J. Ieurol. Neurosurg. Psychiat., 1963, 26, 178

The morphology of Marinesco bodies (paranucleolarcorpuscles) in the melanin-pigmented nuclei

of the brain-stemP. YUEN AND D. W. BAXTER

From the College of Medicine, University of Saskatchewan, Canada

In 1902 Marinesco described the presence ofspherical acidophilic bodies in the nucleoplasm ofthe cells of the human nucleus substantia nigra andthe nucleus locus caeruleus. He wrote: 'The numberof these corpuscles vary from one to six, and generallythere are one, two or often three. When they arenumerous we find them heaped in groups in thenuclear sap, and the position they occupy withregard to the nucleolus is equally variable. Sometimesthey are situated in the neighbourhood of the latter,sometimes they are set aside and can even resideat the two poles of the nucleus. As to their relativesizes, they are seldom larger than the nucleolus,sometimes they can be nearly equal, but generallythey are smaller than the latter, especially when theyare numerous. The diameter of these paranucleolarcorpuscles can reach 7,u'.

'The size of these corpuscles does not appear tobe related with age, for the biggest diameter whichwe found was in a 30-year-old man. Generallyspeaking, they are more numerous in adults and oldpeople than young persons. In the latter, in fact,they are rarely found and are absent in children.'

Marinesco elaborated further on the staining pro-perties of these paranucleolar corpuscles in 1909.Remarkably little attention has been paid to them,at least in the British literature, since that time.Greenfield (1958) and Lipkin (1959) refer briefly tothem, and Wolf and Orton (1932) have describedsimilar intranuclear inclusions in unpigmented cellsof the central nervous system in diverse diseasestates. This paucity of interest is all the more surpris-ing in view of the widespread interest in cytoplasmicinclusions in the cells of the substantia nigra andlocus caeruleus in paralysis agitans and otherextrapyramidal syndromes.The aim of the present study was to determine the

frequency with which Marinesco bodies could befound and whether their presence could be correlatedwith specific or general types of disease or with theage of the patient. We also sought to determinefurther the histochemical properties of the inclusions

and any relationship which the inclusions might haveto melanin pigmentation.

MATERIAL AND METHODS

Cross sections were prepared through the brain-stems of160 randomly chosen cadavers at the levels of the nucleussubstantia nigra and the nucleus locus caeruleus. Thebrain-stems had been fixed in 10% formalin for 10 to 20days and the blocks were embedded in paraffin. Thehistological sections were 6u thick and stained withhaematoxylin and eosin.Each cross section containing cells of the substantia

nigra was scanned for the presence of Marinesco bodies.In most cases only the 350 to 450 cells which form thesubstantia nigra on one side of the midbrain were scannedin detail. If such cells were found they were noted asbeing many (more than 10 cells), few (six to 10 cells),rare (one to five cells), or absent. If absent, the oppositesubstantia nigra was scanned in similar detail and if anyMarinesco bodies were found the classification waschanged from absent to rare. Serial sections were notexamined in those instances in which Marinesco bodieswere not demonstrated in a single cross section. Furthernotes were made concerning the location of the cellscontaining Marinesco bodies within the nucleus sub-stantia nigra, and the presence of any nucleolar, nuclear,or cytoplasmic peculiarities which these cells mightpossess. Attempts were then made to correlate theseobservations with the age of the patient, the cause ofdeath, and the duration of the terminal illness (Tables Iand 11).

Similar observations were made on the cells of thenucleus locus caeruleus in a few cases. In view of thesmall number of cells present in a single cross section itwas necessary to study serial sections in most instances.The following histological techniques were used to

examine in further detail the staining properties of theMarinesco bodies: The Feulgen reaction for D.N.A.,toluidine blue and gallocyanin stains for nucleic acids,Danielli's coupled tetrazonium reaction for aromaticalpha-amino acids, fast-green stain for histones, theperiodic-acid-Schiff reaction for muco- and glyco-proteins, Sudan IV and Sudan black for lipids, VonKossa's stain for calcium, Gomori's stain for iron,Fontana silver stain, Mallory's trichome and Masson's

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The morphology of Marinesco bodies in the melanin-pigmented nuclei of the brain-stem

trichome stains, Heidenhain stain for myelin, Nisslcresyl violet stain, and Van Gieson's stain.

OBSERVATIONS

Marinesco bodies could be demonstrated withinsome cells of the nucleus substantia nigra in themajority of patients over 21 years of age (Table I).Such cells were rare before the age of 30 years butcould be found with relative ease in most olderpatients. There were, however, occasional cases inall age groups in which Marinesco bodies could notbe demonstrated. It seems possible that if serialsections of the substantia nigra had been studied insuch cases Marinesco bodies would have beendemonstrated at least in some of these.There was no correlation between the presence of

Marinesco bodies and the type of terminal illness.They were found as frequentlv in patients killed in

accidents as in those suffering long debilitating ill-nesses. Table II gives the data for the age group,66 years to 70 years.The Marinesco bodies are small, homogenous,

eosinophilic, droplet-like structures which lie withinthe nucleus of the cell (Fig. 1). They vary in diameterfrom 2,u to lOlt with an average of 4,t. As many asfour distinct Marinesco bodies may be seen within asingle nucleus. Their presence does not usuallydistort or displace other intranuclear structures orthe nuclear membrane. However, when theMarinesco bodies are large or numerous they maydisplace the principal nucleolus to one side. Thenucleoplasm of many of these cells is rarefied but thisappearance may be artefactual.Most of the cells in the substantia nigra cells which

contained Marinesco bodies possess two nucleoli.The presence of such double nucleolated cells in the

3LE I

NUMBERS OF MARINESCO BODIES IN SUBSTANTIA NIGRA IN CASES OF PRESENT SERIES

Age Group O=No Marinesco 0 =1- 5 Marinesco 6-10 Mrinesco _Over 10 Morinesco

0-5 00000000000000006-10 0000011-15 0°°16-20 00000l21-25 026-30 000 00031-35 00 @@@ 036-40 OO@OO@) 0

41-46 00 00)46-50 @@oooo@ 0

51-55 cOca @0

56-60 0 0§ 0§ 0s0*-61-65 ©O00@@@@ 06)000*66-70 0 O©@O@@ @0000000 0071-75 0 @@@@ @@ @e00 0 076-80 Go @00

81-85 c)oo ( @0*-86-90 0 0 @091-95 O 0 0 00_

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P. Yuen and D. W. Baxter

TABLE IICAUSE OF DEATH IN SUBJECTS AGED 66 TO 70 YEARS EXAMINED FOR MARINESCO BODIES

Age (yr.) Sex Cause of Death No. of Marinesco Bodies per350-450 Substantia NigraCells

66 M Duodenal ulcer, suppurative gastritis and oesophagitis67 M Coronary thrombosis (sudden death)66 M Carcinoma of stomach, peritonitis70 M Hodgkin's disease67 F Astrocytoma65 M Myelogenous leukaemia69 M Calcific aortic stenosis, coronary thrombosis69 M Coronary thrombosis68 M Brain-stem infarction69 F Head injury (prolonged period of coma)65 F Cerebral air emboli during trigeminal neurectomy70 M Portal cirrhosis68 M Cerebral infarction68 M Head injury (survived 15 days)65 M ? Coronary thrombosis (sudden death)66 M Duodenal and gastric ulcers, massive haematemesis68 M Carcinoma of rectum66 M Haemachro.natosis

6-106-101-56-106-10Over 101-5Over 101-51-51-51-56-10Over 101-51-5None6-10

.

FIG. 1. Typical Marinesco bodies within the nuclei of substantia nigra cells. x 256.

nucleus substantia nigra is neither uncommon norabnormal. These double nucleoli are always dis-similar in size, the smaller being one-quarter to one-third the size of the larger. The smaller nucleoluscan readily be distinguished from the Marinescobodies by its basophilia and the presence of one ormore nucleolar vacuoles (Fig. 2). The Marinescobodies commonly lie in association with, andappear to be derived from, the nucleoli. This isparticularly true of the smaller nucleolus. TwoMarinesco bodies, equal or unequal in size, areoften found lying on either side of the smallernucleolus (Fig. 3). In other cells the smallernucleolus appears to form a basophilic centre of aMarinesco body (Fig. 4). Many of the smallernucleoli which appear to be giving rise to Marinescobodies show morphological changes in the form of

FIG. 2. Substantia nigra cell containing two nucleoli. NoMarinesco bodies are seen. x 256.

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FIG. 3. Cells fromthe substantia nigrashowing the closerelationship whichMarinesco bodiesqften bear to thesecondary nucleolus.x 256.

FIG. 4. Cell from substantia nigrain which the secondary nucleolusforms the basophilic centre of aMarinesco body. x 256.

9FIG. 5.Cells fromsubstantianigracontaining* unusuallylarge singleMarinescobodies.x 256.

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TABLE IIISTAINING PROPERTIES OF MARINESCO BODIES

Fixative Nucleolus

Haematoxylin-eosinVan GiesonMasson trichromeMallory trichromeGomori chrome-alum-haematoxylin-phloxinePeriodic acid SchiffFeulgenGallocyaninToluidine blueCresyl violetHeidenhainSudan IVSudan blackDanielli's stain for amino-acidsFast green stains for histonesVon Kossa stain for CaGomori stain for FeFontana silver stain

FormalinFormalinFormalinFormalinBouinFormalinCarnoyCarnoyFormalinFormalinFormalinFormalin (frozen)Formalin (frozen)AlcoholNeutral formalinFormalinFormalinFormalin

hypertrophy, hyperchromatism, or vacuolization.Vacuolization may progress to the point where onlya ghost-like remnant of the smaller nucleolus can

be identified. Occasional cells are seen in whichthe nucleus contains a single, large nucleolus andone or more Marinesco bodies, which usually liefree and rarely in close association with the nucleo-lus. The foregoing observations have led us tosuspect that the free Marinesco bodies of suchcells have been derived from a secondary nucleoluswhich has either not been included on the tissuesection or has degenerated to a degree that it cannotbe identified. We do not, of course, exclude thepossibility that Marinesco bodies may occasionallybe found in cells with a single nucleolus.There was no correlation between the presence of

Marinesco bodies and the presence or degree ofmelanin pigmentation. Melanin accumulates rapidlyin the cells of the substantia nigra between the age of2 and 20 years and no Marinesco bodies could bedemonstrated in patients of this age group. The cellsof older patients containing Marinesco bodiesshowed a normal degree of pigmentation. Occasionalcells which contain little or no melanin are normallyfound in the adult substantia nigra. Several suchcells with Marinesco bodies in their nuclei were seen.

Marinesco bodies were not seen in the cytoplasm ofcells of the substantia nigra or in any extracellularlocation.The brain-stems of two patients with paralysis

agitans were examined. In each case, both intranu-clear Marinesco bodies and intracytoplasmic Lewybodies were demonstrated in cells of the substantianigra. The Marinesco bodies were no more numerousor larger in these cases than those found in patientsof comparable age not suffering from Parkinson'sdisease. Incidentally, Lewy bodies were not demon-strated in any of the remaining 158 patients.

Marinesco bodies could also be demonstrated inthe cells of the nucleus locus caeruleus. There theywere much more difficult to find than in the sub-stantia nigra of the same brain-stem. This may,however, only be a reflection of the relative ease

with which the two nuclei can be examined.The staining properties of the Marinesco bodies

are detailed in Table III.

DISCUSSION

The presence of intranuclear or cytoplasmic in-clusion bodies is usually considered to be of patho-logical significance. They are known, for example,to appear in association with many viral infections.Electron microscopic studies have shown that theinclusion bodies in cells infected by certain viruses,such as vaccinia and influenza, are formed byaggregates of virus particles. Such inclusions can

also be shown to contain nucleoproteins (Bethlem,1961). On the other hand, there is considerableevidence that inclusion bodies are not necessarilyassociated with virus infections. It is well known thatinclusions can be induced in the cells of certaintissues by exposure to a variety of chemicals(Wolman, 1954), including lead, bismuth, aluminium,and colchicine. Such inclusions are presumablyformed as a consequence of abnormal metabolicactivity in the affected cells. Even the inclusions ofsome known virus infections may reflect abnormalcellular metabolic activity. Bethlem (1961) citesevidence that the Negri bodies of rabies can neitherbe shown to contain virus particles by electronmicroscopy nor nucleoproteins by histochemicaltechniques. Considerable doubt has also been caston the significance of a positive Feulgen reaction as

evidence that certain inclusions are formed by viralaggregates (Bracken, Beaver, and Randall, 1958).

Stain

Deep purpleDark brownDark blueDark blue or redRedUnstainedUnstainedBlueBlueVioletBlackNegativeNegativePositiveNegativeNegativeNegativeBlack

Marinesco Bodies

PinkBrick redLight pinkRed or purpleRedUnstainedUnstainedFaint greyFaint greenUnstainedGreyNegativeNegativePositiveNegativeNegativeNegativeBlack

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The nmorphology of Marinesco bodies in the melanin-pigmented nuclei of the brain-stem1

Marinesco bodies clearly represent a form ofintranuclear inclusion. Our observations suggestthat they are not of pathological significance sincethey are found in the pigmented nerve cells of patientsof widely varying age who died of a variety of diseaseprocesses. These facts would argue against theassociation of Marinesco bodies with virus diseaseor exogenous toxins. It is possible that Marinescobodies reflect an aging or involutional change in thepigmented neurones. If so, it is difficult to under-stand why they are not found with equal ease inneurones elsewhere in the nervous system. We havenot seen typical Marinesco bodies in any cellsexcept those of the substantia nigra and locuscaeruleus. It must be admitted, however, that wehave not searched any other nuclear groups asdiligently. Further, one or two of the illustrations inWolf and Orton's paper (1932) do suggest thatMarinesco bodies may be seen occasionally in non-pigmented cells. If this proves to be true, and ifMarinesco bodies do reflect an involutional change,the implication follows that the melanin-pigmentedcells may have a shorter life span than cells whichare free of this pigment. The fact that the degree of

cell loss in the substantia nigra often seems out ofproportion to the cell loss in other brain-stem nucleiin elderly patients who die of diseases unrelated tothe central nervous system offers support for thisassumption.

SUMMARY

Marinesco bodies are small, intranuclear droplet-like structures found within a proportion of cells ofthe nucleus substantia nigra and the nucleus locuscaeruleus of normal adults. Their presence is thoughtto reflect an involutional change in these cells.

REFERENCES

Bracken, E. C., Beaver, D. L., and Randall, C. C. (1958). J. Path.Bact., 75, 253.

Bethlem, J. (1961). In Virus Meningo-Encephalitis, Ciba FoundationStudy Group No. 7, edited by G. E. W. Wolstenholme andM. P. Cameron, p. 84. Churchill, London.

Greenfield, J. G. (1958). Neuropathology, pp. 8 and 177. Arnold,London.

Lipkin, L. E. (1959). Amer. J. Path., 35, 1117.Marinesco, G. (1902). C.R. Acad. Sci. (Paris), 135, 1000.

, (1909). La Cellule Nerveuse, vol. 1, pp. 195-198.Wolf, A., and Orton, S. T. (1932). Bull. neurol. Inst. N. Y., 2, 194.Wolman, M. (1954). J. Path. Bact., 68, 159.

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