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Brain capillary endothelial cells (BECs) form the ultra-tight blood-brain barrier (BBB). The permeability of the BBB increases with increasing age and neurovascular and neurodegenerative diseases. Major defects of the BBB can be initiated by increased permeability to plasma proteins in small arteriosclerotic arteries and release of proteins from degenerating neurons into the brain extracellular space. These proteins deposit in perivascular spaces, and subsequently negatively influence the BECs leading to decreased expression of barrier proteins. Detection of BBB defects by the use of non-invasive techniques is relevant for clinical use in settings with advanced age and severe brain disorders.

Increased oxidative stress in the brain has consistently been implied in ageing and in several degenerative brain disorders. Acting as a pivotal antioxidant in the brain, vitamin C is preferentially retained during deficiency and may play an essential role in neuroprotection during ageing. Thus, a lack of vitamin C could be associated with an increase in redox imbalance in the ageing brain. The present study compared oxidative stress of ageing to that of a long-term non-scorbutic vitamin C deficiency in guinea pigs. Adults (3-9 months old) were compared to old (36-42 months old) animals during a 6-month dietary intervention by assessing vitamin C transport and redox homoeostasis in the brain. In contrast to our hypothesis, chronic vitamin C deficiency did not affect the measured markers of oxidative stress in the brains of adult and aged animals. However, aged animals generally showed increased lipid oxidation (p < 0.001), decreased glutathione (p < 0.05), increased p53 mRNA expression (p < 0.01) and somewhat elevated DNA oxidation (p = 0.08) compared to adult counterparts irrespective of dietary vitamin C intake. Increased mRNA expression of sod1 (p < 0.05) and svct2 (p = 0.05) was observed in aged animals together with increased superoxide dismutase activity (p < 0.01) and cerebrospinal fluid vitamin C status (p < 0.001) suggesting a compensatory effort that did not counterbalance the effects of ageing. Essentially, no effects of age were observed in the liver demonstrating the brain's unique susceptibility to redox imbalance. Consistent with previous findings, we show that ageing per se constitutes a considerable oxidative insult in the brain. However, our data also suggest that a long-term poor vitamin C status does not accelerate this process., (© 2011 The Authors Basic & Clinical Pharmacology & Toxicology © 2011 Nordic Pharmacological Society.)

Several reports have shown that Vitamin C is depleted in animals with age. Based mainly on comparisons between young animals that have not yet reached maturity and old animals, it appears to be the general assumption that the change in Vitamin C status occurs at a late stage in life and that this phenomenon may either contribute to or result from the ageing process. In the present study, young (3 months old, n = 8) and old (36 months old, n = 8) female guinea pigs were followed for 6 months with monthly blood samplings and monitored for Vitamin C status as measured by plasma ascorbate and erythrocyte ascorbate recycling capacity after which the animals were euthanized. While remaining unchanged in the old animals, plasma Vitamin C status of the young animals significantly declined to that of the old animals within 3 months. During the following 3 months, the Vitamin C status of the young animals remained unchanged. Furthermore, post mortem Vitamin C analyses of the animals now aged 9 and 42 months, respectively, showed no effect of age on Vitamin C in plasma, liver, kidney, heart and brain between the groups while concentrations were significantly increased in cerebrospinal fluid and lung with age (p < 0.05). Moreover, a significantly elevated ascorbic acid oxidation ratio was observed in young compared to old animals (p < 0.05). The present data suggest that the decline in Vitamin C status with age occur early in life and is a phenomenon of maturation rather than of ageing. Data from other species and humans are discussed.

As transferrin in the brain may originate principally from synthesis by three different cell types, i.e. hepatocytes, oligodendrocytes and choroid plexus, this study employed a morphological analysis to specifically address oligodendrocytic expression of transferrin mRNA in young (P17) and adult (P50) rats. In spite of a lowering of the concentration of brain iron by approximately 22% in the young iron deficient rats transferrin mRNA expression in oligodendrocytes was not affected when measured by quantitative densitometry. In adult rats, the baseline transferrin mRNA expression in oligodendrocytes was higher than in the young animals, but did not change in spite of a reduction in brain iron by approximately 19%. Brain iron and transferrin mRNA expression in oligodendrocytes were unaltered in iron overloaded rats when compared to age-matched controls. As transferrin expression was lower in the young rat, when constituents from the blood have a relatively higher concentration in the brain than during adulthood, it seems unlikely that blood-borne factors such as transition metals act as inducers of transferrin gene expression in oligodendrocytes. Instead, the higher but constitutive expression of transferrin mRNA at later ages, when the blood-brain barrier segregates the brain from other body parts, may indicate that molecules released from the brain interior are responsible for regulating transcription of the transferrin gene.

Because zinc (Zn) is a co-factor in enzymes and participates in neurotransmission, it is essential for brain development. However, because excess Zn may cause neuronal injury, cerebral mechanisms for Zn regulation must operate. The metallothionein isoforms I and II (MT I + II) are putative candidates for chelating unbound Zn released from Zn-containing nerve terminals or transported into the brain. Whether vesicular Zn and MT I + II occur in identical regions of the developing brain is unknown. Accordingly, the developmental distribution of MT I + II and vesicular Zn was mapped. By using double-labeling fluorescence histochemistry, MT I + II immunoreactivity (ir) was attributed to astrocytes and cells of myelomonocytic lineage. The cells of the myelomonocytic lineage shared the morphology of monocytes and macrophages but not of microglia and occurred primarily around vessels and ventricles in the brainstem. By contrast, astrocytes were widespread throughout the developing brain. In embryonic and neonatal brain, MT I + IIir astrocytes were almost selectively observed in the septum and fascia dentate hilus (hi) of the hippocampus. With increasing postnatal age, they also occurred in hippocampal cortex, basal forebrain, neocortex, cerebellar cortex, and cranial nerve nuclei. MT I + II mRNAs were detected in regions of the brain that also displayed MT I + IIir, indicating transcriptional events. Vesicular Zn was recorded in neonatal brain solely in the dentate hi of the hippocampus. With increasing age, the amount of vesicular Zn increased in the hippocampus and other forebrain regions. The presence of MT I + II proteins in the developing brain was confirmed by radioimmunoassay. The regional distribution of astrocytic MT I + IIir and vesicular Zn suggests that MT I + II are implicated in Zn metabolism in the developing forebrain., (Copyright 1999 Wiley-Liss, Inc.)

In order to characterize the mechanism by which Iron (Fe) is taken up by neurons, we examined the neuronal expression of transferrin receptor (TR) in rats during development and iron (Fe) deficiency by using immunohistochemistry, in vitro receptor autoradiography and in situ hybridization. In contrast to the continuous expression of TR in brain capillary endothelial cells regardless of the age of the animals studied, the expression of neuronal TR was almost absent at late embryonic and early postnatal ages but increased with increasing age to reach a plateau from postnatal (P) 21 through adulthood as verified by immunohistochemical staining. Reducing the Fe stores potentiated the expression of TR immunoreactivity in neurons of both young and adult rats in several grey matter regions. Increased TR immunoreactivity was also observed in neuronal extensions of neurons of the medial habenular nucleus, reticular neurons of the brainstem, and fibers projecting to the area postrema. TR immunoreactivity was never observed in white matter regions, except for that recorded in brain capillaries. In vitro receptor autoradiography verified the increased capacity for transferrin binding during Fe deficiency. By contrast, TR mRNA expression was not affected by Fe deficiency. These findings demonstrate that the expression of the neuronal TR protein is age dependent and susceptible to Fe deficiency.

The entry of iron from blood into the developing rat brain was studied by means of non-heme iron-histochemistry. The content of non-heme iron in the endothelial cells was manifest already from E14, declined from P3 to P5, and was almost absent on P10-P15. The choroid plexus epithelial cells of either ventricle was non-heme iron-containing from E14. Non-heme iron-containing macrophages situated in the stroma of the choroid plexus were also observed from E14. From E19, the macrophage-like cells tended to invade into (a) regions with transitory structures like the intermediate zone of the cerebral hemisphere, (b) developing axonal tracts like corpus callosum and internal capsule, and (c) deep layers of the tectum, a region with an extensive degree of naturally occurring cell death. The amoeboid macrophage-like cells observed in the brain parenchyma gradually acquired prolonged extensions and apparently differentiated into ramified microglia-like cells, which later lost their non-heme iron-content. Thus, at P70, non-heme iron-positive microglia-like cells were hardly seen reflecting the transitory event of non-heme iron in microglia-like cells. At P200, non-heme iron-containing microglia cells and oligodendrocytes appeared in manifestly higher number than at P70, a phenomenon probably related to aging. These results delineate for the first time the appearance of iron in the developing brain. The results are of relevance for understanding the potential of iron-deficiency for harming the developing central nervous system, generally by decreased transport of iron through brain capillaries and choroid plexus, and specifically by an impaired modulation of the developing brain parenchyma by iron-containing macrophages.

This study presents evidence for retrograde axonal transport of exogenous albumin and transferrin in adult brainstem motor neurons, whereas plasma proteins are not transported in neonatal motor neurons. The plasma protein uptake in motor neurons was dose-dependent, suggesting a nonspecific (fluid-phase) uptake mechanism. Further evidence for nonspecific uptake of exogenous transferrin in the motor neuron was found in the presence of transferrin receptor only on the soma and not on the axon terminal. The immunoreaction product of the exogenous plasma proteins was localized as perinuclear granules in association with the lysosomal system, as verified by staining for the lysosomal marker cathepsin D and by ultrastructural examinations. The results suggest that albumin and transferrin derived from hepatic synthesis gain access to motor neurons nonspecifically by retrograde axonal transport, whereas transferrin derived from intracerebral synthesis specifically gains access to motor neurons due to receptor-mediated uptake at the soma of the neuron. The lack of plasma proteins in developing motor neurons suggests that retrograde axonal transport of plasma proteins has no significance for developing axons. Plasma proteins have a potential for transporting toxic metals to motor neurons. Intraneuronal uptake of aluminum-transferrin either by nonspecific uptake in axon terminals or by receptor-mediated uptake at the soma may have a role in the pathogenesis of the motor neuron disease amyotrophic lateral sclerosis.

The often quoted investigation by Behnsen [4] provides some evidence for an increased permeability of the neonatal mouse blood-brain barrier (BBB) to trypan blue compared to the adult. Trypan blue, which circulates in plasma mainly bound to albumin, is commonly used as a macromolecular tracer, although Behnsen probably injected dyes in amounts exceeding the dye-binding capacity of plasma proteins. The cerebrovascular permeability in neonatal and adult mice and rats was investigated using the visual tracers trypan blue and Evans blue and immunocytochemical staining for endogenous plasma proteins. By administering different concentrations of the dyes, the permeability of the BBB was assessed. The presence of the dyes in plasma as either dye-protein complexes or as free dye was measured by a plasma protein binding assay. When dyes occurred in plasma as macromolecular dye-protein complexes, dyes and plasma proteins were restricted to CNS regions normally devoid of a BBB in both neonates and adults. When unbound (free) dyes occurred in plasma, dyes were observed intraneuronally in regions without projections beyond the BBB in both neonates and adults corresponding to that observed by Behnsen; intraneuronal accumulation of plasma proteins also occurred in regions with projections confined to the BBB but only in neonates. It is concluded that the BBB to macromolecular tracers is fully developed at birth in mouse and rats. However, when free dye is present in plasma, there is a differential permeability to plasma proteins between neonates and adults.