Effects of neonatal systemic inflammation on blood-brain barrier permeability and behaviour in juvenile and adult rats.

Several neurological disorders have been linked to inflammatory insults suffered during development. We investigated the effects of neonatal systemic inflammation, induced by LPS injections, on blood-brain barrier permeability, endothelial tight junctions and behaviour of juvenile (P20) and adult rats. LPS-treatment resulted in altered cellular localisation of claudin-5 and changes in ultrastructural morphology of a few cerebral blood vessels. Barrier permeability to sucrose was significantly increased in LPS treated animals when adult but not at P20 or earlier. Behavioural tests showed that LPS treated animals at P20 exhibited altered behaviour using prepulse inhibition (PPI) analysis, whereas adults demonstrated altered behaviour in the dark/light test. These data indicate that an inflammatory insult during brain development can change blood-brain barrier permeability and behaviour in later life. It also suggests that the impact of inflammation can occur in several phases (short- and long-term) and that each phase might lead to different behavioural modifications.

Effect of minocycline on inflammation-induced damage to the blood-brain barrier and white matter during development.

Damage to white matter in some premature infants exposed to intrauterine infections is thought to involve disruption of the blood-brain barrier. We have examined the effect of minocycline, an agent reported to reduce brain damage resulting from inflammation, on inflammation-induced disruption of the blood-brain barrier and damage to white matter. Post-natal marsupial opossums (Monodelphis domestica) were studied as most brain development in this species occurs after birth. Single intraperitoneal lipopolysaccharide (LPS) injection (0.2 mg/kg) with or without minocycline (45 mg/kg) at post-natal day (P)35 caused short-lasting barrier breakdown to plasma proteins but not to (14)C-sucrose. By P44, blood-brain barrier integrity was intact but a reduced volume of white matter was present. At P44 after prolonged inflammation (5 x 0.2 mg/kg LPS at 48 h intervals), proteins from blood were observed within brain white matter and permeability to (14)C-sucrose in the hindbrain increased by 31%. The volume of the external capsule and the proportion of myelin were 70 and 57%, respectively, of those in control animals. Minocycline administered during prolonged inflammation restored blood-brain barrier integrity but not LPS-induced damage to white matter. These data suggest that long-term changes in blood-brain barrier permeability occur only after a prolonged period of inflammation during development; however, damage to white matter can result from even a short-lasting breakdown of the barrier. Manipulation of the inflammatory response may have implications for prevention of some developmentally induced neurological conditions.

Age-related differences in the local cellular and molecular responses to injury in developing spinal cord of the opossum, Monodelphis domestica.

Immature spinal cord, unlike adult, has an ability to repair itself following injury. Evidence for regeneration, structural repair and development of substantially normal locomotor behaviour comes from studies of marsupials due to their immaturity at birth. We have compared morphological, cellular and molecular changes in spinal cords transected at postnatal day (P)7 or P14, from 3 h to 2 weeks post-injury, in South American opossums (Monodelphis domestica). A bridge between severed ends of cords was apparent 5 days post-injury in P7 cords, compared to 2 weeks in P14. The volume of neurofilament (axonal) material in the bridge 2 weeks after injury was 30% of control in P7- but < 10% in P14-injured cords. Granulocytes accumulated at the site of injury earlier (3 h) in P7 than in P14 (24 h)-injured animals. Monocytes accumulated 24 h post-injury and accumulation was greater in P14 cords. Accumulation of GFAP-positive astrocytes at the lesion occurred earlier in P14-injured cords. Neurites and growth cones were identified ultrastructurally in contact with astrocytes forming the bridge. Results using mouse inflammatory gene arrays showed differences in levels of expression of many TGF, TNF, cytokine, chemokine and interleukin gene families. Most of the genes identified were up-regulated to a greater extent following injury at P7. Some changes were validated and quantified by RT-PCR. Overall, the results suggest that at least some of the greater ability to recover from spinal cord transection at P7 compared to P14 in opossums is due to differences in inflammatory cellular and molecular responses.

Changes in blood-brain barrier permeability to large and small molecules following traumatic brain injury in mice.

The entry of therapeutic compounds into the brain and spinal cord is normally restricted by barrier mechanisms in cerebral blood vessels (blood-brain barrier) and choroid plexuses (blood-CSF barrier). In the injured brain, ruptured cerebral blood vessels circumvent these barrier mechanisms by allowing blood contents to escape directly into the brain parenchyma. This process may contribute to the secondary damage that follows the initial primary injury. However, this localized compromise of barrier function in the injured brain may also provide a 'window of opportunity' through which drugs that do not normally cross the blood-brain barriers are able to do so. This paper describes a systematic study of barrier permeability in a mouse model of traumatic brain injury using both small and large inert molecules that can be visualized or quantified. The results show that soon after trauma, both large and small molecules are able to enter the brain in and around the injury site. Barrier restriction to large (protein-sized) molecules is restored by 4-5 h after injury. In contrast, smaller molecules (286-10,000 Da) are still able to enter the brain as long as 4 days postinjury. Thus the period of potential secondary damage from barrier disruption and the period during which therapeutic compounds have direct access to the injured brain may be longer than previously thought.

Blood-CSF barrier function in the rat embryo.

Blood-cerebrospinal fluid (CSF) barrier function and expansion of the ventricular system were investigated in embryonic rats (E12-18). Permeability markers (sucrose and inulin) were injected intraperitoneally and concentrations measured in plasma and CSF at two sites (lateral and 4th ventricles) after 1 h. Total protein concentrations were also measured. CSF/plasma concentration ratios for endogenous protein were stable at approximately 20% at E14-18 and subsequently declined. In contrast, ratios for sucrose (100%) and inulin (40%) were highest at the earliest ages studied (E13-14) and then decreased substantially. Between E13 and E16 the volume of the lateral ventricles increased over three-fold. Decreasing CSF/plasma concentration ratios for small, passively diffusing molecules during embryonic development may not reflect changes in permeability. Instead, increasing volume of distribution appears to be important in this decline. The intracellular presence of a small marker (3000 Da biotin-dextranamine) in plexus epithelial cells following intraperitoneal injection indicates a transcellular route of transfer. Ultrastructural evidence confirmed that choroid plexus tight junctions are impermeable to small molecules at least as early as E15, indicating the blood-CSF barrier is morphologically and functionally mature early in embryonic development. Comparison of two albumins (human and bovine) showed that transfer of human albumin (surrogate for endogenous protein) was 4-5 times greater than bovine, indicating selective blood-to-CSF transfer. The number of plexus epithelial cells immunopositive for endogenous plasma protein increased in parallel with increases in total protein content of the expanding ventricular system. Results suggest that different transcellular mechanisms for protein and small molecule transfer are operating across the embryonic blood-CSF interface.

Long-term changes in blood-brain barrier permeability and white matter following prolonged systemic inflammation in early development in the rat.

Epidemiological evidence in human fetuses links inflammation during development with white matter damage. Breakdown of the blood-brain barrier has been proposed as a possible mechanism. This was investigated in the present study by inducing a prolonged inflammatory response in newborn rats, with intraperitoneal injections of lipopolysaccharide (LPS; 0.2 mg/kg) given at postnatal (P) day 0, P2, P4, P6 and P8. An acute phase response was present over the whole period of injections. Changes in blood-brain barrier permeability were determined for small (sucrose and inulin) and large (protein) molecules. During and immediately after the inflammatory response, plasma proteins were detected in the brain only within white matter tracts, indicating an increased permeability of the blood-brain barrier to protein during this period. The alteration in permeability to protein was transient. In contrast, the permeability of the blood-brain barrier to 14C-sucrose and 14C-inulin was significantly higher in adult animals that had received serial LPS injections during development. Adult animals receiving a single 1 mg/kg LPS injection at P0 showed no alteration in blood-brain barrier permeability to either small or larger molecules. A significant decrease in the volume of CNPase immunoreactive presumptive white matter tracts occurred in the external capsule and corpus callosum at P9. These results demonstrate that a prolonged systemic inflammatory response in the early postnatal period in rats causes size selective increases in blood-brain barrier permeability at different stages of brain development and results in changes in white matter volume.

Aquaporin-1 in the choroid plexuses of developing mammalian brain.

The normal brain develops within a well-controlled stable internal "milieu" protected by specialised mechanisms referred to collectively as blood-brain barriers. A fundamental feature of this environment is the control of water flow in and out of the developing brain. Because of limited vascularisation of the immature brain, choroid plexuses, via the cerebrospinal fluid, have been proposed as the main route of fluid exchange between the blood and brain interfaces. We describe the temporal expression and appearance of aquaporin-1 (AQP1) which is important for water transfer across adult choroid plexuses. AQP1 expression was studied in rat embryos using real time reverse transcription/polymerase chain reaction. mRNA for AQP1 was present in rat brain at embryonic day 12 (E12) one day before the protein was detectable in the fourth ventricular choroid plexus (the first plexus to appear); its relative levels increased at E13-E14 when more AQP1-immunoreactive cells appeared in all plexuses. The presence of AQP1 was determined immunocytochemically in five different mammalian species (rat, mouse, human, sheep and opossum) in all four choroid plexuses from their earliest appearance. In all five species studied, the appearance of AQP1 immunoreactivity followed the same developmental sequence: the fourth, lateral and, finally, third ventricular choroid plexus. The stage of choroid plexus development when AQP1 was first detected in all five species and in all four choroid plexuses corresponded to the transition between Stages I and II. The cellular localisation of AQP1 in all choroid plexuses, as soon as it was detectable, had the characteristic apical membrane distribution previously described in the adult; a basolateral membrane localisation was also observed.

Breakdown of the blood-brain barrier to proteins in white matter of the developing brain following systemic inflammation.

Compromised blood-brain barrier permeability resulting from systemic inflammation has been implicated as a possible cause of brain damage in fetuses and newborns and may underlie white matter damage later in life. Rats at postnatal day (P) 0, P8 and P20 and opossums (Monodelphis domestica) at P15, P20, P35, P50 and P60 and adults of both species were injected intraperitoneally with 0.2-10 mg/kg body weight of 055:B5 lipopolysaccharide. An acute-phase response occurred in all animals. A change in the permeability of the blood-brain barrier to plasma proteins during a restricted period of postnatal development in both species was determined immunocytochemically by the presence of proteins surrounding cerebral blood vessels and in brain parenchyma. Blood vessels in white matter, but not grey matter, became transiently permeable to proteins between 10 and 24 h after lipopolysaccharide injection in P0 and P8 rats and P35-P60 opossums. Brains of Monodelphis younger than P35, rats older than P20 and adults of both species were not affected. Permeability of the blood-cerebrospinal fluid (CSF) barrier to proteins was not affected by systemic inflammation for at least 48 h after intraperitoneal injection of lipopolysaccharide. These results show that there is a restricted period in brain development when the blood-brain barrier, but not the blood-CSF barrier, to proteins is susceptible to systemic inflammation; this does not appear to be attributable to barrier "immaturity" but to its stage of development and only occurs in white matter.

Permeability and route of entry for lipid-insoluble molecules across brain barriers in developing Monodelphis domestica.

1. We have studied the permeability of blood-brain barriers to small molecules such as [(14)C]sucrose, [(3)H]inulin, [(14)C]L-glucose and [(3)H]glycerol from early stages of development (postnatal day 6, P6) in South American opossums (Monodelphis domestica), using a litter-based method for estimating steady-state cerebrospinal fluid (CSF)/plasma and brain/plasma ratios of markers that were injected I.P. 2. Steady-state ratios for L-glucose, sucrose and inulin all showed progressive decreases during development. The rate of uptake of L-glucose into the brain and CSF, in short time course experiments (7-24 min) when age-related differences in CSF production can be considered negligible also decreased during development. These results indicate that there is a significant decrease in the permeability of brain barriers to small lipid-insoluble molecules during brain development. 3. The steady-state blood/CSF ratio for 3000 Da lysine-fixable biotin-dextran following I.P. injection was shown to be consistent with diffusion from blood to CSF. It was therefore used to visualise the route of penetration for small lipid-insoluble molecules across brain barriers at P0-30. The proportion of biotin-dextran-positive cells in the choroid plexuses declined in parallel with the age-related decline in permeability to the small-molecular-weight markers; the paracellular (tight junction) pathway for biotin-dextran appeared to be blocked, but biotin-dextran was easily detectable in the CSF. A transcellular route from blood to CSF was suggested by the finding that some choroid plexus epithelial cells contained biotin-dextran. 4. Biotin-dextran was also taken up by cerebral endothelial cells in the youngest brains studied (P0), but in contrast to the CSF, could not be detected in the brain extracellular space (i.e. a significant blood-brain barrier to small-sized lipid-insoluble compounds was already present). However, in immature brains (P0-13) biotin-dextran was taken up by some cells in the brain. These cells generally had contact with the CSF, suggesting that it is likely to have been the source of their biotin-dextran. Since the quantitative permeability data suggest that biotin-dextran behaves similarly to the radiolabelled markers used in this study, it is suggested that these markers in the more immature brains were also present intracellularly. Thus, brain/plasma ratios may be a misleading indicator of blood-brain barrier permeability in very immature animals. 5. The immunocytochemical staining for biotin-dextran in the CSF, in contrast to the lack of staining in the brain extracellular space, together with the quantitative permeability data showing that the radiolabelled markers penetrated more rapidly and to a much higher steady-state level in CSF than in the brain, suggests that lipid-insoluble molecules such as sucrose and inulin reach the immature brain predominantly via the CSF rather than directly across the very few blood vessels that are present at that time.