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H l S T O C H E M l C A l H E T H O D S A N D T H E I R A P P L I C A T I O N TO THE S T U D Y OF MlrSCLE S T R U C T U R E

C. E. BODWELL

Histochemical methods have been used since the beginning of the 18th Century a s evidenced by the publication of Raspail's %ssa i -- de Chimie Microscopique &pliqiiee 6 l a Pbysiologie-" i n 1830. A somewhat SLOW develop- ment o f techniques occurred u a t i l m e d i a t e l y pr ior t o World Var I1 when, with t h e rapid advancements of biochemistry, renewed in t e re s t was czeated. Since 1944, very rapid progress has been achieved i n the develo,ment of techniques, types of reactions or substances detectable, and t h e interpretat ion of re- su l t s . In 1950, no journals or periodicals were devoted t o the science of histochemistry. Today, no less than seven sc i en t i f i c journals a r e devoted Solely t o this f ie ld . large proportion re fer t o studies reported since 1950.

Pearse (1960) lists over 2000 references, of which a

Histochemistry involves the application of histological-biochemical techniques t o demonstrate the presence of enzymes or other chemical substances in plant or a n a l t issxes . stances) t o form insoluble, a t the si te of ac t iv i ty (or localization) when reacted wi th specif ic sub- s t r a t e s or reagents, i s u t i l i zed . Three prirnary advantages of his5ochemical techniques E se are2 l a r location ofbiochemically act ive cmpounds, (2) the s t ruc tura l and func- t i o n a l ce l lu la r integri ty can be characterized, and (3) qual i ta t ive t e s t s f o r numerous reactions can be r a p i d b condtlcted on small samples of various t i s sues frm single or multiple sources or treatments. The l a t t e r is of par- t i c u l a r value when used t o supplenent investiGatlons vhich a re pr iaar iJy con- ducted using biochemical methods.

The a b i l i t y of enzymes (or non-enzymic sub- localized, and readily detect ible precipi ta tes

(1) information can be obtained as t o the exact cellu-

T h i s morning, it i s perhaps i n order t o brie2ly review some of t h e principles and techniques involved i n histochemistry and indicate t h e extent of the types of enzpes and substances currently detectable vhen using these techniques. t r y a s a research technique w i l l becoae apparent. areas, per t inent t o the use of these techniques i n meats research, w i l l be suggested.

In so doing, it i s hoped tha t the potent ia l value of histochemis- Lastly, a few specif ic

Mechanical Procedures

The relat ions of the principle methods of microtechnique a re shown in s l ide 1. through the following steps a s shown on the slide, enroute t o the ultimate examination of the t i s sue under the microscope:

In routine histology, a sam2le of f resh t i s sue would be passed

(1) fixation, (2) washing - optional, according t o t h e f ixa t ive used, (3) preparation for em'oedding - dehydration in f i l t r a t ion , etc. , (4) embedding - paraffin or celloidin, (5) sectioning, (61 al'fixing t o s l ides , (7) staining, and (8) dehydration, clearing, and rnomting.

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For a few substances, such as glycogen i n muscle t issue, a similar procedure would be followed i n histochemistry u t i l i z ing a histochemical re- action i n l i e u of the usual s ta ining s tep in t h i s procedure. However, almost a l l histochemical methods, par t icular ly those used i n detecting enzyme act iv- i t y , employ a cold microtome o r cryostat . The cryostat is basical ly a micro- tome contained i n an insulated cabinet which ha8 been sui tably adapted f o r operation a t temperatures f r o m -12 t o -22 degrees Centigrade, and was first developed and used by Linderstqdm-Lang and Mogensen (1938). techniques made possible by a cryostat, materials such as enzymes a r e not subjected t o the denaturing e f fec ts of the procedures of routine histology.

When using

A prepared t i s sue sample, either fresh o r fixed, is rapidly frozen by immersion in a low temperature l iquid, -78 degrees Centigrade (Acetone/COZ) f o r most t issues and -155 t o -160 degrees Centigrade (isopentane/liquid nitrogen) i n the case of muscle t i s sues . ready f o r sectioning w i t h the cryostat e i the r immediately o r a f t e r a short storage period i n the cold. of ice c rys ta l s and loss of enzymic ac t iv i ty a re t o be held t o a minimum. After sectioning, the sections a re affixed t o a cover s l i p ; the cover s l i p and section a re then passed through various reagents, including a histochemi- c a l incubation media, and f i n a l l y mounted t o a s l i d e f o r observation with the microscope. In addition t o enzyme preservation achieved by the cryostat pro- cedure, t he method is also much more rapid than routine p a r a f f i n methods.

After freezing, the t issues a re

The l a t t e r should not exceed 5-10 days if growth

Types - of Reactions

Four types of reactions a re used i n histochemistry as shown i n In each case, a colored o r dense precipi ta te is produced a t the Slide 2.

s i t e of reaction. The four reactions are:

Slide 2. ~-

PRODUCTION OF DEZECTABLE PRM: IPI!L"ES

1. E + S ,-> a l te red substrate"

* 2. E + S --> product >complex

colorless salt

4. Substance + reagent > substance- reagent

complex*

* colored o r dense precipi ta te E = enzyme; S = substrate

In slide 3, an example of the first type of reaction is given.

213.

Slide 3.

I n t h i s case, the subs t ra te i s a co lor less s a l t which i s converted

The com-

However,

i n t o a colored precipi ta te a t the s i t e of react ion w i t h an enz'pe o r other s p e c i f i c reducing substance according t o the p a r t i c u l a r sa l t used. pound above is not s u i t a b l e for a histochCmica1 subs t ra te due t o the ten- dency of the colored product t o d i f f u s e f r o m the s i te of reac t ion . der iva t ives of te t razol ium sal ts , such as Nitro-ET, provide excel lent sub- strates f o r 8 range of s p e c i f i c histochemical react ions.

An example of the second type of react ion is shown i n Slide 4 .

Slide 4

Alkaline Phosphatase

alpha-Napthyl- diazonium chloride

L- - -_I___-

The reac t ion shown here is used t o d e t e c t a l k a l i n e phosphatase a c t i v i t y and i s dependent m the simultaneous coupling of a co lor less salt w i i k t h e product of t h e enzyme-substrate react ion a t the s i t e of react ion t o

214.

form a sui table precipi ta te . similarly detectable precipi ta tes which are localized a t the s i te of re- action.

The remaining two types of reactions resu l t i n

Reaction Mixtures

To detect enzyme ac t iv i ty , histochemically, reaction mixtures which include substrate, act ivators and buffers must be used ju s t as in a biochemi- ca l assay. An example of euch a mixture is shown i n Slide 5.

Slide 5.

LEAD MEZIKID FOR ATPASE React ion Mixture

20 ml. 125 =.$ ATP(di-Na) 20 ml 0.2 M T r i s - E 1 buffer(pH 7.2) 3 m l 2 $ Pb(N03)z 5 ml 0.1 M %SO4 2 m t d i s t i l l e d H20

This is a reaction mixture f o r ATP'aee (adenosine triphosphatase) after Wachstein and Miesel (Pearse, 1960). Here, magnesium ions serve as act ivators f o r the sarcoplasmic ATPase enzyme, the disodium ATP being the substrate. form a lead s a l t a t the s i te of ac t iv i ty , This i s subsequently reacted with ammonium sulfide t o give a brown metall ic precipi ta te , lead sulfide. routine histochemical etudies, the incubation of some sections i n a reaction mixture void of substrate and/or containing specif ic inhibi tors serves a8 a control in detecting non-specific, o r false, reactions.

The phosphate released upon hydrolysis react8 with lead ion to

I n

Confirmation of Results

Where possible, it is also preferable t o u8e a t l e a s t two di f fe r - ent procedures f o r confirming the a c t i v i t y of a given enzyme o r the presence of a par t icu lar non-enzymic substrate. In the next slide, a method is shown which provides a "double reaction" a t the s i t e of ac t iv i ty . This i a a modi- f i ca t ion of the reaction shown i n Slide 4. hydrolysis of napthyl phosphate by alkal ine phosphatase is reacted with calcium ions t o yield calcium phosphate a t the site of the i n i t i a l reaction. This is converted t o a dense precipi ta te , cobaltous sulf ide, by treatment with cobalt and subsequently ammonium sulfide. is also precipitated a t the si te of ac t iv i ty . In a true reaction, the loca- t ions of the two precipitated products w i l l be ident ical .

The phosphate released upon the

An insoluble red-blue pigment

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I.

J.

11. I Phosphatase

\ + HPO4

- CaHPO4

1 Tetra-azotized

0-dianis idine I/ cos

Insoluble Red-Blue Pigplent

MGTBOD FOR CONFILWIIJG THE S D E OF HYDWOLYSI; OF NAPH'.CIMPIiOSPWTE

Analogous Materials

Certain compounds give similar reactions with some histochemical tests. An example of such a series, f o r carbohydrate containing materials, is given i n t h e next slide (Slide 7, Ident i f ica t ion o f Carbohydrate Containing Materials; after Pearse, 1960, p e 236). The series of substances include (among others) glycogen, s tarch, glycoproteins, and glycol ipids . By selec- t i v e use of t he s e r i e s of t e s t s l isted, it is apparent t h a t a4v of the sub- s tances can be ident i f ied . Likewise, another series, using o ther su i t ab le seactiona, must be u t i l i z e d t o d i f f e r e n t i a t e between ce r t a in es te rases and l i pases (Gomori, 1952).

Consequently, a series of d i f f e ren t i a t ing react ions must be used.

Quantlf i ca t ion --.--

Histochemical methods are primarily qua l i t a t ive procedures and biochemical methods must be used f o r quant i f icat ion i n most instances, ever, a l a rge proportion of the current methodology s tudies i n histochem- istry is concerned with t h e development of quant i ta t ive procedures. most successful methods u t i l i z e spectrophotometric procedures (Nachlas and Seligraann, 1950; Doyle, 1950; Doyle - e t a1 ,*, 1951). In thege procedures, a prec ig i ta ted colored react ion product is extracted f r o m t i s s u e sect ions and the concentration of t h e extracted material is determined spectrophotometri- c a l l y using a standard curve of known amounts of t he pmduct extracted. This procedure can only be used when t h e reaction product is su i t ab le f o r spectro- photometric analysis .

How-

The

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Specialized Procedures

Specialized procedures are used i n applying flouorescence micros- copy and autoradiographicel techniques i n histochemistry. of such procedures i n histochemistry has potent ia l usef'ulness and is con- sequently an area of current in te res t . The employment of histochemical techniques i n conjunction with electron microscopy is a fur ther area i n which sui table methods are being developed. e r a l introduction t o these areas of study and refers to several pertinent reviews where detai led information can be found.

The application

Pearse (1960) Includes a gen-

Metabolic Ebzymes

I n addition t o the use of histochemical methods t o c l a r i fy the relat ions o f enzymes and other substances t o structure, these methods can a l so be used t o observe changes i n s t ructure by observing changes i n the flmctional and s t ruc tura l in tegr i ty of the ce l l . which are relevent t o thF6 problem can be extensively followed using histo- chemical method6 .

Two groups of enzymes

The first group include6 cer ta in enzymes of the metabolic cycles, i . e . TCA (tr icarboxylic acid), glycolytic, f a t t y acid oxidation, and hexose monophosphate (pentose ehunt) cycles. These cycles, i n abbreviated form, a r e shown In s l i d e 8 . and succinate dehydrogenases. A side reaction, catalyzed by glutamic de- hydrogenase, yielding products u t i l i zed i n the TCA cycle, can also be de- tected.

In the TCA cycle, these include i soc i t ra te , malate,

In the hexose monophosphate pathway the ac t iv i ty of t w o enzymes, glucose-6-phosphate dehydrogenase and 6-phosphogluconate, can be obaerved histochemically. Three enzymes, whose primary concern is with glycolysis, can be demonstrated. These include alpha-glycerophosphate, alcohol, and l a c t i c acid dehydrogenases. In the fa t ty acid oxidation cycle, only one enzyme, beta-hydroxybutyrate dehydrogenase, can be demonstrated.

By observing the a c t i v i t i e s of these metabolic enzymes, it is possible t o determine t o a considerable extent the general ce l lu l a r metabolic condition and if i r r egu la r i t i e s occur, where the breakdown i n normal metabo- l i s m is present.

Respiratory Euzyrnes

The second group of enzymes demonstrable by histochemical tech- niques which give information concerning the s t ruc tura l and functional ce l lu l a r condition include the respiratory enzymes as shown i n sl ide 9. This is a schematic adaption f r o m Hartree (1957) showing those portions of the electron transport system of in t e re s t t o the histochemist. action6 a re important i n being specif ic indicators of the s t a t e of the mitochondria of the c e l l . TPNB (reduced triphosphopyridine nucleotide) and DPNR (reduced diphosphopyridine nucleotide) related diaphorase and cytochrome a3 oxidase a c t i v i t i e s can be determined d i r ec t ly by histochemical methods. Through u8e of select ive inhibitor6 (amytal, B.A.L,, antimycin A, e tc . ) , it is possible t o histochemically characterize the other portions of t he trans- port system.

These re-

217.

IScampleG of Histochemical Reactions' in tkscle Tissues

In the next few sl ides , examples of histocheniical reactions a re shown as detected in s t r i a t e d muscle t i s sue (larab, beef, poultry) including (1) leucine amino peptidase, a catheptic enzyme, (2) acid phosphatase, (3) succinic dehydmgenase, (4) sarcoplasmic ATPase, and (5) the periodic acid- Schiff reaction f o r carbohydrate containing materials. Lastly, as an example o f the application of histochemical technique i n con junction with electron microscopy, is a photomicrograph of btr ia ted muscle t i s sue which i l l u s t r a t e s the histochemical demonstration of the hydm1ysis"of th io lace t ic acid i n r a t muscle i n the M band (Barnett and Palade, 1959).

Histochemical Methods i n Meats Research

In considering some possible areas of meats research i n which his- tochemical techniques a re of potent ia l value, an area which imedia te ly is of i n t e re s t is tha t of changes occurring i n the muscle during rigor. Particu- l a r l y i n reference t o beef, several questions can be tendered. the enzymes of the metabolic cycles active throughout r igor?

Are a l l of

Andrews, -- e t al. (1951) reported that succinic dehydrogenase, ATPase, and the overal l glycolytic system were active through 2-4 weeks a f t e r slaughter i n in t ac t beef muscle stored a t 2OC. Aldolase, a glycolytic enzyme, was found to decrease markedly during the same period. a c t i v i t y of the other metabolic enzymes during rigor, especially those of the pentose shunt and OP the f a t t y acid oxidation cycle? period a f t e r slaughter is ce l lu l a r respiration operative, as indicated by the ac t iv i ty of the electron transport system? membrane actual ly break down? Do different animals vary in the i n i t i a l l eve ls and/or ra te of decrease i n enzyme ac t iv i ty during r igor of the car- cass? changes o r differences may have considerable e f fec t on pract ical problems such as color and tenderness.

What is the

For how long a

A t w h a t point does the c e l l

These a re a l l questions of basic concern, f o r ( i n beef) such possible

In pork, studies by Bendall and Ilismer-Pederson (1962) have shown that watery pork can be induced with specified treatment6 and that an abnormal accumulation of denatured sarcoplasmic protein on the myofilaments can be detected on the his tological level . comparable t o that of the normal o r non-watery pork under the same condi- t ions? Are the sarcoplasmic and myosin ATPase a c t i v i t i e s normal pr ior t o the accumulation of the sarcoplasmic proteins? Is it possible tha t a break- down i n metabolic o r respiratory enzyme ac t iv i ty is involved i n some manner? These a re a l l pertinent questions t o a histochemical approach t o a problem of current i n t e re s t i n meats research.

Are the enzyme a c t i v i t i e s here

Likewise, histochemical studies would possibly be helpful i n elucidating some of the chemical aspects of muscle t i s sue i n re la t ion t o possible s t ruc tura l changes a6 produced by the use of s t i l bes t e ro l implanta- t i on o r feeding. when using other feeding regimes as well, especially i f biopsy sampling was u t i l i zed during the feeding period.

Histochemically detectable changes would be of i n t e re s t

Lastly, histochemical methods a re of value when observing changes i n various t i s sues of experimental animals on specified d i e t s . In meats

218.

research, two areas of i n t e re s t would be the e f fec ts of feeding smoked products and the feeding of animal f a t s i n the d i e t .

Conclusion

Histochemical methods can be used t o indirect ly study changes i n muscle s t ructure . of grea ter importance, such methods enable the research worker t o r e l a t e chemical changes to muscle structure. This has been a very precursory treatment of a rapidly developing f i e l d of study. The techniques involved would appear t o provide a potent ia l ly useful approach t o cer ta in problems of interest i n meats research.

Andrews, M.M., Guthneck, B. T., McBride, B. H. and Schweigert, B. S. 1951. S t ab i l i t y of cer ta in respiratory and glycolytic enzyme systems i n animal t i s sues . J. of Biol, Chem. 194, - 715-719.

J. Biophysic. and Biochem. Cytol. 5, 163-170. Barrett , Re J. and Palade, G. E. 1959. Enzymatic a c t i v i t y i n the Mband.

Bendall, J. R. and Wismer-Pederson, J. 1962. Some properties of the f i b r i l l a r proteins of normal and watery pork muscle. J. Food Sci. 27, 144-159,

Doyle, W. L. 1950. Quantitative evaluation of Gomri histochemical preparations. Science 111, 64-65.

Doyle, W. L., Ornoto, J. H. and Doyle, M e E. 1951. Estimation of phosphatase

Gormori, G. 1952. Microscopic Histochemistry. Prlnciples - and Practice.

i n his tological preparations. B p . Cell Res. 2, 20-38.

Univ. of Chicago Press.

Hartree, E. F. 1957. Cytochrome i n higher plants. Advances i n Ehzymology, Vole XVIII, p. 1-64,

Linderatx$m-Lang, K. and Mogenson, K. R,

Pearse, A. G. E. 1960. Histochemistry. Theoretical and Applied. L i t t l e ,

Raspail, F. 1830. -I Essai de Chimie Microscopique Appliquee h Phys io lo~ie .

1938. C. R. trav. Lab, Carlsberg serie chim,, - 23, 37.

Brown, and Company, Boston.

(Quoted by Pearse, 1960).

Paris

Seligmn, A. M. and Nachlas, M. N. 1950. The colorimetric determination of Je c l in . Invest. 29, 31-36. l i pase and esterase in human serum.

219.

(Applause )

MR. BRISKZY: Thank you very much. The person preparing our next report is the distinguished international s c i en t i s t , Dr . h j imaki . an associate professor a t the University of Tokyo and since he hasn' t been introduced t h i s morning I would l i ke t o have D r . Fujimaki stand. t a in ly a re pleased tha t he is here.

T-Ie is

\!e cer-

(Applause)

As you bow he is here t o work with Dr. Deatherage. The f a c t that D r . Deatherage has,a reputation t o encourage sc i en t i s t s fmrn a l l over the world t o work with him cer ta inly is a cred i t i n i t s e l f . who, as you know is Chairman of the agricul tural Biochemistry Department, Ohio State, w i l l give the report which Dr. Fujimaki has prepared.

Dr. Deatheraee,

I might add that the reason f o r t h i s is, Dr. Fujimaki has only been here a few weeks and doesn't speak the English language too well. Wet11 hear from him di rec t ly .