Z. Zellforsch. 145, 577--586 (1973) © by Springer-Verlag 1973

The Fine Structure of Intracerebral Vessels

Er ik Dahl

Department of Anatomy, Dental Faculty, University of Oslo, Blindern, Oslo, N~orway

Received September 3, 1973

Summary. Intracerebral vessels of the parietal lobe of the rhesus monkey have been ex- amined by electron microscopy with special reference to the relationship between the lepto- meninges and the cerebral cortex. A "perivascular reticular sheath", in apparent communica- tion with the subarachnoid space surrounds intracerebral arterioles. Myo-endothelial junctions occur in intracerebral arterioles, but no nerve fibres are found in association with such vessels. This indicates that the tone of these vessels may be regulated by chemical mechanisms, possibly mediated through the myo-endothelial junctions.

Key words : Cerebral arteries - - Cerebral cortex - - Arachnoid - - Pin Mater - - Intercellular junctions - - Electron microscopy.

Introduction

I n previous communicat ions , the fine s t ruc ture and the inncrva t ion of cere- bral vessels in the h u m a n (Dahl and Nelson, 1964; Dahl, Nelson and Flora , 1965) and in the m o n k e y have been descr ibed (Dahl, 1973). Much work has been done on the re la t ionship be tween blood vessels, wi th thei r ensheath ing as t rocytes , and the neurons of ve r t eb ra t e nervous sys tem (see Gray, 1964; Wolff, 1964; Mugnaini and Walberg , 1965). Similar s tudies have also been made of inve r t eb ra t e s (Barber and Graziadi , 1967). Studies b y Samaras inghe (1965) and Jones (1970) have de- scr ibed the mode of e n t r y of cerebral blood vessels in the ra t .

However , in view of the increasing current in teres t in the phys io logy of the cerebral circulat ion, and the blood bra in barr ier , fu r ther fine s t ruc tu ra l invest iga- t ions seem worthwhile . The present s t u d y is concerned wi th the re la t ionship of the blood vessels, the leptomeninges and the cerebral cortex. Moreover, in t ra- cerebral vessels have been s tud ied with special references to the organiza t ion of the vessel wall. By using an improved f ixa t ion me thod combined with serial sections for l ight- and e lect ron microscopy, fur ther ana tomica l in format ion has been ob ta ined concerning cerebral b lood vessels.

Materials and Methods

Three Rhesus monkeys about 2 years old were used for this study. Fixation was performed by intraeardial perfusion of dextran, followed by 1.7% glutaraldehyde in 0.1 3¢I phosphate buffer at pH 7.3 (for details, see Kjaerheim, 1969). After perfusion for 10 rain, the brain was removed. Samples from the parietal lobes with the leptomeninges, cerebral cortex and the white matter were cut into slices under the dissecting microscope, then fixed for an additional period of 2 hr by immersion in the perfusion fixative, rinsed for 30-60 rain in 0.15 M phosphate buffer at pH 7.3, postfixed in 2% osmium tetroxide at 4°C for 2 hr, dehydrated in a graded series of acetone, and embedded in Vestopal W (1%yter and Kellenberger, 1958). Ultrathin sections were cut with an LKB Ultratome III , treated with uranyl acetate (3 %) for 30 rain, followed by lead citrate for 5 rain. The sections were examined in a Siemens Elmiskop Ia

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578 E. Dahl

Fig. 1. Survey photomicrograph of superficial cerebral cortex with the leptomeninges. The arachnoid (A) is composed of a dense part which constitutes the outer border, and a more deli- cate inner part which is connected to the pial covering (P) by bridges (arrow). Distributing vessels (V) of different sizes are seen in the subarachnoid space (Sp). Penetrating vessels (P V) are surrounded by ~he pial covering. No perivascular spaces surround the intracerebral

arterioles (a). Ca = capillaries. X 140

Fine Structure of Intracerebral Vessels 579

electron microscope equipped with 50 vm platinum apertures. From the same plastic blocks, sections one micron thick were cut for light microscopy. These sections were stained on ~ heat- ing stage with an aqueous solution of 0.1% toluidine blue adjusted to pH 8.9, with 0.15 ~I Na~HP04.

Results

Light Microscopy I n sections 1 ~m s ta ined for l ight microscopy, the leptomeninges are found to

conta in vessels of different sizes (Fig. 1). A character is t ic fea ture of ex t racerebra l vessels is the i r sha rp ly well defined outer b o u n d a r y which consists of th in cellular processes or iginat ing f rom spindle-shaped e longated cells which possess the struc- tu ra l features of i ibrocytes . The large d i s t r ibu t ing vessels most f r equen t ly lie free in the subarachnoid space (Fig. 1), while thei r pene t ra t ing branches make contac t wi th the cort ical surface. F r o m serial sections, i t is a p p a r e n t t h a t some of the vessels which enter the b ra in subs tance arise f rom the d i s t r ibu t ing vessels a t a p p r o x i m a t e l y 90 °, while others m a y enter more obliquely. They have a very shor t cx t racerebra l course before enter ing the cort ical pa renchyma . A t the poin t of ent ry , the outer b o u n d a r y of the pene t ra t ing blood vessel and the p ia come into very close contac t and no clear per ivascular space can be observed along the i n t r a p a r e n c h y m a l course of the vessels (Fig. 1).

I n the examined samples of the par ie ta l lobes only arterioles, venules and capil laries are seen (Fig. 1). B y serial sections, the capil lar ies seem to be a r ranged as a net , and the grey m a t t e r has a closer mesh t han in the white mat te r .

Electron Microscopy Only arterioles, venules and capil laries are seen in the cerebral cor tex in the

samples examined in this s tudy : only the ar ter ioles and capil laries are descr ibed in the following account . On per fora t ing the pial covering of the cort ical surface, there is a membranous contac t between the outer l ining of the vessel wall and cy top lasmic processes of the pin (Fig. 12). This contac t is es tabl i shed in the small depressions of the b ra in surface in which vessels close to the i r po in t of en t ry lie. I t is a t th is level t h a t t rans i t ion from ext racerebra] to in t racerebra l vessels occurs.

Arterioles The vascu la r endo the l ium forms a complete cont inuous layer wi thou t fene-

s t ra t ion (Fig. 2). A th ick basement membrane , and a f r agmen ted layer of elastic t issue separa te the in t ima from the tun ica media . The media is composed of 1 to

Fig. 2. Survey electron micrograph of the wall of an intracerebral arteriole. The endothelium (Ed) is continuous without fenestrations. In the subendothelial basement membrane (Bme) fragments of elastic tissue (El) are seen. The smooth muscle cells (SM) are bordered by a distinct basement membrane (Bins). Outside this, a perivascular sheath (PVS), composed of collagen (C) and cytoplasmic processes (CP) from the pial covering is seen. A distinct basement membrane (Bran) covers the neuropil (NP) and separates the neural tissue from the perivascular sheath. A myoendothelial junction between a smooth muscle cell and the endothelium is arrowed. Note the ~mount of collagen separating the cytoplasmic processes from the neural tissue, i.e. there is no well defined outer border of the vessel wall or any perivascular space.

× 15000

5

580 E. Dahl

Figs. 3--5

4

Fine Structure of Intraeerebral Vessels 581

3 layers of smooth muscle cells, and contributes most of the thickness of the arterio- lar wall. The outer layer of the intracerebral arterioles is thin and differs to some extent f rom the adventi t ial coat of the extracerebral vessels. F rom the point of ent ry of an arteriole into the cortical parenchyma, the adventi t ia usually contains a single layer of cellular elements which apparent ly are extentions of the lepto- meningeal sheath of the penetrat ing vessel. These cellular processes are separated from the neural tissue by a small amount of collagen and a distinct basement membrane which is continuous with the basement membrane covering the surface of the brain (Fig. 2). Collagen fibres are also found between the cytoplasmic processes of the adventi t ia and the smooth muscle cells of the tuniea media. Thus it seems as if the leptomeningeal cytoplasmic processes at ]east in some places separates the different components of the "adventi t ia l coat" into an "pial layer" (the cerebral basement membrane, collagen fibres and pial processes) and a epi- pial layer (collagen between the pial-proeesses and the smooth muscle cell layer). Within this epi-pial area, foamy cells resembling macrophages are occasionally encountered. A well defined space which separates the penetrat ing vessel f rom the surrounding cerebral tissue, limited on the one side by a distinct outer boundary of the vessel wall, and on the other side by the pial covering of the neuropil is not observed within the cerebral cortex (Fig. 2). I n some sections close to the point of en t ry of a vessel, small extracerebral spaces of variable sizes are seen. By analyzing these sections, it is apparent tha t these "extravascular spaces" are localized to only a small par t of the total circumference of the vessel. Moreover, these perivascular spaces are only seen in vessels entering the cortical surface at an oblique angle. I n other words, this perivascular space is in fact located in the extracerebral area only and not within the parenchyma proper, and intraeerebral vessels do not possess a true, circumferential, perivascular space.

Nerve fibres are never encountered in association with these intraeerebral vessels (Dahl, 1973).

Myo-endothelial Junctions

At regular intervals, endothelial processes penetrate the basement membrane, and the elastic lamina, and come into close contact with the smooth muscle cells to form myo-endothelial junctions (Figs. 2, 3, 5). Similarly, processes of smooth muscle cells of the media break through the basement membrane and establish contacts with the endothelial cells (Fig. 4). Different kinds of such myo-cndo-

Fig. 3. Detail of the myoendothelial junction seen in Fig. 2, demonstrating a membranous contact between these two different cell types. Note the fibrillary material (F) in the endothelial cell (Eel), partly surrounded by a membrane-like structure (arrow). Bm basement membrane.

SM smooth muscle cell. × 60000 Fig. 4. This micrograph demonstrates a foot-like protrusion of the smooth muscle cell (SM) which makes membranous contact (arrows) with the endothelium (Ed). Bm basement mem-

brane. × 60000

Fig. 5. Detail of a protrusion of an endothelial cell (Ed) which makes membranous contact with a smooth muscle cell (arrow and insert). Note the lateral membranous contact between the smooth muscle cells (NM) (open arrow). E1 fragment of elastic tissue. L vessel lumen. × 42000

Insert × 120000

582 E. Dahl

6

1 0

:Figs. 6--10

Fine Structure of Intracerebral Vessels 583

thelial junctions are seen (Fig. 5). Serial sections (Figs. 6-9) indicate that there may be an accumulation of fibrillary material within the endothelial ceils adjacent to the junctions. Occasionally, condensations of several membrane-like structures may be observed at the myo-endothelial junctions (Fig. 5).

Lateral membrane-to-membrane contacts between smooth muscle cells (Fig. 5) are commonly seen (see also Dahl, 1973).

As the arterioles become smaller, the thickness of the outer coat and the number of smooth muscle cells are gradually reduced. These cell layers finally disappear at the capillary level, where only the fused vascular and parenchymal basement membranes separate the neuropil from the endothelium (Fig. 10).

Capillaries

As in the arterioles, the endothelium of the capillaries forms a complete continuous layer with no fenestrations (Fig. 10). The walls of capillaries close to arterio]ar origins may contain pericytes surrounded by basement membrane. Membranous contacts between cytoplasmic processes of the pericytes and the endothelium, similar to the myo-endothelial junctions of the arterioles are rarely seen (Fig. 11). In capillaries without pericytes, the basement membrane is the only structure which separates the endothelium from the surrounding cortical parenchyma.

Discussion

Perivascular spaces lined by elements of pin mater and communicating with the subarachnoid space have long been known from light microscopic studies of the brain and the spinal cord. Woollam and Millen (1954) have reviewed the earlier literature, and their own work has shown that as blood vessels enter nervous tissue, they are accompanied by a reticular perivascular sheath in which two layers, separated by a space, the perivascu]ar space, can be distinguished. Their studies were followed by those of t Iager (1961) and Nelson, Blinzinger and Hager (1961). More recently, Jones (1970) has demonstrated that small arterioles entering the cerebral cortex carry with them to the point at which they become capillaries, an extension of the subarachnoid space which contains electron dense material. Previous electron microscope studies have demonstrated that the extracerebral vessels have a well-defined outer boundary of fibrocytic cellular processes (Dahl, Flora and Nelson, 1965; Dahl, 1973). The present study has revealed that the intracerebral arterioles are invested by a rather complex peripheral coat, different from the adventitial coat of the extracerebral vessels. At the point of entry into the cerebral cortex, there is a contact between the cellular processes of the pial

Figs. 6--9. Serial sections of the myoendotheliaI junction seen in Figs. 2, 3 .Note the fibrillary material (F) in the endothelial cell (Ed) partly surrounded by a membrane-like structure (arrow) and the cytoplasmic protrusion from both of these morphologically difierent cell

types. E1 elastic tissue. Bm basement membrane. SM smooth muscle cell. x 40000

Fig. 10. Survey electron micrograph of a capillary showing a continuous endothelium (Ed) surrounded by a distinct basement membrane (Bin). The pericyte processes (arrows) seem to be

surrounded by the basement membrane common to the whole capillary. X 12000

584 E. Dahl

11

12

Fig. II. In the intracerebral capillaries cytoplasmic processes of the pericytes (arrows) make membranous contact withthe endothelium, similar to the myoendothelial junctions. P pericyte.

Ed endothelium. Bm basement membrane. A8 astrocyte. × 60000

Fig. 12. Part of a penetrating arteriole at the point of entry into the cortical parenchyma. The pial layer (PC) establishes a membranous contact with the outer border of the vessel wail (arrow) and cytoplasmic processes (Cp) continues as a part of the intracerebral vessel (cf. Fig. 2). Note that the basement membrane (Bin) of the surface of the brain continues into the cortex, and separates the perivascular sheath from the neural tissue. L vessel lumen. Ed endothelium.

Ad adventitial coat. SM smooth muscle cell. × 6000

Fine Structure of IntracerebrM Vessels 585

covering and the outer boundary of the vessel wall.Within the brain parenchyma there is no specific coat per Be, but a peripheral sheath, which comprises bundles of collagen, and attenuated cytoplasmic processes similar to those of the lepto- meninges. A well-defined perivaseular space, bounded on one side by the pia and on the other by a distinct outer boundary of the vessel wall can not be demonstrat- ed within the eerebrM cortex. All the components of the peripheral coat of intracerebrM arterioles should be considered together as a complete sheath. Since this sheath is apparently in communication with the subaraehnoid space, it should also be considered as a "reticular sheath" as described by Woollam and Mfllen (1955). Moreover, this sheath may be considered as equivalent to the whole pia-arachnoid in the sense that the latter is probably excavated by the subarach- noid space, rather than forming two distinct limiting membranes (Pease and Sehultz, 1958). This view is supported by the fact that the piM and arachnoid cells are morphologically identical (Jones, 1970). I t should be stressed that the present investigation has confirmed the finding in previous studies that these intraeerebrM vessels do not receive a direct innervation (Samarasinghe, 1965; Dahl, 1973).

In the present study direct membranous contacts between smooth muscle cells and endothelial cells and between pericytes and endothelial cells, and fre- quent regular lateral membrane-to-membrane contacts between smooth muscle cells have also been demonstrated. The significance of these membranous contacts is, at present unknown. However, the possibility exists, that such contacts are related to mechanisms controlling the tone of the vessels (Rhodin, 1967).

In conclusion, the present investigation has demonstrated that the outer coat of the intraeerebral vessels consists of a reticular sheath in communication with the subaraehnoid space. The sheath disappears at the point at which the arteriole becomes a capillary, by fusion of the basement membranes covering the cortical surface and the endothelium.

Moreover, clear evidence has been provided of membranous contact between smooth muscle cells and the endothelinm, and between pericytes and the endo- thelinm. Intracerebral vessels are deprived of innervation. As to the cerebral blood flow, the investigation indicates that while the extraeerebrM vessels may be subjected to both a nervous and chemical control (Dahl, 1973) the tone of the intracerebrM vessels may be regulated by ehemicM mechanism only, most likely mediated through the myo-endotheliM and pericyte-endotheliM junctions.

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Erik Dahl Department of Anatomy Dental Faculty University of Oslo P. O. Box 1052 Blindern Oslo Norway