C J Ek

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.

Functional effectiveness of the blood-brain barrier to small water-soluble molecules in developing and adult opossum (Monodelphis domestica)

We have evaluated a small water‐soluble molecule, biotin ethylenediamine (BED, 286 Da), as a permeability tracer across the blood‐brain barrier. This molecule was found to have suitable characteristics in that it is stable in plasma, has low plasma protein binding, and appears to behave in a similar manner across brain barriers as established by permeability markers such as sucrose. BED, together with a 3000‐Da biotin‐dextran (BDA3000), was used to investigate the effectiveness of tight junctions in cortical vessels during development and adulthood of a marsupial opossum (Monodelphis domestica). Marsupial species are born at an early stage of brain development when cortical vessels are just beginning to appear. The tracers were administered systemically to opossums at various ages and localized in brains with light and electron microscopy. In adults, the tight junctions restricted the movement of both tracers. In neonates, as soon as vessels grow into the neocortex, their tight junctions are functionally restrictive, a finding supported by the presence of claudin‐5 in endothelial cells. However, both tracers are also found within brain extracellular space soon after intraperitoneal administration. The main route of entry for the tracers into immature neocortex appears to be via the cerebrospinal fluid over the outer (subarachnoid) and inner (ventricular) surfaces of the brain. These experiments demonstrate that the previously described higher permeability of barriers to small molecules in the developing brain does not seem to be due to leakiness of cerebral endothelial tight junctions, but to a route of entry probably via the choroid plexuses and cerebrospinal fluid. J. Comp. Neurol. 496:13–26, 2006.

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.

Structural characteristics and barrier properties of the choroid plexuses in developing brain of the opossum (Monodelphis Domestica).

The structural and functional development of the choroid plexuses, the site of the blood‐cerebrospinal fluid (CSF) barrier, in an opossum (Monodelphis domestica) was studied. Marsupial species are extremely immature at birth compared with more conventional eutherian species. Choroid plexus tissue of each brain ventricle, from early stages of development, was collected for light and electron microscopy. During development, the choroidal epithelium changes from a pseudostratified to a cuboidal layer. Individual epithelial cells appear to go through a similar maturation process even though the timing is different between and within each plexus. The ultrastructural changes during development in the choroidal epithelial cells consist of an increase in the number of mitochondria and microvilli, and changes in structure of endoplasmic reticulum. There are also changes in the core of plexuses with age. In contrast, the structure of the tight junctions between epithelial cells does not appear to change with maturation. In addition, the route of penetration for lipid insoluble molecules from blood to CSF across the choroid plexuses was examined using a small biotin‐dextran. This showed that the tight junctions already form a functional barrier in early development by preventing the paracellular movement of the tracer. Intracellular staining shows that there may be a transcellular route for these molecules through the epithelial cells from blood to CSF. Apart from lacking a glycogen‐rich stage, cellular changes in the developing opossum plexus seem to be similar to those in other species, demonstrating that this is a good model for studies of mammalian choroid plexus development. J. Comp. Neurol. 460:451–464, 2003.

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 ∼ 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.

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.

Comparative Aspects of Subplate Zone Studied with Gene Expression in Sauropsids and Mammals

There is currently a debate about the evolutionary origin of the earliest generated cortical preplate neurons and their derivatives (subplate and marginal zone). We examined the subplate with murine markers including nuclear receptor related 1 (Nurr1), monooxygenase Dbh-like 1 (Moxd1), transmembrane protein 163 (Tmem163), and connective tissue growth factor (Ctgf) in developing and adult turtle, chick, opossum, mouse, and rat. Whereas some of these are expressed in dorsal pallium in all species studied (Nurr1, Ctgf, and Tmem163), we observed that the closely related mouse and rat differed in the expression patterns of several others (Dopa decarboxylase, Moxd1, and thyrotropin-releasing hormone). The expression of Ctgf, Moxd1, and Nurr1 in the oppossum suggests a more dispersed subplate population in this marsupial compared with mice and rats. In embryonic and adult chick brains, our selected subplate markers are primarily expressed in the hyperpallium and in the turtle in the main cell dense layer of the dorsal cortex. These observations suggest that some neurons that express these selected markers were present in the common ancestor of sauropsids and mammals.

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.

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.

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 14C‐sucrose. By P44, blood–brain barrier integrity was intact but a reduced volume of white matter was present. At P44 after prolonged inflammation (5 × 0.2 mg/kg LPS at 48 h intervals), proteins from blood were observed within brain white matter and permeability to 14C‐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.