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Neuronal vascular relationships in the zona compacta of normal and parkinsonian substantia nigra
BRAIN RESEARCH
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N E U R O N A L VASCULAR RELATIONSHIPS IN THE ZONA COMPACTA OF N O R M A L AND PARKINSONIAN SUBSTANTIA NIGRA
MARIETTA RADOVICH ISSIDORIDES Department of Biology, University of Athens, Athens (Greece)
(Accepted July 24th, 1970)
INTRODUCTION Although many studies have been concerned with the histological alterations of the substantia nigra in Parkinsonism, there is no general agreement as to which of the lesions observed in this area is the most significant~4, reflecting the pathogenesis of the disease. According to Hassler 15, who is also recently supported by Earle n, the lesions most constantly encountered are gliosis, depigmentation and loss of neurons primarily in the zona compacta. The preferential involvement of the zona compacta in idiopathic Parkinsonism was further established through biochemical investigations, which demonstrated a high dopamine content in the zona compacta of normal brain, actually twice that of the zona reticulata, and its great reduction in Parkinsonism 16. This deficiency in dopamine is considered highly specific for the disease. In the zona compacta of the substantia nigra the capillary network is much denser than in the zona reticulata 7. In the former area the capillaries lie curved in S-shapes between the numerous ganglion cells, while in the latter area they form a loose network in the white substance 7. Spielmeyer stressed the vascular system as an important factor in the pathogenesis of diseases of the central nervous system, certain regions being vulnerable to certain disease processes because of regional differences in blood supply 27. Since the Parkinsonian syndrome may occasionally appear without demonstrable changes in the substantia nigra 14, it was of interest to examine the zona compacta of normal humans and of patients who had died in the early stages of the disease in order to detect possible histological discrepancies, which could account for the faulty metabolism of this area, in the absence of the classical lesions of the disease. MATERIALAND METHODS Brain material of 10 patients with idiopathic Parkinsonism and of 5 healthy, age-matched controls was obtained at autopsy and fixed in 10% formalin. Paraffin Brain Research, 25 (1971) 289-299
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Fig. I. Control human substantia nigra neurons of the zona compacta. Melanin, scant Nissl bodies and unstained capillary with erythrocytes (upper right) are shown in this section stained with toluidine blue. < 2400. Fig. 2. Same as Fig. I, showing capillaries in cross-section. ~ 2400. Fig. 3. Control zona compacta. Melanin was bleached before staining with toluidine blue. Nissl material shows in the lower part and at the right margin of the cell. A capillary is seen in an almost intracellular position. ,~; 2400. Fig. 4. Same as Fig. 3. A narrow band of Nissl material is seen at the lower periphery and around the nucleus. Bleached 'melanin area' is free of Nissl bodies. × 2400. Fig. 5. Control neurons of zona compacta stained with sudan black B showing close investment of capillary on cell surface. ;< 2400. Fig. 6. Same as Fig. 5 showing capillary in contact with one entire side of the cell. x 2400. sections, 4 #m thick, were stained with toluidine blue, sudan G a b e ' s f u c h s i n p a r a l d e h y d e 11, a n d t h e g a l l o c y a n i n - c h r o m a l u m fied-permanganate
oxidation
Brain Research, 25 (1971) 289-299
s t e p , w h i c h is p a r t
of Gabe's
black B-erythrosin, method.
T h e acidi-
fuchsin paraldehyde
CAPILLARIES IN HUMAN SUBSTANTIA NIGRA
291
procedure, was used also with the other methods ~a above, when it was necessary to bleach the melanin granules of the nigral neurons before staining. Photomicrographs were taken under oil immersion with the Orthomat Leica attachment of the Leitz Ortholux microscope. OBSERVATIONS
The study of the control and of the Parkinsonian brains revealed distinct patterns of neuron-capillary associations. In sections of the zona compacta of the normal brains the nigral neurons were seen invested by one or more capillaries, which established very close contacts with the neuronal surface. Although the distribution of melanin, Nissl bodies and capillaries was clearly evident in toluidine blue stained sections (Figs. 1 and 2), it was found that previous treatment with acidified permanganate, used to bleach the melanin, intensified the staining of the Nissl material, the capillary walls and the red blood cells (Figs. 3, 4 and 12). Thus the visualization of the distribution and of the total amount of basophilic substance in the cells as well as that of the vascular relations was made easier. After bleaching no Nissl bodies were found in the areas of the neurons previously occupied by melanin; only dispersed, very fine basophilic granules were present. In the remaining area of the cytoplasm, bands of Nissl substance were seen around the nucleus and against the cellular membrane (Figs. 3, 4 and 12). In Fig. 3 the neuronal vascular contact is so intimate that the capillary appears to have an almost intracellular position. The cytoplasm around this area of contact is free of Nissl bodies. Fig. 4 shows a capillary tangential to the cell body and Fig. 12 a vessel attached to a neuronal process. In order to verify that the observed close contacts were not a preparation artifact, these sections were compared with similarly processed sections of the nucleus supraopticus (Fig. ll), where direct neuronal vascular contact is an established fact. After sudan black B-erythrosin, melanin in the normal nigral neuron retains its natural dark brown color since it is not sudanophilic. Erythrocytes and capillaries, however, are accentuated. They are seen squeezed between adjacent neurons (Figs. 5 and 6) or curved around neuronal surfaces (Fig. 7) where vascular wall and cell membrane appear fused without intervening elements. The same fusion is revealed by the fuchsin paraldehyde method (Figs. 8, 9 and 10). In the zona compacta of the substantia nigra of the Parkinsonian brains neuronal vascular contact was nowhere evident. Although numerous capillaries were found curved around the neurons, they were separated from the neuronal surface by a distinct space. The width of this space varied from barely visible to quite conspicuous and seemed to increase with duration of the disease when judged by this population of patients. Observations of several sections from the brains of patients deceased during the early stages of the disease indicated that, apart from alterations in the staining reactivity of the neurons and their melanosomes to be described below, the most striking feature was the changed relation between capillaries and nigral cells (Figs. 13, 14 and 17). In these brains, while no loss of melanin or other classical lesions were evident, a space between neurons and capillaries was always present. Brain Research, 25 (1971) 289-299
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Fig. 7. Same as Fig. 5, showing capillary wall fused to cell membrane. ,. 2400. Fig. 8. Control neuron from the zona compacta stained with fuchsin paraldehyde. Capillary wall fused to cell surface. ,: 2400. Fig. 9. Control neuron stained with fuchsin paraldehyde after oxidation showing cross-section of capillary and positive staining of the center of each bleached melanin granule. • 2400. Fig. 10. Same as Fig. 9. < 2400. Fig. I 1. Control nucleus supraopticus of the human stained with toluidine blue after oxidation showing neurosecretory neuron in contact with capillary (upper middle), x 2400. Fig. 12. Control nigral neuron bleached and stained with toluidine blue. Wall of capillary in crosssection is seen fused to neuronal process. × 2400.
In a d d i t i o n the capillaries a n d t h e e r y t h r o c y t e s did n o t h a v e t h e i r n o r m a l shape, d i m e n s i o n s a n d stainability. It was f u r t h e r o b s e r v e d t h a t in t h e b r a i n s o f p a t i e n t s h a v i n g suffered a l o n g e r d u r a t i o n o f t h e disease a n d s h o w i n g L e w y b o d i e s (Figs. 15 a n d 16) o r c o m p l e t e l y d e m e l a n i z e d n e u r o n s (Fig. 1 9 ) e x t r a n e u r o n a l p i g m e n t g r a n Brain Research, 25 (1971) 289-299
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Fig. 13. NigralVneuron in zona compacta of Parkinsonian brain showing clear space between cell surface and capillary curving around it, as well as extraneuronal pigment on the left of the cell. Fuchsin paraldehyde after oxidation, x 2400. Fig. 14. Same area and same method as in Fig. 13, but from a different patient. Notice wider space between cell surface and capillary wall. Fig. 15. Nigral neuron in the zona compacta of a case in late stage of Parkinsonism, showing Lewy bodies, and demelanized patches of cytoplasm. Notice capillary, facing the Lewy body, with pigment granules on its wall separated by a fairly wide space from the neuronal surface. Fuchsin paraldehyde after oxidation, x 2400. Fig. 16. Same as Fig. 15, but from a different patient. Three Lewy bodies and a demelanized cytoplasmic area are evident. Notice pigment granules in the 'empty' space between the semi-collapsed capillary and the indented surface of the neuron between the 2 smaller Lewy bodies, x 2400. Fig. 17. Nigral neuron in zona compacta from a case of recent Parkinsonism. Fuchsin paraldehyde after oxidation. This cell resembles most closely control nigral neurons (as in Fig. 9) in all respects including melanin stainability (see text) except for the existing space between neuronal surface and capillary on the left of the figure, z 2400. Fig. 18. Neuron from Parkinsonian zona compacta showing glial pigment very close to a rather healthy looking neuron. Several sudanophilic granules in the brown melanin mass are not evident in black and white photography. Sudan black B-erythrosin. x 2400.
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ules were present on the capillary wall (Fig. 15) or in the 'empty' space between neuronal membrane and capillary (Fig. 16). In other areas extraneuronal pigment was clearly associated with the numerous perineuronal glial nuclei (Fig. 18). The reactivity to stains of the pigment in the Parkinsonian brains could also be differentiated from that of the controls. In the control brains the fuchsin paraldehyde method revealed a component 0f the melanosome substratum which was stained by the dye only after the melanin was bleached by permanganate (Figs. 8, 9 and 10). This reaction is typical and characteristic of lipofuscin in other areas of the brain like the cortex, inferior olive, thalamus, etc. 19. However, in lipofuscin-containing neurons of the areas mentioned the method reveals an almost solid mass of granules with no space between them, while in the melanin-containing neurons which we studied fuchsin paraldehyde stained less material in the bleached 'melanin area'. A comparison can be made between the neurons in Figs. 5, 6 and 7, where melanin granules are unbleached and appear in their natural size and color, and the neurons in Figs. 8, 9 and 10 after oxidation and fuchsin paraldehyde staining, where each body in the 'melanin area' consists of a small positively stained core surrounded by a clear halo. In the nigral neurons of Parkinsonian patients deceased after a short duration of the disease (Figs. 13, 14 and 17) aldehyde fuchsin after oxidation stained more of the melanosome substratum than it did in the control nigral neurons (Figs. 9 and 10). This stain did not differentiate the intraneuronal from the extraneuronal pigment; both were equally stained. However, in sections stained with sudan black B we observed that the glial pigment was sudanophilic while the neuronal pigment remained unstained. In some neurons, especially those surrounded by many glial nuclei, sudanophilic granules were interspersed among the unstained brown melanin granules (Fig. 18). Finally a finding of considerable interest in the nigral neurons of Parkinsonian patients deceased during the early stages of the disease was the inverse relationship observed between melanin content and nucleic acid content. By bleaching sections with acidified permanganate and staining them with gallocyanin-chromalum it was possible to visualize in the same neuron both the 'melanin space', free of formed Nissl bodies, and the total basophilic material in the rest of the cell. The case chosen here to illustrate this finding was a patient with hemiparkinsonism, whose intact nigral zona compacta (Figs. 20-23) served as a control to his affected zona compacta (Figs. 24-27). Classical lesions were also absent in this patient. In the unaffected side nigral neurons after the above method had a typical appearance. The 'melanin area' was almost clear, containing a few fine dots of basophilic substance, Nissl bodies were scant and were found in a few places against the neuronal membrane (Figs. 20 and 23) and/or around the nuclear membrane (Figs. 21 and 23). On the affected side, neurons, although containing more basophilic material than the controls, did not present a typical or uniform appearance. For example, several neurons (Fig. 24) had a recognizable 'melanin area' of varying extent, but showed several coarse Nissl bodies along the periphery of the perikaryon and around the nucleus. In other neurons no distinct 'melanin area' could be identified (Fig. 25) although clear cytoplasmic spaces existed among the coarser, more numerous Nissl bodies which were distribBrain Research, 25 (1971) 289-299
CAPILLARIES IN HUMAN SUBSTANTIA NIGRA
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Fig. 19. Demelanized nigral neuron from a case of advanced Parkinsonism, showing extraneuronal pigment in the space between capillary and cell surface. Fuchsin paraldehyde after oxidation, x 2400. Fig. 20. Nigral neuron in zona compacta from the unaffected nigra of a case of hemiparkinsonism. Bleached and stained with gallocyanin, x 2400. Fig. 21. Same as Fig. 20. x 2400. Fig. 22. Same as Fig. 20. Basophilic substance concentrated at blunt end of the neuron and fine basophilic granules dispersed in the bleached melanin area. x 2400. Fig. 23. Same as Fig. 20 showing peripheral Nissl bodies in some places and nuclear cap of basophilic substance, x 2400. Fig. 24. Nigral neuron in zona compacta from the affected nigra of the patient with hemiparkinsonism of Fig. 20. Notice increased basophilia, peripheral Nissl bodies and restricted 'melanin area' at top corner of cell. Bleached and stained with gallocyanin. × 2400. Fig. 25. Same section as Fig. 24. This neuron shows increase in the number of Nissl bodies, few clear, bleached spaces and more diffuse basophilia. × 2400. Fig. 26. Same section as Fig. 24. This neuron shows still more Nissl material and more intense basophilia than the previous one and a small reduction in size. × 2400. Fig. 27. Same section as Fig. 24. Mostly intense diffuse basophilia is evident with no recognizable Nissl bodies due to the darkness of the staining. Great reduction in size is seen. x 2400.
Brain Research, 25 (1971) 289-299
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uted all over the perikaryon. Another group of neurons (Fig. 26) showed diffuse cytoplasmic and nuclear basophilia in addition to the increased number of Nissl bodies and lack of 'melanin areas'. In this group a slight reduction of neuronal size was also evident. Lastly another group of neurons (Fig. 27) showed almost exclusively intense, diffuse basophilia, which obscured the probable presence of Nissl bodies, and there was a distinct reduction in size. This classification in no way implies spatial groupings of the described neurons. It only facilitates the description of the appearance of the entire population of neurons in the affected zona compacta. DISCUSSION
Our observations indicate that the melanin-containing neurons of the zona compacta in the normal human brain have a close spatial relationship with the blood supply. The capillary walls of this highly vascularized area 7 were found to be fused with the membranes of neuronal perikarya and processes. Such neuronal vascular contacts, as seen here, are characteristic of the nuclei supraopticus and paraventricularis 26, where they are believed to indicate a high dependence of the cells on the blood supply 25 and to serve a chemoreceptor 8 or osmoreceptor 28 function of these neurons. That the neuronal vascular contact in the nigral neurons is effected with exclusion of glial processes is supported by Cammermeyer's observations 3 that neuronal cytoplasm next to neuronal vascular contacts remains free of basophilic deposits, while other parts of the cell periphery covered by glial processes have such deposits. The distribution of Nissl bodies in the capillary-invested nigral neurons in this study was consistent with Cammermeyer's observations. The cytoplasm next to neuronal vascular contacts was packed with melanin, while Nissl bodies were found only in the remaining parts of the cell periphery. In the Parkinsonian brain the normal contact between nigral neuron and capillary is lost. The causative factor appears to be the infiltration of glia between cell surface and capillary wall. The interpretation is based on our present observations and on other reports in the literature. Intense gliosis is characteristic of the zona compacta in Parkinsonism 24 and is especially dense around blood vesselsXL Reserpine, which can produce a Parkinsonian syndrome 1, was shown to cause glial proliferation 21. In our material whenever perineuronal gliosis was evident in the plane of the section (Fig. 18), conspicuous pigment granules were seen investing these glial nuclei. The 'empty' space between neuron and capillary was usually occupied by extraneuronal pigment granules, which in Parkinsonism are known to be contained in the glia 1°. Thus the inference is justified that the 'empty' space actually represents unstained glial processes. The fact that melanin granules stain to a small extent in the control nigral neurons with fuchsin paraldehyde supports the findings of Moses 2° and Barden 2, which indicate that melanin contains a lipofuscin component. The almost total staining of the Parkinsonian melanosomes by the above method lends support from the histochemical level to the EM findings of Duffy and Tennyson 5 that the substructure of the 'altered melanin granules' of the Parkinsonian patients resembles Brain Research, 25 (1971) 289-299
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that of lipofuscin. At this stage it is not clear whether the change in stainability of the Parkinsonian melanosomes indicates an active shift in metabolism from melanogenesis to lipofuscinogenesis or an arrest of melanogenesis uncovering passively a previously masked lipofuscin substratum. Support for the first alternative comes from the fact that permanganate oxidation, which (a) depolymerizes melanin and (b) makes lipofuscin stainable with fuchsin paraldehyde, revealed a dot-like center in the normal nigral pigment granules, while the same procedure stained the entire area of the pigment granules in the Parkinsonian nigra. This speaks for the presence of a larger amount of lipofuscin component in the diseased neuron, which could easily be the wrong substratum for melanin deposition. While the glia in the normal substantia nigra never contains pigment 10 as we have also verified in our controls, glial pigment reacting like lipofuscin with sudan black B and fuchsin paraldehyde was present in the Parkinsonian material. Since melanin granules are not sudanophilic, the glial pigment in Parkinsonism cannot result only from phagocytosed neuronal remnants, as commonly held, especially in the patients where no loss of neurons is evident. It may represent a new product of abnormal glial synthesis for this area. The presence of sudanophilic lipofuscin granules among the melanin granules of the other cases indicates a gradual and progressive shift in metabolism occurring in the nigral neuron. Friede 9 has shown that the melanin-containing neurons of the substantia nigra are devoid of oxidative enzymes while other neurons possess a high content of oxidative enzymes related to a high content of lipofuscin. Therefore the gradual accumulation of lipofuscin components in the glia and nigral neurons of Parkinsonian patients can be considered a marker of some more profound and basic metabolic alteration. That such alteration is active is clearly evident when we consider the changes in basophilia of the Parkinsonian neurons. In the present study the difficulty in determining cytoplasmic basophilia due to overlying melanin granules, as noted by Pakkenberg 22, was circumvented by bleaching the melanin prior to application of the gallocyanin method. The observed increase in Nissl bodies, diffuse cytoplasmic and nuclear basophilia, and reduction in the size of the Parkinsonian nigral neurons were the counterpart of the changes induced by reserpine in rat sympathetic gangliala, 21. These changes are thus interpreted as an expression of reduced firing activity. Since high melanin content parallels function in the nigral neurons, the decrease in melanin and increase in nucleic acids in the Parkinsonian neurons is additional evidence for a deviation of the normal metabolism12 towards non-functional syntheses, which probably antagonize the normal syntheses of these neurons for specific carrier proteins or transmitter production. It is proposed that the close neuronal vascular contact found in the zona compacta of the normal brains must be of importance for the functional metabolism of this area since the interruption of this contact was the only detectable lesion in several patients of idiopathic Parkinsonism. The associated histochemical changes of glia and melanin-containing neurons observed in the Parkinsonian zona compacta support the close metabolic interaction between the two units 17 and emphasize the primary role played by the glial elements in regulatory and barrier mechanisms of the brain 4. Brain Research, 25 (1971) 289-299
298
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SUMMARY
O b s e r v a t i o n s were m a d e on neurons o f the substantia nigra in 10 cases o f i d i o p a t h i c P a r k i n s o n i s m and 5 age-matched controls. It was found that the melaninc o n t a i n i n g neurons o f the zona c o m p a c t a in the n o r m a l brain have a close spatial relationship with the blood circulation. The capillary walls a p p e a r fused to the membranes o f n e u r o n a l p e r i k a r y a and processes. In the P a r k i n s o n i a n zona c o m p a c t a the close c o n t a c t between nigral neuron and capillary is lost. The findings suggest t h a t this is due to the infiltration o f the proliferated glia between cell surface and capillary wall. This neuronal vascular contact in the zona c o m p a c t a o f the n o r m a l brain m u s t be o f i m p o r t a n c e for the functional m e t a b o l i s m o f this area, since interr u p t i o n o f this c o n t a c t was the only detectable nigral lesion in several patients with overt s y m p t o m s o f recent date. Histochemical reactions o f n o r m a l a n d P a r k i n s o n i a n m e l a n i n - c o n t a i n i n g neurons showed t h a t the nigral cells o f the patients have a larger a m o u n t o f lipofuscin c o m p o n e n t in the melanin a r e a t h a n those o f the controls. Increased b a s o p h i l i a was observed in the P a r k i n s o n i a n neurons and was inversely related to their melanin content. It is interpreted as an expression o f reduced firing activity, indicating regression o f function. ACKNOWLEDGEMENTS
This w o r k was s u p p o r t e d by G r a n t No. 733 from The R o y a l Hellenic Research Foundation. The a u t h o r wishes to express her t h a n k s to Dr. J. C o n s t a n t i n i d i s o f the Clinique Psychiatrique Universitaire, Bel Air, G e n e v a for supplying the P a r k i n s o n i a n material.
REFERENCES 1 AYD, F. J., JR., A survey of drug-induced extrapyramidal reactions, J. Amer. rned. Ass., 175 (1961) 1054-1060. 2 BARDEN, H., The histochemical relationship of neuromelanin and lipofuscin, J. Neuropath. exp. Neurol., 28 (1969) 419-441. 3 CAMMERMEYER,J., Differential response of two neuron types to facial nerve transection in young and old rabbits, J. Neuropath. exp. Neurol., 22 (1963) 594-616. 4 CASTAN, P., Les fonctions m6taboliques de l'astroglie c6r6brale, 616ment fondamental de la barri6re h6mato-enckphalique, J. neurol. Sci., 6 (1968) 237-248. 5 DUFFY, P. E., AND TENNYSON, V. M., Phase and electron microscopic observation of Lewy bodies and melanin granules in the substantia nigra and locus coeruleus in Parkinson's disease, J. Neuropath. exp. Neurol., 24 (1965) 398-414. 6 EARLE,K. M., Studies on Parkinson's disease including X-ray fluorescent spectroscopy of formalin fixed brain tissue, J. Neuropath. exp. Neurol., 27 (1968) 1-14. 7 FINLEY,K. H., Angio-architecture of the substantia nigra and its pathogenic significance, Arch. Neurol. Psychiat. (Chic.), 36 (1936) 118-127. 8 FINLEY, K. H., The capillary bed of the paraventricular and supraoptic nuclei of the hypothalamus, Res. Publ. Ass. nerv. merit. Dis., 18 (1938) 94-109. 9 FRIEOE,R. L., The relation of the formation of lipofuscin to the distribution of oxidative enzymes in the human brain, Acta neuropath. (BerL), 2 (1962) 113-125. Brain Research, 25 (1971) 289-299
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10 FRIEDE,R. L., Topographic Brain Chemistry, Academic Press, New York, 1966, p. 322. 11 GABE, M., Sur quelques applications de la coloration par la fuchsine parald6hyde, Bull. MicE. appl., 3 (1953) 153-162. 12 GOMIRATO,G., AND HYD~N, H., A biochemical glia error in the Parkinson disease, Brain, 86 (1963) 773-780. 13 GREENFIELD,J. D., System degenerations in the cerebellum, brain stem and spinal cord. In W. BLACKWOOD,W. H. MCMENEMEY,A. MEYER, R. M. NORMANAND D. S. RUSSELL(Eds.), Greenfield's Neuropathology, Edward Arnold, London, 1963, pp. 581-601. 14 GYBELS,J. M., The Neural Mechanism of Parkinsonian Tremor, Arscia, Brussels, 1963. 15 HASSLER,R., Zur Pathologie dee Paralysis agitans und des postenzephalitischen Parkinsonismus, J. PsychoL Neurol. (Lpz.), 48 (1938) 387-476. 16 HORNYKIEWICZ,O., Die topische Lokalisation und das Verhalten yon Noradrenalin und Dopamin (5-Hydroxytyramin) in der Substantia nigra des normalen und Parkinson-kranken Menschen, Wien. klin. Wschr., 75 (1963) 309-312. 17 HYD~N, H., Dynamic aspects of the neuron-glia relationship. In H. HYD~N (Ed.), The Neuron, Elsevier, Amsterdam, 1967, pp. 179-219. 18 ISSIDORIDES,M. R., AND MYTIL1NEOU'-PRoVELEGIOS,C., Effect of cl'doramphenicol on reserpine treated sympathetic ganglia of the rat, Biol. Psychiat., in press. 19 ISSIDORIDES,M. R., AND SHANKLIN,W. M., Histochemieal reactions of cellular inclusions in the human neurone, J. Anat. (Lond.), 95 (1961) 151-159. 20 MOSES,H. C., GANOTE,C. E., BEAVER,D. L., AND SCHUrEEMAN,S. S., Light and electron microscopic studies of pigment in human and rhesus monkey substantia nigra and locus coeruleus, Anat. Rec., 155 (1966) 155-167. 21 MYTILINEOU,C., Histochemical alterations of sympathetic ganglia of the rat after reserpine administration, BioL Psychiat., 1 (1969) 61-72. 22 PAKKENBERG,H., The pigment in substantia nigra in Parkinsonism. Microspeetrophotometric comparison with other sources of human pigment, Brain Research, 2 (1966) 173-180. 23 PEARSE,A G. E., Histochemistry TheoreticalandApplied, Churchill, London, 1961, pp. 826, 834, 850. 24 RICHARDSON,E. P., Remarks on the pathology of Parkinson's disease. In A. BARBEAU,L. J. DOSHAYAND E. A. SPIEGEL(Eds.), Parkinson's Disease Trends in Research and Treatment, Grune and Stratton, New York, 1965, pp. 63-68. 25 SCHARRER,E., The blood vessels of the nervous tissue, Quart. Rev. Biol., 19 (1944) 308-318. 26 SCHARRER,E., UND GAUPP, R., Neuere Befunde am Nucleus supraopticus und Nucleus paraventricularis des Menschen, Z. Neurol., 148 (1933) 766-772. 27 SPIELMEYER,W., The significance of local factors for the electivity in central nervous system disease processes, Medicine (Baltimore), 10 (1931) 243-256. 28 VERNEV,E. B., Agents determining and influencing the functions of the pars nervosa of the pituitary, Brit. reed. J., 2 (1948) 119-123.
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N E U R O N A L VASCULAR RELATIONSHIPS IN THE ZONA COMPACTA OF N O R M A L AND PARKINSONIAN SUBSTANTIA NIGRA
MARIETTA RADOVICH ISSIDORIDES Department of Biology, University of Athens, Athens (Greece)
(Accepted July 24th, 1970)
INTRODUCTION Although many studies have been concerned with the histological alterations of the substantia nigra in Parkinsonism, there is no general agreement as to which of the lesions observed in this area is the most significant~4, reflecting the pathogenesis of the disease. According to Hassler 15, who is also recently supported by Earle n, the lesions most constantly encountered are gliosis, depigmentation and loss of neurons primarily in the zona compacta. The preferential involvement of the zona compacta in idiopathic Parkinsonism was further established through biochemical investigations, which demonstrated a high dopamine content in the zona compacta of normal brain, actually twice that of the zona reticulata, and its great reduction in Parkinsonism 16. This deficiency in dopamine is considered highly specific for the disease. In the zona compacta of the substantia nigra the capillary network is much denser than in the zona reticulata 7. In the former area the capillaries lie curved in S-shapes between the numerous ganglion cells, while in the latter area they form a loose network in the white substance 7. Spielmeyer stressed the vascular system as an important factor in the pathogenesis of diseases of the central nervous system, certain regions being vulnerable to certain disease processes because of regional differences in blood supply 27. Since the Parkinsonian syndrome may occasionally appear without demonstrable changes in the substantia nigra 14, it was of interest to examine the zona compacta of normal humans and of patients who had died in the early stages of the disease in order to detect possible histological discrepancies, which could account for the faulty metabolism of this area, in the absence of the classical lesions of the disease. MATERIALAND METHODS Brain material of 10 patients with idiopathic Parkinsonism and of 5 healthy, age-matched controls was obtained at autopsy and fixed in 10% formalin. Paraffin Brain Research, 25 (1971) 289-299
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Fig. I. Control human substantia nigra neurons of the zona compacta. Melanin, scant Nissl bodies and unstained capillary with erythrocytes (upper right) are shown in this section stained with toluidine blue. < 2400. Fig. 2. Same as Fig. I, showing capillaries in cross-section. ~ 2400. Fig. 3. Control zona compacta. Melanin was bleached before staining with toluidine blue. Nissl material shows in the lower part and at the right margin of the cell. A capillary is seen in an almost intracellular position. ,~; 2400. Fig. 4. Same as Fig. 3. A narrow band of Nissl material is seen at the lower periphery and around the nucleus. Bleached 'melanin area' is free of Nissl bodies. × 2400. Fig. 5. Control neurons of zona compacta stained with sudan black B showing close investment of capillary on cell surface. ;< 2400. Fig. 6. Same as Fig. 5 showing capillary in contact with one entire side of the cell. x 2400. sections, 4 #m thick, were stained with toluidine blue, sudan G a b e ' s f u c h s i n p a r a l d e h y d e 11, a n d t h e g a l l o c y a n i n - c h r o m a l u m fied-permanganate
oxidation
Brain Research, 25 (1971) 289-299
s t e p , w h i c h is p a r t
of Gabe's
black B-erythrosin, method.
T h e acidi-
fuchsin paraldehyde
CAPILLARIES IN HUMAN SUBSTANTIA NIGRA
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procedure, was used also with the other methods ~a above, when it was necessary to bleach the melanin granules of the nigral neurons before staining. Photomicrographs were taken under oil immersion with the Orthomat Leica attachment of the Leitz Ortholux microscope. OBSERVATIONS
The study of the control and of the Parkinsonian brains revealed distinct patterns of neuron-capillary associations. In sections of the zona compacta of the normal brains the nigral neurons were seen invested by one or more capillaries, which established very close contacts with the neuronal surface. Although the distribution of melanin, Nissl bodies and capillaries was clearly evident in toluidine blue stained sections (Figs. 1 and 2), it was found that previous treatment with acidified permanganate, used to bleach the melanin, intensified the staining of the Nissl material, the capillary walls and the red blood cells (Figs. 3, 4 and 12). Thus the visualization of the distribution and of the total amount of basophilic substance in the cells as well as that of the vascular relations was made easier. After bleaching no Nissl bodies were found in the areas of the neurons previously occupied by melanin; only dispersed, very fine basophilic granules were present. In the remaining area of the cytoplasm, bands of Nissl substance were seen around the nucleus and against the cellular membrane (Figs. 3, 4 and 12). In Fig. 3 the neuronal vascular contact is so intimate that the capillary appears to have an almost intracellular position. The cytoplasm around this area of contact is free of Nissl bodies. Fig. 4 shows a capillary tangential to the cell body and Fig. 12 a vessel attached to a neuronal process. In order to verify that the observed close contacts were not a preparation artifact, these sections were compared with similarly processed sections of the nucleus supraopticus (Fig. ll), where direct neuronal vascular contact is an established fact. After sudan black B-erythrosin, melanin in the normal nigral neuron retains its natural dark brown color since it is not sudanophilic. Erythrocytes and capillaries, however, are accentuated. They are seen squeezed between adjacent neurons (Figs. 5 and 6) or curved around neuronal surfaces (Fig. 7) where vascular wall and cell membrane appear fused without intervening elements. The same fusion is revealed by the fuchsin paraldehyde method (Figs. 8, 9 and 10). In the zona compacta of the substantia nigra of the Parkinsonian brains neuronal vascular contact was nowhere evident. Although numerous capillaries were found curved around the neurons, they were separated from the neuronal surface by a distinct space. The width of this space varied from barely visible to quite conspicuous and seemed to increase with duration of the disease when judged by this population of patients. Observations of several sections from the brains of patients deceased during the early stages of the disease indicated that, apart from alterations in the staining reactivity of the neurons and their melanosomes to be described below, the most striking feature was the changed relation between capillaries and nigral cells (Figs. 13, 14 and 17). In these brains, while no loss of melanin or other classical lesions were evident, a space between neurons and capillaries was always present. Brain Research, 25 (1971) 289-299
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Fig. 7. Same as Fig. 5, showing capillary wall fused to cell membrane. ,. 2400. Fig. 8. Control neuron from the zona compacta stained with fuchsin paraldehyde. Capillary wall fused to cell surface. ,: 2400. Fig. 9. Control neuron stained with fuchsin paraldehyde after oxidation showing cross-section of capillary and positive staining of the center of each bleached melanin granule. • 2400. Fig. 10. Same as Fig. 9. < 2400. Fig. I 1. Control nucleus supraopticus of the human stained with toluidine blue after oxidation showing neurosecretory neuron in contact with capillary (upper middle), x 2400. Fig. 12. Control nigral neuron bleached and stained with toluidine blue. Wall of capillary in crosssection is seen fused to neuronal process. × 2400.
In a d d i t i o n the capillaries a n d t h e e r y t h r o c y t e s did n o t h a v e t h e i r n o r m a l shape, d i m e n s i o n s a n d stainability. It was f u r t h e r o b s e r v e d t h a t in t h e b r a i n s o f p a t i e n t s h a v i n g suffered a l o n g e r d u r a t i o n o f t h e disease a n d s h o w i n g L e w y b o d i e s (Figs. 15 a n d 16) o r c o m p l e t e l y d e m e l a n i z e d n e u r o n s (Fig. 1 9 ) e x t r a n e u r o n a l p i g m e n t g r a n Brain Research, 25 (1971) 289-299
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Fig. 13. NigralVneuron in zona compacta of Parkinsonian brain showing clear space between cell surface and capillary curving around it, as well as extraneuronal pigment on the left of the cell. Fuchsin paraldehyde after oxidation, x 2400. Fig. 14. Same area and same method as in Fig. 13, but from a different patient. Notice wider space between cell surface and capillary wall. Fig. 15. Nigral neuron in the zona compacta of a case in late stage of Parkinsonism, showing Lewy bodies, and demelanized patches of cytoplasm. Notice capillary, facing the Lewy body, with pigment granules on its wall separated by a fairly wide space from the neuronal surface. Fuchsin paraldehyde after oxidation, x 2400. Fig. 16. Same as Fig. 15, but from a different patient. Three Lewy bodies and a demelanized cytoplasmic area are evident. Notice pigment granules in the 'empty' space between the semi-collapsed capillary and the indented surface of the neuron between the 2 smaller Lewy bodies, x 2400. Fig. 17. Nigral neuron in zona compacta from a case of recent Parkinsonism. Fuchsin paraldehyde after oxidation. This cell resembles most closely control nigral neurons (as in Fig. 9) in all respects including melanin stainability (see text) except for the existing space between neuronal surface and capillary on the left of the figure, z 2400. Fig. 18. Neuron from Parkinsonian zona compacta showing glial pigment very close to a rather healthy looking neuron. Several sudanophilic granules in the brown melanin mass are not evident in black and white photography. Sudan black B-erythrosin. x 2400.
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ules were present on the capillary wall (Fig. 15) or in the 'empty' space between neuronal membrane and capillary (Fig. 16). In other areas extraneuronal pigment was clearly associated with the numerous perineuronal glial nuclei (Fig. 18). The reactivity to stains of the pigment in the Parkinsonian brains could also be differentiated from that of the controls. In the control brains the fuchsin paraldehyde method revealed a component 0f the melanosome substratum which was stained by the dye only after the melanin was bleached by permanganate (Figs. 8, 9 and 10). This reaction is typical and characteristic of lipofuscin in other areas of the brain like the cortex, inferior olive, thalamus, etc. 19. However, in lipofuscin-containing neurons of the areas mentioned the method reveals an almost solid mass of granules with no space between them, while in the melanin-containing neurons which we studied fuchsin paraldehyde stained less material in the bleached 'melanin area'. A comparison can be made between the neurons in Figs. 5, 6 and 7, where melanin granules are unbleached and appear in their natural size and color, and the neurons in Figs. 8, 9 and 10 after oxidation and fuchsin paraldehyde staining, where each body in the 'melanin area' consists of a small positively stained core surrounded by a clear halo. In the nigral neurons of Parkinsonian patients deceased after a short duration of the disease (Figs. 13, 14 and 17) aldehyde fuchsin after oxidation stained more of the melanosome substratum than it did in the control nigral neurons (Figs. 9 and 10). This stain did not differentiate the intraneuronal from the extraneuronal pigment; both were equally stained. However, in sections stained with sudan black B we observed that the glial pigment was sudanophilic while the neuronal pigment remained unstained. In some neurons, especially those surrounded by many glial nuclei, sudanophilic granules were interspersed among the unstained brown melanin granules (Fig. 18). Finally a finding of considerable interest in the nigral neurons of Parkinsonian patients deceased during the early stages of the disease was the inverse relationship observed between melanin content and nucleic acid content. By bleaching sections with acidified permanganate and staining them with gallocyanin-chromalum it was possible to visualize in the same neuron both the 'melanin space', free of formed Nissl bodies, and the total basophilic material in the rest of the cell. The case chosen here to illustrate this finding was a patient with hemiparkinsonism, whose intact nigral zona compacta (Figs. 20-23) served as a control to his affected zona compacta (Figs. 24-27). Classical lesions were also absent in this patient. In the unaffected side nigral neurons after the above method had a typical appearance. The 'melanin area' was almost clear, containing a few fine dots of basophilic substance, Nissl bodies were scant and were found in a few places against the neuronal membrane (Figs. 20 and 23) and/or around the nuclear membrane (Figs. 21 and 23). On the affected side, neurons, although containing more basophilic material than the controls, did not present a typical or uniform appearance. For example, several neurons (Fig. 24) had a recognizable 'melanin area' of varying extent, but showed several coarse Nissl bodies along the periphery of the perikaryon and around the nucleus. In other neurons no distinct 'melanin area' could be identified (Fig. 25) although clear cytoplasmic spaces existed among the coarser, more numerous Nissl bodies which were distribBrain Research, 25 (1971) 289-299
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'i
.
.
.
.
.
.
.
,7t
Fig. 19. Demelanized nigral neuron from a case of advanced Parkinsonism, showing extraneuronal pigment in the space between capillary and cell surface. Fuchsin paraldehyde after oxidation, x 2400. Fig. 20. Nigral neuron in zona compacta from the unaffected nigra of a case of hemiparkinsonism. Bleached and stained with gallocyanin, x 2400. Fig. 21. Same as Fig. 20. x 2400. Fig. 22. Same as Fig. 20. Basophilic substance concentrated at blunt end of the neuron and fine basophilic granules dispersed in the bleached melanin area. x 2400. Fig. 23. Same as Fig. 20 showing peripheral Nissl bodies in some places and nuclear cap of basophilic substance, x 2400. Fig. 24. Nigral neuron in zona compacta from the affected nigra of the patient with hemiparkinsonism of Fig. 20. Notice increased basophilia, peripheral Nissl bodies and restricted 'melanin area' at top corner of cell. Bleached and stained with gallocyanin. × 2400. Fig. 25. Same section as Fig. 24. This neuron shows increase in the number of Nissl bodies, few clear, bleached spaces and more diffuse basophilia. × 2400. Fig. 26. Same section as Fig. 24. This neuron shows still more Nissl material and more intense basophilia than the previous one and a small reduction in size. × 2400. Fig. 27. Same section as Fig. 24. Mostly intense diffuse basophilia is evident with no recognizable Nissl bodies due to the darkness of the staining. Great reduction in size is seen. x 2400.
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uted all over the perikaryon. Another group of neurons (Fig. 26) showed diffuse cytoplasmic and nuclear basophilia in addition to the increased number of Nissl bodies and lack of 'melanin areas'. In this group a slight reduction of neuronal size was also evident. Lastly another group of neurons (Fig. 27) showed almost exclusively intense, diffuse basophilia, which obscured the probable presence of Nissl bodies, and there was a distinct reduction in size. This classification in no way implies spatial groupings of the described neurons. It only facilitates the description of the appearance of the entire population of neurons in the affected zona compacta. DISCUSSION
Our observations indicate that the melanin-containing neurons of the zona compacta in the normal human brain have a close spatial relationship with the blood supply. The capillary walls of this highly vascularized area 7 were found to be fused with the membranes of neuronal perikarya and processes. Such neuronal vascular contacts, as seen here, are characteristic of the nuclei supraopticus and paraventricularis 26, where they are believed to indicate a high dependence of the cells on the blood supply 25 and to serve a chemoreceptor 8 or osmoreceptor 28 function of these neurons. That the neuronal vascular contact in the nigral neurons is effected with exclusion of glial processes is supported by Cammermeyer's observations 3 that neuronal cytoplasm next to neuronal vascular contacts remains free of basophilic deposits, while other parts of the cell periphery covered by glial processes have such deposits. The distribution of Nissl bodies in the capillary-invested nigral neurons in this study was consistent with Cammermeyer's observations. The cytoplasm next to neuronal vascular contacts was packed with melanin, while Nissl bodies were found only in the remaining parts of the cell periphery. In the Parkinsonian brain the normal contact between nigral neuron and capillary is lost. The causative factor appears to be the infiltration of glia between cell surface and capillary wall. The interpretation is based on our present observations and on other reports in the literature. Intense gliosis is characteristic of the zona compacta in Parkinsonism 24 and is especially dense around blood vesselsXL Reserpine, which can produce a Parkinsonian syndrome 1, was shown to cause glial proliferation 21. In our material whenever perineuronal gliosis was evident in the plane of the section (Fig. 18), conspicuous pigment granules were seen investing these glial nuclei. The 'empty' space between neuron and capillary was usually occupied by extraneuronal pigment granules, which in Parkinsonism are known to be contained in the glia 1°. Thus the inference is justified that the 'empty' space actually represents unstained glial processes. The fact that melanin granules stain to a small extent in the control nigral neurons with fuchsin paraldehyde supports the findings of Moses 2° and Barden 2, which indicate that melanin contains a lipofuscin component. The almost total staining of the Parkinsonian melanosomes by the above method lends support from the histochemical level to the EM findings of Duffy and Tennyson 5 that the substructure of the 'altered melanin granules' of the Parkinsonian patients resembles Brain Research, 25 (1971) 289-299
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that of lipofuscin. At this stage it is not clear whether the change in stainability of the Parkinsonian melanosomes indicates an active shift in metabolism from melanogenesis to lipofuscinogenesis or an arrest of melanogenesis uncovering passively a previously masked lipofuscin substratum. Support for the first alternative comes from the fact that permanganate oxidation, which (a) depolymerizes melanin and (b) makes lipofuscin stainable with fuchsin paraldehyde, revealed a dot-like center in the normal nigral pigment granules, while the same procedure stained the entire area of the pigment granules in the Parkinsonian nigra. This speaks for the presence of a larger amount of lipofuscin component in the diseased neuron, which could easily be the wrong substratum for melanin deposition. While the glia in the normal substantia nigra never contains pigment 10 as we have also verified in our controls, glial pigment reacting like lipofuscin with sudan black B and fuchsin paraldehyde was present in the Parkinsonian material. Since melanin granules are not sudanophilic, the glial pigment in Parkinsonism cannot result only from phagocytosed neuronal remnants, as commonly held, especially in the patients where no loss of neurons is evident. It may represent a new product of abnormal glial synthesis for this area. The presence of sudanophilic lipofuscin granules among the melanin granules of the other cases indicates a gradual and progressive shift in metabolism occurring in the nigral neuron. Friede 9 has shown that the melanin-containing neurons of the substantia nigra are devoid of oxidative enzymes while other neurons possess a high content of oxidative enzymes related to a high content of lipofuscin. Therefore the gradual accumulation of lipofuscin components in the glia and nigral neurons of Parkinsonian patients can be considered a marker of some more profound and basic metabolic alteration. That such alteration is active is clearly evident when we consider the changes in basophilia of the Parkinsonian neurons. In the present study the difficulty in determining cytoplasmic basophilia due to overlying melanin granules, as noted by Pakkenberg 22, was circumvented by bleaching the melanin prior to application of the gallocyanin method. The observed increase in Nissl bodies, diffuse cytoplasmic and nuclear basophilia, and reduction in the size of the Parkinsonian nigral neurons were the counterpart of the changes induced by reserpine in rat sympathetic gangliala, 21. These changes are thus interpreted as an expression of reduced firing activity. Since high melanin content parallels function in the nigral neurons, the decrease in melanin and increase in nucleic acids in the Parkinsonian neurons is additional evidence for a deviation of the normal metabolism12 towards non-functional syntheses, which probably antagonize the normal syntheses of these neurons for specific carrier proteins or transmitter production. It is proposed that the close neuronal vascular contact found in the zona compacta of the normal brains must be of importance for the functional metabolism of this area since the interruption of this contact was the only detectable lesion in several patients of idiopathic Parkinsonism. The associated histochemical changes of glia and melanin-containing neurons observed in the Parkinsonian zona compacta support the close metabolic interaction between the two units 17 and emphasize the primary role played by the glial elements in regulatory and barrier mechanisms of the brain 4. Brain Research, 25 (1971) 289-299
298
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SUMMARY
O b s e r v a t i o n s were m a d e on neurons o f the substantia nigra in 10 cases o f i d i o p a t h i c P a r k i n s o n i s m and 5 age-matched controls. It was found that the melaninc o n t a i n i n g neurons o f the zona c o m p a c t a in the n o r m a l brain have a close spatial relationship with the blood circulation. The capillary walls a p p e a r fused to the membranes o f n e u r o n a l p e r i k a r y a and processes. In the P a r k i n s o n i a n zona c o m p a c t a the close c o n t a c t between nigral neuron and capillary is lost. The findings suggest t h a t this is due to the infiltration o f the proliferated glia between cell surface and capillary wall. This neuronal vascular contact in the zona c o m p a c t a o f the n o r m a l brain m u s t be o f i m p o r t a n c e for the functional m e t a b o l i s m o f this area, since interr u p t i o n o f this c o n t a c t was the only detectable nigral lesion in several patients with overt s y m p t o m s o f recent date. Histochemical reactions o f n o r m a l a n d P a r k i n s o n i a n m e l a n i n - c o n t a i n i n g neurons showed t h a t the nigral cells o f the patients have a larger a m o u n t o f lipofuscin c o m p o n e n t in the melanin a r e a t h a n those o f the controls. Increased b a s o p h i l i a was observed in the P a r k i n s o n i a n neurons and was inversely related to their melanin content. It is interpreted as an expression o f reduced firing activity, indicating regression o f function. ACKNOWLEDGEMENTS
This w o r k was s u p p o r t e d by G r a n t No. 733 from The R o y a l Hellenic Research Foundation. The a u t h o r wishes to express her t h a n k s to Dr. J. C o n s t a n t i n i d i s o f the Clinique Psychiatrique Universitaire, Bel Air, G e n e v a for supplying the P a r k i n s o n i a n material.
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10 FRIEDE,R. L., Topographic Brain Chemistry, Academic Press, New York, 1966, p. 322. 11 GABE, M., Sur quelques applications de la coloration par la fuchsine parald6hyde, Bull. MicE. appl., 3 (1953) 153-162. 12 GOMIRATO,G., AND HYD~N, H., A biochemical glia error in the Parkinson disease, Brain, 86 (1963) 773-780. 13 GREENFIELD,J. D., System degenerations in the cerebellum, brain stem and spinal cord. In W. BLACKWOOD,W. H. MCMENEMEY,A. MEYER, R. M. NORMANAND D. S. RUSSELL(Eds.), Greenfield's Neuropathology, Edward Arnold, London, 1963, pp. 581-601. 14 GYBELS,J. M., The Neural Mechanism of Parkinsonian Tremor, Arscia, Brussels, 1963. 15 HASSLER,R., Zur Pathologie dee Paralysis agitans und des postenzephalitischen Parkinsonismus, J. PsychoL Neurol. (Lpz.), 48 (1938) 387-476. 16 HORNYKIEWICZ,O., Die topische Lokalisation und das Verhalten yon Noradrenalin und Dopamin (5-Hydroxytyramin) in der Substantia nigra des normalen und Parkinson-kranken Menschen, Wien. klin. Wschr., 75 (1963) 309-312. 17 HYD~N, H., Dynamic aspects of the neuron-glia relationship. In H. HYD~N (Ed.), The Neuron, Elsevier, Amsterdam, 1967, pp. 179-219. 18 ISSIDORIDES,M. R., AND MYTIL1NEOU'-PRoVELEGIOS,C., Effect of cl'doramphenicol on reserpine treated sympathetic ganglia of the rat, Biol. Psychiat., in press. 19 ISSIDORIDES,M. R., AND SHANKLIN,W. M., Histochemieal reactions of cellular inclusions in the human neurone, J. Anat. (Lond.), 95 (1961) 151-159. 20 MOSES,H. C., GANOTE,C. E., BEAVER,D. L., AND SCHUrEEMAN,S. S., Light and electron microscopic studies of pigment in human and rhesus monkey substantia nigra and locus coeruleus, Anat. Rec., 155 (1966) 155-167. 21 MYTILINEOU,C., Histochemical alterations of sympathetic ganglia of the rat after reserpine administration, BioL Psychiat., 1 (1969) 61-72. 22 PAKKENBERG,H., The pigment in substantia nigra in Parkinsonism. Microspeetrophotometric comparison with other sources of human pigment, Brain Research, 2 (1966) 173-180. 23 PEARSE,A G. E., Histochemistry TheoreticalandApplied, Churchill, London, 1961, pp. 826, 834, 850. 24 RICHARDSON,E. P., Remarks on the pathology of Parkinson's disease. In A. BARBEAU,L. J. DOSHAYAND E. A. SPIEGEL(Eds.), Parkinson's Disease Trends in Research and Treatment, Grune and Stratton, New York, 1965, pp. 63-68. 25 SCHARRER,E., The blood vessels of the nervous tissue, Quart. Rev. Biol., 19 (1944) 308-318. 26 SCHARRER,E., UND GAUPP, R., Neuere Befunde am Nucleus supraopticus und Nucleus paraventricularis des Menschen, Z. Neurol., 148 (1933) 766-772. 27 SPIELMEYER,W., The significance of local factors for the electivity in central nervous system disease processes, Medicine (Baltimore), 10 (1931) 243-256. 28 VERNEV,E. B., Agents determining and influencing the functions of the pars nervosa of the pituitary, Brit. reed. J., 2 (1948) 119-123.
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