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Aging in the rat medial nucleus of the trapezoid body. III. Alterations in capillaries
Neurobiology of Aging, Vol. 6, pp. 39-46, 1985. ©Ankho International Inc. Printed in the U.S.A.
0197-4580/85 $3.00 + .00
Aging in the Rat Medial Nucleus of the Trapezoid Body. III. Alterations in Capillaries MICHAEL
A. CASEY l AND MARTIN L. FELDMAN
D e p a r t m e n t o f A n a t o m y , Boston University School o f Medicine, Boston, M A 02118 R e c e i v e d 11 M a y 1984 CASEY, M. A. AND M. L. FELDMAN. Aging in the rat medial nucleus of the trapezoid body. IlL Alterations in capillaries. NEUROBIOL AGING 6(1) 39--46, 1985.--The medial nucleus of the trapezoid body (MTB), a large cell group in the rat brainstem auditory pathway, undergoes significant cell loss and loss of synapses with advancing age [5,6]. The purpose of the present study was to examine the microvasculature of the MTB in rats of the following ages: 3 months (MO), 6 MO, 24 MO, 27 MO, and 33 MO. In rats aged 24 to 33 MO, the following ultrastructural changes were observed in MTB capillaries: (1) large cavitations or spaces within capillary basal laminae, and (2) membranous debris, indicative of cellular degeneration within leaflets of capillary basal lamina. The volume density ratio (VDR) of capillaries decreases significantly between 6 and 33 MO of age. Aging Superior olive Aging auditory system
Nucleus of the trapezoid body
T H E medial nucleus of the trapezoid bo~ly (MTB), the largest cell group of the rat superior olivary complex [14], undergoes significant cell loss and synaptic terminal loss with advancing age in Sprague-Dawley rats [5,6]. We have also observed ultrastructural changes in the neurons, glia, and neuropil of the MTB in aged rats [6,8]. This paper presents the results of our quantitative histological and ultrastructural studies of capillaries in the aging rat MTB. A preliminary report of this work has been pubfished [7].
Capillaries
Microvasculature
verse plane of the MTB. Thick sections for light microscopy were stained with toluidine blue and pyronin B, and thin sections for electron microscopy were stained with uranyl acetate and lead citrate. The point counting method [27] was used to determine the volume density ratio (Va) of capillaries in the MTB. In doing this, we assumed that capillaries are randomly oriented and randomly distributed within the MTB. This is based on our analysis of the MTB in sections cut transversely and sagittally. The shapes o f blood vessel profiles, and their distribution patterns are virtually indistinguishable in the two planes o f section. Blood vessels with diameters of 8/~m or less were identified as capillaries. At 1000x magnification, toluidine blue stained sections containing the MTB were analyzed light microscopically. Using an ocular grid reticle, the number of points (intersections of perpendicular grid lines) lying over the profiles of blood vessels in 0.01 mm ~ fields within the MTB were counted. This value was divided by the total number o f points overlying structures within the MTB (i.e., neurons, gila, neuropil, and blood vessels) to yield Vd. F o r a given section several non-overlapping fields within the MTB were analyzed for capillary Vd. Thus, for each animal a mean capillary V0 was determined by averaging the Vd values from 4 to 6 rostro-candal levels throughout the MTB. A one-way analysis of variance (ANOVA) was used to test the differences in capillary Vd among the age groups. Scheff~ [25] multiple comparison tests based on the F distribution were used to test for significant differences between pairs of age groups.
METHOD Male Sprague-Dawley derived albino rats (Charles River Breeding Laboratories, Wilmington, MA) were sacrificed at the following ages: 3 MO (N=4), 6 MO (N=5), 24 MO (N=2), 27 MO (N=3) and 33 MO (N=3). Since only two animals were studied at 24 MO o f age, they were not included in the quantitative part of this study. Animals aged over 12 months were retired breeders, and kept in a separate aging colony at Charles River Breeding laboratories. F o o d and water were available ad lib, and no animals were given antibiotics. The animals were anesthetized and perfused with aldehydes as previously described [5,21], and appropriate portions of the ventral medulla were blocked for study of the MTB in the transverse plane. The tissue was osmicated, dehydrated, and embedded in Araldite according to standard methods. Two micrometer thick sections and adjacent ultrathin sections were taken at various intervals in the trans-
tRequests for reprints should be addressed to Dr. Michael A. Casey, Department of Anatomy, University of Alabama in Birmingham, University Station, Birmingham, AL 35294.
39
40
CASEY A N D F E L D M A N TABLE 1 VOLUME DENSITY RAT10 (VDR) OF CAPILLARIES IN THE AGING MTB
Age (months)
No. Rats
Mean VDR*
SEM
4 5 3 3
0.0390 0.0404 0.0317 0.0283
0.0027 0.0023 0.0003 0.0015
3 6 27 33
ANOVA: F(3,11)=31.3, p<0.0001. Scheff6 tests: 3 month vs. 33 month, p < 0 . 0 5 ; 6 month vs. 33
month, p<0.05. *Four to six sections of the MTB were analyzed from each animal and then averaged to obtain a mean VDR for each animal.
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TABLE 2 THE EFFECT OF AGE ON THE VOLUME OF THE MTB
Age (months) 3 24
No. Rats
MeanVolume (mma)
SEM
5 4
0.176 0.124
0.006 0.009
Student's t-test: t =3.876, p<0.01.
The volume of the MTB was estimated using a method described by Konigsmark and Murphy [19]. The material analyzed for this purpose was from an earlier study [5], in which transverse sections from blocks of the transversely sectioned MTB were mounted and stained with protargol [3]. Thus, these animals are distinct from those 17 animals listed above which were perfused with aldehydes and analyzed for blood vessel Vd and ultrastructural changes. Using a drawing tube in the light microscope, the perimeter of the MTB was determined by marking on paper the location of peripherally placed MTB ceils, and then connecting these marks with straight lines. The area of the resulting polygon was determined using the point counting method of Weibel [27]. The volume of the MTB in a given section was obtained by multiplying the area by the section thickness (16 /~m). Since every eighth section was analyzed, the total volume of the MTB was calculated by multiplying the sum o f the volumes of the sections analyzed by 8. MTB volume was estimated in animals aged 3 MO (N=5) and 24 MO (N=4). The significance o f the difference between the two age groups was assessed using the Student's t-test. RESULTS
Aging has a marked effect on the vascularity o f the MTB in Sprague-Dawley rats, as illustrated in Fig. 1. The quantitative results of the capillary Vd determinations in the aging rat MTB are shown in Table 1. The Vd of capillaries in the rat MTB decreases significantly with advancing age, F(3,11)=31.3, p<0.0001. The Vd of MTB capillaries in rats aged 33 MO is significantly lower than the Vd in rats aged 3 and 6 MO (Scheff~ multiple comparison test, p<0.05). At 27 MO the mean capillary Va is lower than the values at 3 and 6 MO, but the differences are not statistically significant (Scheff6 test, p>0.05).
FIG. 1. Camera lucida drawings of blood vessel profiles in the MTB of a 3 MO old rat (top figure) and a 33 MO old rat (bottom figure). x200.
Between 3 and 27 MO of age, the volume of the MTB decreases significantly (Table 2). The mean volume of the MTB in rats aged 3 MO is 0.176 mm a (SEM=0.006) and in rats aged 27 MO it is 0.124 mm 8 (SEM=0.009). This represents an approximate 30% reduction between the age groups, and the difference between the means is significant (Student's t-test: t--3 876; p<0.01). Figure 2 is an electron micrograph of a typical capillary in the MTB of a 3 MO old rat. The fine structure of MTB capillaries is similar to that previously described in other parts of the rat nervous system [22]. Briefly, capillary walls consist of endothelial cells which are covered by a basal lamina on their abluminal surface (Fig. 2). The basal lamina also encloses pericyte processes (Fig. 2). In MTB capillaries, the thickness of the basal lamina is variable in young adult
VASCULAR CHANGES IN AGING RAT SUPERIOR OLIVE
41
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D
FIG. 2. An electron micrograph of a capillary profile in the MTB of a 3 MO old rat. The endothelial cell (E) is covered by a basal lamina (arrows) which splits to enclose pericyte processes (P). EN =endothelial cell nucleus; PN =pericyte nucleus. × 18,800.
animals, and the outer surface of the basal lamina is in contact primarily with astrocyte processes (Fig. 2). In aged animals (24-33 MO) ultrastructural changes in MTB capillaries are common. Capillary basal laminae appear to be thickened (Figs. 3, 4, 5, 8), and they commonly display large cavitations (Figs. 3-8). The profdes of these cavitations are typically 0.2-1.5 /~m in width, and contain floccular material resembling basal lamina ultrastructurally (Figs. 3-8). The material in the cavitations often has a "honeycombed" conf~,uration (Figs. 3 and 5). Also present within basal lamina cavitations is electron-dense membranous debris (Figs. 6 and 7). DISCUSSION
The results of this study show a substantial decline in the vascularity of the rat MTB with advancing age. However, age-related changes in the overall volume of the MTB such as that which occurs in the human ventral cochlear nucleus [19] could confound results derived from capillary Vd determinations. Our data show that the volume of the rat MTB in fact decreases significantly between 3 and 27 months of age (Table 2), and this is consistent with the substantial neuron loss in the rat MTB that occurs with aging [5]. Thus, the decreased Vd of capillaries in the MTB observed in this study is the result of age-related devascularization, and is not
the result of an increase in the volume of the MTB with advancing age. The camera lucida drawings of blood vessel prof'des in Fig. 1 support our quantitative data showing a decreased vascularity in the aging rat MTB. The effects of aging on brain microvasculature have received a considerable amount of attention [1, 2, 12, 13, 16, 17, 20, 24], yet vascular aging studies at the level of the brainstem are lacking. In aged humans, a substantial decrease (30.6%) in capillary density in the hippocampus (presubiculum) has been observed [2]. An age-related increase in capillary Vd volume fraction in human putamen was also observed by Meier-Ruge et al. [20], but no change in cortical capillaries was evident. The increase in putamen vasculature was attributed to shrinkage of subcortical structures, and not actual proliferation of the vascular supply [20]. In rats, slight decreases in capillary Vd have been observed in the external plexiform layer of the olfactory bulb [ 12] and in the occipital cortex [l], but no change in the density of capillary profdes was observed between 3 and 24 months in the visual cortex [16]. Using a point counting method similar to the one used in this study, Klein and Michel [15] reported a slight reduction of the Vd volume fraction of "vascular elements" in cerebral cortex of aged Wistar rats. The Vd volume fraction of blood vessels in the induseum griseum of mice does not change between 5 and 18 months of age [26]. Thus, it appears that aging has variable effects on brain vas-
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VASCULAR CHANGES IN AGING RAT SUPERIOR OLIVE
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FIG. 5. A basal laminar cavitation encircling a capillary profile in the MTB of a 24 MO old rat. As in Fig. 3, the substance within the cavitation has a honeycombed appearance (*). A--astrocyte; AF--astrocytic filaments, x21,300.
FACING PAGE FIG. 3 AND 4. Basal laminar cavitations in MTB capillaries of 24 MO old (Fig. 3) and 27 MO old (Fig. 4) rats. Intact portions of the basal laminae are markedly thickened (large arrows). The cavitations contain a floccular material similar in structure to basal lamina (small arrows). In some areas, the material has a "honeycombed" configuration (*). E=endothelial cell; A~-astrocyte process. Figure 3=×23,400, Fig. 4~ x21,400.
44
CASEY AND FELDMAN
FIG. 6 AND 7. Membranous debris within capillary basal laminae (arrows). Figure 6= x22,600, Fig. 7= x 19,980. FIG. 8. Spurs of basal lamina (arrows) extending into adjacent neuropil of the MTB in a 27 MO old rat. ×39,700.
V A S C U L A R C H A N G E S IN A G I N G RAT S U P E R I O R O L I V E culature in humans and laboratory animals, depending on the region studied and the methodology used. This is consistent with the fact that morphological changes in neurons and giia are variable from region to region in aged human and animal brains. Large defects, or " c a v i t a t i o n s " [12], were commonly observed in capillary basal laminae in rats aged 24 months and older. Similar changes were observed in olfactory bulb capillaries [12]. While collagen-like fibrils were occasionally present in basal lamina cavitations in the aged rat olfactory bulb capillaries [12], none were observed in our material. More information on the role o f basal laminae in capillary function is needed before conclusions can be drawn about the physiological significance of age-related defects in capillary basal laminae. Currently, there is no definitive data relating alterations in capillary basal laminae to functional changes in the blood-brain barrier (BBB). Moreover, the BBB in aged rats has been shown to be impermeable to such tracers as horseradish peroxidase [17], sucrose [22], and albumin [23], at least in the cerebral cortex. The stability o f the BBB in the brainstem o f aged animals has not been demonstrated. In this study, and in a study of aged rat cortical capillaries [17], cellular degeneration, as evidenced by deposits of membranous debris, was observed within leaflets of capillary basal laminae. It seems most likely that the debris is a result o f pericyte degeneration, since pericytes are characteristically located within the basal lamina. However, it is also possible that the debris is derived from smooth muscle cells, or some other cell type which has migrated from the neuropii to the capillary basal lamina. If it can be assumed that the debris results from pericyte degeneration, it is then possible to speculate on the origin o f the large basal lamina cavitations that we observed. The cavitations may represent the spaces once occupied by pericytes that have degenerated. At present, the effect of age-related pericyte degeneration on capillary function is unclear. A relationship between age-related neuronal loss and vascular changes has not been demonstrated. While Hinds and McNelly [12] observed a significant decrease in the density
45
of rat olfactory bulb capillaries between 27 and 36 months o f age, it was not considered to be important in the reported age-related loss o f mitral cells [11]. In rat visual cortex, Knox [18] reported a 29.7% decrease in the density of neurons between 3 and 27 months of age, but no change in the density of cortical capillaries was observed [16]. Cell loss in the MTB was found to be statistically significant between 3 and 6 months o f age [5], yet the volume density of capillaries does not change during that period (Table 1). The precise age at which a decline in MTB vascularity occurs was not determined in this study. Because of the small number of animals used, a statistically significant decrease in capillary volume density is not evident until 33 MO of age. A careful study of MTB capillaries in animals at intermediate ages (i.e., 12-18 mo) will be necessary before conclusions can be drawn regarding the relationship between neuronal loss and vascular compromise. It has been suggested that aged rat olfactory bulb neurons are less active metabolically, thus requiring less in the way o f vascular supply [12]. This could also apply to MTB cells. We have found that axosomatic terminals forming synapses with principal cells in the MTB are lost with age [6], and this could alter their net activity in some way. Additionally, the spontaneous activity that MTB cells are known to have [9] may also decline with age. This is a question worth addressing experimentally. In summary, the data from this study indicate that the vascularity of the rat MTB declines with advancing age. Additionally, the following uitrastructural changes in aged rat capillaries were observed, although not quantitatively analyzed: (1) large basal laminar cavitations, and (2) evidence o f cellular degeneration within leaflets o f capillary basal laminae. ACKNOWLEDGEMENTS We thank Carol Craig for technical assistance, and Mrs. Mary AIba for typing the manuscript. This study was supported by grants AG00001, AG00016, and AG03574 from The National Institutes of Health.
REFERENCES 1. Bar, T. Morphometric evaluation of capillaries in different laminae of rat cerebral cortex by automatic image analysis: changes during development and aging. Adv Neurol 20: 1-9, 1978. 2. Bell, M. A. and M. J. Ball. Morphometric comparison of hippocampal microvasculature in ageing and demented people: diameters and densities. Acta Neuropathol (Bed) 53: 299-318, 1981. 3. Bodian, D. A new method for staining nerve fibers and nerve endings in mounted paraffin sections. Anat Rec 65: 89-97, 1936. 4. Bums, E. M., T. W. Kruckenburg and P. K. Gaetano. Changes with age in cerebral capillary morphology. Neurobiol Aging 2: 285-291, 1981. 5. Casey, M. A. and M. L. Feldman. Aging in the rat medial nucleus of the trapezoid body. I. Light Microscopy. Neurobiol Aging 3: 187-195, 1982. 6. Casey, M. A. and M. L. Feldman. UItrastructural studies of aging in the rat medial nucleus of the trapezoid body. Soc Neurosci Abstr 9: 1061, 1983. 7. Casey, M. A. and M. L. Feldman. Age-related vascular alteration in the rat medial nucleus of the trapezoid body: light and electron microscopy. Anat Rec 20g: 28A, 1984.
8. Casey, M. A. and M. L. Feldman. Aging in the rat medial nucleus of the trapezoid body. II. Electron microscopy. J Comp Neurol, in press. 9. Guinan, J. J., B. E. Norris and S. S. Guinan. Single auditory units in the superior olivary complex. II. Locations of unit categories and tonotopic organization. Int J Neurosci 4: 147166, 1972. 10. Hicks, P. C., C. Rolsten, K. Brizzee and T. Samorajski. Agerelated changes in rat brain capillaries. Neurobiol Aging 4: 69-75, 1983. 11. Hinds, J. W. and N. A. McNelly. Aging of the rat olfactory bulb: growth and atrophy of constituent layers and changes in size and number of mitral cells. J Comp Neurol 171: 345-368, 1977. 12. Hinds, J. W. and N. A. McNelly. Capillaries in aging rat olfactory bulb: A quantitative light and electron microscopic analysis. Neurobiol Aging 3: 197-207, 1982. 13. Hunziker, O., S. Abdel'Al and U. Schulz. The aging human cerebral cortex: a stereological characterization of changes in the capillary net. J Gerontol 34: 345-350, 1979. 14. Irving, R. and J. M. Harrison. The superior olivary complex and audition: a comparative study. J Comp Neuro/130: 77-86, 1967.
46 15. Klein, A. W. and M. E. Michel. A morphometric study of the neocortex of young adult and old maze-differentiated rats. Mech Ageing Dev 6: 441--452, 1977. 16. Knox, C. A. and A. Oliveira. Brain aging in normotensive and hypertensive strains of rats. III. A quantitative study of cerebrovasculature. Acta Neuropathol (Bed) 52: 17-25, 1980. 17. Knox, C. A., R. D. Yates, I. Chen and P. M. Klara. Effects of aging on the structural and permeability characteristics of cerebrovasculature in normotensive and hypertensive strains of rats. Acta Neuropathol (Berl) $1: 1-13, 1980. 18. Knox, C. A. Effects of aging and chronic arterial hypertension in the cell population in the neocortex and archicortex of the rat. Acta Neuropathol (Berl) $6: 139-145, 1982. 19. Konigsmark, B. W. and E. A. Murphy. Volume of the ventral cochelar nucleus in man: its relationship to neuronal population and age. J Neuropathol Exp Neurol 31: 304-316, 1972. 20. Meier-Ruge, W., O. Hunziker, U. Schulz, H. J. Tobler and A. Schweizer. Stereological changes in the capillary network and nerve cells of the aging human brain. Mech Ageing Dev 14: 233-243, 1980.
CASEY AND FELDMAN 21. Peters, A. and T. M. Walsh. A study of the organization of apical dendrites in the somatic sensory cortex of the rat. J Comp Neurol 144: 253--268, 1972. 22. Peters, A., S. Palay and H. deF. Webster. The Fine Structure of the Nervous System. Philadelphia: W. B. Saunders, 1976. 23. Rapoport, S. I., K. Ohno and K. D. Pettigrew. Blood-brain barrier permeability in senescent rats. J Gerontol 34: 162-169, 1979. 24. Sankar, R., E. Blossom, K. Clemons and P. Charles. Ageassociated changes in the effects of amphetamine on the bloodbrain barrier of rats. Neurobiol Aging 4: 65-68, 1983. 25. Scheff~, H. A method for judging all contrasts in the analysis of variance. Biometrika 40: 87-104, 1953. 26. Sturrock, R. R. Quantitative and morphological changes in neurons and neuroglia in the indusium griseum of aging mice. J Gerontol 32: 647-658, 1977. 27. Weibel, E. R. Principles and methods for the morphometric study of the lung and other organs. Lab Invest 12: 131-155, 1963.
0197-4580/85 $3.00 + .00
Aging in the Rat Medial Nucleus of the Trapezoid Body. III. Alterations in Capillaries MICHAEL
A. CASEY l AND MARTIN L. FELDMAN
D e p a r t m e n t o f A n a t o m y , Boston University School o f Medicine, Boston, M A 02118 R e c e i v e d 11 M a y 1984 CASEY, M. A. AND M. L. FELDMAN. Aging in the rat medial nucleus of the trapezoid body. IlL Alterations in capillaries. NEUROBIOL AGING 6(1) 39--46, 1985.--The medial nucleus of the trapezoid body (MTB), a large cell group in the rat brainstem auditory pathway, undergoes significant cell loss and loss of synapses with advancing age [5,6]. The purpose of the present study was to examine the microvasculature of the MTB in rats of the following ages: 3 months (MO), 6 MO, 24 MO, 27 MO, and 33 MO. In rats aged 24 to 33 MO, the following ultrastructural changes were observed in MTB capillaries: (1) large cavitations or spaces within capillary basal laminae, and (2) membranous debris, indicative of cellular degeneration within leaflets of capillary basal lamina. The volume density ratio (VDR) of capillaries decreases significantly between 6 and 33 MO of age. Aging Superior olive Aging auditory system
Nucleus of the trapezoid body
T H E medial nucleus of the trapezoid bo~ly (MTB), the largest cell group of the rat superior olivary complex [14], undergoes significant cell loss and synaptic terminal loss with advancing age in Sprague-Dawley rats [5,6]. We have also observed ultrastructural changes in the neurons, glia, and neuropil of the MTB in aged rats [6,8]. This paper presents the results of our quantitative histological and ultrastructural studies of capillaries in the aging rat MTB. A preliminary report of this work has been pubfished [7].
Capillaries
Microvasculature
verse plane of the MTB. Thick sections for light microscopy were stained with toluidine blue and pyronin B, and thin sections for electron microscopy were stained with uranyl acetate and lead citrate. The point counting method [27] was used to determine the volume density ratio (Va) of capillaries in the MTB. In doing this, we assumed that capillaries are randomly oriented and randomly distributed within the MTB. This is based on our analysis of the MTB in sections cut transversely and sagittally. The shapes o f blood vessel profiles, and their distribution patterns are virtually indistinguishable in the two planes o f section. Blood vessels with diameters of 8/~m or less were identified as capillaries. At 1000x magnification, toluidine blue stained sections containing the MTB were analyzed light microscopically. Using an ocular grid reticle, the number of points (intersections of perpendicular grid lines) lying over the profiles of blood vessels in 0.01 mm ~ fields within the MTB were counted. This value was divided by the total number o f points overlying structures within the MTB (i.e., neurons, gila, neuropil, and blood vessels) to yield Vd. F o r a given section several non-overlapping fields within the MTB were analyzed for capillary Vd. Thus, for each animal a mean capillary V0 was determined by averaging the Vd values from 4 to 6 rostro-candal levels throughout the MTB. A one-way analysis of variance (ANOVA) was used to test the differences in capillary Vd among the age groups. Scheff~ [25] multiple comparison tests based on the F distribution were used to test for significant differences between pairs of age groups.
METHOD Male Sprague-Dawley derived albino rats (Charles River Breeding Laboratories, Wilmington, MA) were sacrificed at the following ages: 3 MO (N=4), 6 MO (N=5), 24 MO (N=2), 27 MO (N=3) and 33 MO (N=3). Since only two animals were studied at 24 MO o f age, they were not included in the quantitative part of this study. Animals aged over 12 months were retired breeders, and kept in a separate aging colony at Charles River Breeding laboratories. F o o d and water were available ad lib, and no animals were given antibiotics. The animals were anesthetized and perfused with aldehydes as previously described [5,21], and appropriate portions of the ventral medulla were blocked for study of the MTB in the transverse plane. The tissue was osmicated, dehydrated, and embedded in Araldite according to standard methods. Two micrometer thick sections and adjacent ultrathin sections were taken at various intervals in the trans-
tRequests for reprints should be addressed to Dr. Michael A. Casey, Department of Anatomy, University of Alabama in Birmingham, University Station, Birmingham, AL 35294.
39
40
CASEY A N D F E L D M A N TABLE 1 VOLUME DENSITY RAT10 (VDR) OF CAPILLARIES IN THE AGING MTB
Age (months)
No. Rats
Mean VDR*
SEM
4 5 3 3
0.0390 0.0404 0.0317 0.0283
0.0027 0.0023 0.0003 0.0015
3 6 27 33
ANOVA: F(3,11)=31.3, p<0.0001. Scheff6 tests: 3 month vs. 33 month, p < 0 . 0 5 ; 6 month vs. 33
month, p<0.05. *Four to six sections of the MTB were analyzed from each animal and then averaged to obtain a mean VDR for each animal.
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TABLE 2 THE EFFECT OF AGE ON THE VOLUME OF THE MTB
Age (months) 3 24
No. Rats
MeanVolume (mma)
SEM
5 4
0.176 0.124
0.006 0.009
Student's t-test: t =3.876, p<0.01.
The volume of the MTB was estimated using a method described by Konigsmark and Murphy [19]. The material analyzed for this purpose was from an earlier study [5], in which transverse sections from blocks of the transversely sectioned MTB were mounted and stained with protargol [3]. Thus, these animals are distinct from those 17 animals listed above which were perfused with aldehydes and analyzed for blood vessel Vd and ultrastructural changes. Using a drawing tube in the light microscope, the perimeter of the MTB was determined by marking on paper the location of peripherally placed MTB ceils, and then connecting these marks with straight lines. The area of the resulting polygon was determined using the point counting method of Weibel [27]. The volume of the MTB in a given section was obtained by multiplying the area by the section thickness (16 /~m). Since every eighth section was analyzed, the total volume of the MTB was calculated by multiplying the sum o f the volumes of the sections analyzed by 8. MTB volume was estimated in animals aged 3 MO (N=5) and 24 MO (N=4). The significance o f the difference between the two age groups was assessed using the Student's t-test. RESULTS
Aging has a marked effect on the vascularity o f the MTB in Sprague-Dawley rats, as illustrated in Fig. 1. The quantitative results of the capillary Vd determinations in the aging rat MTB are shown in Table 1. The Vd of capillaries in the rat MTB decreases significantly with advancing age, F(3,11)=31.3, p<0.0001. The Vd of MTB capillaries in rats aged 33 MO is significantly lower than the Vd in rats aged 3 and 6 MO (Scheff~ multiple comparison test, p<0.05). At 27 MO the mean capillary Va is lower than the values at 3 and 6 MO, but the differences are not statistically significant (Scheff6 test, p>0.05).
FIG. 1. Camera lucida drawings of blood vessel profiles in the MTB of a 3 MO old rat (top figure) and a 33 MO old rat (bottom figure). x200.
Between 3 and 27 MO of age, the volume of the MTB decreases significantly (Table 2). The mean volume of the MTB in rats aged 3 MO is 0.176 mm a (SEM=0.006) and in rats aged 27 MO it is 0.124 mm 8 (SEM=0.009). This represents an approximate 30% reduction between the age groups, and the difference between the means is significant (Student's t-test: t--3 876; p<0.01). Figure 2 is an electron micrograph of a typical capillary in the MTB of a 3 MO old rat. The fine structure of MTB capillaries is similar to that previously described in other parts of the rat nervous system [22]. Briefly, capillary walls consist of endothelial cells which are covered by a basal lamina on their abluminal surface (Fig. 2). The basal lamina also encloses pericyte processes (Fig. 2). In MTB capillaries, the thickness of the basal lamina is variable in young adult
VASCULAR CHANGES IN AGING RAT SUPERIOR OLIVE
41
P
D
FIG. 2. An electron micrograph of a capillary profile in the MTB of a 3 MO old rat. The endothelial cell (E) is covered by a basal lamina (arrows) which splits to enclose pericyte processes (P). EN =endothelial cell nucleus; PN =pericyte nucleus. × 18,800.
animals, and the outer surface of the basal lamina is in contact primarily with astrocyte processes (Fig. 2). In aged animals (24-33 MO) ultrastructural changes in MTB capillaries are common. Capillary basal laminae appear to be thickened (Figs. 3, 4, 5, 8), and they commonly display large cavitations (Figs. 3-8). The profdes of these cavitations are typically 0.2-1.5 /~m in width, and contain floccular material resembling basal lamina ultrastructurally (Figs. 3-8). The material in the cavitations often has a "honeycombed" conf~,uration (Figs. 3 and 5). Also present within basal lamina cavitations is electron-dense membranous debris (Figs. 6 and 7). DISCUSSION
The results of this study show a substantial decline in the vascularity of the rat MTB with advancing age. However, age-related changes in the overall volume of the MTB such as that which occurs in the human ventral cochlear nucleus [19] could confound results derived from capillary Vd determinations. Our data show that the volume of the rat MTB in fact decreases significantly between 3 and 27 months of age (Table 2), and this is consistent with the substantial neuron loss in the rat MTB that occurs with aging [5]. Thus, the decreased Vd of capillaries in the MTB observed in this study is the result of age-related devascularization, and is not
the result of an increase in the volume of the MTB with advancing age. The camera lucida drawings of blood vessel prof'des in Fig. 1 support our quantitative data showing a decreased vascularity in the aging rat MTB. The effects of aging on brain microvasculature have received a considerable amount of attention [1, 2, 12, 13, 16, 17, 20, 24], yet vascular aging studies at the level of the brainstem are lacking. In aged humans, a substantial decrease (30.6%) in capillary density in the hippocampus (presubiculum) has been observed [2]. An age-related increase in capillary Vd volume fraction in human putamen was also observed by Meier-Ruge et al. [20], but no change in cortical capillaries was evident. The increase in putamen vasculature was attributed to shrinkage of subcortical structures, and not actual proliferation of the vascular supply [20]. In rats, slight decreases in capillary Vd have been observed in the external plexiform layer of the olfactory bulb [ 12] and in the occipital cortex [l], but no change in the density of capillary profdes was observed between 3 and 24 months in the visual cortex [16]. Using a point counting method similar to the one used in this study, Klein and Michel [15] reported a slight reduction of the Vd volume fraction of "vascular elements" in cerebral cortex of aged Wistar rats. The Vd volume fraction of blood vessels in the induseum griseum of mice does not change between 5 and 18 months of age [26]. Thus, it appears that aging has variable effects on brain vas-
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FIG. 5. A basal laminar cavitation encircling a capillary profile in the MTB of a 24 MO old rat. As in Fig. 3, the substance within the cavitation has a honeycombed appearance (*). A--astrocyte; AF--astrocytic filaments, x21,300.
FACING PAGE FIG. 3 AND 4. Basal laminar cavitations in MTB capillaries of 24 MO old (Fig. 3) and 27 MO old (Fig. 4) rats. Intact portions of the basal laminae are markedly thickened (large arrows). The cavitations contain a floccular material similar in structure to basal lamina (small arrows). In some areas, the material has a "honeycombed" configuration (*). E=endothelial cell; A~-astrocyte process. Figure 3=×23,400, Fig. 4~ x21,400.
44
CASEY AND FELDMAN
FIG. 6 AND 7. Membranous debris within capillary basal laminae (arrows). Figure 6= x22,600, Fig. 7= x 19,980. FIG. 8. Spurs of basal lamina (arrows) extending into adjacent neuropil of the MTB in a 27 MO old rat. ×39,700.
V A S C U L A R C H A N G E S IN A G I N G RAT S U P E R I O R O L I V E culature in humans and laboratory animals, depending on the region studied and the methodology used. This is consistent with the fact that morphological changes in neurons and giia are variable from region to region in aged human and animal brains. Large defects, or " c a v i t a t i o n s " [12], were commonly observed in capillary basal laminae in rats aged 24 months and older. Similar changes were observed in olfactory bulb capillaries [12]. While collagen-like fibrils were occasionally present in basal lamina cavitations in the aged rat olfactory bulb capillaries [12], none were observed in our material. More information on the role o f basal laminae in capillary function is needed before conclusions can be drawn about the physiological significance of age-related defects in capillary basal laminae. Currently, there is no definitive data relating alterations in capillary basal laminae to functional changes in the blood-brain barrier (BBB). Moreover, the BBB in aged rats has been shown to be impermeable to such tracers as horseradish peroxidase [17], sucrose [22], and albumin [23], at least in the cerebral cortex. The stability o f the BBB in the brainstem o f aged animals has not been demonstrated. In this study, and in a study of aged rat cortical capillaries [17], cellular degeneration, as evidenced by deposits of membranous debris, was observed within leaflets of capillary basal laminae. It seems most likely that the debris is a result o f pericyte degeneration, since pericytes are characteristically located within the basal lamina. However, it is also possible that the debris is derived from smooth muscle cells, or some other cell type which has migrated from the neuropii to the capillary basal lamina. If it can be assumed that the debris results from pericyte degeneration, it is then possible to speculate on the origin o f the large basal lamina cavitations that we observed. The cavitations may represent the spaces once occupied by pericytes that have degenerated. At present, the effect of age-related pericyte degeneration on capillary function is unclear. A relationship between age-related neuronal loss and vascular changes has not been demonstrated. While Hinds and McNelly [12] observed a significant decrease in the density
45
of rat olfactory bulb capillaries between 27 and 36 months o f age, it was not considered to be important in the reported age-related loss o f mitral cells [11]. In rat visual cortex, Knox [18] reported a 29.7% decrease in the density of neurons between 3 and 27 months of age, but no change in the density of cortical capillaries was observed [16]. Cell loss in the MTB was found to be statistically significant between 3 and 6 months o f age [5], yet the volume density of capillaries does not change during that period (Table 1). The precise age at which a decline in MTB vascularity occurs was not determined in this study. Because of the small number of animals used, a statistically significant decrease in capillary volume density is not evident until 33 MO of age. A careful study of MTB capillaries in animals at intermediate ages (i.e., 12-18 mo) will be necessary before conclusions can be drawn regarding the relationship between neuronal loss and vascular compromise. It has been suggested that aged rat olfactory bulb neurons are less active metabolically, thus requiring less in the way o f vascular supply [12]. This could also apply to MTB cells. We have found that axosomatic terminals forming synapses with principal cells in the MTB are lost with age [6], and this could alter their net activity in some way. Additionally, the spontaneous activity that MTB cells are known to have [9] may also decline with age. This is a question worth addressing experimentally. In summary, the data from this study indicate that the vascularity of the rat MTB declines with advancing age. Additionally, the following uitrastructural changes in aged rat capillaries were observed, although not quantitatively analyzed: (1) large basal laminar cavitations, and (2) evidence o f cellular degeneration within leaflets o f capillary basal laminae. ACKNOWLEDGEMENTS We thank Carol Craig for technical assistance, and Mrs. Mary AIba for typing the manuscript. This study was supported by grants AG00001, AG00016, and AG03574 from The National Institutes of Health.
REFERENCES 1. Bar, T. Morphometric evaluation of capillaries in different laminae of rat cerebral cortex by automatic image analysis: changes during development and aging. Adv Neurol 20: 1-9, 1978. 2. Bell, M. A. and M. J. Ball. Morphometric comparison of hippocampal microvasculature in ageing and demented people: diameters and densities. Acta Neuropathol (Bed) 53: 299-318, 1981. 3. Bodian, D. A new method for staining nerve fibers and nerve endings in mounted paraffin sections. Anat Rec 65: 89-97, 1936. 4. Bums, E. M., T. W. Kruckenburg and P. K. Gaetano. Changes with age in cerebral capillary morphology. Neurobiol Aging 2: 285-291, 1981. 5. Casey, M. A. and M. L. Feldman. Aging in the rat medial nucleus of the trapezoid body. I. Light Microscopy. Neurobiol Aging 3: 187-195, 1982. 6. Casey, M. A. and M. L. Feldman. UItrastructural studies of aging in the rat medial nucleus of the trapezoid body. Soc Neurosci Abstr 9: 1061, 1983. 7. Casey, M. A. and M. L. Feldman. Age-related vascular alteration in the rat medial nucleus of the trapezoid body: light and electron microscopy. Anat Rec 20g: 28A, 1984.
8. Casey, M. A. and M. L. Feldman. Aging in the rat medial nucleus of the trapezoid body. II. Electron microscopy. J Comp Neurol, in press. 9. Guinan, J. J., B. E. Norris and S. S. Guinan. Single auditory units in the superior olivary complex. II. Locations of unit categories and tonotopic organization. Int J Neurosci 4: 147166, 1972. 10. Hicks, P. C., C. Rolsten, K. Brizzee and T. Samorajski. Agerelated changes in rat brain capillaries. Neurobiol Aging 4: 69-75, 1983. 11. Hinds, J. W. and N. A. McNelly. Aging of the rat olfactory bulb: growth and atrophy of constituent layers and changes in size and number of mitral cells. J Comp Neurol 171: 345-368, 1977. 12. Hinds, J. W. and N. A. McNelly. Capillaries in aging rat olfactory bulb: A quantitative light and electron microscopic analysis. Neurobiol Aging 3: 197-207, 1982. 13. Hunziker, O., S. Abdel'Al and U. Schulz. The aging human cerebral cortex: a stereological characterization of changes in the capillary net. J Gerontol 34: 345-350, 1979. 14. Irving, R. and J. M. Harrison. The superior olivary complex and audition: a comparative study. J Comp Neuro/130: 77-86, 1967.
46 15. Klein, A. W. and M. E. Michel. A morphometric study of the neocortex of young adult and old maze-differentiated rats. Mech Ageing Dev 6: 441--452, 1977. 16. Knox, C. A. and A. Oliveira. Brain aging in normotensive and hypertensive strains of rats. III. A quantitative study of cerebrovasculature. Acta Neuropathol (Bed) 52: 17-25, 1980. 17. Knox, C. A., R. D. Yates, I. Chen and P. M. Klara. Effects of aging on the structural and permeability characteristics of cerebrovasculature in normotensive and hypertensive strains of rats. Acta Neuropathol (Berl) $1: 1-13, 1980. 18. Knox, C. A. Effects of aging and chronic arterial hypertension in the cell population in the neocortex and archicortex of the rat. Acta Neuropathol (Berl) $6: 139-145, 1982. 19. Konigsmark, B. W. and E. A. Murphy. Volume of the ventral cochelar nucleus in man: its relationship to neuronal population and age. J Neuropathol Exp Neurol 31: 304-316, 1972. 20. Meier-Ruge, W., O. Hunziker, U. Schulz, H. J. Tobler and A. Schweizer. Stereological changes in the capillary network and nerve cells of the aging human brain. Mech Ageing Dev 14: 233-243, 1980.
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