J. Anat. (1979), 128, 2, pp. 293-300 With 6 figures Printed in Great Britain
293
An ultrastructural study of the endonasal microcirculation in the Wistar rat during fetal and early postnatal life PATRICIA VAN DIEST AND M. W. KANAN Department of Human Anatomy, University of Oxford
(Accepted 7 March 1978) INTRODUCTION
In spite of the unusual microanatomy of the nasal mucosa, research into the ultrastructure of its microcirculation has been limited. Cauna & Hinderer (1969) described the peculiar ultrastructure of the nasal vascular bed in adult human males and females. They found the large sub-epithelial capillaries of the lateral wall of the nasal cavity to be fenestrated. The fenestrae occurred in the capillary walls facing the ciliated respiratory epithelium. Fenestrae were also seen regularly in blood capillaries of the mixed nasal glands in the lateral wall, but were seldom encountered in the septal capillaries. The basement membrane around the capillaries, venules and veins exhibited fine pores, and in some areas consisted of several incomplete layers. More recently Kanan (1976) has confirmed the overall porosity of the capillary and venular components of the vascular bed in the respiratory nasal mucosa of the Wistar rat, albino mouse, guinea-pig and goat. Endothelial fenestrations were also frequently encountered in the superficial and glandular capillaries under the ciliated mucosa of the lateral nasal wall, less often in the septal mucosa, and hardly ever in the olfactory region. Cauna (1970) believed these fenestrations were specifically designed for rapid passage of fluid and high molecular weight solutes through the vascular walls to meet the functional requirements of the nasal organ. Kanan, Ryan & Weddell (1975) found this view plausible, but in view of the differences between the blood vessels of the respiratory and olfactory mucosae, they speculated that development of fenestrae could be related to the repetitive exposure of the respiratory mucosa to the cool, inspired airstream. As a contribution to the study of the role of environmental factors on the development of these features, it was felt necessary to examine the ultrastructure of the endonasal microvasculature during fetal and early postnatal life. MATERIALS AND METHODS
Eighteen pregnant Wistar rats, time-mated according to the method of Christie (1964), were anaesthetized intraperitoneally with Avertin, and the 83 fetuses obtained were killed by decapitation. Neonates of 1, 2, 4, 12 and 17 days of age from 3 pregnant rats were also killed. The gestation period ranged from 21 to 23 days. The nasal cavities of the animals were flooded with ice cold 3 % glutaraldehyde in 5 % sucrose/ phosphate buffer and 1 % calcium chloride (pH 7.4). Specimens were taken from the vestibular, respiratory, and olfactory regions and fixed for 3 hours at 4 'C. Postfixation was for 2 hours in 1 % osmium tetroxide made up in the sucrose/phosphate buffer. Tissues were dehydrated, embedded in Araldite, sectioned, stained with uranyl acetate and lead citrate, and examined in a Siemens 101 electron microscope.
294
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Fig. 1. Loosely woven mesenchymal tissue (Mc) with few extracellular fibrous elements. Nasal pit of 16 day rat fetus. Ep, epithelium; E, endothelial cell; RBC, red blood corpuscle. x 6000.
Nasal capillary fenestrations
295
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Fig. 2. Details of interendothelial junction (ij) in vessel from nasal pit of 16 day rat fetus. Cytoplasmic plaques (p) and polyribosomes (pr) are seen. x 50000. RESULTS
Capillaries From day 16 after mating, the beginning of the adult design of the nasal fossa and its skeletal structures was already exhibited. The roof, septum, ethmoidal scrolls in the posterior third, and naso- and maxilloturbinals in the anterior two thirds, were evident. In the early stages of the differentiation of the endonasal epithelium, irrespective of region, the corium consisted of loosely woven mesenchymal tissue. The primitive mesenchymal cells, presumably the precursors of future fibroblasts, were associated with very few extracellular fibres (Fig. 1). The vascular bed consisted of a plexus of blood channels, notable for the lack of support in their walls. In their middle perinuclear regions, the endothelial cells were crowded with aggregates of polyribosomes. The attenuated limbs of the endothelial cells formed junctions of simple type, with one or more dense, cytoplasmic plaques along their length (Fig. 2). However, no macula densa could be identified at any point in these junctions. The cytoplasm also included rough endoplasmic reticulum and mitochondria. Little macro- or micropinocytosis was seen on the 16th day of gestation. Between. days 16 and 18 after mating' a vascular basement membrane appeared, together with pericytes. On day 18 of fetal life, banded and unbanded collagen fibres were seen throughout the mesenchymal tissue. Simultaneously, fibroblasts almost completely replaced the primitive mesenchymal cells which had been encountered earlier (Fig. 3).
44
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PATRICIA VAN DIEST AND M. W. KANAN -_ .1'
Fig. 3. Blood vessel in vestibular mesenchyme lacking pericytes and basement membrane. Collagen fibres (col) can be seen for the first time outside fibroblasts (F). 18 day fetal rat nose. x 12500.
Between days 16 and 17 of gestation time the differentiation of the capillary tree into superficial, mid-level and deep plexuses was established. In contradistinction to the olfactory and vestibular capillaries, the superficial capillaries under the epithelium of the lateral nasal wall, and those supplying the serous glands of the respiratory cavity, exhibited endothelial fenestrations in the attenuated segments of their circumference (Fig. 4). The number of fenestrations increased with time. Like the vessels of the mid lamina propria, the perichondrial capillaries were of the closed type, and were surrounded by a loose mesenchyme, richly populated by fibroblasts and moderate networks of collagen fibres. Detailed examination of the endothelium at 18 days of fetal life revealed frequent formation of luminal and adluminal processes with a few vacuoles, presumably formed from the fusion of adjacent endothelial flaps on the luminal surface (Fig. 5). Rod-shaped and round electron-dense bodies, first described in arterial endothelium by Weibel & Palade (1964), were encountered in a few of the endothelial cells of various nasal blood vessels in fetal and postnatal life (Fig. 6). Micropinocytosis was not a salient feature of any of the capillaries during prenatal life. A superficial plexus of capillaries was found close to the olfactory epithelium during fetal life. The plexus was not encountered after the first few postnatal days. On the other hand, close apposition of the capillary loops of the olfactory epithelium forming the medial wall of the vomeronasal organ continued to occur during both fetal and postnatal life.
~ ~ ~ .4
Nasal capillary fenestrations
297
Fig. 4. Part of attenuated endothelial wall of sub-epithelial capillary showing two fenestrations bridged by diaphragms, in lateral wall of nasal fossa of 18 dlay fetal rat. x 50000.
Veins and venules Endonasal venules and veins were observed from the later stages of prenatal development. After birth, micropinocytosis became a salient feature of the venular endothelial cytoplasm, and of the mural smooth muscle cells. In general, the naked endothelial tubes in the nasal mucosa started to develop a tenuous and interrupted basement membrane on the eighteenth day of gestation. Thereafter, the membrane thickened progressively. Arteries and arterioles Muscular arteries developed a well-defined internal elastic lamina only with maturation of the nasal mucosa after the second week of postnatal life. This applied to the respiratory, olfactory and vestibular regions of the nasal fossa. DISCUSSION
The development of fenestrated endonasal capillaries prenatally implies that their formation takes place as part of the contrived design for future physiological requirements in postnatal life. The increase in their number during postnatal life can be explained by the sequential process of maturation with increased physiological requirements. Likewise, the developmental changes of the endothelial basement membranes in the respiratory mucosa may be interpreted as a physiological necessity rather than as a manifestation of microvascular injury resulting from environmental cooling during respiration, as suggested by Kanan, Ryan & Weddell (1975).
298
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Cauna & Hinderer (1969) believed that capillary fenestrations in the nasal respiratory mucosa facilitated rapid exchange of fluids and solutes to and from the capillaries. This function seems likely in view of their relationship to the continuously leaking surface epithelium, and to the serous glands, and in view of their absence from the neuro-olfactory tissues. Wolff & Merker (1966) were able to show fenestrations in the endothelial cells of vaginal capillaries in the rat following treatment with oestrone. They could reverse the phenomenon by treating the animals with progesterone, testosterone, and vitamin A. In fact the oestrogen effect could be prevented in these vessels by prior treatment with these drugs. The latter study suggests that the formation of fenestrations may involve both genetic and humoral factors. A pattern of superficial and deep plexuses of vessels in both the respiratory and olfactory regions was revealed from the seventeenth day of gestation onwards. The capillaries lost their initial contact with the olfactory epithelium soon after birth, but maintained contact with the olfactory epithelium of the vomeronasal organ during postnatal life. A similar observation was made in the mouse by Cuschieri & Bannister (1975 a, b), who believed the difference may be due to the seclusion of the vomeronasal organ from the inspired air stream during postnatal life. The venules beneath the respiratory epithelium are characterized by their large calibre, both near full term and after birth, and especially after the first two weeks of induction of respiration. They presumably serve as a radiator for the inspired air current on leaving the nasal vestibule, as in man and other animals (Slome, 1952; Negus, 1958; Taylor, 1961).
Nasal capillary fenestrations
299
Fig. 6. Details of deep capillary in periosteum of ethmoidal olfactory mucosa, showing WeibelPalade bodies (WP). 19 day fetal rat. x 15000. SUMMARY
Fenestrated capillaries were observed prenatally in the lateral nasal wall and in glandular capillaries of the nasal mucosa of the rat. The number of fenestrations increased towards full term and during postnatal life. Specific ultrastructural features in the vascular bed were found to be primarily determined in utero.
This work was supported by a grant from the Medical Research Council. REFERENCES
CAUNA, N. & HINDERER, K. H. (1969). Fine structure of blood vessels in the human nasal respiratory mucosa. Annals of Otology, Rhinology and Laryngology 78, 865-880. CAUNA, N. (1970). Electron microscopy of the nasal vasculature and its nerve supply. Annals of Otology, Rhinology and Laryngology 79, 443-450. CHRISTIE, G. A. (1964). Developmental stages in somite and pre-somite rat embryos, based on external appearance, and including some features of the macroscopic development of the oral cavity. Journal of Morphology 114, 263-281. CUSCHIERI, A. & BANNISTER, L. H. (1975 a). The development of the olfactory mucosa in the mouse: light microscopy. Journal of Anatomy 119, 277-286. CUSCHIERi, A. & BANNISTER, L. H. (1975 b). The development of the olfactory mucosa in the mouse: electron microscopy. Journal of Anatomy 119, 471-498. KANAN, M. W., RYAN, T. J. & WEDDELL, A. G. M. (1975). The behaviour of the nasal mucosa towards blood borne colloidal carbon in experimental animals. Pathologica Europea 10, 263-276. KANAN, M. W. (1976). The biological behaviour of the nasal mucosa towards blood borne particulate matter. D.Phil. thesis, Oxford University.
300 PATRICIA VAN DIEST AND M. W. KANAN NEGus, V. E. (1958) In The Comparative Anatomy and Physiology of the Nose and Paranasal Sinuses. Edinburgh: E. & S. Livingstone. SiLOME, D. (1952). Diseases of the ear, nose and throat. In Physiology of the Nose and Paranasal Sinuses. (ed. W. G. Scott-Brown). Vol. 1. London: Butterworth & Co. Ltd. TAYLOR, M. (1961). An experimental study of the influence of endocrine system in the nasal respiratory mucosa. Journal of Laryngology and Otology 75, 972-977. WEIBEL, E. R., & PALADE, G. E. (1964). New cytoplasmic components in arterial epithelia. Journal of Cell Biology 23, 101-112. WOLFF, J. & MERKM, H. J. (1966). Ultrastruktur und Bildung von Boren im Endothelien porosen und geschlossenen Kapillaren. Zeitschrift far Zellforschung und mikroskopische Anatomie 73, 174-191.
293
An ultrastructural study of the endonasal microcirculation in the Wistar rat during fetal and early postnatal life PATRICIA VAN DIEST AND M. W. KANAN Department of Human Anatomy, University of Oxford
(Accepted 7 March 1978) INTRODUCTION
In spite of the unusual microanatomy of the nasal mucosa, research into the ultrastructure of its microcirculation has been limited. Cauna & Hinderer (1969) described the peculiar ultrastructure of the nasal vascular bed in adult human males and females. They found the large sub-epithelial capillaries of the lateral wall of the nasal cavity to be fenestrated. The fenestrae occurred in the capillary walls facing the ciliated respiratory epithelium. Fenestrae were also seen regularly in blood capillaries of the mixed nasal glands in the lateral wall, but were seldom encountered in the septal capillaries. The basement membrane around the capillaries, venules and veins exhibited fine pores, and in some areas consisted of several incomplete layers. More recently Kanan (1976) has confirmed the overall porosity of the capillary and venular components of the vascular bed in the respiratory nasal mucosa of the Wistar rat, albino mouse, guinea-pig and goat. Endothelial fenestrations were also frequently encountered in the superficial and glandular capillaries under the ciliated mucosa of the lateral nasal wall, less often in the septal mucosa, and hardly ever in the olfactory region. Cauna (1970) believed these fenestrations were specifically designed for rapid passage of fluid and high molecular weight solutes through the vascular walls to meet the functional requirements of the nasal organ. Kanan, Ryan & Weddell (1975) found this view plausible, but in view of the differences between the blood vessels of the respiratory and olfactory mucosae, they speculated that development of fenestrae could be related to the repetitive exposure of the respiratory mucosa to the cool, inspired airstream. As a contribution to the study of the role of environmental factors on the development of these features, it was felt necessary to examine the ultrastructure of the endonasal microvasculature during fetal and early postnatal life. MATERIALS AND METHODS
Eighteen pregnant Wistar rats, time-mated according to the method of Christie (1964), were anaesthetized intraperitoneally with Avertin, and the 83 fetuses obtained were killed by decapitation. Neonates of 1, 2, 4, 12 and 17 days of age from 3 pregnant rats were also killed. The gestation period ranged from 21 to 23 days. The nasal cavities of the animals were flooded with ice cold 3 % glutaraldehyde in 5 % sucrose/ phosphate buffer and 1 % calcium chloride (pH 7.4). Specimens were taken from the vestibular, respiratory, and olfactory regions and fixed for 3 hours at 4 'C. Postfixation was for 2 hours in 1 % osmium tetroxide made up in the sucrose/phosphate buffer. Tissues were dehydrated, embedded in Araldite, sectioned, stained with uranyl acetate and lead citrate, and examined in a Siemens 101 electron microscope.
294
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44
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Fig. 1. Loosely woven mesenchymal tissue (Mc) with few extracellular fibrous elements. Nasal pit of 16 day rat fetus. Ep, epithelium; E, endothelial cell; RBC, red blood corpuscle. x 6000.
Nasal capillary fenestrations
295
p
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~
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Fig. 2. Details of interendothelial junction (ij) in vessel from nasal pit of 16 day rat fetus. Cytoplasmic plaques (p) and polyribosomes (pr) are seen. x 50000. RESULTS
Capillaries From day 16 after mating, the beginning of the adult design of the nasal fossa and its skeletal structures was already exhibited. The roof, septum, ethmoidal scrolls in the posterior third, and naso- and maxilloturbinals in the anterior two thirds, were evident. In the early stages of the differentiation of the endonasal epithelium, irrespective of region, the corium consisted of loosely woven mesenchymal tissue. The primitive mesenchymal cells, presumably the precursors of future fibroblasts, were associated with very few extracellular fibres (Fig. 1). The vascular bed consisted of a plexus of blood channels, notable for the lack of support in their walls. In their middle perinuclear regions, the endothelial cells were crowded with aggregates of polyribosomes. The attenuated limbs of the endothelial cells formed junctions of simple type, with one or more dense, cytoplasmic plaques along their length (Fig. 2). However, no macula densa could be identified at any point in these junctions. The cytoplasm also included rough endoplasmic reticulum and mitochondria. Little macro- or micropinocytosis was seen on the 16th day of gestation. Between. days 16 and 18 after mating' a vascular basement membrane appeared, together with pericytes. On day 18 of fetal life, banded and unbanded collagen fibres were seen throughout the mesenchymal tissue. Simultaneously, fibroblasts almost completely replaced the primitive mesenchymal cells which had been encountered earlier (Fig. 3).
44
--ir 'k
!iv
296
PATRICIA VAN DIEST AND M. W. KANAN -_ .1'
Fig. 3. Blood vessel in vestibular mesenchyme lacking pericytes and basement membrane. Collagen fibres (col) can be seen for the first time outside fibroblasts (F). 18 day fetal rat nose. x 12500.
Between days 16 and 17 of gestation time the differentiation of the capillary tree into superficial, mid-level and deep plexuses was established. In contradistinction to the olfactory and vestibular capillaries, the superficial capillaries under the epithelium of the lateral nasal wall, and those supplying the serous glands of the respiratory cavity, exhibited endothelial fenestrations in the attenuated segments of their circumference (Fig. 4). The number of fenestrations increased with time. Like the vessels of the mid lamina propria, the perichondrial capillaries were of the closed type, and were surrounded by a loose mesenchyme, richly populated by fibroblasts and moderate networks of collagen fibres. Detailed examination of the endothelium at 18 days of fetal life revealed frequent formation of luminal and adluminal processes with a few vacuoles, presumably formed from the fusion of adjacent endothelial flaps on the luminal surface (Fig. 5). Rod-shaped and round electron-dense bodies, first described in arterial endothelium by Weibel & Palade (1964), were encountered in a few of the endothelial cells of various nasal blood vessels in fetal and postnatal life (Fig. 6). Micropinocytosis was not a salient feature of any of the capillaries during prenatal life. A superficial plexus of capillaries was found close to the olfactory epithelium during fetal life. The plexus was not encountered after the first few postnatal days. On the other hand, close apposition of the capillary loops of the olfactory epithelium forming the medial wall of the vomeronasal organ continued to occur during both fetal and postnatal life.
~ ~ ~ .4
Nasal capillary fenestrations
297
Fig. 4. Part of attenuated endothelial wall of sub-epithelial capillary showing two fenestrations bridged by diaphragms, in lateral wall of nasal fossa of 18 dlay fetal rat. x 50000.
Veins and venules Endonasal venules and veins were observed from the later stages of prenatal development. After birth, micropinocytosis became a salient feature of the venular endothelial cytoplasm, and of the mural smooth muscle cells. In general, the naked endothelial tubes in the nasal mucosa started to develop a tenuous and interrupted basement membrane on the eighteenth day of gestation. Thereafter, the membrane thickened progressively. Arteries and arterioles Muscular arteries developed a well-defined internal elastic lamina only with maturation of the nasal mucosa after the second week of postnatal life. This applied to the respiratory, olfactory and vestibular regions of the nasal fossa. DISCUSSION
The development of fenestrated endonasal capillaries prenatally implies that their formation takes place as part of the contrived design for future physiological requirements in postnatal life. The increase in their number during postnatal life can be explained by the sequential process of maturation with increased physiological requirements. Likewise, the developmental changes of the endothelial basement membranes in the respiratory mucosa may be interpreted as a physiological necessity rather than as a manifestation of microvascular injury resulting from environmental cooling during respiration, as suggested by Kanan, Ryan & Weddell (1975).
298
PATRICIA VAN DIEST AND M. W. KANAN
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.
%
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5 t t.~
Fi.5Xnohlu
facoeailrnrsiaoymcs of 18 day feu exibiin
~lmia flap (bac arrws an a1 fe abuia flp (white arrows)
1000
Cauna & Hinderer (1969) believed that capillary fenestrations in the nasal respiratory mucosa facilitated rapid exchange of fluids and solutes to and from the capillaries. This function seems likely in view of their relationship to the continuously leaking surface epithelium, and to the serous glands, and in view of their absence from the neuro-olfactory tissues. Wolff & Merker (1966) were able to show fenestrations in the endothelial cells of vaginal capillaries in the rat following treatment with oestrone. They could reverse the phenomenon by treating the animals with progesterone, testosterone, and vitamin A. In fact the oestrogen effect could be prevented in these vessels by prior treatment with these drugs. The latter study suggests that the formation of fenestrations may involve both genetic and humoral factors. A pattern of superficial and deep plexuses of vessels in both the respiratory and olfactory regions was revealed from the seventeenth day of gestation onwards. The capillaries lost their initial contact with the olfactory epithelium soon after birth, but maintained contact with the olfactory epithelium of the vomeronasal organ during postnatal life. A similar observation was made in the mouse by Cuschieri & Bannister (1975 a, b), who believed the difference may be due to the seclusion of the vomeronasal organ from the inspired air stream during postnatal life. The venules beneath the respiratory epithelium are characterized by their large calibre, both near full term and after birth, and especially after the first two weeks of induction of respiration. They presumably serve as a radiator for the inspired air current on leaving the nasal vestibule, as in man and other animals (Slome, 1952; Negus, 1958; Taylor, 1961).
Nasal capillary fenestrations
299
Fig. 6. Details of deep capillary in periosteum of ethmoidal olfactory mucosa, showing WeibelPalade bodies (WP). 19 day fetal rat. x 15000. SUMMARY
Fenestrated capillaries were observed prenatally in the lateral nasal wall and in glandular capillaries of the nasal mucosa of the rat. The number of fenestrations increased towards full term and during postnatal life. Specific ultrastructural features in the vascular bed were found to be primarily determined in utero.
This work was supported by a grant from the Medical Research Council. REFERENCES
CAUNA, N. & HINDERER, K. H. (1969). Fine structure of blood vessels in the human nasal respiratory mucosa. Annals of Otology, Rhinology and Laryngology 78, 865-880. CAUNA, N. (1970). Electron microscopy of the nasal vasculature and its nerve supply. Annals of Otology, Rhinology and Laryngology 79, 443-450. CHRISTIE, G. A. (1964). Developmental stages in somite and pre-somite rat embryos, based on external appearance, and including some features of the macroscopic development of the oral cavity. Journal of Morphology 114, 263-281. CUSCHIERI, A. & BANNISTER, L. H. (1975 a). The development of the olfactory mucosa in the mouse: light microscopy. Journal of Anatomy 119, 277-286. CUSCHIERi, A. & BANNISTER, L. H. (1975 b). The development of the olfactory mucosa in the mouse: electron microscopy. Journal of Anatomy 119, 471-498. KANAN, M. W., RYAN, T. J. & WEDDELL, A. G. M. (1975). The behaviour of the nasal mucosa towards blood borne colloidal carbon in experimental animals. Pathologica Europea 10, 263-276. KANAN, M. W. (1976). The biological behaviour of the nasal mucosa towards blood borne particulate matter. D.Phil. thesis, Oxford University.
300 PATRICIA VAN DIEST AND M. W. KANAN NEGus, V. E. (1958) In The Comparative Anatomy and Physiology of the Nose and Paranasal Sinuses. Edinburgh: E. & S. Livingstone. SiLOME, D. (1952). Diseases of the ear, nose and throat. In Physiology of the Nose and Paranasal Sinuses. (ed. W. G. Scott-Brown). Vol. 1. London: Butterworth & Co. Ltd. TAYLOR, M. (1961). An experimental study of the influence of endocrine system in the nasal respiratory mucosa. Journal of Laryngology and Otology 75, 972-977. WEIBEL, E. R., & PALADE, G. E. (1964). New cytoplasmic components in arterial epithelia. Journal of Cell Biology 23, 101-112. WOLFF, J. & MERKM, H. J. (1966). Ultrastruktur und Bildung von Boren im Endothelien porosen und geschlossenen Kapillaren. Zeitschrift far Zellforschung und mikroskopische Anatomie 73, 174-191.
