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Comparisons of capillary maturation in control and hypothyroid rat spinal cord: An ultrastructural study
EXPERIMENTAL
NEUROLOGY
116,96-103
(19%)
Comparisons of Capillary Maturation in Control and Hypothyroid Rat Spinal Cord: An Ultrastructural Study’ ALEYAMMA P. THOMAS AND EDWARD J. H. NATHANIEL Department of Anatomy and Surgery, School of Medicine, University of Manitoba,
INTRODUCTION The vasculature of the central nervous system (CNS) develops from leptomeningeal capillaries that penetrate I This work was supported in part by the Research and Technology Foundation of the Paralysed Veterans of America, the Multiple Sclerosis Society of Canada, and the Medical Research Council of Canada. Thanks are due to C. Andryo and A. C. Eraut for typing the manuscript. A.P.T.-Present address is Department of Biostructure, University of Washington, Seattle, WA. 98195. 96 Inc. reserved.
Canada
the external basal lamina and the marginal glia of the CNS (2,21). At birth, the CNS of altricial species shows poor vascularization (2). Subsequently, by proliferation and progressive branching, a complete vascular bed is developed by the third postnatal week (2,21). Morphological observations of CNS capillaries date back to Ehrlich’s study (10). More recently, postnatal development of capillaries has been studied in various regions of the CNS of several species including the rat (5-7, 30, 33). In the spinal cord of the rat capillary maturation has been examined using the electron microscope (14,31). Phelps (31) described the gliovascular relationships in the cervical rat spinal cord during the prenatal period. It was found that capillaries first appeared on the 11th day and were surrounded by undifferentiated cells and neuroblasts separated from the capillary wall by a perivascular space. At the 13th day the basement membrane began to appear. In prenatal animals the neuroblasts or neurons were in direct contact with the basement membrane. Cells resembling astrocytes were first observed during the 19th day. Eventually astrocytic processes completely invested the capillaries, separating the neurons in the spinal cord of all postnatal animals. Hannah and Nathaniel (14) studied the postnatal development of the blood vessels in the substantia gelatinosa of the rat cervical cord using semithin and thin sections. Light microscopy suggested a qualitative increase in blood vessel profiles between 1 and 3 postnatal weeks. Ultrastructurally, at birth, there were fewer patent vessels but endothelial cells enclosing slit-like lumen were numerous. These primitive vessels displayed endothelial cells rich in free ribosomes and pinocytotic vesicles and numerous pseudopodial projections into the vascular lumen. During the period between 1 and 3 weeks the nuclei of the endothelial cells became less dense; lumen became patent and the endothelial wall became progressively thinner and attenuated. By 3 weeks, most blood vessels resembled blood vessels observed in a mature animal. A light microscopic investigation has shown a reduction in the density of capillaries in the cerebral cortex of a hypothyroid animal (9). Although a number of other
Quantitative and qualitative features of capillary maturation were examined in the ventral horn of the lumbar spinal cord of control and neonatally induced hypothyroid rats from birth to 6 weeks, using light (LM) and electron microscopy (EM). Quantification of the capillary densities by LM in the control animals and their hypothyroid litter mates have shown three- and twofold increases, respectively, from birth to Postnatal (Pn) Day 21. The following features were observed in the control animals at the EM level: (a) The newborn animals showed varying degrees of capillary maturation; (b) The majority of capillaries possessed mature characteristics by Pn Day 21; (c) By Pn Day 42, mature characteristics were found in nearly all capillary profiles. The hypothyroid animals demonstrated: (1) reactive perivascular cells and astrocytes; (2) delayed appearance of glycogen in the early Pn period and its persistence in extensive amounts during the latter part (3-6 weeks) of development; (3) cytoplasmic extensions of endothelial cells and perivascular cells, and (4) the presence of mitotic endothelial cells and perivascular cells even during the latter period of development. The observations suggest that the peak period of vasculogenesis in the lumbar spinal cord of the normal rat occurs during the second and third Pn weeks. The results from the hypothyroid rats point toward a delay in development and maturation of capillaries resulting in a hypoplastic vascular bed of the ventral horn. The reactive cells and the accumulation of glycogen particles could be morphological expressions of biochemical changes in hypothyroidism during the critical period of CNS development. o 1992 Academic PRM, IIW.
0014-48s6/92 $3.00 Copyright 0 1992 by Academic Press, All rights of reproduction in any form
Winnipeg, Manitoba,
CAPILLARY
MATURATION
investigators have dealt with various aspects of neonatal h~othyroi~sm, we are not aware of any work studying the fine structural changes in capillaries of the spinal cord of hypothyroid rats. The emphasis herein is directed to quantitative and qualitative changes in the capillaries of the developing spinal cord in normal and hypothyroid rats from birth to 6 weeks at the light and electron microscopic levels, The results are discussed with respect to previous studies of capillary maturation in the CNS in normal and induced hypothyroid situations. MATERIALS
IN SPINAL
97
CORD
2001
T
180 N=4
160 z F
140
AND METHODS
The pups from 12 litters of Sprague-Dawley rats were pooled, and groups of 10 randomly selected pups were assigned to each of the mothers. The 12 dams were divided into three equal groups. The rats were maintained on regular rat food and water ad Zibitunz. The pups were weaned on Postnatal (Pn) Day 21. Group 1 received daily subcutaneous (SC) injections of propylthiouracil (PTU, Sigma), dissolved in physiological saline per Nicholson and Altman schedule (28): 0.05 ml of 0.2% on Days O-10; 0.1 ml of 0.2% on Days 11-20; 0.1 ml of 0.4% on Days 21-30; and 0.2 ml of 0.4% on Days 31-42. Group II received equivalent amounts of normal saline. The third group was composed of the noninsulted controls. The animals were observed daily and their body weights were recorded weekly. Under deep anesthesia (pentobarbital), four rats from Group I, II, and III were sacrificed by an intracardiac perfusion of Karnovsky’s fixative (17) at weekly intervals. The lumbar spinal cord was dissected out rapidly. The thyroid gland also was removed from each of the animals. From the lumbar expansion of the spinal cord, l-mm-thick transverse sections were slicedusing a dissecting microscope. The sections were fixed in Karnovsky’s fixative for 1 h, postfixed in 1% OsO,, and dehydrated in ascending grades of ethanol followed by embedding in araldite plastic. From the resultant tissue blocks, 0.5 pm sections cut on a Reichert microtome were stained with toluidine blue and examined for quality of fixation and localization of the ventral horn. Thin sections (70 n&f) cut from the desired area were mounted on 2001300 mesh copper grids, counterstained with uranyl acetate and lead citrate, and studied in a Phillips 300 electron microscope. The thyroid glands were processed for routine histological examination. The experimental animals were judged to be hypothyroid on the basis of the absence of colloid and the presence of hyperpiastic follicular epithelium. Quantification of capillary profiles was made from four hypothyroid and four control animals from each age group (3 tissue blocks/animal) examined in this study. The capillary profiles were counted per square millimeter of ventral gray matter using an ocular micrometer disc
t
I
I
t
I
f
1
2
3
4
5
6
Age
Age
in weeks
Controls
Hypothyroid
1 week
15.9
f 0.74
14.9
t 0.62
2 weeks
35.8
t
1.4
29.1
*
3 weeks
50.4
+ 1.8
33.7
it 1.2 **
4 weeks
87.7
k 3
39.5
f: 5 **
5 weeks
111.9
2 3.4
67.9
k 2.6
6 weeks
189.1
2 8.4
74.2
2 3.1 **
Significantly
less
than
controls
1.2 *
**
*(p
FIG. 1. A graph illustrating comparisons of the mean body weights (BW) of the control and h~othyroid rata. From Weeks 2 to 6 there were significant differences between the two groups. The vertical bars denote -+ standard errors of the means (SDM). (P,
(Bausch and Lomb). The mean (X) and standard deviations (SD) for all subjects in each category were calculated. Two-tailed t tests were applied, and the P values were assessed. RESULTS
Light and electron microscopic studies of spinal cord tissue from groups II and III demonstrated no morphological differences. As such, together these two groups constituted the controls. Light microscopic examination of thyroid glands of group I animals showed hyperplastic follicular epithelium and a marked reduction in colloid compared to the control subjects. Hypothyroid animals demonstrated statistically significant reductions (P < 0.02, tO.OO1) in body weight from the beginning of the second Pn week (Fig. 1). Quantification of capillary profile density per square millimeter in the control animals displayed a twofold increase by the Pn Day 14, a threefold increase by Day 21, followed by a
98
THOMAS
AND
n=4 -
Control
-----
Hypothyroid
5o,
6 Age
Week 1
Control 113.3
SD
(weeks)
Hypothyroid
SD
p Values
24.23
100.4
k 3.40
<0.05
2
220.5
26.11
173.0
2 7.43
co.01
3
334.1
+ 6.22
226.5
+ 6.32
CO.01
4
307.7
+ 7.27
2 6.16
5
319.0
f 5.32
224.4
f 5.20
6
330.0
+ 5.24
234.3
f 5.26
201.0
FIG. 2. A graph showing comparisons of the mean capillary densities of the control and hypothyroid rats showing statistically significant differences between the two groups (P,t0.05, ~0.001). The vertical bars are +SDM. N = 4 from each age group of hypothyroid and controls.
plateau. During the same period, the hypothyroid rats showed a twofold increase by Pn Day 21 followed by a plateau, and then a negligible increase by Pn Day 42 (Fig. 2). Electron microscopic examination of the ventral horn at birth in both the control and hypothyroid animals showed nonpatent and patent capillary profiles with varying degrees of maturation. Many of the capillaries with immature characteristics showed an extremely narrow lumen enclosed by endothelial cells whose nuclei were highly irregular and electron dense due to the clumped chromatin, a cytoplasm possessing the usual organelles, numerous free ribosomes that appeared to contribute to the electron density of the cytoplasm, and cell processes presumed to be astrocytic that appeared watery with large amounts of glycogen particles and few organelles particularly in relation to the capillaries showing immature features (Fig. 3A). The capillaries with mature characteristics displayed attenuation of the endothelial cell wall with a concurrent reduction in electron density, more patent lumen with a smooth contour, and a well-defined basal lamina. Between 21 and 42 Pn days, the capillaries gradually took on a more mature appearance. By Pn Day 21, the majority of the capillary profiles in control animals showed mature characteristics that are represented in Fig. 3B. The increasing number of capillary profiles exhibiting mature
NATHANIEL
characteristics were reflected in the three- and twofold increases observed in the control and hypothyroid subjects, respectively (Fig. 2). In the hypothyroid animals, a few instances of mitotic endothelial cells (Fig. 4A) and perivascular cells (Fig. 4B) were noticed on Pn Days 21 and 28. During the same period, no such mitotic activity was seen in the control animals. In the control rats mitotic endothelial cells were observed only during the first 2 Pn weeks. Cytoplasmic extensions or sprouts of endothelial cells and pericytes were observed in the control and hypothyroid animals. In the control animals these sprouts were seen more frequently during the first 3 weeks, and occasionally in the last 3 weeks. In the hypothyroid animals, early observation of this feature was made in the second week with the fourth week showing more than was seen in the controls. In the hypothyroid animals, the presence of numerous perivascular cells and astrocytes constituted the two outstanding morphological features of capillary profiles during the last 3 weeks of Pn development (Figs. 4C, 5A, and 5B). The perivascular cells were characterized by a highly electron-dense nucleus with masses of heterochromatin bordering practically the entire nuclear margin and enclosing a moderately dense karyoplasm. The nucleus also was very irregular, and the cytoplasm was studded with ribosomes (Figs. 4C, 5A, and 5B). The perivascular cells also contained a spectrum of inclusion bodies of variable size and structure, vesicles, and mitochondria (Figs. 4C, 5A, and 5B). Cytoplasmic extensions also were associated with these cells. The astrocytes adjoining the perivascular cells, but separated from them by the basal lamina, contained microfilaments and glycogen. In the control animals, the amount of glycogen granules showed a gradual decrease with increasing age (Figs. 3A and 3B). By Pn Day 21 the astrocytic processes in the control groups demonstrated a few glycogen particles occasionally and much fewer glycogen granules between the fourth and sixth Pn weeks. The situation was different in the hypothyroid animals. In these rats the astrocytic end feet demonstrated strikingly copious increases in glycogen granules during the last three (3-6) weeks (Figs, 50 The relative increases in glycogen concentration observed initially at the end of the third week became extensive in the fourth week and continued throughout the period of study. DISCUSSION
The present developmental study has examined features of capillary maturation in the ventral horn of the lumbar spinal cord in normal and neonatally hypothyroid rats, using quantitative and qualitative measures. The strikingly low body weights seen in this study are one of the clinical manifestations of neonatal hypothyroidism. The P values reflect the highly significant na-
CAPILLARY
MATURATION
IN SPINAL
CORD
FIG. 3. (A) A micrograph of an immature capillary profile from a newborn rat demonstrating a convoluted endothelial cell nucleus (En), a primitive lumen (Lu), and watery astrocytic (As) end feet containing glycogen. X8400. (B) An electron micrograph of a capillary profile from a Zl-day-old control rat displaying mature characteristics such as a uniformally thick basal lamina, attenuated endothelial cell wall (En), profile of a pericyte (P), and a smooth contour of a patent lumen. ~16,380.
ture of the low body weight of hypothyroid rats. Similar reductions in body weight (2535%) have been reported in other studies where hypothyroidism was induced by
neonatal thyroidectomy (3, 32) or PTU treatment (4, 29). The morphological changes in the thyroid gland (hyperplastic thyroid follicular epithelium with mark-
THOMAS
AND NATHANIEL
FIG. 4. (A) An endotbelial ceil nucleus (En) in early prophase bordering a large lumen (Luf in 3-week-old hypothyroid rat. X7960. (B) Observe the well-defined chromosomal masses (Chr) in a perivascular ceil considered to be in the late stage of prophase. The endotheliii cell bordering a large lumen (Lu) is separated from the perivascuiar ceil by a well-defined basal iamina (arrow) in a 3-week-old hypothyroid animal. x14,440. (C!) Micrograph illustrating the reactive nature of the perivascuiar ceils. Several layers of p&vascular ceils (P) are located external to an endotheliai ceil. Basal iamina (arrows) separate the perivascuiar ceils from each other. The cytoplasm of the perivascuiar ceils contain numerous electron dense bodies (db), electron iucent spaces, and numerous vesicles. Profiles of watery astrocytic (As) processes can be seen external to the perivascuiar ceils. Four-week-old hypothyroid rat. ~10,450.
CAPILLARY
FIG. 5. in this figure. perivascular rat. X10,260. rat. X19,150. hypothyroid
MATURATION
IN
SPINAL
CORD
101
(A) The reactive nature of the perivascular cell (P), separated by a well-defined basal lamina from an endothelial cell, is illustrated Note the large numbers of electron dense bodies (db) and electron lucent spaces and numerous vesicles in the cytoplasm of the cell. The nucleus of the perivascular cell is characterized by considerable margination of chromatin. Six-week-old hypothyroid (B) A reactive perivascular cell (P) with electron dense body (db) adjacent to an oligodendrocyte (01). Four-week-old hypothyroid (C) Observe the strikingly large quantities of glycogen (gly) in the perivascular astrocytic (As) processes in a four-week-old rat. A highly attenuated endothelial cell limits a large lumen. X19,600.
102
THOMAS
AND
edly reduced colloid in the follicles), confirmed the occurrence of neonatal hypothyroidism in those rats that received PTU. In normal animals, capillary density showing twoand threefold increases on Pn Days 14 and 21, respectively, indicates that the peak period of vasculogenesis in the ventral horn of the rat lumbar spinal cord is during the second and third Pn weeks. In the rodent, vasculogenesis is much greater in the neonatal period than it is in any other period of development (5-7,30,33). The currently observed peak period of vasculogenesis is similar to other developmental studies reported in other regions of the CNS. This period in the rat cerebral cortex (2,5) and in the olfactory bulb (33) is during the second 10 Pn days; in the cuneate nucleus it is during the first 3 Pn weeks (7). In addition to the developmental period, the capillary density of a given CNS region depends upon the local metabolic activity and oxygen consumption (2, 12). The negligible increase noticed in capillary density between Pn Days 21 and 42, is consistent with reports that a complete vascular bed is formed by the third Pn week (2,21) in the rodent cerebral cortex. The pattern of increases in capillary density by light microscopic quantification appeared similar in both normal and hypothyroid rats. However, the rate of increase as a function of age is considerably lower in the hypothyroid subjects. The disparity is demonstrated mainly between the second and the sixth Pn weeks. Such disparity could result in a reduced level of blood supply in the hypothyroid rats. In the newborn animals a mixture of capillaries showing immature and mature characteristics is a normal developmental phenomenon; this is followed by an increasing number of maturing capillaries during development (2, 21). The present ultrastructural findings are similar to the ultrastructural observations reported in different regions of the CNS, including the rat spinal cord (5, 8, 14, 31). The mitotic division of endothelial cells and perivascular cells is one of the processes involved in vascular growth in the CNS (2, 21, 22). The mitotic activity decreases with increasing age and the morphological maturation of capillary walls (2). In the present study the control animals demonstrated mitotic endothelial cells during the first 2 weeks. In the hypothyroid rats the occurrence of mitotic cells in the third and fourth Pn weeks could indicate a delayed proliferation of new capillaries and/or that the mature characteristics are acquired very slowly, resulting in more capillaries with immature characteristics. In the hypothyroid rat, a delayed migration of external granule cell layer of the cerebellum has been reported (19). In the CNS, capillaries develop from solid vascular cords of endothelial/mesodermal cells, sprouting from existing blood vessels. The sprouts from endothelial cells and perivascular cells penetrate the surrounding
NATHANIEL
neuropil and fuse with afferent capillaries to form the capillary network (2, 5, 17, 21, 22). The sprouts could possess contractile properties since the endothelial cells and pericytes contain actin and myosin-like filaments (18). The endothelial and pericytic sprouts characteristic of immature capillaries (2, 5, 14, 17) were observed frequently in the control rats during the first 3 weeks when the vascular growth is rapid to form a complete bed. In the hypothyroid rats the delayed appearance and the presence of more sprouts in the latter period, compared to the control rats, could indicate a slow or delayed proliferation of new capillaries. This qualitative comparison is based upon several observations where sprouts were noticed. In the hypothyroid animals, the light microscopic quantitative data also show a slight increase in the capillary density for the latter period of development. In the present study, the inclusion-laden cells located within the basal lamina, and their cytoplasmic extensions growing into the neuropil, indicate that these perivascular cells are demonstrating phagocytic reaction. The phagocytic role of pericytes in the CNS has been reported in X-irradiation injury (23) and in hypoxia (23). In traumatic brain lesions, pericytes undergo division and the daughter cells migrate into the surrounding neuropil to become macrophages (23,25). In the current investigation, the age-dependent decline of glycogen particles seen in the control animals is consistent with current biochemical theory that during development, there occurs a progressive addition of aerobic metabolic pathways to anaerobic mechanisms. The glycogen decrease can also be due to its breakdown by the increasing phosphorylase activity in the gray matter of the developing spinal cord (1). Under normal conditions the mammalian brain contains much less (10%) glycogen than does muscle. The low concentration of brain glycogen is adequate to maintain oxidative metabolism for several minutes (1, 20). Any impairment in glycogen synthesis or degradation will result in altered accumulation, reflecting a turnover disorder. In the normal animal, astrocytes contain enzymes that can synthesize glycogen (11). In the hypothyroid rats, it is not clear whether it is the synthetic or the degradative mechanism that is responsible for the present observations of relatively extensive accumulations of glycogen particles in reactive astrocytes. Similar astrocytic responses to CNS insults have been reported in the X-irradiated rat (24) following crushed dorsal root injury (26,27) and in neonatal hypothyroidism (7). Such astrocytic reaction has been described as one of the earliest responses of the CNS to injury, even in the absence of any other morphological changes of the CNS (7, 24, 26, 27). Neonatal hypothyroidism during the critical period of structural development and functional differentiation of the CNS impairs proliferation and maturation of glial
CAPILLARY
cells (3,13). The astrocytic reaction becoming first by the end of the third Pn week could be immaturity of these cells. The immature glial sess a less efficient mechanism for initiating sponses (15).
MATURATION
evident at due to the cells postheir re-
REFERENCES 1. AUSTIN, J. H. 1972. Disorders of glycogen and related macromolecules in the nervous system. In Handbook of Neurochemistry (A. Lajtha Ed.), Vol. 7, pp. 1-15. Pathological chemistry of the Nervous System, New York State Research Institute, Plenum Press. New York. 2. BKR, T. 1980. The vascular system of the cerebral cortex. Adu. Anat. Embryol. Cell Biol. 69: l-62. 3. BASS, N. H., AND E. YOUNG. 1973. Effects of hypothyroidism on the differentiation of neurons and glia in developing rat cerebellum. J. Neurol. Sci. 18: 155-173. 4. BATTIE, C. A., AND M. A. VERITY. 1979. Membrane enzyme development in nerve ending mitochondria during neonatal hypothyroidism. Deu. Neurosci. 2: 139-148. 5. 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. Neurol. 138: 31-48. 6. CRAIGIE, E. H. 1925. Postnatal changes in vascularity in the cerebral cortex of the male albino rat. J. Comp. Neurol. 39: 301-324. 7. DAVID, S., AND E. J. H. NATHANIEL. 1981. Development of capillaries in euthyroid and hypothyroid rats. Exp. Neurol. 73: 243253. 8. DONAHUE, S., AND G. D. PAPPAS. 1961. The fine structure of capillaries in the cerebral cortex of the rat at various stages of development. Amer. J. Anat. 108: 331-347. 9. EAYRS, J. T. 1954. The vascularity of the cerebral cortex in normal and cretinous rats. J. Anat. 88: 164-173. des organisms. Ein 10. EHRLICH, P. 1885. Das Sauerstoff-Bedtirfnis Farbenanalytische. A HirshwaM (Berlin). 11. FRIEDE, R. 1962. The cytochemistry of normal and reactive astrocytes. J. Neuropathol. Exp. Neurol. 21: 471-478. Brain Chemistry. Academic 12. FRIEDE, R. L. 1966. Topographic Press, New York. 13. GEEL, S. E., AND L. W. GONZALES. 1977. Cerebral Cortical ganglioside and glycoprotein metabolism in immature hypothyroidism. Brain Res. 128: 515-525. 14. HANNAH, R. S., AND E. J. H. NATHANIEL. 1974. The postnatal development of blood vessels in the substantia gelatinosa of rat cervical cord-an ultrastructural study. Anat. Rec. 178: 691710. 15. JACOBSON, M. 1978. Developmental Neurobiology, p. 562. Plenum Press, New York. 16. KARNOVSKY, M. J. 1965. A formaldehyde-glutaraldehyde fixa-
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18. 19. 20. 21.
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tive of high osmolality for use in electron microscopy. J. Cell Biol. 27: 137a-138a. KLOSOVSKII, B. N. 1963. The Development of the Brain and its Disturbance by Harmful Factors, pp. 12-14. Macmillian, New York. LEBEAUX, Y. J., AND J. WILLEMOT. 1978. Actin- and myosin-like filaments in rat brain pericytes. Anat. Rec. 190: 811-825. LEGRAND, J. 1967. Analyse de l’action morphogenetique des hormones thyroidiennes sur le cervelet du jeune rat, Arch. Anat. Micr. Morph. Exp. (Paris) 56: 205-244. LIPTON, P. 1988. Regulation of glycogen in the dentate gyrus of the in vitro guinea pig hippocampus: Effect of combined deprovation of glucose and oxygen. J. Neurosci. Methods 28: 147-154. MARIN-PADILLA, M. 1985. Early vascularization of the embryonic cerebral cortex: Golgi and electron microscopic studies. J. Comp.Neurol.
241:237-249.
22. MATO, M., S. OOKAWARA, AND T. NAMIKI. 1989. Studies on the vasculogenesis in rat cerebral cortex. Anat. Rec. 224: 355-364. 23. MAXWELL, D. S., AND L. KRUGER. 1965. The fine structure of astrocytes in the cerebral cortex and their response to focal injury produced by heavy ionizing particles. J. Cell Biol. 26: 141157. 24. MIQUEL, J., AND W. HAYMAKER. 1965. Astroglial reactions to ionizing radiation: With emphasis on glycogen accumulation. Prog.
Brain
Res.
15: 89-114.
25. MORI, S., AND C. P. LEBLOND. 1969. Identification of microglia in light and electron microscopy. J. Comp. Neurol. 135: 57-80. 26. NATHANIEL, E. J. H., AND D. R. NATHANIEL. 1973. Degeneration of dorsal roots in the adult rat spinal cord. Exp. Neurol. 40: 316-332. 27. 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. 28. NICHOLSON, J. L., AND J. ALTMAN. 1972. Synaptogenesis in the rat cerebellum: Effects of early hypo- and hyperthyroidism, Science
176:530-532.
29. OKLUND, S., AND P. S. TIMIRAS. 1977. Influences of thyroid levels in brain ontogenesis in vivo and in vitro. In Thyroid hormones and Brain Development (G. D. Grave, Ed.), pp. 33-45. Raven Press, New York. 30. PETREN, T. 1938. Quantitative studien an den kapillaren des Zentralen Nerven systems bei cavia cobaya. Morphol. Jahrb. 82: 554-562. 31. PHELPS, C. H. 1972. The development of gliovascular relationships in the rat spinal cord. Z. Zellforsch. 128: 558-563. 32. ROSMAN, N. P., AND M. J. MALONE. 1977. Brain myelination in experimental hypothyroidism: Morphological and biochemical observation. In Thyroid Hormones and Brain Development (G. D. Grave, Ed.), pp. 169-198. Raven Press, New York. 33. 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.
NEUROLOGY
116,96-103
(19%)
Comparisons of Capillary Maturation in Control and Hypothyroid Rat Spinal Cord: An Ultrastructural Study’ ALEYAMMA P. THOMAS AND EDWARD J. H. NATHANIEL Department of Anatomy and Surgery, School of Medicine, University of Manitoba,
INTRODUCTION The vasculature of the central nervous system (CNS) develops from leptomeningeal capillaries that penetrate I This work was supported in part by the Research and Technology Foundation of the Paralysed Veterans of America, the Multiple Sclerosis Society of Canada, and the Medical Research Council of Canada. Thanks are due to C. Andryo and A. C. Eraut for typing the manuscript. A.P.T.-Present address is Department of Biostructure, University of Washington, Seattle, WA. 98195. 96 Inc. reserved.
Canada
the external basal lamina and the marginal glia of the CNS (2,21). At birth, the CNS of altricial species shows poor vascularization (2). Subsequently, by proliferation and progressive branching, a complete vascular bed is developed by the third postnatal week (2,21). Morphological observations of CNS capillaries date back to Ehrlich’s study (10). More recently, postnatal development of capillaries has been studied in various regions of the CNS of several species including the rat (5-7, 30, 33). In the spinal cord of the rat capillary maturation has been examined using the electron microscope (14,31). Phelps (31) described the gliovascular relationships in the cervical rat spinal cord during the prenatal period. It was found that capillaries first appeared on the 11th day and were surrounded by undifferentiated cells and neuroblasts separated from the capillary wall by a perivascular space. At the 13th day the basement membrane began to appear. In prenatal animals the neuroblasts or neurons were in direct contact with the basement membrane. Cells resembling astrocytes were first observed during the 19th day. Eventually astrocytic processes completely invested the capillaries, separating the neurons in the spinal cord of all postnatal animals. Hannah and Nathaniel (14) studied the postnatal development of the blood vessels in the substantia gelatinosa of the rat cervical cord using semithin and thin sections. Light microscopy suggested a qualitative increase in blood vessel profiles between 1 and 3 postnatal weeks. Ultrastructurally, at birth, there were fewer patent vessels but endothelial cells enclosing slit-like lumen were numerous. These primitive vessels displayed endothelial cells rich in free ribosomes and pinocytotic vesicles and numerous pseudopodial projections into the vascular lumen. During the period between 1 and 3 weeks the nuclei of the endothelial cells became less dense; lumen became patent and the endothelial wall became progressively thinner and attenuated. By 3 weeks, most blood vessels resembled blood vessels observed in a mature animal. A light microscopic investigation has shown a reduction in the density of capillaries in the cerebral cortex of a hypothyroid animal (9). Although a number of other
Quantitative and qualitative features of capillary maturation were examined in the ventral horn of the lumbar spinal cord of control and neonatally induced hypothyroid rats from birth to 6 weeks, using light (LM) and electron microscopy (EM). Quantification of the capillary densities by LM in the control animals and their hypothyroid litter mates have shown three- and twofold increases, respectively, from birth to Postnatal (Pn) Day 21. The following features were observed in the control animals at the EM level: (a) The newborn animals showed varying degrees of capillary maturation; (b) The majority of capillaries possessed mature characteristics by Pn Day 21; (c) By Pn Day 42, mature characteristics were found in nearly all capillary profiles. The hypothyroid animals demonstrated: (1) reactive perivascular cells and astrocytes; (2) delayed appearance of glycogen in the early Pn period and its persistence in extensive amounts during the latter part (3-6 weeks) of development; (3) cytoplasmic extensions of endothelial cells and perivascular cells, and (4) the presence of mitotic endothelial cells and perivascular cells even during the latter period of development. The observations suggest that the peak period of vasculogenesis in the lumbar spinal cord of the normal rat occurs during the second and third Pn weeks. The results from the hypothyroid rats point toward a delay in development and maturation of capillaries resulting in a hypoplastic vascular bed of the ventral horn. The reactive cells and the accumulation of glycogen particles could be morphological expressions of biochemical changes in hypothyroidism during the critical period of CNS development. o 1992 Academic PRM, IIW.
0014-48s6/92 $3.00 Copyright 0 1992 by Academic Press, All rights of reproduction in any form
Winnipeg, Manitoba,
CAPILLARY
MATURATION
investigators have dealt with various aspects of neonatal h~othyroi~sm, we are not aware of any work studying the fine structural changes in capillaries of the spinal cord of hypothyroid rats. The emphasis herein is directed to quantitative and qualitative changes in the capillaries of the developing spinal cord in normal and hypothyroid rats from birth to 6 weeks at the light and electron microscopic levels, The results are discussed with respect to previous studies of capillary maturation in the CNS in normal and induced hypothyroid situations. MATERIALS
IN SPINAL
97
CORD
2001
T
180 N=4
160 z F
140
AND METHODS
The pups from 12 litters of Sprague-Dawley rats were pooled, and groups of 10 randomly selected pups were assigned to each of the mothers. The 12 dams were divided into three equal groups. The rats were maintained on regular rat food and water ad Zibitunz. The pups were weaned on Postnatal (Pn) Day 21. Group 1 received daily subcutaneous (SC) injections of propylthiouracil (PTU, Sigma), dissolved in physiological saline per Nicholson and Altman schedule (28): 0.05 ml of 0.2% on Days O-10; 0.1 ml of 0.2% on Days 11-20; 0.1 ml of 0.4% on Days 21-30; and 0.2 ml of 0.4% on Days 31-42. Group II received equivalent amounts of normal saline. The third group was composed of the noninsulted controls. The animals were observed daily and their body weights were recorded weekly. Under deep anesthesia (pentobarbital), four rats from Group I, II, and III were sacrificed by an intracardiac perfusion of Karnovsky’s fixative (17) at weekly intervals. The lumbar spinal cord was dissected out rapidly. The thyroid gland also was removed from each of the animals. From the lumbar expansion of the spinal cord, l-mm-thick transverse sections were slicedusing a dissecting microscope. The sections were fixed in Karnovsky’s fixative for 1 h, postfixed in 1% OsO,, and dehydrated in ascending grades of ethanol followed by embedding in araldite plastic. From the resultant tissue blocks, 0.5 pm sections cut on a Reichert microtome were stained with toluidine blue and examined for quality of fixation and localization of the ventral horn. Thin sections (70 n&f) cut from the desired area were mounted on 2001300 mesh copper grids, counterstained with uranyl acetate and lead citrate, and studied in a Phillips 300 electron microscope. The thyroid glands were processed for routine histological examination. The experimental animals were judged to be hypothyroid on the basis of the absence of colloid and the presence of hyperpiastic follicular epithelium. Quantification of capillary profiles was made from four hypothyroid and four control animals from each age group (3 tissue blocks/animal) examined in this study. The capillary profiles were counted per square millimeter of ventral gray matter using an ocular micrometer disc
t
I
I
t
I
f
1
2
3
4
5
6
Age
Age
in weeks
Controls
Hypothyroid
1 week
15.9
f 0.74
14.9
t 0.62
2 weeks
35.8
t
1.4
29.1
*
3 weeks
50.4
+ 1.8
33.7
it 1.2 **
4 weeks
87.7
k 3
39.5
f: 5 **
5 weeks
111.9
2 3.4
67.9
k 2.6
6 weeks
189.1
2 8.4
74.2
2 3.1 **
Significantly
less
than
controls
1.2 *
**
*(p
FIG. 1. A graph illustrating comparisons of the mean body weights (BW) of the control and h~othyroid rata. From Weeks 2 to 6 there were significant differences between the two groups. The vertical bars denote -+ standard errors of the means (SDM). (P,
(Bausch and Lomb). The mean (X) and standard deviations (SD) for all subjects in each category were calculated. Two-tailed t tests were applied, and the P values were assessed. RESULTS
Light and electron microscopic studies of spinal cord tissue from groups II and III demonstrated no morphological differences. As such, together these two groups constituted the controls. Light microscopic examination of thyroid glands of group I animals showed hyperplastic follicular epithelium and a marked reduction in colloid compared to the control subjects. Hypothyroid animals demonstrated statistically significant reductions (P < 0.02, tO.OO1) in body weight from the beginning of the second Pn week (Fig. 1). Quantification of capillary profile density per square millimeter in the control animals displayed a twofold increase by the Pn Day 14, a threefold increase by Day 21, followed by a
98
THOMAS
AND
n=4 -
Control
-----
Hypothyroid
5o,
6 Age
Week 1
Control 113.3
SD
(weeks)
Hypothyroid
SD
p Values
24.23
100.4
k 3.40
<0.05
2
220.5
26.11
173.0
2 7.43
co.01
3
334.1
+ 6.22
226.5
+ 6.32
CO.01
4
307.7
+ 7.27
2 6.16
5
319.0
f 5.32
224.4
f 5.20
6
330.0
+ 5.24
234.3
f 5.26
201.0
FIG. 2. A graph showing comparisons of the mean capillary densities of the control and hypothyroid rats showing statistically significant differences between the two groups (P,t0.05, ~0.001). The vertical bars are +SDM. N = 4 from each age group of hypothyroid and controls.
plateau. During the same period, the hypothyroid rats showed a twofold increase by Pn Day 21 followed by a plateau, and then a negligible increase by Pn Day 42 (Fig. 2). Electron microscopic examination of the ventral horn at birth in both the control and hypothyroid animals showed nonpatent and patent capillary profiles with varying degrees of maturation. Many of the capillaries with immature characteristics showed an extremely narrow lumen enclosed by endothelial cells whose nuclei were highly irregular and electron dense due to the clumped chromatin, a cytoplasm possessing the usual organelles, numerous free ribosomes that appeared to contribute to the electron density of the cytoplasm, and cell processes presumed to be astrocytic that appeared watery with large amounts of glycogen particles and few organelles particularly in relation to the capillaries showing immature features (Fig. 3A). The capillaries with mature characteristics displayed attenuation of the endothelial cell wall with a concurrent reduction in electron density, more patent lumen with a smooth contour, and a well-defined basal lamina. Between 21 and 42 Pn days, the capillaries gradually took on a more mature appearance. By Pn Day 21, the majority of the capillary profiles in control animals showed mature characteristics that are represented in Fig. 3B. The increasing number of capillary profiles exhibiting mature
NATHANIEL
characteristics were reflected in the three- and twofold increases observed in the control and hypothyroid subjects, respectively (Fig. 2). In the hypothyroid animals, a few instances of mitotic endothelial cells (Fig. 4A) and perivascular cells (Fig. 4B) were noticed on Pn Days 21 and 28. During the same period, no such mitotic activity was seen in the control animals. In the control rats mitotic endothelial cells were observed only during the first 2 Pn weeks. Cytoplasmic extensions or sprouts of endothelial cells and pericytes were observed in the control and hypothyroid animals. In the control animals these sprouts were seen more frequently during the first 3 weeks, and occasionally in the last 3 weeks. In the hypothyroid animals, early observation of this feature was made in the second week with the fourth week showing more than was seen in the controls. In the hypothyroid animals, the presence of numerous perivascular cells and astrocytes constituted the two outstanding morphological features of capillary profiles during the last 3 weeks of Pn development (Figs. 4C, 5A, and 5B). The perivascular cells were characterized by a highly electron-dense nucleus with masses of heterochromatin bordering practically the entire nuclear margin and enclosing a moderately dense karyoplasm. The nucleus also was very irregular, and the cytoplasm was studded with ribosomes (Figs. 4C, 5A, and 5B). The perivascular cells also contained a spectrum of inclusion bodies of variable size and structure, vesicles, and mitochondria (Figs. 4C, 5A, and 5B). Cytoplasmic extensions also were associated with these cells. The astrocytes adjoining the perivascular cells, but separated from them by the basal lamina, contained microfilaments and glycogen. In the control animals, the amount of glycogen granules showed a gradual decrease with increasing age (Figs. 3A and 3B). By Pn Day 21 the astrocytic processes in the control groups demonstrated a few glycogen particles occasionally and much fewer glycogen granules between the fourth and sixth Pn weeks. The situation was different in the hypothyroid animals. In these rats the astrocytic end feet demonstrated strikingly copious increases in glycogen granules during the last three (3-6) weeks (Figs, 50 The relative increases in glycogen concentration observed initially at the end of the third week became extensive in the fourth week and continued throughout the period of study. DISCUSSION
The present developmental study has examined features of capillary maturation in the ventral horn of the lumbar spinal cord in normal and neonatally hypothyroid rats, using quantitative and qualitative measures. The strikingly low body weights seen in this study are one of the clinical manifestations of neonatal hypothyroidism. The P values reflect the highly significant na-
CAPILLARY
MATURATION
IN SPINAL
CORD
FIG. 3. (A) A micrograph of an immature capillary profile from a newborn rat demonstrating a convoluted endothelial cell nucleus (En), a primitive lumen (Lu), and watery astrocytic (As) end feet containing glycogen. X8400. (B) An electron micrograph of a capillary profile from a Zl-day-old control rat displaying mature characteristics such as a uniformally thick basal lamina, attenuated endothelial cell wall (En), profile of a pericyte (P), and a smooth contour of a patent lumen. ~16,380.
ture of the low body weight of hypothyroid rats. Similar reductions in body weight (2535%) have been reported in other studies where hypothyroidism was induced by
neonatal thyroidectomy (3, 32) or PTU treatment (4, 29). The morphological changes in the thyroid gland (hyperplastic thyroid follicular epithelium with mark-
THOMAS
AND NATHANIEL
FIG. 4. (A) An endotbelial ceil nucleus (En) in early prophase bordering a large lumen (Luf in 3-week-old hypothyroid rat. X7960. (B) Observe the well-defined chromosomal masses (Chr) in a perivascular ceil considered to be in the late stage of prophase. The endotheliii cell bordering a large lumen (Lu) is separated from the perivascuiar ceil by a well-defined basal iamina (arrow) in a 3-week-old hypothyroid animal. x14,440. (C!) Micrograph illustrating the reactive nature of the perivascuiar ceils. Several layers of p&vascular ceils (P) are located external to an endotheliai ceil. Basal iamina (arrows) separate the perivascuiar ceils from each other. The cytoplasm of the perivascuiar ceils contain numerous electron dense bodies (db), electron iucent spaces, and numerous vesicles. Profiles of watery astrocytic (As) processes can be seen external to the perivascuiar ceils. Four-week-old hypothyroid rat. ~10,450.
CAPILLARY
FIG. 5. in this figure. perivascular rat. X10,260. rat. X19,150. hypothyroid
MATURATION
IN
SPINAL
CORD
101
(A) The reactive nature of the perivascular cell (P), separated by a well-defined basal lamina from an endothelial cell, is illustrated Note the large numbers of electron dense bodies (db) and electron lucent spaces and numerous vesicles in the cytoplasm of the cell. The nucleus of the perivascular cell is characterized by considerable margination of chromatin. Six-week-old hypothyroid (B) A reactive perivascular cell (P) with electron dense body (db) adjacent to an oligodendrocyte (01). Four-week-old hypothyroid (C) Observe the strikingly large quantities of glycogen (gly) in the perivascular astrocytic (As) processes in a four-week-old rat. A highly attenuated endothelial cell limits a large lumen. X19,600.
102
THOMAS
AND
edly reduced colloid in the follicles), confirmed the occurrence of neonatal hypothyroidism in those rats that received PTU. In normal animals, capillary density showing twoand threefold increases on Pn Days 14 and 21, respectively, indicates that the peak period of vasculogenesis in the ventral horn of the rat lumbar spinal cord is during the second and third Pn weeks. In the rodent, vasculogenesis is much greater in the neonatal period than it is in any other period of development (5-7,30,33). The currently observed peak period of vasculogenesis is similar to other developmental studies reported in other regions of the CNS. This period in the rat cerebral cortex (2,5) and in the olfactory bulb (33) is during the second 10 Pn days; in the cuneate nucleus it is during the first 3 Pn weeks (7). In addition to the developmental period, the capillary density of a given CNS region depends upon the local metabolic activity and oxygen consumption (2, 12). The negligible increase noticed in capillary density between Pn Days 21 and 42, is consistent with reports that a complete vascular bed is formed by the third Pn week (2,21) in the rodent cerebral cortex. The pattern of increases in capillary density by light microscopic quantification appeared similar in both normal and hypothyroid rats. However, the rate of increase as a function of age is considerably lower in the hypothyroid subjects. The disparity is demonstrated mainly between the second and the sixth Pn weeks. Such disparity could result in a reduced level of blood supply in the hypothyroid rats. In the newborn animals a mixture of capillaries showing immature and mature characteristics is a normal developmental phenomenon; this is followed by an increasing number of maturing capillaries during development (2, 21). The present ultrastructural findings are similar to the ultrastructural observations reported in different regions of the CNS, including the rat spinal cord (5, 8, 14, 31). The mitotic division of endothelial cells and perivascular cells is one of the processes involved in vascular growth in the CNS (2, 21, 22). The mitotic activity decreases with increasing age and the morphological maturation of capillary walls (2). In the present study the control animals demonstrated mitotic endothelial cells during the first 2 weeks. In the hypothyroid rats the occurrence of mitotic cells in the third and fourth Pn weeks could indicate a delayed proliferation of new capillaries and/or that the mature characteristics are acquired very slowly, resulting in more capillaries with immature characteristics. In the hypothyroid rat, a delayed migration of external granule cell layer of the cerebellum has been reported (19). In the CNS, capillaries develop from solid vascular cords of endothelial/mesodermal cells, sprouting from existing blood vessels. The sprouts from endothelial cells and perivascular cells penetrate the surrounding
NATHANIEL
neuropil and fuse with afferent capillaries to form the capillary network (2, 5, 17, 21, 22). The sprouts could possess contractile properties since the endothelial cells and pericytes contain actin and myosin-like filaments (18). The endothelial and pericytic sprouts characteristic of immature capillaries (2, 5, 14, 17) were observed frequently in the control rats during the first 3 weeks when the vascular growth is rapid to form a complete bed. In the hypothyroid rats the delayed appearance and the presence of more sprouts in the latter period, compared to the control rats, could indicate a slow or delayed proliferation of new capillaries. This qualitative comparison is based upon several observations where sprouts were noticed. In the hypothyroid animals, the light microscopic quantitative data also show a slight increase in the capillary density for the latter period of development. In the present study, the inclusion-laden cells located within the basal lamina, and their cytoplasmic extensions growing into the neuropil, indicate that these perivascular cells are demonstrating phagocytic reaction. The phagocytic role of pericytes in the CNS has been reported in X-irradiation injury (23) and in hypoxia (23). In traumatic brain lesions, pericytes undergo division and the daughter cells migrate into the surrounding neuropil to become macrophages (23,25). In the current investigation, the age-dependent decline of glycogen particles seen in the control animals is consistent with current biochemical theory that during development, there occurs a progressive addition of aerobic metabolic pathways to anaerobic mechanisms. The glycogen decrease can also be due to its breakdown by the increasing phosphorylase activity in the gray matter of the developing spinal cord (1). Under normal conditions the mammalian brain contains much less (10%) glycogen than does muscle. The low concentration of brain glycogen is adequate to maintain oxidative metabolism for several minutes (1, 20). Any impairment in glycogen synthesis or degradation will result in altered accumulation, reflecting a turnover disorder. In the normal animal, astrocytes contain enzymes that can synthesize glycogen (11). In the hypothyroid rats, it is not clear whether it is the synthetic or the degradative mechanism that is responsible for the present observations of relatively extensive accumulations of glycogen particles in reactive astrocytes. Similar astrocytic responses to CNS insults have been reported in the X-irradiated rat (24) following crushed dorsal root injury (26,27) and in neonatal hypothyroidism (7). Such astrocytic reaction has been described as one of the earliest responses of the CNS to injury, even in the absence of any other morphological changes of the CNS (7, 24, 26, 27). Neonatal hypothyroidism during the critical period of structural development and functional differentiation of the CNS impairs proliferation and maturation of glial
CAPILLARY
cells (3,13). The astrocytic reaction becoming first by the end of the third Pn week could be immaturity of these cells. The immature glial sess a less efficient mechanism for initiating sponses (15).
MATURATION
evident at due to the cells postheir re-
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