formalin-fixed material,
provided that the tissue is not immersed in a hot clearing agent (xylol, benzol,
&c.) and the period of immersion in the mixture of alcohol-clearing agent be
kept short. The period in which paraffin replaces the clearing agent should be
as short as possible.
The impregnation is markedly improved if sections of paraffin-embedded
tissue are left for 24 hours in 10% formalin, then rinsed before the standard
Bielschowsky procedure is carried out.
This study was supported by the Robert Moses Wecksler Memorial Research Scholarship. The author is grateful to Miss H. Tryfus and Mr. Y.
Gutman, medical students, for the technical assistance rendered.
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