вход по аккаунту


Proliferation of epithelial cells in the adult primate choroid plexus.

код для вставкиСкачать
THE ANATOMICAL RECORD 197: 495-502 (1980)
Proliferation of Epithelial Cells in the Adult Primate
Choroid Plexus
Boston University School of Medicine, Department of Anatomy, Boston, Massachusetts
An adult rhesus 'monkey was injected intraperitoneally with
[H3]thymidine (2.3 pCi/gram body weight) and perfused 90 minutes later with
a mixture of aldehydes. One and a half micrometer plastic sections were then
cut and dipped into liquid emulsion for radioautography. Labeled cells were
observed in the choroid plexus of the anterior lateral ventricle; cell identification
was evaluated using electron micrographs taken from serial thin sections of reembedded. radioautographic 1.5-pm sections. The ultrastructure and location of
both mitotic figures and labeled cells confirmed the presence of undifferentiated
basal choroid plexus epithelial cells in the adult primate central nervous system.
Although many principles underlying the
dynamic state of the choroid plexus have been
investigated (Brightman, '68; Milhorat, '76;
Peters and Swan, '79), very little information
is available on the capacity of the plexus
epithelium to proliferate. In thick radioautographic sections of the choroid plexus, it is
difficult to differentiate labeled cell types, and
therefore, in the immature mouse (Miale and
Sidman, '61; Mares et al., '75) and in the adult
rat and mouse (Messier and Leblond, '60;
Schultze and Oehlent, '60; Mares et al., '75),
investigators have not speculated what type
of the choroid plexus cells (Kolmer cells, epithelial cells, fibroblasts -Peters et al., '76)
have incorporated intraperitoneal injections
of [H3] thymidine. To date, only two light
microscopic studies have reported labeled epithelial cells: (1)in a 43-year-old man, in both
neoplastic and non-neoplastic regions (Johnson et al., '60) after eight intravenous injections of [H3]thymidine, and (2) in the adolescent and adult mouse (MareEi and Lodin, '74).
If in the adult there are indeed newly
formed epithelial cells in the choroid plexus,
then one might expect to see immature forms
in the epithelial layer. However, immature
plexus cells have never been reported in the
adult, nor has the identification of labeled
epithelial cells been resolved with the electron
microscope. In the present study on the Rhesus
monkey, epithelial stem cell proliferation has
been demonstrated by ultrastructural identification of labeled cells.
A male rhesus monkey (Macaca mulatta)
was caged in an animal room until 4 years, 8
months after birth. He was then injected in0003-276X/80/1974-0495$01.70
traperitoneally with [H3] thymidine (2.29 pc
per gram body weight, New England Nuclear,
20 Ci/m mole); care was taken not to leak any
[H3] thymidine from the injection site. One
and a half hours later the animal was perfused
through the heart with 1,200 ml of a solution
consisting of 1% Paraformaldehyde and 1%
glutaraldehyde in 0.08 M cacodylate buffer.
The head was then removed and placed in the
refrigerator until the next morning, when the
brain was put into concentrated fixative. Coronal slices (1.5 mm) of a small region including the anterior lateral ventricle were washed
with four changes of 0.1 M cacodylate buffer
for 1 hour, postfixed in 1% OsO, at room
temperature for 2 hours, washed again in
buffer, dehydrated in a graded series of alcohols and propylene oxide, and embedded in
Araldite 502. Sections (1.5 pm) were cut with
%"-thick glass knives on a JB-4 microtome.
These large sections (6 mm wide x 9 mm
long) were dried onto glass slides in a way
that flattened the sections and reduced the
formation of air bubbles under the section: the
slide was placed on a 300C hot-plate for several seconds and then transferred to a 60C
Slides were dipped into Kodak NTB-2 emulsion a t 40C to 42"C, exposed for 1 month a t
4C (Cowan et al., '721, and stained with 1%
toluodine blue and 0.4% sodium borate. In the
light microscope, labeled cells were identified
and the number of grains over the nuclei were
Reeeived January 21, 1980; accepted February 29, 1980.
Michael S. Kaplan's present address is: Florida State University,
Biology Unit One, Tallahassee, Florida 32306.
counted the location of each labeled cell was
recorded using a Lovin microslide field finder.
After an entire section was examined, labeled
cells were chosen for re-embedding, and the
1.5-pm radioautographic section was lifted off
the glass slide with a plastic block (Kaplan
and Hinds, '77). A diamond knife was aligned
to the flat face of the re-embedded block for
serial thin sectioning.
The technique of picking up thin sections is
adapted from a method first used by McCarthy
and Peters (Hinds and Hinds, '72; Vaughan
and Peters, '73). These thin sections were
stained for 6 minutes in a solution of saturated
uranyl acetate diluted 1:l with 95% ethanol,
followed by an aqueous 0.2% solution of lead
citrate for 2 minutes. With the aid of a camera
lucida drawing at 40 x and 100 x and photographs of the original radioautographic section, the same cell was located in the electron
microscope and photographed.
All of the choroid plexus cells appeared
heavily labeled, with 15-28 grains over the
nucleus, as would be expected from the short
survival interval of 90 minutes after injection,
which is not enough time for mitosis to have
occurred (Lewis, '68a; Sidman, '70). Apparent
epithelial cells were labeled in the base of the
choroid plexus epithelium throughout the level of the anterior lateral ventricle. Four labeled cells and one mitotic cell were re-embedded for electron microscopy. Labeled epithelial
cells of the adult monkey resembled immature
epithelial cells previously described in the
newly weaned pig (Davis et al., '73) and perinatal rabbit (Tennyson and Pappas, '68) as
follows (Figs. 1,2,3,4): (1)Relatively straight
membrane borders between epithelial cells,
with very little basal infolding; (2) rough
endoplasmic reticulum throughout the cytoplasm; (3) finely fibrillar ground substance
and scattered microtubules; (4) numerous mitochondria; (5)small vesicles and larger vesicles which sometimes contained dense spherical inclusions or granules; and (6) nuclei that
are large and elongated. Furthermore, these
labeled cells possess three features characteristic of mature choroid plexus epithelial cells
(Peters et al., '76); they are located superficial
to the basement membrane; they may touch
the basal lamina (Fig. l), and they have short,
irregular, dilated cisternae of rough endoplasmic reticulum. The morphology of the
mitotic cell in the epithelial cell layer (Fig. 4)
is similar to the labeled cells just described.
The percentage of labeled epithelial cells
found in the choroid plexus was calculated by
estimating the total number of epithelial cells
per unit length and multiplying this number
by the total length of choroid plexus examined
(1.14 x lo5 pm), to obtain a value of 6,472
epithelial cells. The number of labeled epithelial cells (five) was then divided by 6,472 to
obtain 0.077% labeled choroid plexus epithelial cells.
Labeled cells found in this study resemble
immature epithelial cells (Davis et al., '73;
Tennyson and Pappas, '68), possess features
characteristic of mature choroid plexus epithelial cells (Peters e t al., '761, and appear
pseudostratified. Thus, as in the olfactory
(Graziadei and Graziadei, '79) and respiratory
(Gordon and Lane, '77) epithelium, the labeled
choroid plexus cells and mitotic figures probably represent stem cells which do not reach
the apical surface of the epithelial layer. However, the low percentage of labeled epithelial
cells observed indicates a slow proliferation of
epithelial cells.
Although the observed labeled cells and the
mitotic cell found in the base of the choroid
plexus epithelium are indicative of their role
as an epithelial stem cell, other possibilities
need to be discussed. Cells which morphologically resemble epiplexus (Kolmer) cells have
been reported between the epithelial cells of
the choroid plexus (Merker, '72; Sturrock, '78)
and are thought to be monocytes migrating
into the ventricle, where they become epiplexus (Kolmer) cells. Netsky and Shuangshoti
('701, however, have not found monocytes in
the epithelial layer; instead, they reported
that swollen epithelial cells may be detached
into the CSF or into the stroma and act as
mobile macrophages. Since epiplexus epithelial cell types (Merker, '72; Sturrock, '78) appear either very vacuolated or with a large
space separating the plasma membrane of the
cell from that of adjacent cells, their macrophage-like appearance may be a result of a
fixation artifact. In addition, other ultrastructural investigations have not reported any
C, Capillary
EC, Unlabeled Epithelial Cell
F, Filaments
Fb, Fibroblast
Fig. 1. Immature epithelial cell of the choroid plexus in the adult monkey; C, capillary. Asterisk identifies the labeled
cell in the 1.5-pm light radioautograph (lower left inset, x 1,500) and in the electron micrograph ( X 7,700) of the reembedded 1.5-pm section. In the large electron micrograph, the labeled epithelial cell contacts the basal lamina; this
region (in box) is shown at higher magnification ( x 12,000) in lower right inset of an adjacent thin section. Note the
epithelial cell contact (between arrows) with the basal lamina.
Fig. 2. Immature epithelial cell of the choroid plexus in the adult monkey; EC, unlabeled epithelial cell; Fb, fibroblast.
Asterisk identifies the labeled cell in the 1.5-pm light radioautograph (lower left inset, x 2,300) and in the electron
micrograph ( X 21,000) of the re-embedded 1.5-pm section. Note the relatively straight membrane borders, numerous
mitochondria, and lysosomal bodies.
Fig. 3. Immature epithelial cell of the choroid plexus in the adult monkey; thin section adjacent to that of Figure 2;
asterisk identifies the labeled cell with that of Figure 2. For orientation between Figures 2 and 3, note the fibroblast (Fb)
and basement membrane in the light and electron micrograph of these figures. Figure 3 demonstrates the basal infoldings
(arrowheads) of the labeled epithelial cell. Also note the finely fibrillar ground substance and scattered microtubules. x
evidence of macrophage cells in the epithelial
layer superficial to the choroidal epithelium
basal lamina (Carpenter et al., '70; Peters et
al., '76; Peters and Swan, '79). Nevertheless,
the mitotic and labeled epithelial cells in the
present study are lighter than their non-labeled counterpart; thus, they should not be
confused with the dark cells reported by other
investigators (reviewed in Stumock, '79).
In the rat there appears to be a small population of epithelial cells that are morphologically similar (Peters, personal communication) to those labeled in this study of the
monkey. In both species the cells appear to
have a light cytoplasm, with some cells lighter
than others. One could speculate that the light
cells which appear somewhat darker than others may correspond to transitional elements
to mature forms.
The number of labeled epithelial cells was
calculated to be about 0.077% of the total
epithelial cell population. With this labeling
index one may attempt to assess the rate of
cell proliferation. The length of the DNA synthetic phase(s) in another stem cell population
in the brain has been estimated to be 8.5
hours long (Lewis, '68a). Since tritiated thymidine is available for incorporation into DNA
for less than a n hour (Sidman, '70), at 8.5
hours after injection, almost all the cells labeled would have left S phase and another
equally large population of cells could be labeled if another injection was given, doubling
the number compared with only a single injection. On the assumption that choroid plexus
epithelial cell production continues at the
same rate throughout life, one could calculate
how many injections, if given once every 8.5
hours, are required for 100% of the cells to be
labeled: 10@%/.077%= 1,300 injections given
every 8.5 hours. Thus, the choroid plexus
epithelium might undergo a complete turnover after 1.3 years (1300 x 8.5 hr.). These
numbers suggest that during the life of the
rhesus monkey, the entire choroid plexus population turns over several times. However,
the exact turnover time is subject to considerable error because of diurnal variations
(Messier and Leblond, '60) and lack of knowledge of the exact DNA synthetic time. In any
case, it is interesting that the choroid plexus,
a neuroepithelial derivative, could undergo
complete turnover even once in a lifetime. In
addition, a certain number of labeled cells
may be adding to the total cell population and
possibly result in considerable growth.
The author wishes to thank Drs. James W.
Hinds and Alan Peters for their help in all
phases of this research and for their critical
review of this manuscript. This work was
supported by USPHS grant AGOOOO1.
BrightMan, M.W. (1968) The intracerebd movement of
proteins injected into blood and cerebrospinal fluid of
mice. In: Brain Barrier Systems, Progress in Brain Fksearch. A. Lajtha and D. H. Fod, eds. Elsevier Amsterdam,Vol. 29,pp. 19-37.
Carpenter, S.J., L.E. McCarthy, and H.L.
Borison (1970) Electrun microscope study of the epiplexus (Kolmer) cells of the cat choroid plexus. Z. Gll-
forsch., 110:471-486.
Cowan, WM., D.L. Guttlieb, AX. Hendrickson, J.L. Price,
and TA. Woolsey (1972) The autoradiographic demonstration of axonal wnnections in the central nervous
system. Brain Fks., 372-51.
Davis, DA.,B.J. Lloyd, Jr., a d T.H. Milhorat (1973) A
comparative ultrastructural study of the choroid plexuses
of the immature pig. Anat. Rw.,176:-454.
Godon, R.E., and B.P. h e (1977) cytokinetics of rat
trachael epithelium stimulated by mechanical trauma.
Cell Tissue Kinet., 10:171-181.
Grazkki, PP.C., and GAM. Grazladei (1979) Neuqemsis
and neuron regeneration in the olfactory system of mammals. I. Morphological aspects of differentiation and
structural organization of the olfactory sensory neurons.
J. Neurocytol.,8:1-18.
Hinds, J.W., and P.L. Hinds (1972) Reconstruction of
dendritic growth cones in neonatal mouse olfactory bulb.
J . N e d l . , 1:16%187.
Johnson. HA.. W.E. Havmaker. J.R. Rubini, T.M. Fliedner,
V.P. &nd, E.P. Cronkte, a& W.L. Hughe8 (1960) A
radioautographic study of a human brain and glioblastom multifome after the in vivo uptake of tritiated
thymidine. Cancer, 13:636-642.
Kaplan, M.S., and J.W. Hinds (1977) Newgenesis in
the adult rat Electron microscopic analysis of light
radioautographs. Science, 197:1092-1094.
Lewis, P.D. (1968a) A quantitative study of cell proliferation in the subependymal layer of the adult rat brain.
Exp. Neurol., 20:20%207.
Mareg, V., and Z. Lodin (1974) An autoradiographic
study of DNA synthesis in adolescent and adult mouse
forebrain. Brain Res., 76:557-561.
Man%, V.,Z.Lodin, and M. Jilek (1975) An estimate of
the number of cells arising by division in mouse cerebral
Fig. 4. Mitotic epithelial cell of the choroid plexus in the adult monkey; asterisk identifies the mitotic cell
in the lower power electron micrograph (upper left inset, x 5,200)and the corresponding high magnification
electron micrograph ( X 30,000). For orientation note the membranous body in both micrographs (open arrow).
The mitotic cell displays free chromosomes in the cytoplasm. Note the filaments (F), lysosomes, and apparent
cytoplasmic blebs extending into the extracellular space surrounding the mitotic cell.
hemispheres from age one to 12 months: an autoradiographic study of DNA synthesis. J . Comp. Neurol.,
Merker, G. (1972) Einige Feinstrukturbefunde an den
Plexus chorioidei von M e n . Z. Zellforsch., 134:56&5&1.
Messier, B., and C.P. Leblond (1960) Cell proliferation
and migration as revealed by radioautography after injection of W-thymidine into male rats and mice. Am. J.
Anat., 106r247-285.
Miale, I.L., and R.L. Sidman (1961) An autoradiographic
analysis of histogenesis in the mouse cerebellum. Exp.
Milhorat, T.H. (1976) Structure and function of the choroid plexus and other sites of cerebrospinal fluid formation. Intl. Rev. Cytol., 47:226288.
Netsky, M.G., and S. Shuangshoti (1970) Studies on the
choroid plexus. In: Neuroscience Research. S. Ehrenpreis
and 0.C: SolNtzky, eds. Academic Press, New York and
London, Vol. 3, pp. 131-173.
Peters, A., S.L. Palay, and H. deF. Webster (1976) The
fine structure of the nervous system: The neurons and
supporting cells. W.B. Saunders Company, Philadelphia.
Peters,A,, and R.C. Swan (1979) The chomid plexus of
the mature and aging rat: The choroidal epithelium,
Anat. Rec., 1943326354.
schultze, B., and W. Oehlent (1960) Autoradiographic
investigation of incorporation of H3-thymidine into cells
of the rat and mouse. Science, 131:737-738.
Sidman, R.L. (1970) Autoradiographic methods and
principles for study of the nervous system with H3-thymidine. In: Contemporary &search Methods in Neuroanatomy. W.J.H. Nauta and S.O.E. Ebbeson, eds. Springer,
Berlin, pp. 252-274.
Sturrock, R.R. (1978) A developmental study of the epiplexus cells and supraependymal cells and their possible
relationship to microglia. Neuropathol. and Applied Neurobiol., 4:307-322.
Sturrock, R.R. (1979) A morphological study of the development of the mouse choroid plexus. J . Anat.,
129: 777-793.
Tennyson, VM., and G.D. Pappas (1968) The fine structure of the choroid plexus: adult and development stages.
Progress in Brain &s.,29:63-85.
Vaughan, D.W., and A. Peters (1973) A threedimensional study of layer I of the rat parietal cortex. J . Comp.
Neurol., 149:35&370.
Без категории
Размер файла
1 144 Кб
adults, epithelium, primate, proliferation, choroid, plexus, cells
Пожаловаться на содержимое документа