C. Joakim Ek

Barriers in the brain: a renaissance?

Barrier mechanisms regulate the exchange of molecules between the brains internal milieu and the rest of the body. Correct functioning of these mechanisms is critical for normal brain activity, maintenance and development. Dysfunctional brain barrier mechanisms contribute to the pathology of neurological conditions, ranging from trauma to neurodegenerative diseases, and provide obstacles for successful delivery of potentially beneficial pharmaceutical agents. Previous decades of research have yielded insufficient understanding for solving brain barrier problems in vivo. However, an awakening of interest and novel approaches are providing insight into these mechanisms in developing and dysfunctional brain, as well as suggesting new approaches to circumventing brain barrier mechanisms to get therapeutic agents into the central nervous system.

Efflux mechanisms at the developing brain barriers: ABC-transporters in the fetal and postnatal rat

Proteins of the ATP-binding cassette (ABC) family, present at the blood-brain barrier interfaces, have been shown to reduce the entry of compounds from blood into the brain by active efflux. Their substrates are diverse including many drugs and toxins and therefore provide an important mechanism for brain neuroprotection. However, knowledge of their presence and function in the developing brain is very limited. We have used qPCR and immunocytochemistry to determine gene expression and localisation of four main barrier ABC-transporters (pgp/ABCB1, MRP1/ABCC1, MRP4/ABCC4 and BCRP/ABCG2) in the fetal and neonatal rat brain cerebral blood vessels (site of blood-brain barrier) and choroid plexus (site of blood-CSF barrier). The study shows that ABC-transporters localise to the brain barriers even at early fetal stages and although pgp expression was lower in the fetus, the other transporters were expressed at comparable levels in fetal and adult brains suggesting direct neuroprotection of the brain in addition to that provided by the placenta. BCRP was expressed at higher levels in developing choroid plexus and was only detected at fetal stages on the blood-facing side of epithelial cells indicating a particular role of this transporter for early brain efflux mechanisms.

Spatio-Temporal Progression of Grey and White Matter Damage Following Contusion Injury in Rat Spinal Cord

Cellular mechanisms of secondary damage progression following spinal cord injury remain unclear. We have studied the extent of tissue damage from 15 min to 10 weeks after injury using morphological and biochemical estimates of lesion volume and surviving grey and white matter. This has been achieved by semi-quantitative immunocytochemical methods for a range of cellular markers, quantitative counts of white matter axonal profiles in semi-thin sections and semi-quantitative Western blot analysis, together with behavioural tests (BBB scores, ledged beam, random rung horizontal ladder and DigiGait™ analysis). We have developed a new computer-controlled electronic impactor based on a linear motor that allows specification of the precise nature, extent and timing of the impact. Initial (15 min) lesion volumes showed very low variance (1.92±0.23 mm3, mean±SD, n = 5). Although substantial tissue clearance continued for weeks after injury, loss of grey matter was rapid and complete by 24 hours, whereas loss of white matter extended up to one week. No change was found between one and 10 weeks after injury for almost all morphological and biochemical estimates of lesion size or behavioural methods. These results suggest that previously reported apparent ongoing injury progression is likely to be due, to a large extent, to clearance of tissue damaged by the primary impact rather than continuing cell death. The low variance of the impactor and the comprehensive assessment methods described in this paper provide an improved basis on which the effects of potential treatment regimes for spinal cord injury can be assessed.

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 [14C]sucrose, [3H]inulin, [14C]l‐glucose and [3H]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 P 0–30. 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 2source 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.

Age-Dependent Changes in the Proteome Following Complete Spinal Cord Transection in a Postnatal South American Opossum (Monodelphis domestica)

Recovery from severe spinal injury in adults is limited, compared to immature animals who demonstrate some capacity for repair. Using laboratory opossums (Monodelphis domestica), the aim was to compare proteomic responses to injury at two ages: one when there is axonal growth across the lesion and substantial behavioural recovery and one when no axonal growth occurs. Anaesthetized pups at postnatal day (P) 7 or P28 were subjected to complete transection of the spinal cord at thoracic level T10. Cords were collected 1 or 7 days after injury and from age-matched controls. Proteins were separated based on isoelectric point and subunit molecular weight; those whose expression levels changed following injury were identified by densitometry and analysed by mass spectrometry. Fifty-six unique proteins were identified as differentially regulated in response to spinal transection at both ages combined. More than 50% were cytoplasmic and 70% belonged to families of proteins with characteristic binding properties. Proteins were assigned to groups by biological function including regulation (40%), metabolism (26%), inflammation (19%) and structure (15%). More changes were detected at one than seven days after injury at both ages. Seven identified proteins: 14-3-3 epsilon, 14-3-3 gamma, cofilin, alpha enolase, heart fatty acid binding protein (FABP3), brain fatty acid binding protein (FABP7) and ubiquitin demonstrated age-related differential expression and were analysed by qRT-PCR. Changes in mRNA levels for FABP3 at P7+1day and ubiquitin at P28+1day were statistically significant. Immunocytochemical staining showed differences in ubiquitin localization in younger compared to older cords and an increase in oligodendrocyte and neuroglia immunostaining following injury at P28. Western blot analysis supported proteomic results for ubiquitin and 14-3-3 proteins. Data obtained at the two ages demonstrated changes in response to injury, compared to controls, that were different for different functional protein classes. Some may provide targets for novel drug or gene therapies.

Mechanisms That Determine the Internal Environment of the Developing Brain: A Transcriptomic, Functional and Ultrastructural Approach

We provide comprehensive identification of embryonic (E15) and adult rat lateral ventricular choroid plexus transcriptome, with focus on junction-associated proteins, ionic influx transporters and channels. Additionally, these data are related to new structural and previously published permeability studies. Results reveal that most genes associated with intercellular junctions are expressed at similar levels at both ages. In total, 32 molecules known to be associated with brain barrier interfaces were identified. Nine claudins showed unaltered expression, while two claudins (6 and 8) were expressed at higher levels in the embryo. Expression levels for most cytoplasmic/regulatory adaptors (10 of 12) were similar at the two ages. A few junctional genes displayed lower expression in embryos, including 5 claudins, occludin and one junctional adhesion molecule. Three gap junction genes were enriched in the embryo. The functional effectiveness of these junctions was assessed using blood-delivered water-soluble tracers at both the light and electron microscopic level: embryo and adult junctions halted movement of both 286Da and 3kDa molecules into the cerebrospinal fluid (CSF). The molecular identities of many ion channel and transporter genes previously reported as important for CSF formation and secretion in the adult were demonstrated in the embryonic choroid plexus (and validated with immunohistochemistry of protein products), but with some major age-related differences in expression. In addition, a large number of previously unidentified ion channel and transporter genes were identified for the first time in plexus epithelium. These results, in addition to data obtained from electron microscopical and physiological permeability experiments in immature brains, indicate that exchange between blood and CSF is mainly transcellular, as well-formed tight junctions restrict movement of small water-soluble molecules from early in development. These data strongly indicate the brain develops within a well-protected internal environment and the exchange between the blood, brain and CSF is transcellular and not through incomplete barriers.

Cellular transfer of macromolecules across the developing choroid plexus of Monodelphis domestica

Choroid plexus epithelial cells secrete cerebrospinal fluid (CSF) and transfer molecules from blood into CSF. Tight junctions between choroidal epithelial cells are functionally effective from early in development: the route of transfer is suggested to be transcellular. Routes of transfer for endogenous and exogenous plasma proteins and dextrans were studied in Monodelphis domestica (opossum). Pups at postnatal (P) days 1–65 and young adults were injected with biotinylated dextrans (3–70 kDa) and/or foetal protein fetuin. CSF, plasma and brain samples were collected from terminally anaesthetized animals. Choroid plexus cells containing plasma proteins were detected immunocytochemically. Numbers of plasma protein‐positive epithelial cells increased to adult levels by P28, but their percentage of plexus cells declined. Numbers of cells positive for biotinylated probes increased with age, while their percentage remained constant. Colocalization studies showed specificity for individual proteins in some epithelial cells. Biotinylated probes and endogenous proteins colocalized in about 10% of cells in younger animals, increasing towards 100% by adulthood. Injections of markers into the ventricles demonstrated that protein is transferred only from blood into CSF, whereas dextrans pass in both directions. These results indicate that protein and lipid‐insoluble markers are transferred by separate mechanisms present in choroid plexuses from the earliest stage of brain development, and transfer of proteins from plasma across choroid plexus epithelial cells contributes to the high protein concentration in CSF in the immature brain.

The neonatal blood-brain barrier is functionally effective, and immaturity does not explain differential targeting of AAV9

The neonatal blood-brain barrier is functionally effective, and immaturity does not explain differential targeting of AAV9

Pathological Changes in the White Matter after Spinal Contusion Injury in the Rat

It has been shown previously that after spinal cord injury, the loss of grey matter is relatively faster than loss of white matter suggesting interventions to save white matter tracts offer better therapeutic possibilities. Loss of white matter in and around the injury site is believed to be the main underlying cause for the subsequent loss of neurological functions. In this study we used a series of techniques, including estimations of the number of axons with pathology, immunohistochemistry and mapping of distribution of pathological axons, to better understand the temporal and spatial pathological events in white matter following contusion injury to the rat spinal cord. There was an initial rapid loss of axons with no detectable further loss beyond 1 week after injury. Immunoreactivity for CNPase indicated that changes to oligodendrocytes are rapid, extending to several millimetres away from injury site and preceding much of the axonal loss, giving early prediction of the final volume of white matter that survived. It seems that in juvenile rats the myelination of axons in white matter tracts continues for some time, which has an important bearing on interpretation of our, and previous, studies. The amount of myelin debris and axon pathology progressively decreased with time but could still be observed at 10 weeks after injury, especially at more distant rostral and caudal levels from the injury site. This study provides new methods to assess injuries to spinal cord and indicates that early interventions are needed for the successful sparing of white matter tracts following injury.

Factors involved in inflammation-induced developmental white matter damage

Developmental white matter damage is a brain pathology associated with several long-term neurological disorders. An inflammatory insult has been suggested as the major instigating event. This study investigated the relative influence of inflammation, blood-brain barrier permeability and glial ontogeny in white matter damage. Systemic inflammation was induced in Monodelphis domestica (opossum) by serial intraperitoneal injections of lipopolysaccharide at different stages of brain development. Volume of white matter was estimated for the external capsule. Blood-brain barrier permeability was assessed immunocytochemically. Quantitative RT-PCR was used to measure relative levels of mRNA for IL-1beta, IL-6 and COX-2. Developmental changes in numbers and appearance of microglia and astrocytes were estimated. Results showed that in response to systemic inflammation, white matter was reduced in the external capsule during a circumscribed period only. At the same developmental stage blood-brain barrier permeability was altered, cerebral inflammatory response was present and numbers of microglia increased. However, the periods of altered blood-brain barrier permeability and the cerebral inflammatory response were longer than the period of the external capsules susceptibility to white matter damage, which coincided with the developmental increase in the number of astrocytes in this tract. Thus, the mechanism of white matter damage following systemic inflammation is multifactorial, including cerebral inflammation and breakdown of brain barriers occurring simultaneously at specific stages of glial cell development.