Structure and histochemistry of corpora amylacea in the brain of an orangutan (Pongo pygmaeus)

J. COYP.

PATH.

1971. VOL.

STRUCTURE AMYLACEA

81.

89

AND IN

HISTOCHEMISTRY THE BRAIN OF

(PONGO

AK

OF CORPORA ORANGUTAN

PYGMAECS.’ BY

W. B. Department

of ZJoology, Uniuersity

QUAY

of California,

Berkelg,

California,

U.S.A.

INTRODUCTIOS

Corpora amylacea are tissue deposits composed of concentric layers of complex and insoluble organic materials which generally give histochemical reactions for polysaccharide constituents. Although known from brain and some other human organs for over a century (note historical reviews of Puchtler and Sweat, 1966; Jaspar and Prick, 1969), there are still contradictions and inconsistencies concerning their etiology, precise site(s) of origin, chemical composition and relation to pathological deposits of somewhat similar nature (e.g., amyloid, paramyloid). In view of the diversity of these structures one can appreciate Weil’s (1945) conclusion that, ‘it would be advisable to drop the term corpora amylacea altogether, or at least to refrain from the attempt to reconstruct a common etiology.’ Careful systematic study of corpora amylacea and similar tissue deposits in other species of animals, and especially in individuals with well-known or well-controlled histories, will help to clarify the issues of their origin, significance and classification, and lead to selection of species and conditions for then possible experimental production as has been reported recently in the case of amyloidosis (Cohen, 1965; Pearse, 1968). The present report describes the results of a detailed histochemical analysis of corpora amylacea in the brain of an orangutan. Such lesions do not seem to have been analysed previously in non-human primates, although many other kinds of cerebral and vascular lesions have received such attention (Lucke, 1923; Fox, 1933; Innes and Saunders, 1962). C om p arisons of the histochemical and staining characteristics of the orangutan corpora and those of human corpora amylacea and amyloid reveal points both of similarity and difference. Furthermore, the structure of the orangutan corpora is distinctive and suggests gradual development in vivo rather than by means of post mortem or fixation artifact. MATERIALS

AND

METHODS

Case. The animal was a male orangutan, weighing 34 kg. and estimated from radiological examination at the National Centre for Primate Biology (Davis, California) to be approximately 7 years old. Available information suggests that its entire life span was spent in captivity following birth reputedly in a primate colony in Japan. Accumulative effects of intestinal obstruction and perforation, and subsequent abdominal exploratory surgery necessitated euthanasia on August 18, 1969. Antibiotics and a tranquilizer* had been administered from the time of surgery to euthanasia three weeks later bv nembutal and bleeding. The autopsy revealed an probably caused by atrophic thymus, extensive peritonitis, intestinal perforations impacted peach stones; the kidneys, lungs and liver were normal. *Sernalyn,

Parke,

Davis

& Co.

90

CORPORA

AMYLACE.

IN

BRAIN

OF

ORANGUTAN

Tissue fechniques. Craniotomy was performed immediately after death and the brain was removed and fixed at once in 10 per cent. formalin. Routine histological techniques were applied to a block of medial brain tissues including the corpus collosum, epithalamus, dorsal thalamus, anterior tectum and the intervening meningeal and cerebrovascular structures. Dehydration with ethanol clearing in cedarwood oil and benzene, vacuum infiltration with paraplast and serial sectioning at 7 ,u were followed by preparation of 473 slides, two sections per slide. Slides at intervals through the series were stained by the following procedures : (1) Ehrlich’s

acid alum

haematoxylin

and eosin Y (Bensley

and Bensley,

1938).

nuclear stain, (2) Periodic acid-Schiff (PAS) (Lillie, 1954) with : (a) haematoxylin or (b) 5 hours at 25OC. in 0.1 per cent. alpha-amylase (Nutritional Biochemicals Corp., B. subtilis derivative) in pH6 buffer with NaCl (=8.0 g. NaCl, 1.3 g. Na,HPO,, 0.8 g. NaH,PO, * H20/1.), or (c) 5 hours at 25’ in hyaluronidase (Nutritional Biochemicals Corp., beef testis derivative, about 2 USP units/ml.) in the same buffer, or (d) 5 hours at 25°C. in the same buffer alone, or (e) 5 hours at 25°C. in 0.1 per cent. carboxypeptidase (A Worthington Biochemical Corp., from bovine pancreas) in pH 7.9 buffer (552 mg. NaH2P0, * H,O + 13.6 g. Na2HP0,/l.), or (f) 12 hours in 1 N HCl, or (g) 12 hours in demineralized distilled water. (3) Chrome

alum haematoxylin

(4) Modified 1956).

chrome

(5) Aldehyde

fuchsin

and phloxine

alum

haematoxylin

(Halmi,

(Gijmijri, with

phloxine

haematoxylin

(8) von K6ssa’s method (9) Perls’ ferrocyanide (10) DMAB-nitrite (11) DDD 1952).

followed

for calcium reaction

method

reaction

and

Mallory

treatment 1954).

by saturated salts (Lillie,

for ferric iron

for tryptophan

for protein-bound

Oil

with

(15) Mallory’s

iodine

reaction

(Lille,

Aniline,

fluid

and

black

B

1954).

(Pearse,

1968). groups

(Barrnett

cert. No.

7) (Matheson, to Schwartsz,

for amyloid

Congo red (Nat. to Lillie (1954).

Bouin’s

1954).

sulfhydryl

blue (Nat. Aniline, (Pearse, 1968).

(14) Thioflavine S (=Direct Yellow C. I. No. 49010) for amyloid according KelCnyi (1967). (16) Bennhold’s amyloid according

(Quay,

red 0 or Sudan

(12) Highman’s crystal violet (Nat. .4niline,, cert. No. NC-38; or 42555 [new]) method as given by Lillie (1954). (13) ‘Standard’ toIuidine method for metachromasia

II

1952).

(6) 1 per cent. Azure A at 60°C. after overri$lt with differentiation in colophonium alcohol (Lllhe, (7) Acid alum (Lillie, 1954).

1941).

and Seligman,

C. I. No. 681 [old]

NU-17;

C. I. No.

52040)

Coleman and Bell No. TX510, Kurucz and Kurucz (1964) and

as emended

by Lillie

cert. No. NQ-13;

(1954). C. I. No. 370 [old]) for

(17) Puchtler, Sweat and Levine’s (1962) ‘ a lk a 1ine Congo red method for amyloid (using Chroma Congorot, No. 10460). (18) Casella reaction with 1 per cent. potassium permanganate for 20 minutes and 20 minutes in Schiff reagent (Lillie, 1954). (19) Bauer reaction reagent.

with 5 per cent. chromic

anhydride

1 hour and 1 hour in Schiff

W.

B. QUAY

91

RESULTS

Location, Size and Structure Corpora amylacea occurred in two sites, both within well-vascularized connective tissue regions in or near the roof of the diencephalon. One site was the ventral and posterior wall of the dorsal sac (or suprapineal recess). Corpora were found between the ependymal lining epithelium of the dorsal sac and the parenchymal lobules of the pineal gland (Fig. 1) and, more anteriorly, between this ependymal lining and the habenular commissure and the stria medullaris thalami. They varied from about 5 p to over 150 p in diameter and were similar structurally, tinctorially and histochemically. Corpora amylacea of somewhat different structure and staining characteristics were found in the other sites, which may be described as paravascular. They were adjacent to or near venous sinusesof both large and small calibre in and beyond the anterior and dorsal wall of the dorsal sac and continuing along the walls of the venae cerebri internae, the vena cerebri magna and their tributaries (Figs. 2-4). These also varied from about 5 p to over 150 f~ in diameter. The lamination and composition of these paravascular corpora were more complex and variable than in the subependymal corpora. Corpora amylacea or similar types of tissue deposits were not found in other regions of the central brain tissue blocks. Thus, within the pineal itself there were no corpora amylacea or arenacea and within the portions of the brain studied there were no amyloid, corpora amylacea or other well-defined pathological deposits. The concentration of glycogen may have been increased in perivascular cells, especially astrocytes, within different parts of the brainstem and most markedly around capillaries and venules in a subpial zone dorsally. These perivascular, finely granular and mostly intracellular deposits were PAS-positive and were totally removed only by prior treatment with amylase. Subependymal Corpora Due to the more limited and more medial distribution of corpora of this category, fewer slides contained them and so fewer staining and histochcmical techniques could be employed in their study. Five characteristics were, however, noted : (1) They were intensely PAS-positive throughout (Fig. l), but were not numerous enough for analysis by extraction methods prior to staining by PAS. (2) They were polychromatophilic by various histological staining techniques, the different laminae or regions of a deposit staining to different degrees by different stains of a staining mixture or sequence, such as, (a) Ehrlich’s haematoxylin and eosin, (b) chrome alum haematoxylin and phloxine, (c) Mallory II counterstain, and (d) aldehyde fuchsin and Light Green SF and Orange G counterstains. No well-defined chemical significance need be implied by these tinctorial effects. (3) Metachromasia throughout the corpora was shown by crystal violet. (4) Some laminae especially the outer ones were coloured grey by Sudan black, but were uncoloured by Oil red 0. (5) A moderately strong, but variable staining by the DDD reaction for sulfhydryl groups in proteins was shown. Perls’ reaction for ferric salts and von Kc%w.‘s reaction for calcium salts were entirely negative. Neither birefringence nor significant fluorescence were demonstrable.

92

CORPORA

AMYLACEA

IN

BRAIN

OF

ORANGUTAN

Paravascular Corpora Like the subependymal corpora, the structures showed the following characters : they were intensely PAS-positive (Fig. 2); they were polychromatophilic with the same histological staining methods; they were strongly metachmmatic showing rose to deep red after the standard toluidine blue method; and they were isotropic and not significantly fluorescent. They differed from the subependymal corpora by being less strongly stained and more variably positive by the method for sulfhydryl groups; variably stained with Sudan black, but usually primarily and most strongly in the central portion (Fig. 3), though still negative throughout for Oil red 0; frequently strongly stained blue in the central portion by the method for ferric salts (Fig. 4). An additional feature of the paravascular, as compared with the subependymal corpora, was the frequent participation in their outer lamination by what appear to be collagenous fibres and fibrocytes (note especially Figs. 2 and 3). These visibly more fibrous laminae stained similarly in comparison with irregular collagenous fibres in the immediately adjacent connective tissue, as for example, strongly with aniline blue in the Mallory II counterstain and weakly with PAS (Fig. 2). Other techniques applied to the paravascular corpora revealed resistance of the PAS-positive materials to amylase, hyaluronidase, carboxypeptidase and 1N HCl; equivalent strong reactions to the Casella and Bauer oxidation-Schiff methods; selective and strongly fluorescent staining (yellow) with Thioflavine S; variably weak to strong staining by the congo red methods, but without birefringence, dichroism or fluorescence; and no staining by the Mallory iodine reaction. TABLE CHARACTERISTICS

OF ORANGUTAN

OF PREVIOUSLY

DESCRIBED

I

PARAVASCULAR BRAIN

Characteristics

CORPORA

CORPORA

Orangutan

corpora ~~

Natural birefringence Natural fluoresence Soluble in IN HCI PAS after diastase or amylase Glycogen content Casella reaction Bauer reaction Metachromasia with toluidine Sudanophilia Iodine staining Congo red staining birefringence Thioflavine S-fluroescence staining Perls (ferric salts) van K&a (calcium salts) *0 = negative; (+) = weakly **Data derived and summarized Prick (1969), Kahn and Santos (1963, 1968), Puchtler and Sweat (1952) and Wagner, Damodoran

COMPARED

AMYLACEA

AND

Brain corpora amylacea * *

WITH

THOSE

AhnLOID*

Amyloid*

00 0

0-q 0 -

i

0

()I-

z

*

0 blue

7Oi-(0

&l-L O--

0

($\I 0--+

02+ 0

3 ‘0 0

+ J : t 0

positive; + = usually moderately to strongly positive. from Braunstein and Buerger (1959), Diezel (1956), Jaspar and (1963), KelCnyi (1967), Lillie (1951, 1954), Molnar (1951), Pearse (1966), Ramsey (1965), Steele, Kinley, Leuchtenberger and Lieb and Feigin (1957).

W.

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93

DISCUSSION

Data summarized in Table 1 suggest that so far as optical, staining and h&ochemical properties are concerned the paravascular corpora in the orangutan are similar to mme kinds of brain corpora amylacea and to amyloid. The resemblance is greater with the usually laminated and primarily polysaccharide-containing corpora amylacea. Some of the differences suggested by the arbitrary scoring in Table 1 may be due to technical factors or to chemical peculiarities of the local tissue sites of the genesis of the deposits. From the technical standpoint further studies would be necessary before much reliance can be placed on the lack of solubility in HCl, the failure of staining with iodine and the chemical significance of staining with Sudan black. The lack of birefringence and the consistently strong Casella reaction in the corpora are more difficult to explain. These results and recent pathohistochemical studies of human corpora amylacea, amyloid and paramyloid reinforce the evident heterogeneity and lack of co&tent characterization of these complex organic deposits. Even within the sections of orangutan brain the structure and composition of the corpora vary considerably within the paravascular category and dier consistently when corpora of subependymal and paravascular types are compared. The latter difference is probably due to different degrees of incorporation of connective tissue elements and ground substances, such incorporation being greatest in the paravascular corpora and minimal in the subependymal. The composition and size ranges of both types of orangutan brain corpora appear, however, to extend beyond the relatively small and diffuse corpora amylacea studied in human brains recently by Ramsey (1965) and Jaspar and Prick (1969). The size and complex structure of the corpora in the orangutan brain appear to rule out the possibility of their sudden genesis post mortem by acute changes in vascular permeability, precipitation of ground substances or exudate, or fixation artifact. A more gradual and chronic genesis in vivo seems most likely. In this connection it should be pointed out that in none of the sites of corpora formation were there any signs of an old or recent inflammatory response or haemorrhage. The general concept of corpora amylacea as a complex polysaccharide or glycoprotein metabolic product deposited in laminated form, especially in older individuals, would seem to cover the orangutan corpora. The possible origins of brain corpora amylacea from breakdown of myelin sheaths or other components of nervous tissue or their origins within glia have been suggested by some investigators. These possibilities are not readily supported in the case of the orangutan corpora. The orangutan corpora amylacea appear to have developed in certain vascularized meningeal connective tissue layers, possibly commencing as accumulations of material within perivascular or subependymal phagocytes. There is some support for this suggestion in the following observations derived from the same orangutan brain sections. In both subependymal and paravascular sites phagocytes can be found with small cytoplasmic granules that are PAS-positive, resistant to the enzymes amylase, hyaluronidase and carboxypeptidase, and metachromatic. In the perivascular connective tissue of the meninges in particular, adjacent and possibly transitional connective tissue cells are found with hypertrophic cytoplasm

94

CORPORA

AMYLACEA

Ii% BRAIN

OF

ORANGUTAN

containing homogeneous PAS-positive material. With adjacent fibroblasts and the forementioned granular cells, these form a perivascular sleeve of oedematous cells. Less commonly adjacent to neighbouring vessels in the same regions, bodies of a possible transitional character are seen in which the structure of the phagocytic cells is obscured by fusion of the cytoplasmic granules, loss of visible organelles and circumferential deposition of ground substance; and, in the paravascular corpora, of connective tissue. In relation to some of the larger and more diverse paravascular corpora, two other possible tissue derivations which might be considered are thrombosed and fibrotic vascular segments, or degenerated epithelial extensions of the dorsal sac surrounded by fibrosis. Support for the first of these possibilities might be the frequently positive Perls’ reaction of the core material in some of these corpora : on the other hand, most of the corpora are spherical and seem to be isolated from direct vascular connections. The second possibility, while theoretically possible from similar appearances seen in avian brains (Quay and Renzoni, 1967), is difficult to support without any vestiges of epithelial cells in their cores. SUMMARY

The structure and histochemistry of corpora amylacea in the brain of a 7-yearold orangutan are described. Within the same tissue slices of this specimen, structural and histochemical differences occurred between subependymal and paravascular corpora amylacea. In most, but not all, of their optical, tinctorial and chemical properties, the corpora resembled those described by others in human brains. Postulated modes of their genesis and development are discussed; there is evidence that phagocytes in oedematous perivascular zones may form foci leading to their formation. ACKKOWLEDGMENTS

I wish to thank Drs. Tetsuo Hayashida and Raymond Young for the orangutan and for services rendered at the time of its autopsy, Mrs. Joan F. Quay, Mr. David McKasson and Mr. Peter Witte for laboratory assistance, Mr. James Hendel for photomicrography and Miss Helen Sherry for biblographic and office assistance. REFERENCES

Barrnett, R. J., and Seligman, A. M. (1952). Science, 116, 323. Bensley, R. R., and Bensley, S. H. (1938). Handbook of Histological and Cytological Technique, Univ. Chicago Press;Chicago. Braunstein, H., and Buerger, L. (1959). Amer. 1. Path., 35, 791. Cohen, A. S. (1965). Int. Rev. exp. Path., 4, 159. Diezel, P. B. (1956). Verb. dtsch, path. Ges., 39, 199. Fox, H. (1933). Arteriosclerosis, Ed. Cowdry, E. V., Macmillan Co.; New York. Gomori, G. (1941). Amer. J. Path., 17, 395. Halmi, N. S. (1952). Stain Tech., 27,61. Innes, J. R. M., and Saunders, L. Z. (1962). Com parative Neuropathology, Academic Press: New York. Jaspar, H.‘H. J.. and Prick, J. J. G. (1969). P rot. Koninkl. Nederl. Akad. Wetenschap., Ser. C 72: 385.

W.

B. QUAY

Fig.

1.

Subependymal and partly fused corpora amylacea (arrows) in the connective tissue bet tween the dorsal sac with its recesses (at top) extending into the pineal and the pineal parenc hyma (at bottom). The corpora are shown here intensely and uniformly positive to the PAS technique; cell nuclei are stained with haematoxylin. x 315.

Fig.

2.

Corpora amylacea (arrows) in the vicinity of dorsal sac and vena cerebri magna (of G, alen). Central portions and inner laminae of the corpora are intensely positive here to the PAS technique; outer collagenous and fibroblast laminae are negative or weakly positive ‘; cell nuclei are stained with haematoxylin. x 255.

CORPORA

AMYLACEA

IN

BRAIN

OF

ORANGUTAN

Fig.

3.

The Corpora amylacea in thr vicinity of dorsal sac and vena cerebri magna (of Galen). central masses (arrows) of many but not all corpora are stained blue here by Perls’ rnc :thod pink for ferric salts; some of the lammae of the corpora, as well as all cell nuclei, are stained to red by the safranin counterstain. x 330.

Fig.

4.

Corpora amylacea in the vicinity of dorsal sac and vena cerebri magna (of Galen). portions and inner laminae of some corpora (arrows) are coloured here dark grey by Sudan black B; cell nuclei are lightly stained by haematoxylin. ;< 190.

Ct mtral to Iblack

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95

Kahn, H. L., and Santos, J. C. (1963). Acta neuropath., suppl. 2,66. Kelenyi, G. (1967). 1. Hzstochem., 15, 172. Lillie, R. D. (1951). Stain Tech., 26, 123; (1954). Histopathologic Technic and Practical Histochemistry ; Blakiston Co.; New York & Toronto. Lucke, B. (1923). Arch. NeuroE. Psychiat., Chicago, 10,212. Molnar, J. (1951). Nature, London, 168, 39. Pearse,A. G. E. (1963). Acta neuropath., suppl. 2, 100; (1968). Histochemistry Theoretical and Applied, 1, 3rd edition, Little, Brown & Co.; Boston. Puchtler, H., and Sweat, F. (1966). J. Histochem., 14, 123. Puchtler, H., Sweat, F., and Levine, M. (1962). Ibid., 10, 355. Quay, W. B. (1956). Exp. Cell Res., 10, 541. Quay, W. B., and Renzoni, A. (1967). Riv. biol., 60, 9. Ramsey, H. J. (1965).J. Neuropath., 24,25. Schwartz, P., Kurucz, J., and Kurucz, A. (1964). Zbl. Path., 106, 320. Steele, H. D., Kinley, G., Leuchtenberger, Cl., and Lieb, E. (1952). Arch. Path., 54, 94. Wagner, B. M., Damodoran, V. N., and Feigin, I. (1957). Lab. Invest., 6, 259. Weil, A. (1945). Textbook of Neuropathology, 2nd edition, Grune & Stratton; New York. [Received for publication, April 20th, 19701

G