Technical variation isn Koelle's histochemical method for … · 2006-05-26 · Technical variation isn Koelle's histochemical method for demonstrating cholinesterase activity By

Technical variations in Koelle's histochemical method fordemonstrating cholinesterase activity

By N. T. NAIK(From the Department of Anatomy, King's College, University of Durham,

Newcastle-upon-Tyne)

With 4 plates (figs, i to 4)

SummaryKoelle's histochemical method for demonstrating cholinesterase activity and theliterature on its subsequent modifications have been reviewed. Experiments werecarried out on the effect on the cholinesterase reaction of formaldehyde fixation, coldstorage of tissues, pH of incubation solution, and progressive increase of incubationtime. A series of experiments was also carried out in testing the specificity of sub-strates and selective inhibitors used in the Koelle method. Enzyme reaction was visual-ized by the ammonium sulphide method. As a result of these experiments thefollowing technical desiderata have been established:

1. Fixation of tissues for 3 h in neutral formaldehyde solution at 40 C preserved themorphology of the tissues without appreciably affecting the histochemical results.Fixation for more than 6 h produced definitive inhibition of cholinesterases,especially AChE, in most tissues.

2. Periods of up to 24 h of cold storage before fixation had no appreciable effect on thecholinesterase reaction.

3. Incubation at pH values between 50 and 6-o produced neither significant diffusionartifacts nor loss of enzyme activity. Below pH 5 the AChE reaction was affected toa varying extent according to the tissues used.

4. B.W. 284 at a concentration of 5 X icr5 M and ethopropazine hydrochloride at aconcentration of 1 X io~4 M were found to be suitable selective inhibitors for AChEand ChE respectively.

5. Visualization of results by means of ammonium sulphide method was found to bepreferable to phase-contrast microscopy.

IntroductionT H E acetylcholine-cholinesterase system, which involves the enzymatichydrolysis of acetylcholine (ACh), forms an important part of the mechanismof nervous activity. Dale (1914), studying the pharmacological properties ofACh, suggested that an enzyme was responsible for its removal. Loewi (1921)showed that stimulation of the vagus nerve caused the formation or liberationof a substance which he called Vagusstoff. Later, Loewi and Navratil (1926 a,b) identified the 'vagus substance' as a derivative of choline, possibly acetyl-choline. They also showed that the vagus substance and ACh were destroyedby an enzyme present in aqueous extracts of the frog's heart. Thus, not onlywas the original suggestion of Dale (1914) confirmed, but the theory of neuro-humoral transmission (Langley, 1901; Elliot, 1905) was significantly extended.

Final agreement is lacking on the details of the role of cholinesterases in

[Quart. J. micr. Sci., Vol. 104, pt. 1, pp. 89-100, 1963.]

90 Naik—Koelle's method for cholinesterase

nervous activity. Some authorities even suggest that this enzyme is essentialnot only in all synaptic activities but also in nervous conduction (Nachman-sohn, 1959). In general, it is believed that ACh is the active agent of nervoustransmission and that cholinesterase, especially acetylcholinesterase (AChE),acts by removing the released agent to restore the nervous mechanism forrepeated activity (Eccles, 1957; Nachmansohn, 1959).

Before the introduction of a histochemical method the distribution ofcholinesterases was studied biochemically (Glick, 1937, 1938; Nachmansohn,1937, 1938, 1940; Mendel and associates, 1943; Augustinsson, 1948).Gomori (1948) proposed a histochemical method for the direct visualizationof cholinesterase activity in tissues with higher fatty acid esters of choline assubstrates. The method did not, however, give satisfactory results.

Koelle and Friedenwald (1949) introduced a method with acetylthio-choline as a substrate. This is hydrolysed by acetylcholinesterase (AChE) andnon-specific cholinesterase (ChE) more rapidly than is ACh itself. Themethod involved the incubation of fresh frozen sections or teased preparationsin a medium containing acetylthiocholine iodide, copper sulphate, and glycineat pH 8-o. On hydrolysis the liberated thiocholine was presumed to act withcopper salt to give a rather insoluble product, copper thiocholine. To facilitateprecipitation and to prevent diffusion the incubating medium was saturatedwith copper thiocholine before the sections were placed in it. After incuba-tion the sections were treated with ammonium sulphide solution to convert thewhite precipitate of copper thiocholine into a brown amorphous deposit ofcopper sulphide. This produced massive diffusion of the precipitate because,at pH 8-o, the products of hydrolysis diffused into adjacent regions untilsufficient acidity was built up locally (Koelle, 1950). To reduce the diffusionartifacts, Koelle (1950) lowered the pH of the incubation solution to 6-4 withphosphate buffer. He also introduced butyrylthiocholine as substrate forChE. Koelle (1951) further modified the method by incorporating sodiumsulphate in the storage and incubating solutions and by replacing the phosphatebuffer by the maleate buffer. By means of recovery tests he showed thatcholinesterases were completely precipitated at pH 6-o in the presence ofsodium sulphate. The phosphate buffer used in the original method was re-placed by maleate buffer because of the greater buffering capacity of the latterat pH 6-o and because it did not precipitate cupric or magnesium ions in theconcentrations employed. Gomori (1952), working on Koelle's thiocholinemethod, used tissues fixed in neutral cold formaldehyde solution before incu-bation and omitted the storage solution.

Couteaux (1951), Taxi (1952), and Couteaux and Taxi (1952), tryingKoelle's method on fixed and unfixed tissues, varied the pH between 7-0 and4-0 and found that pH between 5-3 and 5-6 gave the best results in tissues fixedwith formaldehyde. They also observed that the acetate buffer gave the bestresults at pH 5-3 and 5-6.

The mechanism of the thiocholine method was re-examined by Zajicek,Sylven, and Datta (1954) and by Malmgren and Sylven (1955). They observed

Naik—Koelk's method for cholinesterase 91

that during reaction, minute rod-like crystals of copper thiocholine weredeposited at the sites of cholinesterase activity initially. If the medium wassaturated with copper thiocholine, as in the Koelle method, the crystals in-creased in size. Malmgren and Sylven, therefore, considered that only theinitial deposit could be regarded as a direct result of enzyme activity and thatseveral factors other than enzyme activity must influence the amount of depositat local sites. Similarly, they studied the effect of treatment of sections withammonium sulphide after incubation. When a purified sample of copperthiocholine was placed on a coverslip and treated with dilute ammoniumsulphide, the crystals were dissolved and were replaced by an amorphousdeposit of copper sulphide. These authors considered that the process ofconversion might produce distortion of the true picture. On this suggestion,Holmstedt (19576) introduced a modification of Koelle's method. He omittedthe saturation of the incubating medium with copper thiocholine and the con-version of copper thiocholine crystals into copper sulphide, and visualizedthe results by phase-contrast microscopy. Pearse (i960) agreed that displace-ment of the original precipitate of copper thiocholine by the copper sulphidemight produce some inaccuracies, but suggested that this could be toleratedsince visualization by means of phase-contrast microscopy might produceother difficulties.

In addition to the various modifications of Koelle's thiocholine method, nowin general use, other methods are sometimes employed for demonstration ofcholinesterase activity (Nachlas and Seligman, 1949; Crevier and Belanger,1955). It should be emphasized that it is the products of enzyme activity andnot the enzymes themselves that are revealed by the present histochemicalmethods, but the thiocholine method is more physiological than proceduresbased on hydrolysis of other compounds employed in certain alternativemethods (Holmstedt and Sjoqvist, 1961).

Materials and methodsThe main aim of this investigation has been to assess critically the following

variables in the technique: (a) the duration of fixation, (b) the pH of the in-cubating solution, (c) the specificity of substrates and inhibitors, (d) visualiza-tion methods of the results, (e) length of incubation, and (/) the effect of coldstorage of tissues before fixation.

The material studied came from man and from a number of other mam-malian species. Human material was collected from 35 individuals rangingfrom late foetal stages to 81 years of age. Comparative material is representedby the following species: 62 cats, 30 rats (hooded), 30 guinea-pigs (red andblack), 10 moles (Talpa europaea), and 5 hedgehogs (Erinaceus europaeus).Human tissues were obtained during surgical operations and were fixedimmediately, or they were stored at 4° C from a few minutes to 6 h, or longerif the effect of cold storage was studied on the tissues used for the cholin-esterase reaction. Animals were killed by an overdose of chloroform or byintra-peritoneal injection of sodium pentobarbitone (nembutal).


This paper reports only the results of the technical studies: other findingsbased on the application of these methods will be reported elsewhere.

The tissues were fixed in 10% neutral formalin at 4° C adjusted to pH 7-0with sodium acetate. Fixation time ranged from 30 min to 3 h for routinestudies; in a special study of the effect of the fixative it was extended up to 72 h.Incubation was carried out with floating sections by the revised Koelle tech-nique (Koelle, 1951, 1955) with certain simplifications and modifications. Theroutine procedure was as follows:

(1) Fix small blocks of tissues or whole ganglia in 10% neutral formalin at40 C for 30 min to 3 h.

(2) Cut frozen sections at 15 /u. or 20 (j. and collect them in distilled water.(3) Wash in distilled water for 30 min. Place the sections intended for

eserine controls in 3 X icr5 M eserine sulphate solution for 30 min be-fore incubation (1-9 mg of eserine sulphate in 100 ml of distilled water).

(4) Incubate the sections for 15 min to 6 h (or longer) at 370 C and pH 5-3.

The basis of the incubation solution was prepared as follows (for the totalquantity of 10 ml of incubation solution):

1. 2-5% copper sulphate (CuSO4, 5H2O) 0-2 ml2. 3-7% glycine (amino-acetic acid) 0-2 ml3. o-i N sodium acetate 7-3 ml4. o-i N acetic acid 1-5 ml

Items 3 and 4 are constituents of the acetate buffer solution adjusted topH 5-3. For eserine controls add 0-19 mg of eserine sulphate for every 10 ml ofincubation solution.

To the above basis add freshly prepared substrate. The substrate was pre-pared as follows: 15-0 mg of acetylthiocholine iodide or 16-5 mg of butyrylthiocholine iodide was dissolved in 0-75 ml of distilled water in a centrifugetube. To this was added 0-3 ml of 2-5% copper sulphate solution; a brownprecipitate was formed. The contents of the tube were centrifuged for 10 to15 min at 2,000 r.p.m., and the clear supernatant fluid was added to the basis,of the incubation solution, bringing the total volume to io-o ml. The sub-strates were used with and without inhibitors. The different combina-tions of substrates and inhibitors and the enzymes visualized are shown intable r.

After incubation, take samples at suitable intervals, wash in distilled waterfor 1 min, transfer to approximately 5% ammonium sulphide solution for 30sec to 1 min, wash again in distilled water, mount on slides, dry and mountthrough xylene in neutral Canada balsam. If required, counterstain somesections before dehydration.

The tissues intended for neurohistological studies were fixed in 10% com-mercial formalin solution for 3 to 10 days or longer. Nerve-fibres werestained in frozen sections, 15 to 20 /n thick, by a simplified Bielschowsky-Gros silver impregnation method (Cauna, 1959).

Naik—Koelle's method for cholinesterase 93

TABLE I

Demonstration of cholinesterase activity by the use of substrates and theircombinations with inhibitors

Substrates

(a) acetylthiocholineiodide

(6) butyrylthiocholineiodide

(c) acetylthiocholineiodide or butyrylthio-choline iodide

{d) acetylthiocholineiodide

(e) acetylthiocholineiodide

J) acetylthiocholineiodide

(g) butyrylthiocholineiodide

(h) butyrylthiocholineiodide

Inhibitors

eserine sulphate 3 X icr5 M (1-9 mgin 100 ml)

ethopropazine hydrochloride 1Xio"4 M (3'I2 mg in 100 ml)

B. W. 284 5 X icr5 M (28-0 mg in1000 ml)

B. W. 284+ethopropazine hydro-chloride

B. W. 284

ethopropazine hydrochloride

Enzyme visualized

AChEChEChE

AChE

ChE

ChE

AChE (after pro-longed incubation)

Observations and discussionMost histochemical studies of cholinesterase activity reported in the litera-

ture are based on various modifications of Koelle's technique. The resultsobtained by different workers are basically similar, but in certain instancesdifferences have been reported which have been partly attributed to variationsin the techniques employed. The importance of critical application of thetechnique was emphasized at the symposium on histochemistry of cholines-terase held in Basel in i960 (Schwarzacher, 1961). With this view in mind itwas decided to study the effect of fixation, pH, cold storage, incubation time,and specific inhibitors on the reaction, and also the visualization of results bymeans of phase contrast and ammonium sulphide treatment.

The effect of formaldehyde fixation on the cholinesterase reaction

The effect of formaldehyde fixation was studied on a variety of tissues fromhuman and animal material. The tissues were fixed in 10% neutral formalinbuffered with sodium acetate at 40 C for 3, 6, 18, 24, 64, and 72 h by im-mersing the whole ganglia or thin slices of other tissues. After fixation thetissues were cut on the freezing microtome, washed for 30 min in distilledwater, and then incubated and compared with unfixed controls under similarconditions.

It was found that fixed specimens required longer incubation time to obtainthe same intensity of reaction as in unfixed specimens. The activity of ChEwas less affected by formaldehyde fixation than that of AChE. Fixation forup to 6 h did not appreciably change the pattern of histochemical distributionof the enzymes. After fixation for from 6 h to 18 h definite loss was


progressively observed in tissues of low enzyme activity. Further fixationreduced the intensity of the reaction even in sites of high enzyme activity.The main findings are illustrated in fig. i, which shows the stellate ganglion ofthe cat incubated for AChE activity, A is a section of unfixed tissue. B is asection incubated after 3 h of formalin fixation; it shows all the features of A.In both A and B three types of nerve-cells can be distinguished: heavilystained (a), moderately stained (b), and unstained or lightly stained (c). c is asection incubated after 24 h of fixation. The cells of high enzyme activityshow no noticeable change, but all the remaining cells appear cholinesterase-negative. This means that the enzyme has been inactivated in these cells byprolonged fixation.

These experiments show that fixation for 3 h preserves the structure of thetissues adequately, the specimens can be cut easily on the freezing microtome,and incubation can be carried out by the floating-section method. Thehistochemical distribution of cholinesterase activity in such tissues is similarto, and the cy tological localization is better than, that in sections incubated with-out fixation. In addition, fixed tissues are easier to handle, they are lesscytolysed by reagents, and their soluble proteins are prevented from diffusion.For this reason fixed specimens give a better morphological picture and moreaccurate localization of enzyme activity (Gomori, 1952; Pearse, i960). Thiscan be seen if A and B (fig. 1) are compared. These findings are in agreementwith a number of investigators who have studied the question by biochemicalas well as by histochemical methods (Taxi, 1952; Couteaux and Taxi, 1952;Holmstedt, 1957a; Eranko, 1959, Fukuda and Koelle, 1959; Lehrer and Orn-stein, 1959).

The effect of variation of pH on the cholinesterase reaction

It has been established biochemically that pH 8 is optimal for cholinester-ase activity (Bernheim and Bernheim, 1936; Easson and Stedman, 1936;Glick, 1937, 1938; Alles and Hawes, 1940). However, when this pH isemployed for histochemical demonstration of the enzyme, massive diffusionresults. (See, for example, the paper of Koelle and Friedenwald, 1949, whichintroduced the thiocholine method.) To reduce diffusion, most investigatorshave lowered the pH to 6 (Koelle, 1950, 1951; Gomori, 1952; Holmstedt,1957&). Others have reduced it further, between the values of 5 and 6(Couteaux and Taxi, 1952; Coupland and Holmes, 1957; Lewis, 1961). Excep-tionally the very low pH of 4-2 has been used (Snell, 1958).

The effect of pH on the cholinesterase reaction was mainly studied on thesympathetic ganglia of the cat and on human skin. The hydrogen-ion

F I G . I (plate). Photomicrographs of sections of the stellate ganglion of the cat incubated forAChE activity, showing the effect of formalin fixation, a, heavily stained nerve-cells;b, moderately stained nerve-cells; c, unstained nerve-cells. Frozen sections, 20 ft.. pH 5 3 .

A, incubated for 45 min without fixation.B, incubated for 1 h 30 min after fixation in neutral formalin for 3 h.C, incubated for 1 h 30 min after fixation in neutral formalin for 24 h.

...V > i

B

Lv.

, 100//'

FIG. I

N. T. NAIK

FIG. 2

N. T. NAIK


concentration was varied between pH 7-0 and 4-2. It was found that at pH yothe enzyme reaction was fast, and reasonably sharp localization of the coppersulphide precipitate was obtained after short periods of incubation. However,as the incubation period was progressively extended the staim diffused from theperikarya of the nerve-cells into the nuclei and surrounding tissues, and theenzyme could not be localized cytologically (fig. 2, A). At pH 6-o the reactionwas slower but the stain remained better localized (fig. 2, B). Some diffusiontook place, however, especially into the nuclei of the satellite cells after longerincubation. Further reduction of the pH to 5-0 or 5-3 did not affect the patternof enzyme distribution, and cytological localization was further improved(fig. 1, B; also fig. 3, B). Under these conditions it was possible to carry outincubation for a long period without much diffusion artifact (see fig. 4, A to c).At pH 4-0 or 4-2 the increased acidity inhibited the reaction, especially thatof AChE, in many tissues. The ganglia of the sympathetic trunk of the catincubated at pH 4-2 showed a negative reaction in nerve-cells which did infact contain AChE, and showed a positive reaction only in tissues surroundingthe nerve-cells, where ChE was mainly present (compare fig. 2, c with figs.1, B; 2, B). Sections incubated with butyryl substrate at pH 4-2 showed adistribution of ChE similar to that in sections incubated at higher pH(compare fig. 2, D with fig. 3, c). Sections incubated with acetyl substrate plusethopropazine hydrochloride as an inhibitor of ChE on the contrary remainedblank or showed very little reaction even after long periods of incubation.These experiments demonstrate that in the sympathetic ganglia of the catalmost complete inactivation of AChE and slight inactivation of ChE takesplace at pH 4-2. A similar finding was reported by Lewis (1961) in thecholinesterase of rat brain.

Sections of human digital skin incubated at pH 5-3 or higher showed astrong positive cholinesterase reaction in various sites: Meissner's corpusclesand Pacinian corpuscles showed ChE, while the adventitia of the sweat-glandsand blood-vessels contained AChE. When the skin was incubated at pH 4-2it failed to show any enzyme reaction in Meissner's corpuscles (arrows in fig.2, F; compare with arrows in fig. 2, E) or in the adventitia of the sweat-glandsor blood-vessels. But Pacinian corpuscles still showed a positive ChE reaction.

FIG. 2 (plate). Photomicrographs of sections incubated for cholinesterase activity, showingthe effect of variations in pH. Frozen sections, 15 ji.

A, stellate ganglion of the cat, incubated with acetyl thiocholine iodide plus ethopropazinehydrochloride for 1 h. pH 70.

B, stellate ganglion of the cat, incubated with acetyl thiocholine idodide plus ethopropazinefor 1 h 5 min. pH 6-o.

C, stellate ganglion of the cat, incubated with acetyl thiocholine iodide for zz h. pH 42.D, stellate ganglion of the cat incubated with butyryl thiocholine iodide for 22 h. pH 42.E, human digital skin incubated with acetyl thiocholine iodide for 1 h 20 min. pH 5-3.

Meissner's corpuscles (arrows) give a strong positive cholinesterase reaction. Male, 42 years.Frozen sections, 20 ft.

F, human digital skin incubated with acetyl thiocholine iodide for 22 h. pH 42. Meissner'scorpuscles (arrows) give negative cholinesterase reaction. (Compare with fig. 2, E.) Male, 19years. Frozen sections, 20 /i.


These experiments show that the hydrogen-ion concentration of the incuba-tion medium is an important factor. Incubation at pH 6-o or higher producesconsiderable diffusion artifact, whereas incubation at pH 4-2 or lower usuallyfails to demonstrate AChE and, in certain situations, ChE as well. Thesefindings are in general agreement with the biochemical observations ofAugustinsson (1948), who stated that the AChE of horse erythrocytes wasdestroyed at pH 4-5 and the ChE of horse serum at pH 2-0. The best cytolo-gical and histochemical results were obtained at between pH 5 and pH 6 in alltissues examined. For this reason pH 5-3 was chosen in this research forroutine purposes, although a higher pH (6-o) was also used for comparison.

The specificity of substrates and inhibitors

The substrates, acetylthiocholine iodide and butyrylthiocholine iodide, usedin Koelle's histochemical method, are rather specific for cholinesterases.Occasionally they may be hydrolysed by some simple esterases, but these canbe identified by the use of eserine controls (Easson and Stedman, 1937;Richter and Croft, 1942; Mendel and Rudney, 1943 a, b). Acetyl substrate ishydrolysed by both AChE and ChE (Koelle, 1950, 1951; Holmstedt, 1957a).Therefore, the positive reaction obtained by this substrate shows the totalcholinesterase (fig. 3, A). In order to differentiate between the two enzymesselective inhibitors are used.

Koelle (1950, 1951) recommended DFP as an inhibitor of ChE. However,it is not possible to select a concentration of this compound that gives completeinhibition of ChE without affecting AChE (Holmstedt, 1957a). Besides, DFPis inconvenient to handle and highly toxic; the prepared solutions are unstable.Instead of DFP, Holmstedt (1957 a, b) recommended Mipafox, which has ahigh specificity and is easily handled (Holmstedt and Sjoqvist, 1961). Theinhibitor sold under the name of Mipafox in this country (Light & Co.) has aslightly different composition (Holmstedt and Sjoqvist, personal communica-tion), and in our hands did not produce complete inhibition of ChE. Similarexperience is reported by Pepler and Pearse (1957) in their trials of this com-pound on the rat-brain cholinesterase. Bayliss and Todrick (1956) haverecommended the use of ethopropazine methosulphate as inhibitor of ChE.According to Pearse (i960) this compound may produce precipitation in thesubstrate solution. In the present work ethopropazine hydrochloride wasused instead of ethopropazine methosulphate. It does not affect the solutionsand its inhibitory action is reliable. Fig. 3, B shows the stellate ganglion of the

FIG. 3 (plate). Photomicrographs of sections of the stellate ganglion of the cat, fixed inneutral formalin at 40 C and incubated for cholinesterase activity at pH 53. The plate de-monstrates the specificity of substrates and selective inhibitors. Frozen sections, 15^.

A, incubated with acetylthiocholine iodide for 1 h 45 min to show the total cholinesterase.B, incubated with acetyl thiocholine iodide plus ethopropazine hydrochloride for 1 h 20 min

to show AChE.c, incubated with butyrylthiocholine for 1 h 50 min to show ChE.D, incubated with acetyl thiocholine iodide plus B.W. 284 for 1 h 20 min to show ChE.

Fie. 3

N. T. NAIK


cat incubated with acetyl substrate plus ethopropazine hydrochloride as an in-hibitor of ChE. AChE is localized mainly in certain nerve-cells and to a smalldegree in other tissue elements (cf. fig. 3, A).

As an inhibitor of AChE, B.W. 284 has been found effective (Denz, 1953;Koelle, 1955; Bayliss and Todrick, 1956; Holmstedt, 1957a). This was foundreliable in the present work. Fig. 3, D shows the stellate ganglion of the catincubated with acetyl substrate plus B.W. 284 as an inhibitor of AChE. As aresult, the distribution of ChE is shown: all nerve-cells in A and B (fig. 3) givea negative reaction, but the tissue elements surrounding the nerve-cellsare strongly positive (compare with fig. 3, c). When A, B, and c (fig. 3) arecompared, the effect of selective inhibitors is evident, B and c show eachenzyme separately whereas A shows the combined picture, namely the totalcholinesterase.

In comparison with acetyl substrate, butyryl substrate may be considered asspecific for ChE for all practical purposes and is being used as such withoutinhibitors (Koelle, 1950, 1951; Couteaux and Taxi, 1952; Holmstedt, 1957^)-The section in fig. 3, c has been incubated with butyryl substrate withoutinhibitor. When the incubation is prolonged, however, some traces of staincorresponding to the distribution of AChE can be detected.

As a result of these experiments, it can be seen that AChE can be demon-strated by means of incubation of sections with acetyl substrate plus etho-propazine hydrochloride (fig. 3, B). If ChE is not present in the tissuesconcerned, acetyl substrate will show AChE without the use of the inhibi-tor. ChE can be demonstrated either by acetyl substrate plus B.W. 284 or bybutyryl substrate with or without B.W. 284 (fig. 3, c, D). It will be noted,however, that butyryl substrate shows ChE in a shorter period of incubationand therefore should be preferred.

The specificity of ethopropazine hydrochloride and B.W. 284 as selectiveinhibitors was further confirmed by incubating tissues containing bothenzymes with acetyl substrate to which both inhibitors were added. Suchsections remained blank, and this demonstrates that both enzymes presentwere completely inhibited by B.W. 284 and ethopropazine hydrochloride.

Visualization methods of the results

Most histochemists visualize the results of the histochemical cholinesterasereaction by treatment of the sections with diluted ammonium sulphidesolution after incubation. In this way, the deposit of the colourless copperthiocholine crystals is replaced by the brown copper sulphide crystals.Holmstedt (1957&), however, recommends the study of the results directly bythe use of phase-contrast microscopy without treating with dilute ammoniumsulphide solution.

A series of experiments was carried out to assess the advantages and dis-advantages of the two methods. Sections incubated for cholinesterase activitywere mounted in an aqueous mounting medium without treatment withammonium sulphide solution, and the localization of copper thiocholine


crystals was studied by phase-contrast microscopy. Subsequently, some ofthese sections were removed from the slides and immersed in ammoniumsulphide solution; after mounting they were re-examined by ordinary trans-mitted illumination. On comparing the results obtained with and without theuse of ammonium sulphide, it was found that the localization of the crystalswas the same in both cases. It was, however, difficult and sometimes impos-sible to identify the deposit of copper thiocholine in sites of low concentration.On the contrary, the stained sections showed clear localization even at sites ofvery low concentration without difficulty. It was decided, therefore, not to usephase-contrast microscopy in this work.

A further experiment was carried out with ammonium sulphide visualiza-tion to determine the effect of the time factor in its application. A number ofsections was simultaneously placed in the ammonium sulphide solution andtaken out one by one at intervals from 15 sec to 5 min. No difference inresults was obtained. Similarly, no difference was observed when the con-centration of ammonium sulphide solution was varied. The stock ammoniumsulphide solution has a clear yellow appearance and it can be stored in therefrigerator for several months. When, however, the solution becomesopaque it should not be used. If incubated sections were placed in sucha solution the copper thiocholine was replaced by copper sulphide, but thelatter was then dissolved, giving false negative results.

The effect of progressive increase of incubation time on cholinesterase reaction

When a standard method is used the optimum time required for demon-stration of cholinesterase activity depends upon the concentration of theenzyme and also upon intrinsic factors including species differences. For thatreason no standard incubation time can be laid down, but it has to be deter-mined empirically.

The effect of progressive increase of incubation time was studied in thestellate ganglion of the rat. The nerve-cells of this ganglion contain enzymesin different concentration. In the early stages of incubation only some nerve-cells give a positive reaction (fig. 4, A) ; later all cells show a positive reaction(fig. 4, B, c). If A and c (fig. 4) are compared it becomes clear that A shows anincomplete picture and c shows an excessive reaction, masking the true dis-tribution of cholinesterase. In both cases misinterpretation of the findingsmay result. Hence the optimum incubation time to demonstrate AChE in thiscase is in the region of 1 to 2 h (fig. 4, B). Similarly, in the tissues supplied bythe autonomic nerves, variations in incubation time produce equally markeddifferences. For instance, in the parotid gland of the guinea-pig, after a short

FIG. 4 (plate). Photomicrographs of the sections of the stellate ganglion of the rat, fixed inneutral formalin for 3 h at 4° C and incubated for AChE activity at pH 5-3 to show the effect ofvariation in incubation time. Frozen sections, 25 /x.

A, incubated for 25 min.B, incubated for 1 h 10 min.C, incubated for 4 h.

FIG. 4

N. T. NAIK


period of incubation, only the nerve-bundles give strong positive cholinester-ase reaction. Later the finer branches and also the adventitia surroundingthe acini of the gland show a definite positive reaction as well.

The experiments show that it is not advisable to prejudge the incubationtime. For each newly studied tissue a range of incubation time should be used.This is more easily done when incubation is carried out by the floating sectionmethod.

The effect of cold storage of the tissues on cholinesterase reaction

The routine procedure for cholinesterase reaction requires continuous atten-tion for a period of approximately 5 to 10 h, and cold storage of the tissuesenables one to time the beginning of the process. This is especially importantin the case of human material obtained during operations, sometimes late inthe day.

The effect of cold storage on cholinesterase reaction was studied on avariety of tissues of various species. The tissues were closely wrapped in poly-thene sheets to avoid evaporation and stored in the refrigerator at 4° C forperiods up to 70 h. The tissues were then processed in a uniform manner andcompared with tissues not subjected to cold storage.

It was found that short periods of cold storage had no appreciable effect onthe cholinesterase reaction. Even after storage for 70 h the pattern of distri-bution and localization of the stain was not substantially affected as comparedwith specimens incubated without cold storage. However, the reaction wasslower and required longer incubation time. These findings are in harmonywith those of Glick (1938), Augustinsson (1948), Koelle (1950, 1951), Gomori(1952), and Gerebtzoff(i959) and it was considered that tissues obtained in theafternoon could safely be stored overnight at 40 C if the need arose.

The author wishes to thank Dr. N. Cauna for his guidance and ProfessorR. J. Scothorne, Head of the Department of Anatomy, for his criticism of themanuscript and for the facilities offered. The author also wishes to thank Mr.C. J. Duncan and the staff of the Department of Photography for their help inphotomicrography and Mrs. M. Young for typing the manuscript. Thispublication is part of the work done in partial fulfilment of requirements forthe degree of Ph.D. in the University of Durham.

ReferencesALLES, G. A., and HAWES, R. C, 1940. J. biol. Chem., 133, 375.AUGUSTINSSON, K. B., 1948. Acta physiol. scand., 15, suppl., 52, 1.BAYLISS, B. J., and TODRICK, A., 1956. Biochem. J., 62, 62.BERNHEIM, F., and BERNHEIM, M. L. C, 1936. J. Pharmacol., 57, 427.CAUNA, N., 1959. J. comp. Neurol., 113, 169.COUPLAND, R. E., and HOLMES, R. L., 1957. Quart. J. micr. Sci., 98, 327.COUTEAUX, R., 1951. Arch. int. Physiol., S9» 526.

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ioo Naik—Koelle's method for cholinesterase

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Technical variation isn Koelle's histochemical method for … · 2006-05-26 · Technical variation isn Koelle's histochemical method for demonstrating cholinesterase activity By