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Development of brain capillaries in euthyroid and hypothyroid rats
EXPERIMENTAL
73, 243-253 (1981)
NEUROLOGY
Development
of Brain Capillaries in Euthyroid and Hypothyroid Rats
S. DAVID AND E. J. H. NATHANIEL’ Department
Received
of Anatomy. Faculty of Medicine. University Winnipeg, Manitoba. Canada August
29, 1980; revision
received
December
of Manitoba,
3. 1980
The number of blood vessel profiles per square millimeter in the cuneate nucleus of normal rats, as estimated by light microscopy, doubled between 1 and 3 weeks postnatum (pn), and plateaued thereafter. In hypothyroid rats this postnatal increase occurred only until 2 weeks pn. There was also a significant reduction in blood vessel numbers in hypothyroid rats (P < 0.01) observed as early as 2 weeks pn. Ultrastructurally, capillary maturation in normal rats consisted of the following: the earliest identifiable capillaries displayed a slit-like lumen enclosed by thick endothelial cells and surrounded by an unevenly thick basal lamina. The astrocytic endfeet of these capillaries appeared as pale, dilated profiles. With maturation, the capillary wall became attenuated with a concomitant increase in lumen size. Mature capillaries were surrounded by an evenly thick basal lamina and narrow astrocytic end-feet containing large bundles of filaments. Outward cytoplasmic projections of pericytes and endothelial cells were seen as long as 3 weeks pn. Fine structural alterations in hypothyroid rats consisted of pericytic hypertrophy at 2 weeks pn, followed by the appearance of large inclusion-laden cells, situated within the vessel wall. At 4 weeks pn the astrocytic end-feet of mature capillaries lacked filament bundles but contained numerous glycogen granules. Edema of the astrocytic endfeet was noted frequently at 6 weeks pn.
INTRODUCTION Several quantitative studies on blood vessels during postnatal development of the murine (11, 29) and rodent (4, 6, 30) central nervous system Abbreviations: LM-light microscope, PTU-propylthiouracil, pn-postnatum. ’ This work was supported in part by the Research and Technology Foundation of the Paralysed Veterans of America, Multiple Sclerosis Society of Canada, Mrs. James A. Richardson Foundation, and the University of Manitoba Grant-in-Aid, and Medical Research Council of Canada student fellowship to SD., whose present address is Department of Neurology, McGill University and Montreal General Hospital, Montreal, P.Q., Canada. 243 0014-4886/81/070243-11$02.00/0 Copyright 0 1981 by Academic Press. Inc. All rights of repraduclion in any form reserved
244
DAVID
AND
NATHANIEL
indicated a rapid and dramatic increase in vascularization during the second 10 days of life, with little change beyond this period. Fine structural changes accompanying capillary maturation were reported in the rat cerebral cortex (4, 9) in the rat spinal cord (13, 27,. 33), in the rat olfactory bulb (30) in the avian brain (8, 28, 36), and in the human brain (14). Neonatal hypothyroidism is known to result in alterations in neurons (17) and glia (3, 26). However, except for the quantitative light microscope (LM) study by Eayrs (lo), no literature exists on the vascular changes in this condition. This paper documents quantitative light microscopic and fine structural alterations of capillaries in the cuneate nucleus, an important relay station, during postnatal development of euthyroid and hypothyroid rats. Alterations in the growth and development of dorsal column nuclei in neonatal hypothyroidism could possibly result in faulty information processing and subsequent sensory and even motor deficits. MATERIALS
AND
METHODS
Twelve litters of Sprague-Dawley rats from a highly inbred colony were divided into three groups of four litters each. Pups in group lreceived daily subcutaneous injections of propylthiouracil (PTU, Sigma), dissolved in physiological saline according to the following schedule: 0.05 ml 0.2% PTU on days 0 to 10; 0.1 ml 0.2% PTU on days 11 to 20; 0.1 ml 0.4% PTU on days 21 to 30; and 0.2 ml 0.4% PTU on days 3 1 to 42. Pups in group 2 received physiologic saline and pups in group 3 served as noninsulted controls. Four pups in each group (one from each litter) were fixed at weekly intervals from birth to 6 weeks under pentobarbital anaesthesia by intracardiac perfusion with Karnovsky’s fixative at pH 7.2. Thin slices of the medulla oblongata were removed and further fixed 1 h in Karnovsky’s fixative followed by postfixation in 1% osmium tetroxide and were eventually embedded in araldite after dehydration in ascending grades of ethanol. Thick sections (0.5 pm) were stained with toluidine blue for localization of the cuneate nucleus and for quantitative LM studies of the number of blood vessel profiles per square millimeter. Thin sections through the cuneate nucleus were stained with uranyl acetate and lead citrate and viewed with a Philips EM 300 electron microscope. At the time of killing the thyroid glands were removed for routine LM studies. The animals were judged to be hypothyroid on the basis of lack of colloid and presence of hyperplastic follicular epithelium. Estimation of the number of blood vessel profiles per square millimeter were obtained from 0.5Frn-thick toluidine blue-stained cross sections of
DEVELOPMENT
OF BRAIN CAPILLARIES
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cuneate nucleus situated in the lower medulla. Counts were made using a micrometer disk (Bausch and Lomb) on sections obtained from four to six blocks from each animal. All quantitative measurements were made on a sample size of four animals. It may be relevant to mention that the capillaries did not have any specific preferred orientation. The quantitative LM data were analyzed using a two-sample t test. RESULTS Light and electron microscopy revealed no differences between the saline-injected controls of group 2 and the noninsulted controls of group 3. The findings in these two groups will be discussed under the subtitle normal rats. Normal Rats Light Microscopy. Quantitative studies revealed a doubling of blood vessel profiles per square millimeter between 1 and 3 weeks postnatum (pn), with no further increase thereafter (Fig. 1). Electron Microscopy. Blood vessels displaying various degrees of maturation were encountered in animals to the third postnatal week. The earliest identifiable capillary possessed a slit-like lumen, surrounded by a thick endothelial cell wall (Fig. 2A). The endothelial nuclei were irregularly shaped with masses of peripherally clumped chromatin. Their cytoplasm contained abundant free ribosomes, mitochondria, rough endoplasmic reticulum, pinocytotic vesicles, as well as multivesicular bodies and Golgi complexes (Figs, 2A, B). Junctional complexes between adjacent endothelial cells were seen in the earliest nonpatent vessels. A basal lamina of uneven thickness surrounded the endothelial cell and split to enclose pericytes (Fig. 2A). The astrocytic investment was almost complete at birth, even in the earliest non-
FIG.
1. Graph showing growth of brain capillaries in hypothyroid and control rats
246
DAVID AND NATHANIEL
FIG. 2. (A) Micrograph of an immature capillary of a normal rat presenting a slit-like lumen (arrowheads). The endothelial cell has a large irregular-shape nucleus (Nu) and a cytoplasm containing numerous organelles. Note the pale, dilated astrocytic end-feet (As) surrounding the capillary. A small portion of pericyte (P) is seen surrounded by the basal lamina. The basal lamina surrounding the endothelial cell is uneven in thickness (arrows). X16.181.25. (B) Micrograph of an immature patent capillary from a normal rat. The thick endothelial cell cytoplasm (E) contains numerous polyribosomes and segments of rough endoplasmic reticulum. Portions of pericytic cytoplasm (P) are seen adjacent to the endothelial ceil. Observe the uneven thickness of the surrounding basal lamina (arrows). X13.237.5. (C) A mature capillary displaying a wide lumen and an attenuated vessel wall. The basal lamina is well defined and uniform in thickness and splits to enclose a pericyte (P). The astrocytic end-feet (As) are compact and contain large amounts of astrocytic filaments. Normal rat. X10.665. (D) Micrograph of an immature capillary demonstrating a cytoplasmic projection of the endothelial cell (arrowheads), passing through a break in the basal lamina (arrows). Normal rat. X27.720.
DEVELOPMENT
OF BRAIN
CAPILLARIES
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patent capillaries where they appeared as large, pale, dilated profiles, containing few organelles or inclusions (Fig. 2A). Capillary maturation was accompanied by attenuation of the endothelial wall, with an increase in the patency of the lumen. The endothelial cell nucleus was thinner with an irregular profile and a cytoplasm with many organelles. The basal lamina remained uneven in thickness (Fig. 2B). With further maturation there was attenuation of the endothelial cell, decrease in both pseudopodia and pinocytotic vesicles, and widening of the lumen (Fig. 2C). The basal lamina was uniform in thickness and well defined. The astrocytic end-feet were reduced in size and contained large bundles of filaments. Mitotic pericytes and endothelial cells were sometimes encountered. In animals between 1 and 3 weeks of age, outward cytoplasmic projections of endothelial cells (Fig. 2D) and pericytes were occasionally found insinuating between elements of the surrounding neuropil. Hypothyroid
Rats
Light Microscopy. Quantitative studies revealed that in hypothyroid rats the postnatal increase in blood vessel profiles per square millimeter occurred only until 2 weeks pn, in contrast to control animals in which such an increase continued until 3 weeks pn. In addition, beyond the second postnatal week, there was a significant reduction in blood vessel profiles in hypothyroid rats compared with controls (P < 0.01) (Fig. 1). Electron Microscopy. The fine structural features of the vessels at birth and 1 week were similar to that of controls. From 2 weeks onward, there appeared to be a hypertrophy of pericytes (Fig. 3A) characterized by a large nucleus with deep nuclear invaginations and a cytoplasm packed with organelles. In hypothyroid animals of 3 weeks of age and older, cells with various kinds of inclusions were frequently found lying external to the pericyte but still enclosed within the basal lamina (Figs. 3B, C). In addition, the cytoplasm also contained numerous smooth and coated vesicles: a few segments of rough endoplasmic reticulum and unusually long segments of smooth-surfaced endoplasmic reticulum with coated vesicles budding from their ends (Fig. 3C). The chromatin pattern of these cells resembled that of pericytes. Mature capillaries of the 4-week-old hypothyroid animals displayed attenuated walls and other ultrastructural features similar to those in control animals. However, the astrocytic end-feet seemed to contain fewer filaments and an increased amount of glycogen granules (Fig. 3D) compared with controls. In contrast to controls, immature, nonpatent vessels were still encountered in 5- and 6-week-old hypothyroid rats (Fig. 3E). The
248
DAVID AND NATHANIEL
FIG. 3. (A) Part of a blood vessel from a 4-week-old hypothyroid rat displaying a hypertrophied pericyte (P). Note the numerous glycogen granules in the adjacent astrocytic processes (As). X10.296. (8) Micrograph from a Sweek-old hypothyroid rat illustrating part of a blood vessel (bv), a pericyte (P), and an inclusion-laden cell (IC) enclosed within the basal lamina. X8700. (C) Micrograph illustrating an inclusion-laden cell situated external to the pericyte (P) but still surrounded by the basal lamina (arrows). Observe the three types of cyto-
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outstanding feature in such rats was edema of the astrocytic end-feet (Fig. 3E). This swelling appeared to be part of a more generalized swelling of a large number of astrocytic processes throughout the neuropil. DISCUSSION The quantitative studies of blood vessel profiles indicate that the maximum period of vasculogenesis resulting in maximal density of vascular profiles occurs in the rat cuneate nucleus between 1 and 3 weeks after birth. In the rat cerebral cortex (4) and olfactory bulb (30), vasculogenesis was reported to take place during the second 10 days of life. It therefore appears that vascular proliferation culminating in maximal vascular density occurs in various regions of the central nervous system generally during the first 3 postnatal weeks. Endothelial cells of immature, nonpatent capillaries of the cuneate nucleus exhibit nuclear and cytoplasmic features suggestive of increased synthetic activity which are in keeping with findings of several investigations (4, 9, 13, 30). As these capillaries mature there is some attenuation of the endothelial cells with a concomitant increase in the size and patency of the lumen. Such capillaries, nonetheless, still displayed immature characteristics, such as an irregular endothelial nucleus, a cytoplasm containing abundant organelles, and an uneven ill-defined basal lamina. Thus maturation of the various components of the capillary is not completed with the opening of its lumen, but continues for some time thereafter. Similar observations were also made in the developing substantia gelatinosa (13). Decrease in the number of pinocytotic vesicles with maturation of capillaries in the central nervous system was reported by most investigators and was related to the development of a more complete and discriminatory blood-brain barrier (13). On the other hand, pinocytosis in immature capillaries might reflect an increased metabolic demand of the endothelial cell itself. Investment of the capillary surface by astrocytic end-feet appears to be almost complete at birth in the rat cuneate nucleus. The rate of acquisition plasmic inclusions: homogeneous dense bodies (DB), medium-dense granular bodies (GB), and electron-lucent bodies containing a flocculent material (LB). Three-week-old hypothyroid rat. X8648. (D) A mature capillary from a 4-week-old hypothyroid rat, demonstrating a wide lumen (L) and an attenuated endothelial cell cytoplasm (E). Some hypertrophy of the pericyte (P) is also evident. Note the abundance of glycogen granules in the surrounding astrocytic end-feet (As). X 13,240. (E) An immature, nonpatent capillary from a 5-week-old hypothyroid rat. Observe the slit-like lumen (arrowheads) and the surrounding basal lamina (arrows). The nucleus (Nu) of the endothelial cell is large displaying pronounced imaginations. Swollen astrocytic processes (As) form perivascular end-feet. X 1I, 140.
250
DAVID
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of the astrocytic investment varies in different parts of the central nervous system. It is complete at birth in the ventral horn of the rat spinal cord (27, 33), whereas in the rat cerebral cortex (4) and substantia gelatinosa (30) it occurs between 6 and 9 days pn. In the present investigation, to the third postnatal week, astrocytic endfeet often appeared as large, pale profiles, especially surrounding immature capillaries. This somewhat edematous appearance might reflect the state of development of astrocytes and/or a poorly developed fluid transport mechanism around immature capillaries. Outward cytoplasmic extensions of endothelial cells and pericytes were found in the present study, insinuating between elements of the neuropil. These processes contained filaments or mitochondria, and appeared in animals between 1 and 3 weeks pn, which is the period of maximum vasculogenesis. Recent studies revealed the presence of actin- and myosin-like filaments in endothelial cells and pericytes of rat brain capillaries ( 18, 19). If such pseudopod-like cytoplasmic extensions possess contractile properties, it is possible they represent early stages of an infiltrative process, making the way for the developing vascular cords sprouting from afferent vessels ( 16). In the current investigation a drastic reduction in the number of blood vessels was observed in the cuneate nucleus of hypothyroid rats. In the only other quantitative study reported in the literature, a reduction in capillaries was also noted in the cerebral cortex of hypothyroid rats (10). It thus appears that reduction in vascularization of the central nervous system, may occur as a generalized reaction to neonatal hypothyroidism. Fine structural alterations of blood vessels in the hypothyroid animals consisted of hypertrophy of pericytes followed at later time sequences by the appearance of large inclusion-laden cells lying external to the pericytes but still contained within the basal lamina. The hypertrophy of pericytes preceding the appearance of inclusion-laden cells raises the possibility that the latter cells may arise from pericytes and might represent the granular pericytes of light microscopy (5). The pagocytic role of pericytes was emphasized by Majno (20) and was demonstrated in neoplasms of the central nervous system (34), Tay-Sachs disease (32), Krabbe’s disesase (l), and after irradiation injury (23). Maxwell and Kruger (23) suggested that under certain conditions pericytes may divide, and the daughter cells then migrate into the nervous tissue to become macrophages. Activation of pericytes and their transformation into microglial cells was also claimed by Hager (12) in traumatic brain lesions. Migration of pericytes or pericytal microglia through breaks in the basal lamina has also been reported (2, 12, 21). Ultrastructurally however, these inclusion-laden cells also resemble the round and ameboid
DEVELOPMENT
OF BRAIN CAPILLARIES
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microglia seen in the neonatal rabbit brain (31), and ameboid cells in the neonatal rat corpus callosum (15). The latter investigators considered the ameboid cells to arise from monocytes, and to transform into microglia. Two possibilities therefore exist as to the origin of the inclusion-laden cells observed in the hypothyroid animals. They may either arise from pericytes or from monocytes. It is possible that after these cells break through the confines of the basal lamina, they come to lie in the neuropil and function as microglia. The presence of these cells may be related to the low-grade degeneration encountered in the cuneate nucleus of neonatally hypothyroid rats (7). The accumulation of glycogen granules within astrocytic end-feet observed after the fourth postnatal week is considered to be a generalized reaction of the astrocytes to insult of the central nervous system. A similar response was reported in rats to follow X-irradiation (22) and crushing of the dorsal roots (24, 25), and was found to be the earliest response of the central nervous system even in the absence of any morphologic alteration in other nervous elements. Swelling of the astrocytic end-feet which was seen in 5- and 6-week-old hypothyroid rats, is also a common feature of several experimental conditions such as traumatic brain lesions ( 12), cerebral ischemia (37), as well as after intravascular infusion of ouabain (38). The astrocytic swelling caused by ouabain, an inhibitor of Na-K adenosine triphosphatase, is particularly interesting, because the activity of this enzyme has been shown to be reduced in neonatal hypothyroidism (35). REFERENCES I. ANDREWS, J. M., P. A. CANCILLA, J. GRIPPO, AND J. H. MENKES. 1971. Globoid cell leukodystrophy (Krabbe’s disease): morphological and biochemical studies. Neurology (Minneapolis) 21: 337-352. 2. BARON, M., AND A. GALLEGO. 1972. The relation of the microglia with the pericyte in the cat cerebral cortex. Z. Zellforsch. 12% 42-57. 3. BASS, N. H., AND E. YOUNG. 1973. Effects of hypothyroidism on the differentiation of neurons and glia in developing rat cerebrum. J. Neurof. Sci. 18: 155-173. 4. CALEY, D. W., AND D. S. MAXWELL. 1970. Development of the blood vessels and extracellular spaces during postnatal maturation of the rat cerebral cortex. J. Comp. Neural. 138~ 31-48. 5. CAMMERMEYER. J. 1970. The life history of the microglial cell: a light microscopic study. Pages 43-129 in S. EHRENPREIS AND 0. C. SOLNITZY, Eds., Neurosciences Research, Vol. 3. Academic Press, New York. 6. CRAIGIE, E. H. 1925. Postnatal changes in vascularity in the cerebral cortex of the male albino rat. J. Comp. Neural. 39: 301-324. 7. DAVID, S. 1979. Postnatal Development of the Cuneate Nucleus in Euthyroid and Hypothyroid Rats-An Ultrastructural Study, Ph.D. Thesis. University of Manitoba. 8. DELORME, P., G. GRIGNON, AND J. GAYET. 1968. Ultrastructure des capillaries darts le
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9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30.
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t61Cc6phale du poulet au tours de I’embryogenbe et de la croissance postnatale. 2. Zellforsch. 87: 592602. DONAHUE, S., AND G. D. PAPPAS. 1961. The fine structure of capillaries in the cerebral cortex of the rat at various stages of development. Am. J. Anat. 108: 331-347. EAYRS, J. T. 1954. The vascularity of the cerebral cortex in normal and cretinous rats. J. Anat. 88: 164-173. GYLLENSTEN, L. 1959. Influence of oxygen exposure on the postnatal vascularization of the cerebral cortex in mice. AcIa Morph. Neerl. Stand. 2: 289-310. HAGER, H. 1975. EM findings on the source of reactive mircroglia on the mammalian brain. Acta Neuropathol. (Berlin) Suppl. VI: 279-283. HANNAH, R. S., AND E. J. H. NATHANIEL. 1974. The postnatal development of blood vesselsin the substantia gelatinosa of rat cervical cord-an ultrastructural study. Anat. Rec. 178: 691-710. HAUW, J.-J., B. BERGER, AND R. ESCOUROLLE. 1975. Electron microscopic study of the developing capillaries of human brain. Acta Neuropathol. (Berlin) 31: 229-242. IMAMOTO, K., AND C. P. LEBLOND. 1978. Radioautographic investigation of gliogenesis in the corpus callosum of young rats. II. Origin of microglial cells. J. Comp. Neurol. 180: 139-164. KLOSOVSKII, B. N. 1963. The Development of the Brain, p. 275. Pergamon, New York. LAUDER, J. M. 1977. Effects of thyroid state on development of rat cerebellar cortex. Pages 235-254 in GILMAN D. GRAVE, Ed., Thyroid Hormonesand Brain Development. Raven Press, New York. LE BEUX, Y. J., AND J. WILLEMOT. 1978. Actin-like filaments in the endothelial cells of adult rat brain capillaries. Exp. Neurol. 58: 446-454. LE BEUX, Y. J., AND J. WILLEMOT. 1978. Actin- and myosin-like filaments in rat brain pericytes. Anat. rec. 190: 81 I-825. MAJNO, G. 1965. Ultrastructure of the vascular membrane. Page 2293 W. F. HAMILTON AND P. Dow, Eds., Handbook of Physiology. Amer. Physiol. Sot., Washington, DC. MORI, S., AND C. P. LEBLOND. 1969. Identification of microglia in light and electron microscopy. J. Comp. Neurol. 135: 57-80. MIQUEL, J., AND W. HAYMAKER. 1965. Astroglial reactions to ionizing radiation: with emphasis on glycogen accumulation. Prog. Brain Res. IS: 89-l 14. MAXWELL, D. S., AND L. KRUGER. 1965. Small blood vesselsand the origin of phagocytes in the rat cerebral cortex following heavy particle irradiation. Bxp. Neural. 12: 33-54. NATHANIEL, E. J. H., AND D. R. NATHANIEL. 1973. Degeneration of posterior roots in the spinal cord of adult rats. An electron microscopic study. Exp. Neural. 40: 316332. NATHANIEL, E. J. H., AND D. R. NATHANIEL. 1977. Astroglial response to degeneration of dorsal root fibers in adult rat spinal cord. Exp. Neural. 54: 60-76. PESETSKY, I. 1973. The development of abnormal cerebellar astrocytes in young hypothyroid rats. Brain Res. 63: 456-460. PHELPS, C. H. 1972. The development of glio-vascular relationships in the rat spinal cord. Z. Zellforsch. 128: 555-563. ROY, S. A., A. HIRANO. J. A. KOCHEN, AND H. M. ZIMMERMAN. 1974. The fine structure of cerebral blood vessels in chick embryo. Acra Neuropathof. (Berlin) 30: 277-285. SAKLA, F. B. 1965. Post-natal growth of neuroglia cells and blood vessels of the cervical spinal cord of the albino mouse. J. Comp. Neural. 124: 189-202. SINGH, D. N. P., AND E. J. H. NATHANIEL. 1975. Postnatal development of blood vessels (capillaries) in the rat olfactory bulb: a light and ultrastructural study. Neurosci. Lett. 1: 203-208.
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31. STENSAAS, L. J., AND W. H. REICHERT. 1971. Round and ameboid microglial cells in the neonatal rabbit brain. Z. Zellforsch. 119: 147-163. 32. TERRY, R. D., AND M. WEISS. 1963. Studies on Tay-Sachs disease. II. Ultrastructure of the cerebrum. J. Neuropathol. Exp. Neurol. 22: 18-55. 33. THOMAS, A. P., AND E. J. H. NATHANIEL. 1980. Postnatal developmental study of blood vessels in the ventral horn of euthyroid and hypothyroid rat lumbar spinal cord-an ultrastructural study. J. Cell Biol. 87, 7 1A. (abstract). 34. TORACK, R. M. 1961. Ultrastructure of capillary reaction to brain tumors. Arch. Neural. 5: 416-428. 35.
VALCANA, T., AND P. S. TIMIRAS. 1969. Effect of hypothyroidism on ionic metabolism and Na-K activated ATP phosphohydrolase activity in the developing rat brain. J. Neurochem.
16: 935-943.
36. WECHSLER, W. 1965. Entwicklung der Gefabe and perivascularen Gewebsraume im Zentralnervensystem von HUhnern. Z. Anat. Entwickl. Gesch. 124: 367-395. 37. WESTERGAARD, E.. G. Go, I. KLATZO, AND M. SPATZ. 1976. Increased permeability of cerebral vessels to horseradish peroxidase induced by ischemia in mongolian gerbils. Acta
Neuropathol.
(Berlin)
35: 307-325.
38. WOLFF, J. R., C. SCHIEWECK, H. EMMENEGGER, AND W. MEIER-RUGE. 1975. Cerebrovascular ultrastructural alterations after intra-arterial infusion of ouabain, scillaglycosides, heparin and histamine. Acta Neuropathol. (Berlin) 31: 45-58.
73, 243-253 (1981)
NEUROLOGY
Development
of Brain Capillaries in Euthyroid and Hypothyroid Rats
S. DAVID AND E. J. H. NATHANIEL’ Department
Received
of Anatomy. Faculty of Medicine. University Winnipeg, Manitoba. Canada August
29, 1980; revision
received
December
of Manitoba,
3. 1980
The number of blood vessel profiles per square millimeter in the cuneate nucleus of normal rats, as estimated by light microscopy, doubled between 1 and 3 weeks postnatum (pn), and plateaued thereafter. In hypothyroid rats this postnatal increase occurred only until 2 weeks pn. There was also a significant reduction in blood vessel numbers in hypothyroid rats (P < 0.01) observed as early as 2 weeks pn. Ultrastructurally, capillary maturation in normal rats consisted of the following: the earliest identifiable capillaries displayed a slit-like lumen enclosed by thick endothelial cells and surrounded by an unevenly thick basal lamina. The astrocytic endfeet of these capillaries appeared as pale, dilated profiles. With maturation, the capillary wall became attenuated with a concomitant increase in lumen size. Mature capillaries were surrounded by an evenly thick basal lamina and narrow astrocytic end-feet containing large bundles of filaments. Outward cytoplasmic projections of pericytes and endothelial cells were seen as long as 3 weeks pn. Fine structural alterations in hypothyroid rats consisted of pericytic hypertrophy at 2 weeks pn, followed by the appearance of large inclusion-laden cells, situated within the vessel wall. At 4 weeks pn the astrocytic end-feet of mature capillaries lacked filament bundles but contained numerous glycogen granules. Edema of the astrocytic endfeet was noted frequently at 6 weeks pn.
INTRODUCTION Several quantitative studies on blood vessels during postnatal development of the murine (11, 29) and rodent (4, 6, 30) central nervous system Abbreviations: LM-light microscope, PTU-propylthiouracil, pn-postnatum. ’ This work was supported in part by the Research and Technology Foundation of the Paralysed Veterans of America, Multiple Sclerosis Society of Canada, Mrs. James A. Richardson Foundation, and the University of Manitoba Grant-in-Aid, and Medical Research Council of Canada student fellowship to SD., whose present address is Department of Neurology, McGill University and Montreal General Hospital, Montreal, P.Q., Canada. 243 0014-4886/81/070243-11$02.00/0 Copyright 0 1981 by Academic Press. Inc. All rights of repraduclion in any form reserved
244
DAVID
AND
NATHANIEL
indicated a rapid and dramatic increase in vascularization during the second 10 days of life, with little change beyond this period. Fine structural changes accompanying capillary maturation were reported in the rat cerebral cortex (4, 9) in the rat spinal cord (13, 27,. 33), in the rat olfactory bulb (30) in the avian brain (8, 28, 36), and in the human brain (14). Neonatal hypothyroidism is known to result in alterations in neurons (17) and glia (3, 26). However, except for the quantitative light microscope (LM) study by Eayrs (lo), no literature exists on the vascular changes in this condition. This paper documents quantitative light microscopic and fine structural alterations of capillaries in the cuneate nucleus, an important relay station, during postnatal development of euthyroid and hypothyroid rats. Alterations in the growth and development of dorsal column nuclei in neonatal hypothyroidism could possibly result in faulty information processing and subsequent sensory and even motor deficits. MATERIALS
AND
METHODS
Twelve litters of Sprague-Dawley rats from a highly inbred colony were divided into three groups of four litters each. Pups in group lreceived daily subcutaneous injections of propylthiouracil (PTU, Sigma), dissolved in physiological saline according to the following schedule: 0.05 ml 0.2% PTU on days 0 to 10; 0.1 ml 0.2% PTU on days 11 to 20; 0.1 ml 0.4% PTU on days 21 to 30; and 0.2 ml 0.4% PTU on days 3 1 to 42. Pups in group 2 received physiologic saline and pups in group 3 served as noninsulted controls. Four pups in each group (one from each litter) were fixed at weekly intervals from birth to 6 weeks under pentobarbital anaesthesia by intracardiac perfusion with Karnovsky’s fixative at pH 7.2. Thin slices of the medulla oblongata were removed and further fixed 1 h in Karnovsky’s fixative followed by postfixation in 1% osmium tetroxide and were eventually embedded in araldite after dehydration in ascending grades of ethanol. Thick sections (0.5 pm) were stained with toluidine blue for localization of the cuneate nucleus and for quantitative LM studies of the number of blood vessel profiles per square millimeter. Thin sections through the cuneate nucleus were stained with uranyl acetate and lead citrate and viewed with a Philips EM 300 electron microscope. At the time of killing the thyroid glands were removed for routine LM studies. The animals were judged to be hypothyroid on the basis of lack of colloid and presence of hyperplastic follicular epithelium. Estimation of the number of blood vessel profiles per square millimeter were obtained from 0.5Frn-thick toluidine blue-stained cross sections of
DEVELOPMENT
OF BRAIN CAPILLARIES
245
cuneate nucleus situated in the lower medulla. Counts were made using a micrometer disk (Bausch and Lomb) on sections obtained from four to six blocks from each animal. All quantitative measurements were made on a sample size of four animals. It may be relevant to mention that the capillaries did not have any specific preferred orientation. The quantitative LM data were analyzed using a two-sample t test. RESULTS Light and electron microscopy revealed no differences between the saline-injected controls of group 2 and the noninsulted controls of group 3. The findings in these two groups will be discussed under the subtitle normal rats. Normal Rats Light Microscopy. Quantitative studies revealed a doubling of blood vessel profiles per square millimeter between 1 and 3 weeks postnatum (pn), with no further increase thereafter (Fig. 1). Electron Microscopy. Blood vessels displaying various degrees of maturation were encountered in animals to the third postnatal week. The earliest identifiable capillary possessed a slit-like lumen, surrounded by a thick endothelial cell wall (Fig. 2A). The endothelial nuclei were irregularly shaped with masses of peripherally clumped chromatin. Their cytoplasm contained abundant free ribosomes, mitochondria, rough endoplasmic reticulum, pinocytotic vesicles, as well as multivesicular bodies and Golgi complexes (Figs, 2A, B). Junctional complexes between adjacent endothelial cells were seen in the earliest nonpatent vessels. A basal lamina of uneven thickness surrounded the endothelial cell and split to enclose pericytes (Fig. 2A). The astrocytic investment was almost complete at birth, even in the earliest non-
FIG.
1. Graph showing growth of brain capillaries in hypothyroid and control rats
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FIG. 2. (A) Micrograph of an immature capillary of a normal rat presenting a slit-like lumen (arrowheads). The endothelial cell has a large irregular-shape nucleus (Nu) and a cytoplasm containing numerous organelles. Note the pale, dilated astrocytic end-feet (As) surrounding the capillary. A small portion of pericyte (P) is seen surrounded by the basal lamina. The basal lamina surrounding the endothelial cell is uneven in thickness (arrows). X16.181.25. (B) Micrograph of an immature patent capillary from a normal rat. The thick endothelial cell cytoplasm (E) contains numerous polyribosomes and segments of rough endoplasmic reticulum. Portions of pericytic cytoplasm (P) are seen adjacent to the endothelial ceil. Observe the uneven thickness of the surrounding basal lamina (arrows). X13.237.5. (C) A mature capillary displaying a wide lumen and an attenuated vessel wall. The basal lamina is well defined and uniform in thickness and splits to enclose a pericyte (P). The astrocytic end-feet (As) are compact and contain large amounts of astrocytic filaments. Normal rat. X10.665. (D) Micrograph of an immature capillary demonstrating a cytoplasmic projection of the endothelial cell (arrowheads), passing through a break in the basal lamina (arrows). Normal rat. X27.720.
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patent capillaries where they appeared as large, pale, dilated profiles, containing few organelles or inclusions (Fig. 2A). Capillary maturation was accompanied by attenuation of the endothelial wall, with an increase in the patency of the lumen. The endothelial cell nucleus was thinner with an irregular profile and a cytoplasm with many organelles. The basal lamina remained uneven in thickness (Fig. 2B). With further maturation there was attenuation of the endothelial cell, decrease in both pseudopodia and pinocytotic vesicles, and widening of the lumen (Fig. 2C). The basal lamina was uniform in thickness and well defined. The astrocytic end-feet were reduced in size and contained large bundles of filaments. Mitotic pericytes and endothelial cells were sometimes encountered. In animals between 1 and 3 weeks of age, outward cytoplasmic projections of endothelial cells (Fig. 2D) and pericytes were occasionally found insinuating between elements of the surrounding neuropil. Hypothyroid
Rats
Light Microscopy. Quantitative studies revealed that in hypothyroid rats the postnatal increase in blood vessel profiles per square millimeter occurred only until 2 weeks pn, in contrast to control animals in which such an increase continued until 3 weeks pn. In addition, beyond the second postnatal week, there was a significant reduction in blood vessel profiles in hypothyroid rats compared with controls (P < 0.01) (Fig. 1). Electron Microscopy. The fine structural features of the vessels at birth and 1 week were similar to that of controls. From 2 weeks onward, there appeared to be a hypertrophy of pericytes (Fig. 3A) characterized by a large nucleus with deep nuclear invaginations and a cytoplasm packed with organelles. In hypothyroid animals of 3 weeks of age and older, cells with various kinds of inclusions were frequently found lying external to the pericyte but still enclosed within the basal lamina (Figs. 3B, C). In addition, the cytoplasm also contained numerous smooth and coated vesicles: a few segments of rough endoplasmic reticulum and unusually long segments of smooth-surfaced endoplasmic reticulum with coated vesicles budding from their ends (Fig. 3C). The chromatin pattern of these cells resembled that of pericytes. Mature capillaries of the 4-week-old hypothyroid animals displayed attenuated walls and other ultrastructural features similar to those in control animals. However, the astrocytic end-feet seemed to contain fewer filaments and an increased amount of glycogen granules (Fig. 3D) compared with controls. In contrast to controls, immature, nonpatent vessels were still encountered in 5- and 6-week-old hypothyroid rats (Fig. 3E). The
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FIG. 3. (A) Part of a blood vessel from a 4-week-old hypothyroid rat displaying a hypertrophied pericyte (P). Note the numerous glycogen granules in the adjacent astrocytic processes (As). X10.296. (8) Micrograph from a Sweek-old hypothyroid rat illustrating part of a blood vessel (bv), a pericyte (P), and an inclusion-laden cell (IC) enclosed within the basal lamina. X8700. (C) Micrograph illustrating an inclusion-laden cell situated external to the pericyte (P) but still surrounded by the basal lamina (arrows). Observe the three types of cyto-
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outstanding feature in such rats was edema of the astrocytic end-feet (Fig. 3E). This swelling appeared to be part of a more generalized swelling of a large number of astrocytic processes throughout the neuropil. DISCUSSION The quantitative studies of blood vessel profiles indicate that the maximum period of vasculogenesis resulting in maximal density of vascular profiles occurs in the rat cuneate nucleus between 1 and 3 weeks after birth. In the rat cerebral cortex (4) and olfactory bulb (30), vasculogenesis was reported to take place during the second 10 days of life. It therefore appears that vascular proliferation culminating in maximal vascular density occurs in various regions of the central nervous system generally during the first 3 postnatal weeks. Endothelial cells of immature, nonpatent capillaries of the cuneate nucleus exhibit nuclear and cytoplasmic features suggestive of increased synthetic activity which are in keeping with findings of several investigations (4, 9, 13, 30). As these capillaries mature there is some attenuation of the endothelial cells with a concomitant increase in the size and patency of the lumen. Such capillaries, nonetheless, still displayed immature characteristics, such as an irregular endothelial nucleus, a cytoplasm containing abundant organelles, and an uneven ill-defined basal lamina. Thus maturation of the various components of the capillary is not completed with the opening of its lumen, but continues for some time thereafter. Similar observations were also made in the developing substantia gelatinosa (13). Decrease in the number of pinocytotic vesicles with maturation of capillaries in the central nervous system was reported by most investigators and was related to the development of a more complete and discriminatory blood-brain barrier (13). On the other hand, pinocytosis in immature capillaries might reflect an increased metabolic demand of the endothelial cell itself. Investment of the capillary surface by astrocytic end-feet appears to be almost complete at birth in the rat cuneate nucleus. The rate of acquisition plasmic inclusions: homogeneous dense bodies (DB), medium-dense granular bodies (GB), and electron-lucent bodies containing a flocculent material (LB). Three-week-old hypothyroid rat. X8648. (D) A mature capillary from a 4-week-old hypothyroid rat, demonstrating a wide lumen (L) and an attenuated endothelial cell cytoplasm (E). Some hypertrophy of the pericyte (P) is also evident. Note the abundance of glycogen granules in the surrounding astrocytic end-feet (As). X 13,240. (E) An immature, nonpatent capillary from a 5-week-old hypothyroid rat. Observe the slit-like lumen (arrowheads) and the surrounding basal lamina (arrows). The nucleus (Nu) of the endothelial cell is large displaying pronounced imaginations. Swollen astrocytic processes (As) form perivascular end-feet. X 1I, 140.
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of the astrocytic investment varies in different parts of the central nervous system. It is complete at birth in the ventral horn of the rat spinal cord (27, 33), whereas in the rat cerebral cortex (4) and substantia gelatinosa (30) it occurs between 6 and 9 days pn. In the present investigation, to the third postnatal week, astrocytic endfeet often appeared as large, pale profiles, especially surrounding immature capillaries. This somewhat edematous appearance might reflect the state of development of astrocytes and/or a poorly developed fluid transport mechanism around immature capillaries. Outward cytoplasmic extensions of endothelial cells and pericytes were found in the present study, insinuating between elements of the neuropil. These processes contained filaments or mitochondria, and appeared in animals between 1 and 3 weeks pn, which is the period of maximum vasculogenesis. Recent studies revealed the presence of actin- and myosin-like filaments in endothelial cells and pericytes of rat brain capillaries ( 18, 19). If such pseudopod-like cytoplasmic extensions possess contractile properties, it is possible they represent early stages of an infiltrative process, making the way for the developing vascular cords sprouting from afferent vessels ( 16). In the current investigation a drastic reduction in the number of blood vessels was observed in the cuneate nucleus of hypothyroid rats. In the only other quantitative study reported in the literature, a reduction in capillaries was also noted in the cerebral cortex of hypothyroid rats (10). It thus appears that reduction in vascularization of the central nervous system, may occur as a generalized reaction to neonatal hypothyroidism. Fine structural alterations of blood vessels in the hypothyroid animals consisted of hypertrophy of pericytes followed at later time sequences by the appearance of large inclusion-laden cells lying external to the pericytes but still contained within the basal lamina. The hypertrophy of pericytes preceding the appearance of inclusion-laden cells raises the possibility that the latter cells may arise from pericytes and might represent the granular pericytes of light microscopy (5). The pagocytic role of pericytes was emphasized by Majno (20) and was demonstrated in neoplasms of the central nervous system (34), Tay-Sachs disease (32), Krabbe’s disesase (l), and after irradiation injury (23). Maxwell and Kruger (23) suggested that under certain conditions pericytes may divide, and the daughter cells then migrate into the nervous tissue to become macrophages. Activation of pericytes and their transformation into microglial cells was also claimed by Hager (12) in traumatic brain lesions. Migration of pericytes or pericytal microglia through breaks in the basal lamina has also been reported (2, 12, 21). Ultrastructurally however, these inclusion-laden cells also resemble the round and ameboid
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microglia seen in the neonatal rabbit brain (31), and ameboid cells in the neonatal rat corpus callosum (15). The latter investigators considered the ameboid cells to arise from monocytes, and to transform into microglia. Two possibilities therefore exist as to the origin of the inclusion-laden cells observed in the hypothyroid animals. They may either arise from pericytes or from monocytes. It is possible that after these cells break through the confines of the basal lamina, they come to lie in the neuropil and function as microglia. The presence of these cells may be related to the low-grade degeneration encountered in the cuneate nucleus of neonatally hypothyroid rats (7). The accumulation of glycogen granules within astrocytic end-feet observed after the fourth postnatal week is considered to be a generalized reaction of the astrocytes to insult of the central nervous system. A similar response was reported in rats to follow X-irradiation (22) and crushing of the dorsal roots (24, 25), and was found to be the earliest response of the central nervous system even in the absence of any morphologic alteration in other nervous elements. Swelling of the astrocytic end-feet which was seen in 5- and 6-week-old hypothyroid rats, is also a common feature of several experimental conditions such as traumatic brain lesions ( 12), cerebral ischemia (37), as well as after intravascular infusion of ouabain (38). The astrocytic swelling caused by ouabain, an inhibitor of Na-K adenosine triphosphatase, is particularly interesting, because the activity of this enzyme has been shown to be reduced in neonatal hypothyroidism (35). REFERENCES I. ANDREWS, J. M., P. A. CANCILLA, J. GRIPPO, AND J. H. MENKES. 1971. Globoid cell leukodystrophy (Krabbe’s disease): morphological and biochemical studies. Neurology (Minneapolis) 21: 337-352. 2. BARON, M., AND A. GALLEGO. 1972. The relation of the microglia with the pericyte in the cat cerebral cortex. Z. Zellforsch. 12% 42-57. 3. BASS, N. H., AND E. YOUNG. 1973. Effects of hypothyroidism on the differentiation of neurons and glia in developing rat cerebrum. J. Neurof. Sci. 18: 155-173. 4. CALEY, D. W., AND D. S. MAXWELL. 1970. Development of the blood vessels and extracellular spaces during postnatal maturation of the rat cerebral cortex. J. Comp. Neural. 138~ 31-48. 5. CAMMERMEYER. J. 1970. The life history of the microglial cell: a light microscopic study. Pages 43-129 in S. EHRENPREIS AND 0. C. SOLNITZY, Eds., Neurosciences Research, Vol. 3. Academic Press, New York. 6. CRAIGIE, E. H. 1925. Postnatal changes in vascularity in the cerebral cortex of the male albino rat. J. Comp. Neural. 39: 301-324. 7. DAVID, S. 1979. Postnatal Development of the Cuneate Nucleus in Euthyroid and Hypothyroid Rats-An Ultrastructural Study, Ph.D. Thesis. University of Manitoba. 8. DELORME, P., G. GRIGNON, AND J. GAYET. 1968. Ultrastructure des capillaries darts le
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38. WOLFF, J. R., C. SCHIEWECK, H. EMMENEGGER, AND W. MEIER-RUGE. 1975. Cerebrovascular ultrastructural alterations after intra-arterial infusion of ouabain, scillaglycosides, heparin and histamine. Acta Neuropathol. (Berlin) 31: 45-58.