AJH
1992;5:851-856
Ultrastructural Study of the Choroid Plexus of Spontaneously Hypertensive Rats MM. Ruchoux, C. Rosati, A. Gelot, Y. Lhuintre, and R. Garay
driae had increased in number and tended to fill the cytoplasma while the nuclei had returned to a resting level. These ultrastructural changes further more suggest an increased secretory activity in the choroid plexus in spontaneously hypertensive rats. Am J Hypertens 1992;5:851-856
We previously gave an account of an increased ion transport activity in choroid plexus from spontane ously hypertensive rats. We have since examined this organ in scanning and transmission electronic microscopy. In the choroid plexus from young spontaneously hypertensive rats, the epithelial cells showed the following: a partial loss of the brush border and infoldings of basolateral mem branes, an increased number of Golgi apparatus, vesicles, and mitochondriae, and an activated nu cleus. In adult hypertensive rats, the mithochon1
KEY WORDS: Choroid plexus, spontaneously hyper tensive rat, stroke-prone spontaneously hyperten sive rat, ultrastructure, ion transport.
D
espite the many studies already carried out, the mechanism of hypertension in spontane ously hypertensive rats from the Okamoto strain (SHR) remains an open question. In deed, most of these studies have dealt with biochemical and physiological aspects of blood pressure regulation in this model, while morphological studies are scarce in the literature. Previous studies have shown abnormal fluxes by the Na ,K ,Cr-cotransport system and the Na ,K -pump in red blood cells from S H R . Further studies have revealed abnormal cotransport (and pump) fluxes in vas cular smooth muscle and choroid plexus from SHR. Interestingly, increased cotransport and pump fluxes in the choroid plexus would reflect an increased secretory and noradrenergic activity of SHR. The choroid plexus (CP) consists of a core of highly +
+
+
+
2 - 4
5
1
Received July 3, 1991. Accepted August 18, 1992. From the Neuropathology Department (MMR, AG), and Pathology Department (YL), CHU Bretonneau, Tours, and INSERM U7, Hopital Necker, Paris (CR, RG), France. Address correspondance and reprint requests to M.M. Ruchoux, Neuropathology Department, CHU Bretonneau, Bd Tonnelle, 37 000 Tours, France.
© 1992 by the American Journal of Hypertension, Inc.
vascularized connective tissue, interfaced at a basement membrane with a cuboidal epithelium. This epithelium is extensively folded, covered with microvilli, and there fore extends over a large surface. The capillaries are of a larger sinusoidal type (20 μτη) and their endothelium is fenestrated, contrary to the blood vessels in most parts of the brain, which are lined with continuous endothe lial cells with tight endothelial junctions. Such morpho logical features are consistent with its commonly ac cepted function of producing a considerable proportion of cerebrospinal fluid (CSF). Besides the CP acts as a selective barrier separating the fluctuating fluid constitu ents of the brain vascular compartiment from the stable, more precisely defined C S F . An increased secretory activity of the choroid plexus is characterized by typical morphological changes. ' Surprisingly, very little is known concerning morpho logical aspects of the choroid plexus of SHR. So we undertook to examine this organ by means of scanning and transmission electronic microscopy. 6
7 - 8
9
10
METHODS Male Okamoto SHR, and their normotensive controls (Wistar-Kyoto rats, WKY, derived from the original Okamoto stock and supplied by Centre d'Elevage Roger 0895-7061/92/$5.00
852
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microscope processing, fragments were dehydrated in an alcohol series, thereafter in propylene oxide and em bedded in Epon resin. After 24 h Epon polymerization at 60 °C, blocks were cut and sections were contrasted with uranyl acetate and lead citrate before observation with Philips CM 12 and Jeol 100 Β electron microscopes. Basement membrane measurements were made with the Philips CM 12 microscope, which has an online measurement device. Suitable areas were recorded, then accurate and reproducible measurements were made immediately without taking any photographs. In view of having as precise a figure as possible, the thick ness of the basement membrane was recorded 15 times. For the study of apical microvilli evaluation, three samples were examined in six rats of each strain. The difference between SHR and WKY was obvious. How ever, there were two difficulties in quantification: the SHR brush border may show both a slight homoge neous decrease of microvilli and marqued losses, and the light and dark staining cells do not have the same microvilli. The dark staining cells had filliform micro villi. In addition, SHR possessed an increased number of
FIGURE 1. Electron micrograph of choroid plexus from 6week-old WKY. A) Aspect of normal epithelial cell. At the apical site, the brush border was dense and regular. The nucleus (N) presented with homogeneous chromatin. In the cytoplasm, mitochondriae (short arrow), vacuoles and Golgi apparatus (G) were observed. X 10,000. B) Higher magnification of the brush border. Tight junction on the left side (long arrow) was observed, as were microvacuoles (star), mitochondriae (short arrow), and Golgi appa ratus (G). X 20,000.
Janvier, Le Genest, France) were studied at 4 to 20 weeks of age. Immediately after cervical dislocation, the whole CP (from the lateral, third, and fourth ventricles) of six ani mals in each strain was removed with the aid of a dis secting microscope. We did not use perfuse fixation be cause samples of fresh tissue were also obtained from these animals for ionic transport studies. Samples were fixed in Trump's liquid ( 2 % parafor maldehyde and 2 % glutaraldehyde) for ultrastructural study. For scanning electron microscope processing, the CP were desiccated with C 0 at the critical point in a Bazers device, sprinkled with gold under a rarefied argon atmosphere and observed with a scanning elec tron microscope (JSM 35C). For transmission electron 1
2
FIGURE 2. Electron micrograph of choroid plexus from 20week-old WKY. A) Normal light and dark staining cells with dense filiform microvilli. Multiple infoldings of the basolateral mem brane (anow) were observed. X8000. B) Higher magnification of the basolateral membrane. X30,000.
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dark staining cells compared to WKY. In order to obtain the optimum evaluation we decided to count the micro villi where the junctions with cytoplasm were clearly identifiable and not the apices of microvilli that we can perceive on the picture. Each count was made using the same magnification (X 6000). We analysed a total length of approximately 5000 μτη for each rat. Five blocks of each plexus were examined. Identical measurements were carried out for the same number (n = 6) of SHR and WKY. RESULTS From the opening of the ventricles, the choroid plexus from SHR seemed larger than those from WKY and their vascularization more visible. WKY (4,6,8, and 20 Weeks of Age) The choroid plexus exhibited normal ultrastructural morphology with light cells and unfrequent dark cells distinguished by their dense cytoplasm (Figure 1A). Mitochondriae and vesi cles still had their structural integrity. The apical tight junction (zona occludens) remained unaltered (Figure IB). So did the typical filiform and predominantly bul bous microvilli (Figure 1 A, B), emerging from the apical surface membrane as a narrow stalk enlarging into an ovoid blob with a greatly infolded basolateral labyrinth (Figures 2A, 3). The basement membrane thickness averaged 89 nm. The perivascular space was very tight and the capillary basement membrane was approxi mately 130 nm thick at 6 weeks (Table 1). Scanning electron microscope examination showed the typical long cauliflower shaped structure of a nor mal choroid plexus formed of polygonal cells covered
CHOROID PLEXUS ULTRASTRUCTURE
853
with a long-pile carpet structure with even microvilli at their convex apical surface. Microvillar density (Figure 4A, B) made it difficult to tell one cell from another. No remarkable changes were observed between 4 to 6 weeks to 20 weeks, except for the cell and capillary basement membranes, which had thickened (respec tively by 133 and 199 nm) (Figure 3), and the dark staining cells, whose number had increased (Figure 2A). SHR (4, 6, and 8 Weeks of Age) Widespread changes were observed unevenly throughout the choroid plexus. The most striking change was a partial loss of microvilli. (Figures 3, 5A, and 5B). Two morphological types of microvilli were present as in normal choroidal epithe lium: filiform and bulbous or club-shaped. But from week 4, we noted alterations in the microvilli shape and multiple large and bulbous microvilli were observed in decreasing numbers over time. In these areas, numerous microvesicles were found close to the apical epithelial surface or merging with the apical membrane. The pres ence of a great number of merged vesicles with the api cal membrane could explain the partial loss of microvilli. These losses were more marked on the apices of the light staining cells. The surface area partially lost corre sponded to approximately 1 5 % of total microvillar sur face (Figure 3). The dark staining cells only showed filiform dark mi crovilli and these were seen in growing numbers in SHR, while their density varied according to areas. They showed dense cytoplasm filled with osmiophilic drop lets and numerous mitochondriae with dense matrices. In the light staining cells, Golgi apparatus was very well developed (Figure 5 A). The irregular nuclei showed
FIGURE 3. Schematic diagram corresponding to choroid plexus cell in WKY and SHR at 6 weeks. In SHR, the nucleus (N) showed a heteroge neous chromatin. Golgi apparatus were more numerous, as were mito chondriae (mi). On the other hand, we observed a clear loss of microvilli and a decrease in infoldings of the basal membrane.
WKY
SHR
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TABLE 1. THICKNESS (nm) OF THE CELL BASAL MEMBRANE AND THE VASCULAR BASAL MEMBRANE IN WKY AND SHR AT 6 AND 20 WEEKS 6 weeks
WKY SHR
20 weeks
Cell Basal Membrane
Vascular Basal Membrane
Cell Basal Membrane
Vascular Basal Membrane
89 160
132 140
133 302
199 322
All measurements in nanometers. These results corresponded to the average of ten values.
heterogenous chromatin and a distinct nucleoli. Modifications were also marked at the basolateral membrane level where the infolded labyrinth was less regularly distributed with large defects (Figure 3). More or less plane areas were noted where mitochondriae had gathered (Figure 5B). The basement membrane had markedly thickened: 160 nm at 6 weeks. The capil-
FIGURE 5. Electron micrograph of choroid plexus from 6-week old SHR. A) Epithelial cells with a loss of surface microvilli. The tight junction (small medium-weight arrow), the microvesicles close to the apical surface (thin long arrow), mitochondriae (short arrow) and the Golgi apparatus (G) were all observed. The nucleus (N) showed a heterogeneous chromatin. Basolateral membrane exhibited rare infoldings. (large thick arrow) X 10,000 B) Apical light staining cell surface with partial loss of the microvilli. Small vesicles (thin long arrow) were observed close to the plane apical surface. The mitochondriae (short arrow) were more or less gathered together. X20,000.
laries showed fewer modifications at 6 weeks with a lesser thickening (140 nm) of the basement membrane (Table 1). Scanning electron microscope examination provided confirmation of the microvilli loss. The cauliflower shape was maintained, but we could now distinguish the apex of each cell and a sort of disjunction between FIGURE 4. Electron micrograph of choroid plexus from 6- them due to a decrease in microvillar density (Figure 6A, B). Some areas were more involved, in which small week-old WKY. A) Scanning electron micrograph of the choroid plane and bald surfaces could be observed (Figure 6B). plexus, which shows as a long cauliflower-shaped structure covered with regular and dense microcilli. X1000. B) With higher At 20 weeks, the choroid plexus from SHR presented magnification, the different apical surfaces (star) were identified exactly the same appearance in scanning electron microonly with difficulty because of the micovilli density. X4800. scope pictures.
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scattered chromatin in the light staining cells and very irregular with a voluminous nucleolus in the dark staining cells (Figure 7). The SHR cell basement membrane was two-fold thicker (302 nm) than WKY's. The SHR membrane was planar with a small number of thickened infoldings. The capillary basement membrane was 322 nm thick (Table 1). DISCUSSION The choroid plexus is the most important site of CSF production, secreting roughly 9 0 % of the total CSF, thus playing a major role in the maintenance of homeostasis of the central nervous system microenvironment. The complete integrity of its lining is essential to maintaining its functions. Interestingly, ultrastructural examination of choroid plexus from young SHR revealed an irregular nucleus with heterogenous chromatin and a 7,8
9,10
FIGURE 6. Electron micrograph of choroid plexus from 6week-old SHR. A) In scanning electron micrograph, the choroid plexus showed a long and large cauliflower shape structure. The different apical surfaces (star) were clearly identified. X1000. B) Higher magnification showed the partial loss of microvilli and a space between the cell apical surface (star). Little bald areas were noticed (arrow). X4800.
At 8 weeks, the choroid plexus from SHR showed the same modifications with some more clusters of mitochondriae and osmiophilic droplets in transmission electron microscope examination. On the other hand, transmission electron microscope examination of 20 week old SHR choroidal epithelium and capillaries showed deep modifications. Two-thirds of the cells were of the dark staining type with dense cytoplasm full of mitochondriae, dense vesicles, and osmiophilic droplets. (Figure 7A). Their apical surface was covered with thin and filiform microvilli (Figure 7A, B). The light stained cells presented a loss of bulbous microvilli and some of them showed a striking abundance of mitochondriae filling the cytoplasm, which gave the cell the aspect of an oncocyte. Fewer micro vesicles were observed in the basement membrane than in the apical membrane. The nuclei were regular with delicately
FIGURE 7. Electron micrograph of choroid plexus from 20week-old SHR. A) The epithelial cells showed an insignificant loss of microvilli. The dark staining cells showedfiliformmicrovilli. Their nuclei (N) were inegular with a heterogeneous chromatin and a voluminous nucleolus. X4000. B) Higher magnification of a dark staining cell with its heterogeneous nucleus (N) and numerous and large mitochondriae (M) with a dense matrix. X 16,000.
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distinct nucleolus, which are typical morphologic changes of increased secretory activity. This increased secretory activity is accountable for a partial loss of apical and basolateral membranes and it brought about so many metabolic modifications that, at the age of 20 weeks, the cells showed signs of overwork, such as numerous mitochondriae of more or less regular shape and size, filling the cytoplasma, and giving the cell the characteristic aspect of an oncocyte. These pictures are evidence of a long increased secretory activity. This is in accordance with our previous results showing increased cotransport and pump activities, particularly in young SHR, in spite of the diminished basolateral and apical membrane surface. By the same token, this increased secretory and transport activity seemed to utterly exhaust the cell capacities, since the adult choroidal epithelium presented a more decreased basolateral membrane surface and, above all, a less functional cytoplasm. We observed a good correlation between the ultrastructural morphological changes and the cotransport and pump activities. However, we do not know whether these features of an intense secretory activity result in increased CSF formation and may be somehow related to blood pressure regulation. In SHR, the rise in blood pressure is linked to an increase in total peripheral resistance, the cause of which is neurogenic during the initiation of hypertension. SHR have elevated plasma catecholamine levels and the reactivity of vessel resistance to norepinephrine is increased. The present work is, to the best of our knowledge, the first to demonstrate there are morphological abnormalities in the young and adult SHR choroid plexus. Similar data have been recently reported in the intestinal and renal epithelium in SHR. Unfortunately, little is known about regulation of blood flow to the choroid plexus. Our findings might suggest there is a relation between increased catecholamine level and increased activities of transport and secretion. Elsewhere Lindvall and recently Faraci have shown that intravenous norepinephrine decreases blood flow to choroid plexus and CSF production as well. The question remains as to whether the abnormalities in choroid plexus can be linked to the increased noradrenergic activity or hypertensive mechanism in SHR.
2.
Duhm J, Gobel BO, Beck FX: Sodium and potassium ion transport accelerations in erythrocytes of CDOC, DOCsalt, one-clip, two kidney and spontaneously hypertensive rats: role of hypokalemia and cell volume. Hypertension 1983;5:642-652.
3.
Feig UP, Michell PP, Boylan JW: Erythrocyte membrane transport in hypertensive humans and rats: effect of sodium depletion and excess. Hypertension 1985;7:423429.
4.
Saitta MN, Haennaert PA, Rosati C, et al: A kinetic analysis of inward Na , K cotransport in erythrocytes from spontaneously hypertensive rats. J Hypertens 1987: 5(suppl 5):285-286.
1
+
O'Donnel ME, Owen NE: Reduced Na-K-Cl-cotransport in vascular smooth muscle cell from spontaneously hypertensive rats. Am J Physiol 1 9 8 8 ; 2 5 5 : 0 6 9 - 0 8 0 .
6.
MacKinley MJ, McAllen RM, Mendelsohn FAO, et al: Circumventricular organs: neuroendocrine interfaces between the brain and the hemal milieu, in Ganong WF, Martini L (eds): Frontiers in Neuroendocrinology. New York, Raven Press, 1990, pp 91-127.
7.
Cserr HF: Physiology of the choroid plexus. Physiol Rev 1974;51:273-311.
8.
Rapoport SI: Blood Brain Barrier Physiology and Medicine. New York, Raven Press, 1976.
9.
McComb JG: Recent research into the nature of cerebrospinal fluid formation and absorption. J Neurosurg 1983;59:369-383.
10.
Wright EM: Transport processes in the formation of cerebrospinal fluid. Rev Physiol Biochem Pharmacol 1978; 83:3-34.
11.
Weiss L: Histology: Cell and Tissue Biology, 6th ed. New York, Elsevier Science Publishing Co., 1983, pp 25-31.
12.
Kovacs K, Horvath E, Bilbao JM: Oncocyte in the anterior lobe of the human pituitary gland. A light and electron microscopy study. Acta Neuropathol (Berlin) 1974;2 7: 43-54.
13.
Nakamura K, Nakamura K: Cerebral noradrenergic and adrenergic neuronal mechanisms in the development and maintenance of hypertension in spontaneously hypertensive rats. Jpn Heart J 1979;20(suppl 1):101-103.
14.
Yamori Y: Neural and non-neural mechanisms in spontaneous hypertension. Clin Sci Mol Med 1976;51(suppl): 431s-434s.
15.
Drueke T, Hennessen U, Nabarra B, et al: Ultrastructural and functional abnormalities of intestinal and renal epithelium in the SHR. Kidney Int 1990;37:1438-1448.
16.
Lindvall M, Owman C: Autonomic nerves in the mammalian choroid plexus and their influence on the formation of cerebrospinal fluid. J Cereb Blood Flow Metab 1981;1:245-266.
17.
Faracci FM, Mayhan WG, Williams JK, Heistad DD: Effects of vasoactive stimuli on blood flow to choroid plexus. Am J Physiol 1988;254:H286-H291.
9
16
ACKNOWLEDGMENTS We thank Miss Genevieve Chauveaud and Mr. Daniel Ekmekdjian for their technical assistance. REFERENCES 1.
Rosati C, Ruchoux MM, et al: Cellular aspects of [Na ,K ,Cl~]-cotransport system in primary hypertension studies in red cells and in the choroid plexus of +
+
+
5.
13,14
15
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spontaneously rats (SHR), in Bruschi G, Borghetti A (eds): Cellular Aspects of Hypertension. BerlinHeidelberg-New York, Springer-Verlag, 1992, pp 257261.
11
12
1992-VOL
1992;5:851-856
Ultrastructural Study of the Choroid Plexus of Spontaneously Hypertensive Rats MM. Ruchoux, C. Rosati, A. Gelot, Y. Lhuintre, and R. Garay
driae had increased in number and tended to fill the cytoplasma while the nuclei had returned to a resting level. These ultrastructural changes further more suggest an increased secretory activity in the choroid plexus in spontaneously hypertensive rats. Am J Hypertens 1992;5:851-856
We previously gave an account of an increased ion transport activity in choroid plexus from spontane ously hypertensive rats. We have since examined this organ in scanning and transmission electronic microscopy. In the choroid plexus from young spontaneously hypertensive rats, the epithelial cells showed the following: a partial loss of the brush border and infoldings of basolateral mem branes, an increased number of Golgi apparatus, vesicles, and mitochondriae, and an activated nu cleus. In adult hypertensive rats, the mithochon1
KEY WORDS: Choroid plexus, spontaneously hyper tensive rat, stroke-prone spontaneously hyperten sive rat, ultrastructure, ion transport.
D
espite the many studies already carried out, the mechanism of hypertension in spontane ously hypertensive rats from the Okamoto strain (SHR) remains an open question. In deed, most of these studies have dealt with biochemical and physiological aspects of blood pressure regulation in this model, while morphological studies are scarce in the literature. Previous studies have shown abnormal fluxes by the Na ,K ,Cr-cotransport system and the Na ,K -pump in red blood cells from S H R . Further studies have revealed abnormal cotransport (and pump) fluxes in vas cular smooth muscle and choroid plexus from SHR. Interestingly, increased cotransport and pump fluxes in the choroid plexus would reflect an increased secretory and noradrenergic activity of SHR. The choroid plexus (CP) consists of a core of highly +
+
+
+
2 - 4
5
1
Received July 3, 1991. Accepted August 18, 1992. From the Neuropathology Department (MMR, AG), and Pathology Department (YL), CHU Bretonneau, Tours, and INSERM U7, Hopital Necker, Paris (CR, RG), France. Address correspondance and reprint requests to M.M. Ruchoux, Neuropathology Department, CHU Bretonneau, Bd Tonnelle, 37 000 Tours, France.
© 1992 by the American Journal of Hypertension, Inc.
vascularized connective tissue, interfaced at a basement membrane with a cuboidal epithelium. This epithelium is extensively folded, covered with microvilli, and there fore extends over a large surface. The capillaries are of a larger sinusoidal type (20 μτη) and their endothelium is fenestrated, contrary to the blood vessels in most parts of the brain, which are lined with continuous endothe lial cells with tight endothelial junctions. Such morpho logical features are consistent with its commonly ac cepted function of producing a considerable proportion of cerebrospinal fluid (CSF). Besides the CP acts as a selective barrier separating the fluctuating fluid constitu ents of the brain vascular compartiment from the stable, more precisely defined C S F . An increased secretory activity of the choroid plexus is characterized by typical morphological changes. ' Surprisingly, very little is known concerning morpho logical aspects of the choroid plexus of SHR. So we undertook to examine this organ by means of scanning and transmission electronic microscopy. 6
7 - 8
9
10
METHODS Male Okamoto SHR, and their normotensive controls (Wistar-Kyoto rats, WKY, derived from the original Okamoto stock and supplied by Centre d'Elevage Roger 0895-7061/92/$5.00
852
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AJH-NOVEMBER
1992-VOL
5, NO. 11
microscope processing, fragments were dehydrated in an alcohol series, thereafter in propylene oxide and em bedded in Epon resin. After 24 h Epon polymerization at 60 °C, blocks were cut and sections were contrasted with uranyl acetate and lead citrate before observation with Philips CM 12 and Jeol 100 Β electron microscopes. Basement membrane measurements were made with the Philips CM 12 microscope, which has an online measurement device. Suitable areas were recorded, then accurate and reproducible measurements were made immediately without taking any photographs. In view of having as precise a figure as possible, the thick ness of the basement membrane was recorded 15 times. For the study of apical microvilli evaluation, three samples were examined in six rats of each strain. The difference between SHR and WKY was obvious. How ever, there were two difficulties in quantification: the SHR brush border may show both a slight homoge neous decrease of microvilli and marqued losses, and the light and dark staining cells do not have the same microvilli. The dark staining cells had filliform micro villi. In addition, SHR possessed an increased number of
FIGURE 1. Electron micrograph of choroid plexus from 6week-old WKY. A) Aspect of normal epithelial cell. At the apical site, the brush border was dense and regular. The nucleus (N) presented with homogeneous chromatin. In the cytoplasm, mitochondriae (short arrow), vacuoles and Golgi apparatus (G) were observed. X 10,000. B) Higher magnification of the brush border. Tight junction on the left side (long arrow) was observed, as were microvacuoles (star), mitochondriae (short arrow), and Golgi appa ratus (G). X 20,000.
Janvier, Le Genest, France) were studied at 4 to 20 weeks of age. Immediately after cervical dislocation, the whole CP (from the lateral, third, and fourth ventricles) of six ani mals in each strain was removed with the aid of a dis secting microscope. We did not use perfuse fixation be cause samples of fresh tissue were also obtained from these animals for ionic transport studies. Samples were fixed in Trump's liquid ( 2 % parafor maldehyde and 2 % glutaraldehyde) for ultrastructural study. For scanning electron microscope processing, the CP were desiccated with C 0 at the critical point in a Bazers device, sprinkled with gold under a rarefied argon atmosphere and observed with a scanning elec tron microscope (JSM 35C). For transmission electron 1
2
FIGURE 2. Electron micrograph of choroid plexus from 20week-old WKY. A) Normal light and dark staining cells with dense filiform microvilli. Multiple infoldings of the basolateral mem brane (anow) were observed. X8000. B) Higher magnification of the basolateral membrane. X30,000.
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dark staining cells compared to WKY. In order to obtain the optimum evaluation we decided to count the micro villi where the junctions with cytoplasm were clearly identifiable and not the apices of microvilli that we can perceive on the picture. Each count was made using the same magnification (X 6000). We analysed a total length of approximately 5000 μτη for each rat. Five blocks of each plexus were examined. Identical measurements were carried out for the same number (n = 6) of SHR and WKY. RESULTS From the opening of the ventricles, the choroid plexus from SHR seemed larger than those from WKY and their vascularization more visible. WKY (4,6,8, and 20 Weeks of Age) The choroid plexus exhibited normal ultrastructural morphology with light cells and unfrequent dark cells distinguished by their dense cytoplasm (Figure 1A). Mitochondriae and vesi cles still had their structural integrity. The apical tight junction (zona occludens) remained unaltered (Figure IB). So did the typical filiform and predominantly bul bous microvilli (Figure 1 A, B), emerging from the apical surface membrane as a narrow stalk enlarging into an ovoid blob with a greatly infolded basolateral labyrinth (Figures 2A, 3). The basement membrane thickness averaged 89 nm. The perivascular space was very tight and the capillary basement membrane was approxi mately 130 nm thick at 6 weeks (Table 1). Scanning electron microscope examination showed the typical long cauliflower shaped structure of a nor mal choroid plexus formed of polygonal cells covered
CHOROID PLEXUS ULTRASTRUCTURE
853
with a long-pile carpet structure with even microvilli at their convex apical surface. Microvillar density (Figure 4A, B) made it difficult to tell one cell from another. No remarkable changes were observed between 4 to 6 weeks to 20 weeks, except for the cell and capillary basement membranes, which had thickened (respec tively by 133 and 199 nm) (Figure 3), and the dark staining cells, whose number had increased (Figure 2A). SHR (4, 6, and 8 Weeks of Age) Widespread changes were observed unevenly throughout the choroid plexus. The most striking change was a partial loss of microvilli. (Figures 3, 5A, and 5B). Two morphological types of microvilli were present as in normal choroidal epithe lium: filiform and bulbous or club-shaped. But from week 4, we noted alterations in the microvilli shape and multiple large and bulbous microvilli were observed in decreasing numbers over time. In these areas, numerous microvesicles were found close to the apical epithelial surface or merging with the apical membrane. The pres ence of a great number of merged vesicles with the api cal membrane could explain the partial loss of microvilli. These losses were more marked on the apices of the light staining cells. The surface area partially lost corre sponded to approximately 1 5 % of total microvillar sur face (Figure 3). The dark staining cells only showed filiform dark mi crovilli and these were seen in growing numbers in SHR, while their density varied according to areas. They showed dense cytoplasm filled with osmiophilic drop lets and numerous mitochondriae with dense matrices. In the light staining cells, Golgi apparatus was very well developed (Figure 5 A). The irregular nuclei showed
FIGURE 3. Schematic diagram corresponding to choroid plexus cell in WKY and SHR at 6 weeks. In SHR, the nucleus (N) showed a heteroge neous chromatin. Golgi apparatus were more numerous, as were mito chondriae (mi). On the other hand, we observed a clear loss of microvilli and a decrease in infoldings of the basal membrane.
WKY
SHR
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TABLE 1. THICKNESS (nm) OF THE CELL BASAL MEMBRANE AND THE VASCULAR BASAL MEMBRANE IN WKY AND SHR AT 6 AND 20 WEEKS 6 weeks
WKY SHR
20 weeks
Cell Basal Membrane
Vascular Basal Membrane
Cell Basal Membrane
Vascular Basal Membrane
89 160
132 140
133 302
199 322
All measurements in nanometers. These results corresponded to the average of ten values.
heterogenous chromatin and a distinct nucleoli. Modifications were also marked at the basolateral membrane level where the infolded labyrinth was less regularly distributed with large defects (Figure 3). More or less plane areas were noted where mitochondriae had gathered (Figure 5B). The basement membrane had markedly thickened: 160 nm at 6 weeks. The capil-
FIGURE 5. Electron micrograph of choroid plexus from 6-week old SHR. A) Epithelial cells with a loss of surface microvilli. The tight junction (small medium-weight arrow), the microvesicles close to the apical surface (thin long arrow), mitochondriae (short arrow) and the Golgi apparatus (G) were all observed. The nucleus (N) showed a heterogeneous chromatin. Basolateral membrane exhibited rare infoldings. (large thick arrow) X 10,000 B) Apical light staining cell surface with partial loss of the microvilli. Small vesicles (thin long arrow) were observed close to the plane apical surface. The mitochondriae (short arrow) were more or less gathered together. X20,000.
laries showed fewer modifications at 6 weeks with a lesser thickening (140 nm) of the basement membrane (Table 1). Scanning electron microscope examination provided confirmation of the microvilli loss. The cauliflower shape was maintained, but we could now distinguish the apex of each cell and a sort of disjunction between FIGURE 4. Electron micrograph of choroid plexus from 6- them due to a decrease in microvillar density (Figure 6A, B). Some areas were more involved, in which small week-old WKY. A) Scanning electron micrograph of the choroid plane and bald surfaces could be observed (Figure 6B). plexus, which shows as a long cauliflower-shaped structure covered with regular and dense microcilli. X1000. B) With higher At 20 weeks, the choroid plexus from SHR presented magnification, the different apical surfaces (star) were identified exactly the same appearance in scanning electron microonly with difficulty because of the micovilli density. X4800. scope pictures.
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scattered chromatin in the light staining cells and very irregular with a voluminous nucleolus in the dark staining cells (Figure 7). The SHR cell basement membrane was two-fold thicker (302 nm) than WKY's. The SHR membrane was planar with a small number of thickened infoldings. The capillary basement membrane was 322 nm thick (Table 1). DISCUSSION The choroid plexus is the most important site of CSF production, secreting roughly 9 0 % of the total CSF, thus playing a major role in the maintenance of homeostasis of the central nervous system microenvironment. The complete integrity of its lining is essential to maintaining its functions. Interestingly, ultrastructural examination of choroid plexus from young SHR revealed an irregular nucleus with heterogenous chromatin and a 7,8
9,10
FIGURE 6. Electron micrograph of choroid plexus from 6week-old SHR. A) In scanning electron micrograph, the choroid plexus showed a long and large cauliflower shape structure. The different apical surfaces (star) were clearly identified. X1000. B) Higher magnification showed the partial loss of microvilli and a space between the cell apical surface (star). Little bald areas were noticed (arrow). X4800.
At 8 weeks, the choroid plexus from SHR showed the same modifications with some more clusters of mitochondriae and osmiophilic droplets in transmission electron microscope examination. On the other hand, transmission electron microscope examination of 20 week old SHR choroidal epithelium and capillaries showed deep modifications. Two-thirds of the cells were of the dark staining type with dense cytoplasm full of mitochondriae, dense vesicles, and osmiophilic droplets. (Figure 7A). Their apical surface was covered with thin and filiform microvilli (Figure 7A, B). The light stained cells presented a loss of bulbous microvilli and some of them showed a striking abundance of mitochondriae filling the cytoplasm, which gave the cell the aspect of an oncocyte. Fewer micro vesicles were observed in the basement membrane than in the apical membrane. The nuclei were regular with delicately
FIGURE 7. Electron micrograph of choroid plexus from 20week-old SHR. A) The epithelial cells showed an insignificant loss of microvilli. The dark staining cells showedfiliformmicrovilli. Their nuclei (N) were inegular with a heterogeneous chromatin and a voluminous nucleolus. X4000. B) Higher magnification of a dark staining cell with its heterogeneous nucleus (N) and numerous and large mitochondriae (M) with a dense matrix. X 16,000.
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distinct nucleolus, which are typical morphologic changes of increased secretory activity. This increased secretory activity is accountable for a partial loss of apical and basolateral membranes and it brought about so many metabolic modifications that, at the age of 20 weeks, the cells showed signs of overwork, such as numerous mitochondriae of more or less regular shape and size, filling the cytoplasma, and giving the cell the characteristic aspect of an oncocyte. These pictures are evidence of a long increased secretory activity. This is in accordance with our previous results showing increased cotransport and pump activities, particularly in young SHR, in spite of the diminished basolateral and apical membrane surface. By the same token, this increased secretory and transport activity seemed to utterly exhaust the cell capacities, since the adult choroidal epithelium presented a more decreased basolateral membrane surface and, above all, a less functional cytoplasm. We observed a good correlation between the ultrastructural morphological changes and the cotransport and pump activities. However, we do not know whether these features of an intense secretory activity result in increased CSF formation and may be somehow related to blood pressure regulation. In SHR, the rise in blood pressure is linked to an increase in total peripheral resistance, the cause of which is neurogenic during the initiation of hypertension. SHR have elevated plasma catecholamine levels and the reactivity of vessel resistance to norepinephrine is increased. The present work is, to the best of our knowledge, the first to demonstrate there are morphological abnormalities in the young and adult SHR choroid plexus. Similar data have been recently reported in the intestinal and renal epithelium in SHR. Unfortunately, little is known about regulation of blood flow to the choroid plexus. Our findings might suggest there is a relation between increased catecholamine level and increased activities of transport and secretion. Elsewhere Lindvall and recently Faraci have shown that intravenous norepinephrine decreases blood flow to choroid plexus and CSF production as well. The question remains as to whether the abnormalities in choroid plexus can be linked to the increased noradrenergic activity or hypertensive mechanism in SHR.
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ACKNOWLEDGMENTS We thank Miss Genevieve Chauveaud and Mr. Daniel Ekmekdjian for their technical assistance. REFERENCES 1.
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