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Potential mechanisms of the age-related changes in the blood-brain barrier
Neurobiologyof Aging,Vol. 15, No. 6, pp. 751-755, 1994 Copyright © 1994ElsevierScienceLtd Printed in the USA. All rights reserved 0197-4580/94$6.00 + .00
Pergamon 0197-4580(94)00074-3
PEER COMMENTARY
Potential Mechanisms of the Age-Related Changes in the Blood-Brain Barrier A R S H A G D. M O O R A D I A N
St. Louis V.A. Medical Center and the Division of Endocrinology, Department of lnternal Medicine, St. Louis University Health Sciences Center, St. Louis, MO 63104 MOORADIAN, A. D. Potential mechanisms of the age-related changes in the blood-brain barrier. NEUROBIOL AGING 15(6) 751-755, 1994.--A variety of age-related changes in the blocxt-brain barrier transport processes have been identified. These include reduced hexose and butyrate transport, reduced choline transport, reduced triiodothyronine transport without a change in the transport of neutral and basic amino acids. The potential mechanisms underlying these age-related changes include hemodynamic alterations in the cerebral circulation of aged rats, notably increased occurrence of arteriovenous shunting. Additional age-related changes in cerebral microvessels include alterations in protein composition, and increased accumulation of lipid peroxidation byproducts, along with changes in membrane fluidity of isolated cerebral microvessels. In addition, neurotransmitter activity notably beta adrenergic neurotransmission, is significantly reduced in cerebral microvessels of aged rats. These alterations taken together may account for some of the age-related changes in the blood-brain barrier. Aging
Blood-brain barrier
Hemodynamic changes
Lipid order
INTRODUCTION
Neurotransmitter activity
the blood-brain barrier may be influenced by pathologic changes to a greater extent compared to the effects of the changes in biochemical parameters of cerebral microvessels.
The blood-brain barrier (BBB) is the primary interface between the central nervous system (CNS) and the environment. Considering the differences in the surface areas, the two other interfaces between the CNS and the environment namely, bloodcerebrospinal fluid (CSF) barrier and possibly the olfactory nerve terminals are relatively minor compared to the BBB. In addition, to its function as a barrier to circulating toxic agents and antibodies, the BBB through specialized transport processes assures adequate delivery of nutrients, hormones, and precursors of neurotransmitters to the CNS (for review see ref. 39). Under certain conditions, those transport processes can be rate limiting to various key metabolic pathways of neuronal cells. Hence, it is possible that some of the age-related degenerative changes in the CNS are partly related to the deterioration in the functional and biochemical integrity of the BBB (18). In the present communication, the age-related changes in the BBB transport processes is briefly summarized and the potential underlying mechanisms that may account for the changes observed in the aging animal are discussed. For an overview of the agerelated changes in the barrier function of the BBB, and the histologic changes in cerebral circulation the reader is referred to a previous review (18) and some additional studies published recently (6,43,44,51). It should be acknowledged at the outset that although the age-related changes in microvascular histology is not the focus of this communication, the transport of substances across
AGE-RELATEDCHANGESIN THE BBB TRANSPORTFUNCTION Eight independent transport systems assure the availability of metabolic substrates and precursors of neurotransmitters in the brain (18,39). There are also specific transport systems for hormones, neuropeptides, vitamins, and minerals. These transport processes are modulated by nutritional and metabolic factors (18, 20,39). In addition, aging, both during development and senescence is associated with significant changes in some BBB transport processes (Table 1). Although all the transport carriers are operational at birth, significant developmental and maturational changes have been observed (3). The developmental decline in choline transport appears to be further aggravated with senescence (19). Thus, 24-month-old Fischer 344 rats compared to 3-month-old (mature young) rats have reduced BBB choline transport as a result of reduced choline transporter capacity (V,,,~). On the other hand, the developmental decrease in the capacity of neutral and basic amino acid transporter is not accentuated with further aging (18). There are also developmental changes in the BBB hexose and monocarboxylic acid carriers (3,4,42). The 28-day-old rabbits compared to newborn rabbits have reduced capacity of glucose 751
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TABLE 1 AGE-RELATEDCHANGESIN THE BLOOD-BRAINBARRIERTRANSPORTPROCESSES Transport System Hexose Neutral amino acid Basic amino acid Acidic amino acid Monocarboxylic acid Purine Nucleoside Amine Others
Representative Nutrients
Changes With Age
glucose, galactose, vitamin C phenylalanine, leucine, tyrosine, isoleucine, methionine, tryptophan, valine, DOPA, cysteine Iysine, arginine, ornithine glutamate, aspartate lactate, pyruvate, ketone bodies adenine, guanine adenosine, guanidine, uridine choline neuropeptides thiamine folate electrolytes thyroid hormone steroid hormones
decreased no change or decreased* no change or decreased* no change decreased unknown unknown decreased some decreased unknown unknown unknown 6ecreased unknown
* May be decreased if 28 to 31-month-old rats are compared to intermediate age rats. transporter and increased capacity of monocarboxylic acid carrier (3). Our studies in aging rats indicate that 24-month-old rats, compared to 3-month-old rats have reduced BBB transport of glucose (29) and butyrate (23). The decrease in BBB glucose transport in aged rats was the result of reduced transporter capacity (V,,,~) which correlated with reduced cytochalasin B binding sites in isolated microvessels (29). However, immunoreactive glucose transporter (Glut 1) mass in isolated cerebral microvessels was not significantly reduced in aged animals (29). The discrepancy between the cytochalasin B binding data and immunologically detected Glut 1 concentrations could not be explained. The observation that vitamin C is transported to the brain via the hexose carrier (16) suggests that the transport of this vitamin may also be reduced with aging. The potential biochemical consequences of such a change can not be predicted. It is possible~ however, that some of the age-related changes in enzymes involved in neurotransmitter synthesis such as tyrosin hydroxylase, may be affected by the reduction in the BBB transport of vitamin C, an important modulator of tyrosine hydroxytase (17). When old animals are compared to young animals the Brain Uptake Index (BUI) of various amino acids do not appear to be altered (18). However, when 28- to 31-month-old rats are compared to intermediate age rats (7- to 11-month-old), the BU1 of methionine and possibly lysine is reduced whereas the BUI of glutamate is not different (2). However, the latter study did not rigorously control for the age-related changes in cerebral blood flow. Aging in rats is also associated with reduced BBB transport of thyroid hormone (triiodothyronine, T3) (21,22). The BUI of both levo and dextro enantiomers of T 3 is reduced in 25-month-old rats compared to 3-month-old rats (22). However, the volume of distribution of T 3 in the brain measured at steady-state conditions is not altered with age (22). Similar age-related changes in T 3 transport in hepatic tissue has been found (21). Finally, it appears that aging can also alter BBB transport of peptides such as Tyr-MIF-I(I). Both the Vm,,x and K,,, of the transport of this peptide were proportionally decreased in 24month-old rats compared to younger controls (1). The effect of aging on BBB transport of purine, nucleoside, and various vitamins or electrolytes remains unknown. The scarcity of aged animals and their cost impose significant limitations on the studies of BBB transport kinetics.
POTENTIAL MECHANISMSOF AGE-RELATEDCHANGES IN THE BBB For simplification purposes, the potential mechanisms of agerelated changes in the BBB can be separated into four general categories (Table 2). The first category of histopathologic changes in cerebral microcirculation is not discussed here. The other four categories of potential mechanisms are discussed separately although the multiplicity of underlying mechanisms and the possible overlap between some of those mechanisms should be acknowledged at the outset,
Age-Related Changes in Hemodynamic Variables In addition to the well documented structural changes in cerebral microvessels with age (6,18,51), hemodynamic changes in cerebral microcirculation may contribute to the age-related changes in the BBB. These potential hemodynamic alterations include leukocyte plugging, attachment of blood cells to endothelial walls of vessels, arteriovenous shunting, and changes in vascular reactivity altering regional blood flow. To evaluate these parameters the cerebral microvessels of Fischer 344 rats at different ages were studied using intravital fluorescence microscopy (27). Aging in this rat model was associated with significant arteriovenous shunting without alterations in blood flow characteristics, blood cell margination at the endothelial walls of the vessels and without changes in vascular permeability to 150 KDa FITC dextran (27). TABLE 2 POSSIBLE MECHANISMSUNDERLYINGTHE AGE-RELATEDCHANGES IN THE BLOOD-BRAINBARRIER Pathological changes in cerebral microcirculation (a) capillary dropout; (b) capillary atrophy Hemodynamic changes in the cerebral microcireulation: (a) arteriovenous shunting; (b) altered vascular reactivity Changes in biophysical properties of cerebral capillaries: (a) altered membrane fluidity; (b) altered surface change of endothetium Compositional changes: (a) changes in protein compositions; (b) changes in lipid composition; (c) accumulation of lipid peroxidation byproducts Alterations in neurotransmitter activity: (a) altered beta-adrenergic receptor number; (b) altered adenylate cyclase activity
BLOOD-BRAIN BARRIER AND AGING Aging is also associated with significant alterations in cerebral microvascular reactivity. The initial brief constriction of arterioles in response to BaC12 was practically eliminated in aged rats. The subsequent dilatory response however was similar in all agegroups (27). This is in agreement with a previous study of isolated basilar artery in vitro where age-related selective decreases in contractile responses were found without a change in dilatory responses (10). This notion is further supported by the observation that aging is associated with increased expression of neuropeptidergic vasodilatory cerebrovascular nerves whereas vasoconstrictor nerve density is either reduced or unaltered with age (15,41). These changes, therefore, would favor the maintenance of vasodilatory responses in aging rats with a loss of vasoconstrictor responsiveness. However, the age-related changes in cerebrovascular reactivity depend on the stimulus. Thus, the dilatation of cerebral arterioles in response to EDTA may be reduced in aged rats (9). This age-related reduction in distensibility was attributed to reduced proportion of distensible elements of the microvasculature such as the elastin and smooth muscle relative to nondistensible components of cerebral areteries such as collagen and basement membrane (9). In another study using intravital microscopy adenosine-induced vasodilatation was greater in young than in aged (24-month-old) rats (48). The reduced distensibility of cerebral vasculature in aging limits the dilatory response of these vessels in response to hypercapnia, hypoxia, or hypotension and may predispose aged animals to cerebral ischemia (9). In addition to the structural changes the reactivity of the microvessels also depends on receptor and second messenger systems related to sympathetic innervation. These changes are discussed in subsequent sections. The increased arteriovenous shunting in aged rats in itself would reduce the delivery of nutrients to the capillary bed and impair the metabolism of cerebral tissue. The kinetic parameters of various transport systems at the blood-brain barrier of aging rats should be interpreted cautiously because the changes in Vm~ (transport capacity) may well be secondary to arteriovenous shunting or capillary drop out. These changes however should not alter the K,,, of the transport system studied. The lack of extravasation of FITC dextran suggest that aging is not associated with significant changes in BBB leakiness. Similar conclusions have been previously drawn by others using different techniques (40,52). In a study utilizing et-aminoisobutyric acid, which is only minimally transported across the normal cerebral microvessels while it is actively transported in cerebral parenchymal cells, Saija et al. concluded that the BBB permeability of 28to 30-week-old rats is increased compared to younger rats (43). Moreover, older rats appeared to have increased vulnerability of the BBB to haloperidol-induced enhancement of permeability (44). It also appears that there is a maturational increase of susceptibility of the BBB permeability to electro convulsive shocks (37). However, recent studies with intravital fluoresent microscopy indicate that the susceptibility of the BBB to distuption during acute hypertension is not increased with aging (14). For complete assessment of BBB leakiness in aged rats, FITC dextran of various molecular weights, rather than a single FITC dextran should be studied in various regions of the brain since the age-related changes can be region specific (8).
Age-Related Changes in Biochemical Composition of Cerebral Microvessels A potential cause for the age-related changes in BBB transport is an alteration in biochemical composition of the cerebral microvessels. Aging is associated with a variety of modifications of gene products either because of altered gene expression, altered protein turnover, or as a result of post translational modifications
753 of proteins (35). In Fischer 344 rat the activity of two BBB enzymes, alkaline phosphatase, and gamma glutamyl transpeptidase, is not altered with age (29). The Na ÷- K ÷-ATPase activity of cerebral microvessels also is not altered with age although Ca ÷ ÷ Mg ÷ ÷- ATPase activity and Mg + + independent Ca ÷ ÷- ATPase activity may be reduced in 24-month-old rats compared to 3-month-old rats (49). We have recently found that the activity of vacuolar H÷-ATPase activity in cerebral microvessels(24) may also be reduced with age (25). The lack of a change in Na +K+-ATPase activity with age is consistent with the observation that aging is not associated with a change in ouabain binding sites of cerebral microvessels (11). The activity of glutathione peroxidase and glutathione reductase of cerebral microvessels does not change with age. However, the catalase activity may decrease and superoxide dismutase activity of cerebral microvessels is increased with age (50). Overall, a deficiency in enzymes protective against free radicals can not account for the observed age-related accumulation of lipid peroxidation byproducts in cerebral microvessels (32). The effect of various other enzymes known to be localized at the BBB is not studied. The age-related changes in adenylate cyclase activity is discussed in a subsequent section. More recently the age-related changes in a BBB specific antigen (47), the endothelial barrier antigen (EBA), was quantitated using immunohistologic techniques (26). The anti-EBA-stained microvessels were normalized against the total microvessels identified with either anti-EBA binding or anti Glut-1 (glucose transporter 1) binding. The anti-EBA stained microvessels of the hippocampus, but not of other cerebral areas, were reduced in aged rats (26). This is in agreement with the notion that hippocampus microvessels are more likely to be affected with aging changes (18). The changes in EBA do not appear to correlate with the BBB permeability in aged rats since the aging in rats is not asociated with increased permeability of the BBB in the hippocampal area (8,40,43). To gain further insight into the age-related changes in protein composition of cerebral microvessels, two-dimensional electrophoretic protein profile of cerebral microvessels isolated from rats at different ages was quantitatively evaluated (28). Of the 26 proteins in cerebral microvessels consistently identified on the gel, 10 showed significant age-related changes. A large acidic protein with a molecular weight of 144,000 and an isoelectric point of 5.4 was found exclusively in aged rats. Attempts at sequencing this protein was unsuccessful because it was N-terminally blocked and the amount of protein available was exceedingly small (28). The identity of the proteins altered in aged rat cerebral microvessels was not known and therefore the functional significance of the observation can not be precisely ascertained. Nevertheless an alteration in a relatively large proportion (38%) of the protein constituents of cerebral microvessels is likely to result in significant physiological changes in the BBB. The most widely recognized post translational modifications of proteins with age are glycosylation and protein conjugation with malondialdehyde (MDA), a byproduct of free radical-induced lipid peroxidation (35). The cerebral microvessel content of advanced glycosylation endproducts as well as pentosidine content are not affected with age (28). It is possible, however, individual glycoproteins may be increased or decreased with age whereas the average amount remains unchanged. Similarly, the amount of proteins conjugated with MDA as measured with a specific antiMDA-protein antibodies (13), were also not altered in the cerebral microvessels of aged rats (unpublished observations). This is in contrast to increased glycosylation endproducts and MDAmodified proteins found in cerebral microvessels of diabetic rats (30). However, lipid peroxidation byproducts, such as conjugated dienes, are increased in cerebral microvessels of both aged (32)
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MOORADIAN
and diabetic rats (33). This suggests that the interactions of proteins with lipid peroxidation byproducts in aging may be different from that found in diabetic rats. Some aspects of the lipid composition of cerebral microvessels of aging rats has been studied previously (32,50). Tayarani et al. (50) found that aging in Sprague-Dawley rats is associated with increased monounsaturated fatty acids and reduced polyunsaturated fatty acids in cerebral microvessels. The percent of fatty acids in isolated cerebral capillaries did not change with age (50). In Fischer 344 rats, we could not observe any significant agerelated change in fatty acid composition or in cholesterol and phospholipid content of cerebral microvessels. The differences between these two studies can not be readily explained. They may however be related to the differences in rat strains used. Of interest is that the concentration of various minerals in cerebral microvessels may also change with age (50). Thus, in Sprague-Dawley rats, aging is associated with increased content of copper and zinc in cerebral microvessels, whereas manganese content is not altered (50). Overall it appears that aging is associated with significant alterations in biochemical composition of cerebral microvessels. It is likely that these changes may contribute to the age-related alterations in BBB function, either through changes in specific transporter molecules or through changes in the biophysical properties of endothelial cell membranes thereby altering the mobility of various transporters.
Age-Related Changes in Biophysical Properties of Cerebral Microvessels Membrane microviscosity and surface charges of endothelial cells are important determinants of transport across the BBB (5,36). In one study, the increased permeability of cerebral endothelium was associated with a reduction in surface charge (36). In a study of age-related alterations in the permeability properties of the aortic endothelium it was found that positively charged cationized ferritin was more rapidly transported through the endothelium of aged rat aorta compared to young rats (5). Aged rat aorta was found to have a decreased density of cell-surface sialic acids. It remains to be shown whether similar changes occur in the surface charge of endothelial cell lining of the cerebral microvessels of aged animals. Alterations in membrane microviscosity with aging have been found in various tissues. The changes in membrane microviscosity are tissue specific. In some the microviscosity is increased wheres in other cell types microviscosity may be reduced with age (32). Recently, we have found that the steady state fluorescence polarization of diphenylhexatriene, a membrane core probe incorporated into isolated cerebral microvessel membranes at 35°C, was not different in aged rats compared to young rats (32). The thermotropic transition temperature of these membranes was not altered with age either (32). However, the fluorescence polarization of 1-(4-tri-melthylammoniumphenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH), a probe for the surface region of the membrane, was significantly reduced in aged rats (34). Thus, aging in rats is
associated with a significant decrease in lipid order near the membrane surface of cerebral microvessels. Future studies should attempt to correlate the age-related changes in BBB permeability with changes in biophysical properties of cerebral endothelial cell membranes.
Age-Related Changes in Neurotransmitter Activi~ of Cerebral Microvessels Cerebral microvessels are rich in receptors for various neurotransmitters (38). With the exception of beta adrenergic receptors ( 12,31), the age-related changes in neurotransmitter receptor density of cerebral microvessels has not been adequately addressed. One study using a single preparation of cerebral microvessels found that beta-adrenergic receptor binding sites (B,,,~) in aged rats was 21% lower than that of mature rats (12). This observation was confirmed in a subsequent study (31). In addition, the basal adenylate cyclase activity (AC) as well as isoproterenol or sodium fluoride-stimulated AC activity in cerebral microvessels were significantly reduced with age (31). This is in contrast to the changes in AC activity in cerebral tissue. In this tissue AC is not altered with age although beta adrenergic receptor number is reduced (46). Thus, the age-related changes in betaadrenergic neurotransmission of cerebral microvessels are not identical to those seen in cerebral tissue. A direct quantitation of norepinephrine content of isolated microvessels of rat brain has also demonstrated an age-related reduction (7). These findings taken together suggest that adrenergic neurotransmission in cerebral microvessels is significantly reduced with age. The extent of the contribution of these changes to the alterations in BBB function remains to be demonstrated. CONCLUDING REMARKS
The functional integrity of the BBB is of utmost importance in maintaining the optimal milieu for the CNS. It is well established that some aspects of the BBB function are compromised with age. The underlying mechanisms for these age-related changes are multifactorial. The relative importance of each factor can not be estimated at the present time. The degree of the contribution of the changes in BBB to the age-related degenerative diseases of the CNS is not known. However, considering the central importance of the BBB in various aspects of CNS metabolism,it is highly likely that the changes in BBB found with aging contribute to the deterioration of the CNS function commonly found in aged animals. Future research should focus on identifying additional specific markers of the BBB and evaluate their relevance to the BBB function. ACKNOWLEDGEMENT
This review is dedicated to the memory of Dr. William Oldendorf for his pioneering work in blood-brain barrier research and for his advice to me. This work was supported by the Medical Research of the Department of Veterans Affairs.
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BLOOD-BRAIN BARRIER AND AGING
7. Embree, L. J.; Roubein, I.F.; Jackson, D. W.; Ordway, F. Aging effect on the noradrenaline content of rat brain microvessels. Exp. Aging Res. 7:215-224; 1981. 8. Goldman, H.; Berman, R. F.; Gershon, S.; Murphy, S.; Morehead, M.; Altman, H. J. Cerebrovascular permeability and cognition in the aging rat. Neurobiol. Aging 13:57-62; 1992. 9. Hajdu, M. A.; Heistad, D. D.; Siems, F. E.; Baumbach, G. L. Effects of aging on mechanics and composition of cerebral arterioles in rats. Circ. Res. 66:1747-1754; 1990. 10. Hamel, E.; Assumel-Lurdin, C.; Bouloy, M.; Mackenzie, E. T. Selective age-related changes in neuronal markers and smooth muscle reactivity in cerebrovascular beds of Fischer 344 rats. Neurobiol. Aging 11:631-639; 1990. 11. Harik, S. I.; Mitchell, M. J.; Kalaria, R. N. Ouabain binding in the human brain. Effects of Alzheimer's disease and aging. Arch. Neurol. 46:951-954; 1989. 12. Kobayashi, H.; Maoret, T.; Spano, P. F.; Trabucchi, M. Effect of age on 13-adrenergic receptors on cerebral microvessels. Brain Res. 244: 374-377; 1982. 13. Lung, C. C.; Pinnas, J. L.; Yahya, M. D.; Meinke, G. C.; Mooradian, A. D. Malondialdehyde modified proteins and their antibodies in the plasma of control and streptozotocin induced diabetic rats. Life Sci. 52:329-337; 1992. 14. Mayhan, W. G. Disruption of blood-brain barrier during acute hypertension in adult and aged rats. Am. J. Physiology 258:H1735H1738; 1990. 15. Mione, M. C.; Dhital, K. K.; Amenta, F.; Burnstock, G. An increase in the expression of neuropeptidergic vasodilator, but not vasoconstrictor, cerebrovascular nerves in aging rats. Brain Res. 460:103113; 1988. 16. Mooradian, A. D. The effect of ascorbate and dehydroascorbate on tissue uptake of glucose. Diabetes 36:1001-1004; 1987. 17. Mooradian, A. D. Diabetic complications of the central nervous system. Endocrine Rev. 9:346-356; 1988. 18. Mooradian, A. D. Effect of aging on the blood-brain barrier. Neurobiol. Aging 9:31-39; 1988. 19. Mooradian, A. D. Blood-brain barrier transport of choline is reduced in the aged rat. Brain Res. 440:328-332; 1988. 20. Mooradian, A. D. Metabolic fuel and amino acid transport into the brain in experimental hypothyroidism. Acta. Endocrinol. 122:156162; 1990. 21. Mooradian, A. D. The hepatic transcellular transport of 3,5,3'- triiodothyronine is reduced in aged rats. Biochem. Biophys. Acta. 1054: 1-7; 1990. 22. Mooradian, A. D. Blood-brain transport of triiodothyronine is reduced in aged rats. Mech. Aging Dev. 52:141-147; 1990. 23. Mooradian A. D. The blood-brain barrier transport of glucose and butyrate is reduced in aged rats. Clin. Res. 38:81A; 1990. 24. Mooradian A. D.; Bastani, B. Identification of proton-translocating adenosine tripbosphatases in rat cerebral microvessels. Brain Res. 629:128-132; 1993. 25. Mooradian A . D . ; Bastani, B. The blood-brain barrier protontranslocating ATPases in diabetic and aged rats. Clin. Res. 42:13A; 1994. 26. Mooradian A. D.; Grabau, G.; Uko-Eninn, A. In situ quantitative estimates of the age-related and diabetes-related changes in cerebral endothelial barrier antigen. Brain Res. 309:41--44; 1993. 27. Mooradian A. D.; McCuskey, R. S. In vivo microscopic studies of age-related changes in the structure and the reactivity of cerebral microvessels. Mech. Aging Dev. 64:247-254; 1992. 28. Mooradian A. D.; Meredith, K. E. The effect of age on protein composition of rat cerebral microvessels. Neurochem. Res. 17:665-670; 1992. 29. Mooradian, A. D.; Morin, A. M.; Cipp, L. J.; Haspel, H. C. Glucose transport is reduced in the blood-brain barrier of aged rats. Brain Res. 551:145-149; 1991. 30. Mooradian, A. D.; Pinnas, J. L.; Lung, C. C.; Yahya, M. D.;
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PEER COMMENTARY
Potential Mechanisms of the Age-Related Changes in the Blood-Brain Barrier A R S H A G D. M O O R A D I A N
St. Louis V.A. Medical Center and the Division of Endocrinology, Department of lnternal Medicine, St. Louis University Health Sciences Center, St. Louis, MO 63104 MOORADIAN, A. D. Potential mechanisms of the age-related changes in the blood-brain barrier. NEUROBIOL AGING 15(6) 751-755, 1994.--A variety of age-related changes in the blocxt-brain barrier transport processes have been identified. These include reduced hexose and butyrate transport, reduced choline transport, reduced triiodothyronine transport without a change in the transport of neutral and basic amino acids. The potential mechanisms underlying these age-related changes include hemodynamic alterations in the cerebral circulation of aged rats, notably increased occurrence of arteriovenous shunting. Additional age-related changes in cerebral microvessels include alterations in protein composition, and increased accumulation of lipid peroxidation byproducts, along with changes in membrane fluidity of isolated cerebral microvessels. In addition, neurotransmitter activity notably beta adrenergic neurotransmission, is significantly reduced in cerebral microvessels of aged rats. These alterations taken together may account for some of the age-related changes in the blood-brain barrier. Aging
Blood-brain barrier
Hemodynamic changes
Lipid order
INTRODUCTION
Neurotransmitter activity
the blood-brain barrier may be influenced by pathologic changes to a greater extent compared to the effects of the changes in biochemical parameters of cerebral microvessels.
The blood-brain barrier (BBB) is the primary interface between the central nervous system (CNS) and the environment. Considering the differences in the surface areas, the two other interfaces between the CNS and the environment namely, bloodcerebrospinal fluid (CSF) barrier and possibly the olfactory nerve terminals are relatively minor compared to the BBB. In addition, to its function as a barrier to circulating toxic agents and antibodies, the BBB through specialized transport processes assures adequate delivery of nutrients, hormones, and precursors of neurotransmitters to the CNS (for review see ref. 39). Under certain conditions, those transport processes can be rate limiting to various key metabolic pathways of neuronal cells. Hence, it is possible that some of the age-related degenerative changes in the CNS are partly related to the deterioration in the functional and biochemical integrity of the BBB (18). In the present communication, the age-related changes in the BBB transport processes is briefly summarized and the potential underlying mechanisms that may account for the changes observed in the aging animal are discussed. For an overview of the agerelated changes in the barrier function of the BBB, and the histologic changes in cerebral circulation the reader is referred to a previous review (18) and some additional studies published recently (6,43,44,51). It should be acknowledged at the outset that although the age-related changes in microvascular histology is not the focus of this communication, the transport of substances across
AGE-RELATEDCHANGESIN THE BBB TRANSPORTFUNCTION Eight independent transport systems assure the availability of metabolic substrates and precursors of neurotransmitters in the brain (18,39). There are also specific transport systems for hormones, neuropeptides, vitamins, and minerals. These transport processes are modulated by nutritional and metabolic factors (18, 20,39). In addition, aging, both during development and senescence is associated with significant changes in some BBB transport processes (Table 1). Although all the transport carriers are operational at birth, significant developmental and maturational changes have been observed (3). The developmental decline in choline transport appears to be further aggravated with senescence (19). Thus, 24-month-old Fischer 344 rats compared to 3-month-old (mature young) rats have reduced BBB choline transport as a result of reduced choline transporter capacity (V,,,~). On the other hand, the developmental decrease in the capacity of neutral and basic amino acid transporter is not accentuated with further aging (18). There are also developmental changes in the BBB hexose and monocarboxylic acid carriers (3,4,42). The 28-day-old rabbits compared to newborn rabbits have reduced capacity of glucose 751
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TABLE 1 AGE-RELATEDCHANGESIN THE BLOOD-BRAINBARRIERTRANSPORTPROCESSES Transport System Hexose Neutral amino acid Basic amino acid Acidic amino acid Monocarboxylic acid Purine Nucleoside Amine Others
Representative Nutrients
Changes With Age
glucose, galactose, vitamin C phenylalanine, leucine, tyrosine, isoleucine, methionine, tryptophan, valine, DOPA, cysteine Iysine, arginine, ornithine glutamate, aspartate lactate, pyruvate, ketone bodies adenine, guanine adenosine, guanidine, uridine choline neuropeptides thiamine folate electrolytes thyroid hormone steroid hormones
decreased no change or decreased* no change or decreased* no change decreased unknown unknown decreased some decreased unknown unknown unknown 6ecreased unknown
* May be decreased if 28 to 31-month-old rats are compared to intermediate age rats. transporter and increased capacity of monocarboxylic acid carrier (3). Our studies in aging rats indicate that 24-month-old rats, compared to 3-month-old rats have reduced BBB transport of glucose (29) and butyrate (23). The decrease in BBB glucose transport in aged rats was the result of reduced transporter capacity (V,,,~) which correlated with reduced cytochalasin B binding sites in isolated microvessels (29). However, immunoreactive glucose transporter (Glut 1) mass in isolated cerebral microvessels was not significantly reduced in aged animals (29). The discrepancy between the cytochalasin B binding data and immunologically detected Glut 1 concentrations could not be explained. The observation that vitamin C is transported to the brain via the hexose carrier (16) suggests that the transport of this vitamin may also be reduced with aging. The potential biochemical consequences of such a change can not be predicted. It is possible~ however, that some of the age-related changes in enzymes involved in neurotransmitter synthesis such as tyrosin hydroxylase, may be affected by the reduction in the BBB transport of vitamin C, an important modulator of tyrosine hydroxytase (17). When old animals are compared to young animals the Brain Uptake Index (BUI) of various amino acids do not appear to be altered (18). However, when 28- to 31-month-old rats are compared to intermediate age rats (7- to 11-month-old), the BU1 of methionine and possibly lysine is reduced whereas the BUI of glutamate is not different (2). However, the latter study did not rigorously control for the age-related changes in cerebral blood flow. Aging in rats is also associated with reduced BBB transport of thyroid hormone (triiodothyronine, T3) (21,22). The BUI of both levo and dextro enantiomers of T 3 is reduced in 25-month-old rats compared to 3-month-old rats (22). However, the volume of distribution of T 3 in the brain measured at steady-state conditions is not altered with age (22). Similar age-related changes in T 3 transport in hepatic tissue has been found (21). Finally, it appears that aging can also alter BBB transport of peptides such as Tyr-MIF-I(I). Both the Vm,,x and K,,, of the transport of this peptide were proportionally decreased in 24month-old rats compared to younger controls (1). The effect of aging on BBB transport of purine, nucleoside, and various vitamins or electrolytes remains unknown. The scarcity of aged animals and their cost impose significant limitations on the studies of BBB transport kinetics.
POTENTIAL MECHANISMSOF AGE-RELATEDCHANGES IN THE BBB For simplification purposes, the potential mechanisms of agerelated changes in the BBB can be separated into four general categories (Table 2). The first category of histopathologic changes in cerebral microcirculation is not discussed here. The other four categories of potential mechanisms are discussed separately although the multiplicity of underlying mechanisms and the possible overlap between some of those mechanisms should be acknowledged at the outset,
Age-Related Changes in Hemodynamic Variables In addition to the well documented structural changes in cerebral microvessels with age (6,18,51), hemodynamic changes in cerebral microcirculation may contribute to the age-related changes in the BBB. These potential hemodynamic alterations include leukocyte plugging, attachment of blood cells to endothelial walls of vessels, arteriovenous shunting, and changes in vascular reactivity altering regional blood flow. To evaluate these parameters the cerebral microvessels of Fischer 344 rats at different ages were studied using intravital fluorescence microscopy (27). Aging in this rat model was associated with significant arteriovenous shunting without alterations in blood flow characteristics, blood cell margination at the endothelial walls of the vessels and without changes in vascular permeability to 150 KDa FITC dextran (27). TABLE 2 POSSIBLE MECHANISMSUNDERLYINGTHE AGE-RELATEDCHANGES IN THE BLOOD-BRAINBARRIER Pathological changes in cerebral microcirculation (a) capillary dropout; (b) capillary atrophy Hemodynamic changes in the cerebral microcireulation: (a) arteriovenous shunting; (b) altered vascular reactivity Changes in biophysical properties of cerebral capillaries: (a) altered membrane fluidity; (b) altered surface change of endothetium Compositional changes: (a) changes in protein compositions; (b) changes in lipid composition; (c) accumulation of lipid peroxidation byproducts Alterations in neurotransmitter activity: (a) altered beta-adrenergic receptor number; (b) altered adenylate cyclase activity
BLOOD-BRAIN BARRIER AND AGING Aging is also associated with significant alterations in cerebral microvascular reactivity. The initial brief constriction of arterioles in response to BaC12 was practically eliminated in aged rats. The subsequent dilatory response however was similar in all agegroups (27). This is in agreement with a previous study of isolated basilar artery in vitro where age-related selective decreases in contractile responses were found without a change in dilatory responses (10). This notion is further supported by the observation that aging is associated with increased expression of neuropeptidergic vasodilatory cerebrovascular nerves whereas vasoconstrictor nerve density is either reduced or unaltered with age (15,41). These changes, therefore, would favor the maintenance of vasodilatory responses in aging rats with a loss of vasoconstrictor responsiveness. However, the age-related changes in cerebrovascular reactivity depend on the stimulus. Thus, the dilatation of cerebral arterioles in response to EDTA may be reduced in aged rats (9). This age-related reduction in distensibility was attributed to reduced proportion of distensible elements of the microvasculature such as the elastin and smooth muscle relative to nondistensible components of cerebral areteries such as collagen and basement membrane (9). In another study using intravital microscopy adenosine-induced vasodilatation was greater in young than in aged (24-month-old) rats (48). The reduced distensibility of cerebral vasculature in aging limits the dilatory response of these vessels in response to hypercapnia, hypoxia, or hypotension and may predispose aged animals to cerebral ischemia (9). In addition to the structural changes the reactivity of the microvessels also depends on receptor and second messenger systems related to sympathetic innervation. These changes are discussed in subsequent sections. The increased arteriovenous shunting in aged rats in itself would reduce the delivery of nutrients to the capillary bed and impair the metabolism of cerebral tissue. The kinetic parameters of various transport systems at the blood-brain barrier of aging rats should be interpreted cautiously because the changes in Vm~ (transport capacity) may well be secondary to arteriovenous shunting or capillary drop out. These changes however should not alter the K,,, of the transport system studied. The lack of extravasation of FITC dextran suggest that aging is not associated with significant changes in BBB leakiness. Similar conclusions have been previously drawn by others using different techniques (40,52). In a study utilizing et-aminoisobutyric acid, which is only minimally transported across the normal cerebral microvessels while it is actively transported in cerebral parenchymal cells, Saija et al. concluded that the BBB permeability of 28to 30-week-old rats is increased compared to younger rats (43). Moreover, older rats appeared to have increased vulnerability of the BBB to haloperidol-induced enhancement of permeability (44). It also appears that there is a maturational increase of susceptibility of the BBB permeability to electro convulsive shocks (37). However, recent studies with intravital fluoresent microscopy indicate that the susceptibility of the BBB to distuption during acute hypertension is not increased with aging (14). For complete assessment of BBB leakiness in aged rats, FITC dextran of various molecular weights, rather than a single FITC dextran should be studied in various regions of the brain since the age-related changes can be region specific (8).
Age-Related Changes in Biochemical Composition of Cerebral Microvessels A potential cause for the age-related changes in BBB transport is an alteration in biochemical composition of the cerebral microvessels. Aging is associated with a variety of modifications of gene products either because of altered gene expression, altered protein turnover, or as a result of post translational modifications
753 of proteins (35). In Fischer 344 rat the activity of two BBB enzymes, alkaline phosphatase, and gamma glutamyl transpeptidase, is not altered with age (29). The Na ÷- K ÷-ATPase activity of cerebral microvessels also is not altered with age although Ca ÷ ÷ Mg ÷ ÷- ATPase activity and Mg + + independent Ca ÷ ÷- ATPase activity may be reduced in 24-month-old rats compared to 3-month-old rats (49). We have recently found that the activity of vacuolar H÷-ATPase activity in cerebral microvessels(24) may also be reduced with age (25). The lack of a change in Na +K+-ATPase activity with age is consistent with the observation that aging is not associated with a change in ouabain binding sites of cerebral microvessels (11). The activity of glutathione peroxidase and glutathione reductase of cerebral microvessels does not change with age. However, the catalase activity may decrease and superoxide dismutase activity of cerebral microvessels is increased with age (50). Overall, a deficiency in enzymes protective against free radicals can not account for the observed age-related accumulation of lipid peroxidation byproducts in cerebral microvessels (32). The effect of various other enzymes known to be localized at the BBB is not studied. The age-related changes in adenylate cyclase activity is discussed in a subsequent section. More recently the age-related changes in a BBB specific antigen (47), the endothelial barrier antigen (EBA), was quantitated using immunohistologic techniques (26). The anti-EBA-stained microvessels were normalized against the total microvessels identified with either anti-EBA binding or anti Glut-1 (glucose transporter 1) binding. The anti-EBA stained microvessels of the hippocampus, but not of other cerebral areas, were reduced in aged rats (26). This is in agreement with the notion that hippocampus microvessels are more likely to be affected with aging changes (18). The changes in EBA do not appear to correlate with the BBB permeability in aged rats since the aging in rats is not asociated with increased permeability of the BBB in the hippocampal area (8,40,43). To gain further insight into the age-related changes in protein composition of cerebral microvessels, two-dimensional electrophoretic protein profile of cerebral microvessels isolated from rats at different ages was quantitatively evaluated (28). Of the 26 proteins in cerebral microvessels consistently identified on the gel, 10 showed significant age-related changes. A large acidic protein with a molecular weight of 144,000 and an isoelectric point of 5.4 was found exclusively in aged rats. Attempts at sequencing this protein was unsuccessful because it was N-terminally blocked and the amount of protein available was exceedingly small (28). The identity of the proteins altered in aged rat cerebral microvessels was not known and therefore the functional significance of the observation can not be precisely ascertained. Nevertheless an alteration in a relatively large proportion (38%) of the protein constituents of cerebral microvessels is likely to result in significant physiological changes in the BBB. The most widely recognized post translational modifications of proteins with age are glycosylation and protein conjugation with malondialdehyde (MDA), a byproduct of free radical-induced lipid peroxidation (35). The cerebral microvessel content of advanced glycosylation endproducts as well as pentosidine content are not affected with age (28). It is possible, however, individual glycoproteins may be increased or decreased with age whereas the average amount remains unchanged. Similarly, the amount of proteins conjugated with MDA as measured with a specific antiMDA-protein antibodies (13), were also not altered in the cerebral microvessels of aged rats (unpublished observations). This is in contrast to increased glycosylation endproducts and MDAmodified proteins found in cerebral microvessels of diabetic rats (30). However, lipid peroxidation byproducts, such as conjugated dienes, are increased in cerebral microvessels of both aged (32)
754
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and diabetic rats (33). This suggests that the interactions of proteins with lipid peroxidation byproducts in aging may be different from that found in diabetic rats. Some aspects of the lipid composition of cerebral microvessels of aging rats has been studied previously (32,50). Tayarani et al. (50) found that aging in Sprague-Dawley rats is associated with increased monounsaturated fatty acids and reduced polyunsaturated fatty acids in cerebral microvessels. The percent of fatty acids in isolated cerebral capillaries did not change with age (50). In Fischer 344 rats, we could not observe any significant agerelated change in fatty acid composition or in cholesterol and phospholipid content of cerebral microvessels. The differences between these two studies can not be readily explained. They may however be related to the differences in rat strains used. Of interest is that the concentration of various minerals in cerebral microvessels may also change with age (50). Thus, in Sprague-Dawley rats, aging is associated with increased content of copper and zinc in cerebral microvessels, whereas manganese content is not altered (50). Overall it appears that aging is associated with significant alterations in biochemical composition of cerebral microvessels. It is likely that these changes may contribute to the age-related alterations in BBB function, either through changes in specific transporter molecules or through changes in the biophysical properties of endothelial cell membranes thereby altering the mobility of various transporters.
Age-Related Changes in Biophysical Properties of Cerebral Microvessels Membrane microviscosity and surface charges of endothelial cells are important determinants of transport across the BBB (5,36). In one study, the increased permeability of cerebral endothelium was associated with a reduction in surface charge (36). In a study of age-related alterations in the permeability properties of the aortic endothelium it was found that positively charged cationized ferritin was more rapidly transported through the endothelium of aged rat aorta compared to young rats (5). Aged rat aorta was found to have a decreased density of cell-surface sialic acids. It remains to be shown whether similar changes occur in the surface charge of endothelial cell lining of the cerebral microvessels of aged animals. Alterations in membrane microviscosity with aging have been found in various tissues. The changes in membrane microviscosity are tissue specific. In some the microviscosity is increased wheres in other cell types microviscosity may be reduced with age (32). Recently, we have found that the steady state fluorescence polarization of diphenylhexatriene, a membrane core probe incorporated into isolated cerebral microvessel membranes at 35°C, was not different in aged rats compared to young rats (32). The thermotropic transition temperature of these membranes was not altered with age either (32). However, the fluorescence polarization of 1-(4-tri-melthylammoniumphenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH), a probe for the surface region of the membrane, was significantly reduced in aged rats (34). Thus, aging in rats is
associated with a significant decrease in lipid order near the membrane surface of cerebral microvessels. Future studies should attempt to correlate the age-related changes in BBB permeability with changes in biophysical properties of cerebral endothelial cell membranes.
Age-Related Changes in Neurotransmitter Activi~ of Cerebral Microvessels Cerebral microvessels are rich in receptors for various neurotransmitters (38). With the exception of beta adrenergic receptors ( 12,31), the age-related changes in neurotransmitter receptor density of cerebral microvessels has not been adequately addressed. One study using a single preparation of cerebral microvessels found that beta-adrenergic receptor binding sites (B,,,~) in aged rats was 21% lower than that of mature rats (12). This observation was confirmed in a subsequent study (31). In addition, the basal adenylate cyclase activity (AC) as well as isoproterenol or sodium fluoride-stimulated AC activity in cerebral microvessels were significantly reduced with age (31). This is in contrast to the changes in AC activity in cerebral tissue. In this tissue AC is not altered with age although beta adrenergic receptor number is reduced (46). Thus, the age-related changes in betaadrenergic neurotransmission of cerebral microvessels are not identical to those seen in cerebral tissue. A direct quantitation of norepinephrine content of isolated microvessels of rat brain has also demonstrated an age-related reduction (7). These findings taken together suggest that adrenergic neurotransmission in cerebral microvessels is significantly reduced with age. The extent of the contribution of these changes to the alterations in BBB function remains to be demonstrated. CONCLUDING REMARKS
The functional integrity of the BBB is of utmost importance in maintaining the optimal milieu for the CNS. It is well established that some aspects of the BBB function are compromised with age. The underlying mechanisms for these age-related changes are multifactorial. The relative importance of each factor can not be estimated at the present time. The degree of the contribution of the changes in BBB to the age-related degenerative diseases of the CNS is not known. However, considering the central importance of the BBB in various aspects of CNS metabolism,it is highly likely that the changes in BBB found with aging contribute to the deterioration of the CNS function commonly found in aged animals. Future research should focus on identifying additional specific markers of the BBB and evaluate their relevance to the BBB function. ACKNOWLEDGEMENT
This review is dedicated to the memory of Dr. William Oldendorf for his pioneering work in blood-brain barrier research and for his advice to me. This work was supported by the Medical Research of the Department of Veterans Affairs.
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