THE ANATOMICAL RECORD 197: 495-502 (1980) Proliferation of Epithelial Cells in the Adult Primate Choroid Plexus MICHAEL S. KAPIAN Boston University School of Medicine, Department of Anatomy, Boston, Massachusetts 02118 ABSTRACT 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. MATERIALS AND METHODS 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 1980 ALAN R. LISS, INC. 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 hot-plate. 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. 495 496 MICHAEL S. KAPLAN 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. RESULTS 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. DISCUSSION 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 Abbreviations C, Capillary EC, Unlabeled Epithelial Cell F, Filaments Fb, Fibroblast PROLIFERATION IN THE ADULT PRIMATE CHOROID PLEXUS 497 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. 498 MICHAEL S. KAPLAN 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. PROLIFERATION IN THE ADULT PRIMATE CHOROID PLEXUS 499 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 35,000. 500 MICHAEL S. KAPLAN PROLIFERATION IN THE ADULT PRIMATE CHOROID PLEXUS 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 501 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. 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