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Pericytes in human cerebral microvasculature.

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THE ANATOMICAL RECORD 218~466-469(1987)
Pericytes in Human Cerebral Microvasculature
C.R. FARRELL, P.A. STEWART, C.L. FARRELL, AND R.F. DEL MAESTRO
Department of Anatomy, University of Toronto, Toronto, Ontario M5S 1A8 (C.
R.F., PA.S.),
and Brain Research Laboratory, Departments of Clinical Neurological Sciences and
Biophysics, University of Western Ontario, Victoria Hospital, London, Ontario N6A 4G5
(C.L.F., R.F.D.), Canada
ABSTRACT
Two classes of pericytes are thought to exist in cerebral microvasculature, granular and agranular. This classification is based on the presence or
absence of cytoplasmic, lysosome-like granules of variable size and appearance. The
pericytes in structurally normal human brain tissue from 17 patients, male and
female, ranging in age from 14 to 77, were examined. Light microscopic examination
of single sections revealed that 67% of pericyte profiles contained granules in both
sexes, and this ratio did not change with age. Following the serial reconstructions of
80 individual pericytes, it was found that all contained characteristic cytoplasmic
granules. These data show that if truly agranular pericytes do exist in human
cerebral microvasculature, they must constitute less than 5% of the population.
Pericytes are a class of perivascular cells enclosed
within the basement membrane of capillaries. Pericyte
function is not well understood even though they have
been identified in a number of tissues and are probably
common to all microvascular beds. A great deal of confusion regarding the form and distribution of pericytes
in the central nervous system (CNS) is evident in the
literature. Some authors make no distinction among
different types of pericytes (Castejon, 1984; Kristensson
and Olsson, 1973; Le Beux and Willemot, 1980). Others
refer to two types of pericytes, one containing numerous
large, dense granules, which are autofluorescent, acid
phosphatase positive, and PAS positive (Jeynes, 19851,
and the other apparently without granules, although
there is no consistency in the terminology used by different authors. Various distinctions between these two
types include: agranular vs. granular pericytes (Jeynes,
1985; Mat0 and Ookawara, 1981); “ordinary” vs. phagocytic pericytes (van Deurs, 1976); pericytes containing
numerous primary and secondary lysosomes vs. those
with few lysosomes (Lafarga and Palacios, 1975); and
those laden with granules vs. unladen pericytes (Sumner, 1982).
The idea that there are two classes of pericytes came
from the observation that, in single sections, a little
more than half of the pericyte profiles contain granules
and the rest do not. An alternative explanation, however, is that granules may be present in every pericyte
but are not evenly distributed in the cytoplasm, and so
would not be seen in every section. The form and distribution of pericytes in human brain are not known. The
current study was undertaken to describe pericytes in
human cerebral cortex and to re-examine the hypothesis
that there are two classes of pericytes with the use of
serial reconstruction.
MATERIALS AND METHODS
Structurally normal cerebral cortex was obtained a t
biopsy from patients undergoing lobectomy for removal
0 1987 ALAN R. LISS, INC.
of a tumor or corticectomy for treatment of intractable
epilepsy.’ The samples were chosen as far from the site
of pathology as possible (Stewart et al., 1986) and were
judged to be normal. Standard light microscopic neuropathological evaluation of tissue texture, cell density,
and organization of appropriate cells for the sites selected was performed. There was a n absence of vacuolization in the tissue around the cells indicating that
edema (cytotoxic or vasogenic) was absent. No abnormal
cell infiltration or alteration of vessels and endothelial
cells in number or topography was judged to be present.
Tissue was obtained from 14 tumor patients (13 glial
tumors and 1 lymphoma) and 3 epileptic patients. Samples were obtained from the frontal lobe (5 patients),
frontal-temporal area (2 patients), parietal lobe (1 patient), occipital lobe (2 patients), parieto-occipital area (2
patients), and the temporal lobe (5 patients). Patients
ranged in age from 14 to 77 and consisted of 6 males and
11females. None of the patients had received prior chemotherapy or radiotherapy. Tissue samples were immediately immersed in fixative and cut into small
fragments of approximately 1 mm3 within one minute
of removal from the brain during surgery. Tissue was
fixed in 2% glutaraldehyde and 2% paraformaldehyde
in 0.1 M phosphate buffer (pH 7.4) and postfixed in
osmium tetraoxide and embedded in epon (JBS). Thin
sections were cut and stained with toluidine blue for
light microscopic observations.
Pericytes are undifferentiated mesenchymal cells associated with capillaries and enclosed within the vascu-
Received October 2, 1986; accepted March 4, 1987.
Address reprint requests to C.R. Farrell, Dept. of Anatomy, University of Toronto, Toronto, Ontario M5S 1A8, Canada.
’Human tissue was obtained through informed consent, and its use
in this project was approved by the University of Toronto Human
Rights Review Committee, February 27, 1984.
PERICYTES IN HUMAN CEREBRAL MICROVASCULATURE
lar basal lamina. In the light microscopic preparations
used in this study, pericytes were identified by their
close association with capillary walls (Fig. la,b). They
were scored as granular or agranular depending on the
presence or absence of granules. Control studies showed
that the characteristic lysosome-like granules that
stained dark purple in toliudine blue are also PAS positive. A second, more variable type of granule present in
pericytes stained green in toluidine blue and were
golden-brown in unstained sections indicating a lipofuscin component (Fig. lb). The effects of age and sex on
the mean percentage of granular pericytes were examined using linear regression analysis.
To test the hypothesis that there are two classes of
pericytes, a total of 80 pericytes were serially reconstructed from 3 patients. Each series consisted of 30-32
continuous sections. Statistical procedures were based
on the binomial distribution. The null hypothesis, that
the agranular pericytes constitute at least 5% of the
TABLE 1. Distribution of granular pericyte profiles in
human cerebral cortex
patients
No. of
Total no.
of pericytes
Mean%’ profiles
w i t h granules
Females’
6
11
124
236
65.89 3.26
67.33 f 2.64
Total
17
360
66.98 rf: 1.88
Males2
‘Mean % k standard error of the mean.
2No difference was found in % pericyte profiles with granules between
males and females (p > 0.05).
467
pericyte population, was tested. Power was calculated
using P = 100 (l-pn)%where p, representing the proportion of granular pericytes, was set at .95.
RESULTS
In single sections we found that 66.98% k 1.88 (S.E.M.)
of pericyte profiles contained granules and the rest were
apparently agranular. There were no significant (p >
0.05) differences due to age or sex in the distribution of
granular or agranular profiles in the tissues studied
(Table 1). A slight but not statistically significant increase in the percentage of granular pericyte profiles
with increasing age was observed (Fig. 2). No qualitative
differences in the pericytes were observed among the
different patients.
We serially reconstructed 80 pericytes and found that
they all contained granules. Two basic types of inclusion
bodies were observed: dense, amorphous, lysosome-like
granules that stained purple in toluidine blue (Fig. la,
arrowhead), and large green staining bodies that displayed a greater diversity in morphology (Fig. lb, arrowhead). Both types often occurred together in the same
cell. When we began reconstructing pericytes from serial sections and failed to find any agranular ones, it
became apparent that if “agranular” pericytes do exist,
they must constitute only a small percentage of the
population. We arbitrarily chose 5% as a working hypothesis and tested this using the formula shown above.
This hypothesis was rejected at p < 0.02. Statistically
this means more than 95% of the pericytes in the cortical tissue studied were granular and that if “agranular”
pericytes do exist, they could constitute no more than
5% of the pericyte population. Figure 3a-c. shows a
Fig. 1 . These micrographs show the close association of pericytes and capillary profiles in human
cerebral cortex. Two types of granules are present in the cytoplasm of the pericytes in a and b. Dense,
lysosome-like granules are indicated in a (arrowhead), and the larger, more heterogeneous variety of
inclusion bodies indicated in b (arrowhead). The brilliant green color of this second type of body is not
obvious from the black-and-white micrograph in b (1,200X 1.
C.R. FARRELL, P.A. STEWART, C.L. FARRELL, AND R.F. DEL MAESTRO
468
"
o
%
Pericyte 6o
Profiles
with
Granules 4 0
e
0
tb
20
30
40
'-
m
0 0
50
0
0
60
7.0
80
Age of Patients [years)
Fig. 2. Graph showing the distribution of granular pericytes in
human cerebral cortex. Male and female patients are represented by
closed and open dots, respectively. The change in the percentage of
pericytes containing granules is represented, and although a slight
increase with age is evident, it is nonsignificant.
sequential series of sections of the same pericyte in
which granules are present in two sections but not in
the third.
DISCUSSION
Two types of inclusion bodies in pericytes of the CNS
have been described: large, spherical, lysosome-like electron dense bodies with homogeneous matrices (Cancilla
et al., 1972; Castejon, 1984; Jeynes, 1985; Lafarga and
Palacios, 1975; Mat0 and Ookawara, 1981), and large
bodies with polymorphic matrices including dense granulations and possibly lipid vacuoles (Jeynes, 1985; Lafarga and Palacios, 1975) and lipofuscin aggregates
(Kristensson and Olsson, 1973). In the present study
both of these types were observed. The larger heterogeneous type of inclusion body displayed considerable diversity. Pale areas were sometimes observed in these
bodies and they often assumed irregular shapes. In some
cells these inclusion bodies were very large, occupying
much of the cytoplasm and in some cases obscuring part
of the nucleus. These two types of inclusion bodies occurred together in many of the cells analyzed.
Pericytes may be a n important part of the blood-brain
barrier acting as a "second line of defense" by phagocytosing molecules that pass the endothelial layer when it
is stressed (Cancilla et al., 1972; van Deurs, 1976). Our
finding that the majority (> 95%) of CNS pericytes are
of the granular form strengthens this hypothesis that
pericytes play a n important back-up role in the bloodbrain barrier. The phagocytic properties of pericytes in
the brain have been well documented. Under experimental conditions such as acute hypertension (van
Deurs, 19761, freeze-injury (Baker et al., 1971; Cancilla
et al., 19721, and portocaval anastomosis (Sumner, 1982),
in which the blood-brain barrier becomes leaky, granulecontaining pericytes have been observed to be actively
involved in the phagocytosis of foreign proteins that
entered the brain. The phagocytic role of CNS pericytes
under pathological conditions such as neoplasms and
allergic encephalomyelitis has also been established (Torack, 1961). Furthermore under such conditions peri-
Fig. 3. A series of 3 micrographs of the same pericyte associated with
a capillary from human cerebral cortex. In a, numerous granules
(arrowhead) are present in the pericyte cytoplasm; in b, one granule
may be present; in c, granules are completely lacking (1,200x).
PERICYTES IN HUMAN CEREBRAL MICROVASCULATURE
cytes and their lysosomes increase in number (Cancilla
et al., 1972; Mat0 and Ookawara, 1981; Van Deurs,
1976).
With advancing age granules have been observed by
others to become more closely packed or replaced by
large vesicles (Cammermeyer, 1970),but the phagocytic
capabilities of pericytes also declines in aging (Mato and
Ookawara, 1981). In a previous study we found a decrease in the number of pericytes with increasing age in
human cerebral white matter but not gray matter (Stewart et al., 1986). Such factors may contribute to the
pathology of aging by decreasing the ability to compensate for transient leaks in the blood-brain barrier. Since
the number and size of lysosomelike granules in pericytes increases with age (Cammermeyer, 1970; Mat0
and Ookawara, 19811, the chances of sectioning a pericyte through a granule should also increase. The slight
(but not statistically significant) increase in the percentage of pericyte profiles with granules may reflect this
change and should be further investigated.
Another possible function attributed to pericytes in
the CNS is that of mechanical support of small blood
vessels to counteract hydrostatic pressure. In one study
acute hypertension caused the blood-brain barrier to
break down, whereas the blood-retina1 barrier, which
contains a higher density of pericytes, remained intact
and undamaged (Laties et al., 1979).
A contractile function of pericytes has also been postulated. Actin and myosin have been identified in the
pericytes of rat brains (Le Beux and Willemot, 1980).
Actin has been identified in retinal pericytes, and although it was also found to be present in retinal endothelial cells, filaments were more numerous in pericytes
(Wallow and Burnside, 1980). Thus, if pericytes have the
ability to contract, then the filaments may be involved
in the regulation of capillary blood flow. However, the
mere presence of actin and myosin is not proof of cellular
contractility since these proteins play a central role in
the phagocytic process (Silverstein et al., 1977).
We have shown that the majority (> 95%)of pericytes
in the CNS contain cytoplasmic granules, which are
known to be associated with the phagocytic role of pericytes (as above). That we were unable to identify any
“agranular” pericytes casts doubt on the hypothesis that
they exist as a separate class from granular ones. If they
do exist, they are rare. We suggest that the presence of
filaments in pericytes may be a reflection of their phagocytic capabilities. In addition it is possible that CNS
pericytes are also contractile as they are in skeletal
muscle (Tilton et al., 1979) and so may play a role in
vasoconstriction.
Our failure to find any “agranular” pericytes also has
implications for future work elucidating the functions of
cerebral pericytes since interpretation of data is no
longer constrained bv the necessity to explain two different-morphological types of CNS pericyt;
469
ACKNOWLEDGMENTS
This work was supported by the National Cancer Institute (Canada) and the Brain Research Fund Foundation. R.F.D. is a recipient of the Canadian Life Insurance
Medical Scholarship. The authors wish to thank Dr. J.
Girvin for supplying tissue from epilepsy patients and
Mrs. K. Hayakawa for her excellent technical assistance. The authors are grateful to Drs. J.J. Gilbert and
J.F.C. Kaufmann for their neuropathological expertise.
LITERATURE CITED
Baker, R.N., P.A. Cancilla, P.S. Pollock, and S.P. Frommes (1971) The
movement of exogenous protein in experimental cerebral edema.
J. Neuropathol. Exp. Neurol., 80:668-679.
Cammermeyer, J. (1970) The history of the microglial cell: A light
microscopic study. In: Neurosciences Research, vol. 3. S. Ehrenpreis and O.C. Solnitzky, eds. New York, Academic Press, pp. 44170.
Cancilla, P.A., R.N. Baker, P.S. Pollock, and S.P. Fommes (1972) The
reaction of the central nervous system to exogenous protein. Lab.
Invest., 26:376-383.
Castejon, O.J. (1984) Submicroscopic changes of cortical capillary pericytes in human perifocal brain edema. J. Submicrosc. Cytol.,
16:601-618.
Jeynes, B. (1985) Reactions of granular pericytes in a rabbit cerebrovascular ishemia model. Stroke, 16:121-125.
Kristensson, K., and Y. Olsson (1973) Accumulation of protein tracers
in pericytes of the central nervous system following systemic injection in immature mice. Acta Neurol. Scand., 49:189-194.
Lafarga, M., and G. Palacios (1975) Ultrastructural study of pericytes
in the rat supraoptic nucleus. J. Anat., 120:433-438.
Laties, A.M., S.I., Rapoport, and A. McGlinn (1979) Hypertensive
breakdown of cerebral but not retinal blood vessels in rhesus monkey. Arch Opthalamol., 79:1511-1514.
Le Beux, Y.V.I., and J. Willemot (1980) Actin and myosin-like filaments in retinal pericytes and endothelial cells. Invest. Opthalamol. Vis. Sci., 19:1433-1441.
Mato, M., and S. Ookawara (1981) Influences of age and vasopresson
on the uptake capacity of fluorescent granular perithelial cells
(FGP) of small cerebral vessels of the rat. Amer. J. Anat., 162:4553.
Silverstein, S.C., R.M. Steinman, and Z.A. Cohn (1977) Endocytosis.
Ann. Rev. Biochem., 46:699-722.
Stewart, P.A., M. Magliocco, K. Hayakawa, C.L. Farrell, R.F. Del
Maestro, J. Girvin, J. Kaufmann, H. Vinters, and J. Gilbert (1987)
Blood-brain barrier ultrastructural changes in the aging human.
Microvasc. Res. (in press).
Sumner, B.E.H. (1982) A quantitative study of vascular permeability
to horseradish peroxidase, and the subsequent fate of ihe tracer, in
rat brains after postcaval anastomosis. Neuropathol. Appl. Neurobiol., 8:117-133.
Tilton, R.G., C. Kilo, J.R. Williamson, and D.W. Muich (1979) Differences in pericyte contractile function in rat cardiac and skeletal
muscle microvasculature. Microvasc. Res. 18:336-352.
Torack, R.M. (1961) Ultrastructure of capillary reaction to brain tumors. Arch Neurol., 5:416-428.
van Deurs, B. (1976) Observations on the blood-brain barrier in hypertensive rats, with particular reference to phagocytic pericytes. J.
Ultrastr. Res., 56:65-77.
Wallow, H.I., and B. Burnside (1980) Actin filaments in retinal pericytes and endothelial cells. Invest. Opthalmol. Vis. Sci. 19:14331441.
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