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During development, embryos and foetuses may be exposed to maternally ingested antiseizure medications (ASM), valproate and lamotrigine, essential in some patients to control their epilepsy symptoms. Often, the two drugs are co-administered to reduce required doses of valproate, a known potential teratogen. This study used Genetic Absence Epilepsy Rat from Strasbourg to evaluate transfer of valproate and lamotrigine across late gestation placenta and their entry into cerebrospinal fluid (CSF) and brain of developing rats, in mono- and combination therapies. Animals at embryonic day (E) 19, postnatal day (P) 0, 4 and 21, and adults were administered valproate (30 mg/kg) or lamotrigine (6 mg/kg) with their respective [ 3 H]-tracers, either alone or in combination. In chronic experiments, females consumed valproate-containing diet from 2 weeks prior to mating until offspring were used at E19 and P0. Drugs were injected 30 min before blood, CSF and brain samples were collected from terminally anaesthetised animals. Radioactivity in samples was measured. In acute monotherapy brain entry of valproate was higher in foetal than postnatal animals, correlating with its plasma protein binding. Brain entry of lamotrigine was not age-dependent. Combination therapy enhanced entry of lamotrigine into the adult brain but had no effects on brain and CSF entry of valproate. Following chronic valproate exposure, placental transfer of valproate decreased in combination therapy; however, foetal brain entry increased. Results suggest that during pregnancy, the use of combination therapy of valproate and lamotrigine may mitigate overall foetal exposure to valproate but potential risks to foetal brain development are less clear., (© 2024 The Author(s). European Journal of Neuroscience published by Federation of European Neuroscience Societies and John Wiley & Sons Ltd.)

This study used a marsupial Monodelphis domestica, which is born very immature and most of its development is postnatal without placental protection. RNA-sequencing (RNA-Seq) was used to identify the expression of influx and efflux transporters (ATP-binding cassettes [ABCs] and solute carriers [SLCs]) and metabolizing enzymes in brains of newborn to juvenile Monodelphis. Results were compared to published data in the developing eutherian rat. To test the functionality of these transporters at similar ages, the entry of paracetamol (acetaminophen) into the brain and cerebrospinal fluid (CSF) was measured using liquid scintillation counting following a single administration of the drug along with its radiolabelled tracer [ 3 H]. Drug permeability studies found that in Monodelphis, brain entry of paracetamol was already restricted at P5; it decreased further in the first week of life and then remained stable until the oldest age group tested (P110). Transcriptomic analysis of Monodelphis brain showed that expression of transporters and their metabolizing enzymes in early postnatal (P) pups (P0, P5, and P8) was relatively similar, but by P109, many more transcripts were identified. When transcriptomes of newborn Monodelphis brain and E19 rat brain and placenta were compared, several transporters present in the rat placenta were also found in the newborn Monodelphis brain. These were absent from E19 rat brain but were present in the adult rat brain. These data indicate that despite its extreme immaturity, the newborn Monodelphis brain may compensate for the lack of placental protection during early brain development by upregulating protective mechanisms, which in eutherian animals are instead present in the placenta., (© 2024 The Author(s). The Journal of Comparative Neurology published by Wiley Periodicals LLC.)

Cannabidiol is a major component of cannabis but without known psychoactive properties. A wide range of properties have been attributed to it, such as anti-inflammatory, analgesic, anti-cancer, anti-seizure and anxiolytic. However, being a fairly new compound in its purified form, little is known about cannabidiol brain entry, especially during development. Sprague Dawley rats at four developmental ages: embryonic day E19, postnatal day P4 and P12 and non-pregnant adult females were administered intraperitoneal cannabidiol at 10 mg/kg with [ 3 H] labelled cannabidiol. To investigate the extent of placental transfer, the drug was injected intravenously into E19 pregnant dams. Levels of [ 3 H]-cannabidiol in blood plasma, cerebrospinal fluid and brain were estimated by liquid scintillation counting. Plasma protein binding of cannabidiol was identified by polyacrylamide gel electrophoresis and its bound and unbound fractions measured by ultrafiltration. Using available RNA-sequencing datasets of E19 rat brain, choroid plexus and placenta, as well as P5 and adult brain and choroid plexus, expression of 13 main cannabidiol receptors was analysed. Results showed that cannabidiol rapidly entered both the developing and adult brains. Entry into CSF was more limited. Its transfer across the placenta was substantially restricted as only about 50% of maternal blood plasma cannabidiol concentration was detected in fetal plasma. Albumin was the main, but not exclusive, cannabidiol binding protein at all ages. Several transcripts for cannabidiol receptors were expressed in age- and tissue-specific manner indicating that cannabidiol may have different functional effects in the fetal compared to adult brain., (© 2024. The Author(s).)

Solute carriers (SLCs) regulate transfer of a wide range of molecules across cell membranes using facilitative or secondary active transport. In pregnancy, these transporters, expressed at the placental barrier, are important for delivery of nutrients to the fetus, whilst also limiting entry of potentially harmful substances, such as drugs. In the present study, RNA-sequencing analysis was used to investigate expression of SLCs in the fetal (embryonic day 19) rat brain, choroid plexus and placenta in untreated control animals and following maternal paracetamol treatment. In the treated group, paracetamol (15 mg/kg) was administered to dams twice daily for 5 days (from embryonic day 15 to 19). In untreated animals, overall expression of SLCs was highest in the placenta. In the paracetamol treatment group, expression of several SLCs was significantly different compared with control animals, with ion, amino acid, neurotransmitter and sugar transporters most affected. The number of SLC transcripts that changed significantly following treatment was the highest in the choroid plexus and lowest in the brain. All SLC transcripts that changed in the placenta following paracetamol treatment were downregulated. These results suggest that administration of paracetamol during pregnancy could potentially disrupt fetal nutrient homeostasis and affect brain development, resulting in major consequences for the neonate and extending into childhood., (© 2023 The Authors. Experimental Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.)

Binding of therapeutics to proteins in blood plasma is important in influencing their distribution as it is their free (unbound) form that is able to cross cellular membranes to enter tissues and exert their actions. The concentration and composition of plasma proteins vary during pregnancy and development, resulting in potential changes to drug protein binding. Here, we describe an ultrafiltration method to investigate the extent of protein binding of six drugs (digoxin, paracetamol, olanzapine, ivacaftor, valproate and lamotrigine) and two water soluble inert markers (sucrose and glycerol) to plasma proteins from pregnant and developing rats. Results showed that the free fraction of most drugs was lower in the non-pregnant adult plasma where protein concentration is the highest. However, plasma of equivalent protein concentration to younger pups obtained by diluting adult plasma did not always exhibit the same extent of drug binding, reinforcing the likelihood that both concentration and composition of proteins in plasma influence drug binding. Comparison between protein binding and brain drug accumulation in vivo revealed a correlation for some drugs, but not others. Results suggests that plasma protein concentration should be considered when using medications in pregnant and paediatric patients to minimise potential for fetal and neonatal drug exposure., (© 2023. The Author(s).)

Studies of the choroid plexus lag behind those of the more widely known blood-brain barrier, despite a much longer history. This review has two overall aims. The first is to outline long-standing areas of research where there are unanswered questions, such as control of cerebrospinal fluid (CSF) secretion and blood flow. The second aim is to review research over the past 10 years where the focus has shifted to the idea that there are choroid plexuses located in each of the brain's ventricles that make specific contributions to brain development and function through molecules they generate for delivery via the CSF. These factors appear to be particularly important for aspects of normal brain growth. Most research carried out during the twentieth century dealt with the choroid plexus, a brain barrier interface making critical contributions to the composition and stability of the brain's internal environment throughout life. More recent research in the twenty-first century has shown the importance of choroid plexus-generated CSF in neurogenesis, influence of sex and other hormones on choroid plexus function, and choroid plexus involvement in circadian rhythms and sleep. The advancement of technologies to facilitate delivery of brain-specific therapies via the CSF to treat neurological disorders is a rapidly growing area of research. Conversely, understanding the basic mechanisms and implications of how maternal drug exposure during pregnancy impacts the developing brain represents another key area of research.

Background: Apparent permeability of the blood brain barrier to hydrophilic markers has been shown to be higher in the developing brain. Apart from synthesis in situ, any substance detected in the brain parenchyma can originate from two sources: directly through blood vessels of brain vasculature and/or indirectly by entry from the cerebrospinal fluid (CSF) after transfer across the choroid plexuses. The relative quantitative contribution of these two routes to the overall brain entry remains unclear., Methods: In rats at embryonic day 16, 19 and postnatal day 4 and young adults, a small (sucrose, mw. 342 Da) or a large (dextran, mw. 70 kDa) radiolabelled hydrophilic marker was injected intravenously for very short periods of time (30 s to 5 min) before collection of plasma, cerebrospinal fluid (CSF) and brain samples. Results are presented as concentration ratios between radioactivity measured in CSF or brain and that in plasma (%)., Results: The dextran brain/plasma ratio five minutes post injection was similar (2-4%) from E16 to adulthood whereas the sucrose brain/plasma ratio was significantly higher in fetal brains, but was comparable to dextran values in the adult. Sucrose CSF/plasma ratios were also significantly higher in fetal animals and decreased with age. In very short experiments involving fetal animals, entry of sucrose into the CSF after only 30 s was similar to that of dextran and both markers showed similar brain/plasma ratios., Conclusions: In the developing brain the apparent higher brain entry of a small hydrophilic marker such as sucrose can be attributed to its higher entry into the CSF and subsequent diffusion into the brain. By contrast, movement of a larger marker like 70 kDa dextran is restricted firstly by choroid plexus epithelial tight junctions and secondly by specialised junctions in the neuroependymal interface between the CSF and brain. Brain/plasma ratios of 70 kDa dextran were similar in fetal and adult rats. Therefore 70 kDa dextran should be considered an appropriate marker if brain residual vascular space is to be measured, especially in younger animals., (© 2022. The Author(s).)

Background: Little is known about the extent of drug entry into developing brain, when administered to pregnant and lactating women. Lithium is commonly prescribed for bipolar disorder. Here we studied transfer of lithium given to dams, into blood, brain and cerebrospinal fluid (CSF) in embryonic and postnatal animals as well as adults., Methods: Lithium chloride in a clinically relevant dose (3.2 mg/kg body weight) was injected intraperitoneally into pregnant (E15-18) and lactating dams (birth-P16/17) or directly into postnatal pups (P0-P16/17). Acute treatment involved a single injection; long-term treatment involved twice daily injections for the duration of the experiment. Following terminal anaesthesia blood plasma, CSF and brains were collected. Lithium levels and brain distribution were measured using Laser Ablation Inductively Coupled Plasma-Mass Spectrometry and total lithium levels were confirmed by Inductively Coupled Plasma-Mass Spectrometry., Results: Lithium was detected in blood, CSF and brain of all fetal and postnatal pups following lithium treatment of dams. Its concentration in pups' blood was consistently below that in maternal blood (30-35%) indicating significant protection by the placenta and breast tissue. However, much of the lithium that reached the fetus entered its brain. Levels of lithium in plasma fluctuated in different treatment groups but its concentration in CSF was stable at all ages, in agreement with known stable levels of endogenous ions in CSF. There was no significant increase of lithium transfer into CSF following application of Na + /K + ATPase inhibitor (digoxin) in vivo, indicating that lithium transfer across choroid plexus epithelium is not likely to be via the Na + /K + ATPase mechanism, at least early in development. Comparison with passive permeability markers suggested that in acute experiments lithium permeability was less than expected for diffusion but similar in long-term experiments at P2., Conclusions: Information obtained on the distribution of lithium in developing brain provides a basis for studying possible deleterious effects on brain development and behaviour in offspring of mothers undergoing lithium therapy., (© 2021. The Author(s).)

Adenosine triphosphate binding cassette (ABC) transporters transfer lipid-soluble molecules across cellular interfaces either directly or after enzymatic metabolism. RNAseq analysis identified transcripts for ABC transporters and enzymes in rat E19, P5 and adult brain and choroid plexus and E19 placenta. Their functional capacity to efflux small molecules was studied by quantitative analysis of paracetamol (acetaminophen) and its metabolites using liquid scintillation counting, autoradiography and ultra-performance liquid chromatography coupled with tandem mass spectrometry (UPLC-MS/MS). Animals were treated acutely (30 min) and chronically (5 days, twice daily) with paracetamol (15 mg/kg) to investigate ability of brain and placenta barriers to regulate ABC transport functionality during extended treatment. Results indicated that transcripts of many efflux-associated ABC transporters were higher in adult brain and choroid plexus than at earlier ages. Chronic treatment upregulated certain transcripts only in adult brain and altered concentrations of paracetamol metabolites in circulation of pregnant dams. Combination of changes to metabolites and transport system transcripts may explain observed changes in paracetamol entry into adult and fetal brains. Analysis of lower paracetamol dosing (3.75 mg/kg) indicated dose-dependent changes in paracetamol metabolism. Transcripts of ABC transporters and enzymes at key barriers responsible for molecular transport into the developing brain showed alterations in paracetamol pharmacokinetics in pregnancy following different treatment regimens., (© 2021. The Author(s).)

Background: The potential effects of ivacaftor during pregnancy and breastfeeding on the offspring are still unknown. This study aimed to investigate pre-/postnatal age-related entry into the brain and lungs and transfer of maternally administered drug by the placental and via the milk., Methods: In acute experiments Sprague Dawley rats at embryonic day (E) 19, postnatal days (P) 4, 9, 16, and adult were administered an intraperitoneal injection of ivacaftor (40 mg/kg) traced with [3H] ivacaftor. To determine tissue entry, plasma, cerebrospinal fluid (CSF), lungs and brains were collected, and radioactivity measured using liquid scintillation counting. For long term experiments pregnant dams were orally treated at 25 mg/kg/day for 7 days and pups collected at E19. For postnatal pups, dams received treatment for 7 or 14 days and pups were collected at P6, 9, 13 and 16. To estimate placental and milk transfer concentration of ivacaftor in pup & maternal plasma was determined by liquid chromatography-mass spectrometry., Results: At all ages, entry of ivacaftor into lungs, following either acute or prolonged exposure, was much higher than into brain & CSF. Brain entry appeared higher at earlier ages. Transfer across the placenta and breast milk. was estimated to be around ~40% of maternal plasma., Conclusions: Fetal and postnatal rats were exposed to maternally administered ivacaftor via placental and milk transfer. Preferential entry in the lungs at all ages suggests the possibility that exposing CF babies to maternally administered ivacaftor could be beneficial for limiting progression of CF pathology in early development., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Background: Women with epilepsy face difficult choices whether to continue antiepileptic drug treatment during pregnancy, as uncontrolled seizures carry great risk to mother and fetus but continuing treatment may have adverse effects on baby's development. This study aimed at evaluating antiepileptic drug entry into developing brain. Methods: Anaesthetised pregnant, non-pregnant adult females, postnatal and fetal rats were injected intraperitoneally with different doses, single or in combinations, of valproate and lamotrigine, all within clinical range. Injectate included 3 H-labelled drug. After 30min, CSF, blood and brain samples were obtained; radioactivity was measured using liquid scintillation counting. Some animals were also exposed to valproate in feed throughout pregnancy and into neonatal period. Drug levels were measured by liquid chromatography coupled to mass spectrometry (LC-MS). Results are given as CSF or tissue/plasma% as index of drug entry. Results: Entry of valproate into brain and CSF was higher at E19 and P4 compared to adult but was not dose-dependent;  placental transfer increased significantly at highest dose of 100mg/Kg. Lamotrigine entry into the brain was dose dependent only at E19. Chronic valproate treatment, or combination of valproate and lamotrigine had little effect on either drug entry, except for reduced valproate brain entry in adult brain with chronic treatment. Placental transfer decreased significantly after chronic valproate treatment. LC-MS measurement of valproate in adults confirmed that rat plasma values were within the clinical range and CSF/plasma and brain/plasma ratios for LC-MS and 3 H-valproate were similar. Conclusion: Results suggest that entry of valproate may be higher in developing brain, the capacity of barrier mechanism is mostly unaffected by doses within the clinical range, with or without addition of lamotrigine. Chronic valproate exposure may result in upregulation in cellular mechanisms restricting its entry into the brain. Entry of lamotrigine was little different at different ages and was not dose dependent., Competing Interests: No competing interests were disclosed., (Copyright: © 2021 Toll SJ et al.)

Background: Adenosine triphosphate binding cassette transporters such as P-glycoprotein (PGP) play an important role in drug pharmacokinetics by actively effluxing their substrates at barrier interfaces, including the blood-brain, blood-cerebrospinal fluid (CSF) and placental barriers. For a molecule to access the brain during fetal stages it must bypass efflux transporters at both the placental barrier and brain barriers themselves. Following birth, placental protection is no longer present and brain barriers remain the major line of defense. Understanding developmental differences that exist in the transfer of PGP substrates into the brain is important for ensuring that medication regimes are safe and appropriate for all patients., Methods: In the present study PGP substrate rhodamine-123 (R123) was injected intraperitoneally into E19 dams, postnatal (P4, P14) and adult rats. Naturally fluorescent properties of R123 were utilized to measure its concentration in blood-plasma, CSF and brain by spectrofluorimetry (Clariostar). Statistical differences in R123 transfer (concentration ratios between tissue and plasma ratios) were determined using Kruskal-Wallis tests with Dunn's corrections., Results: Following maternal injection the transfer of R123 across the E19 placenta from maternal blood to fetal blood was around 20 %. Of the R123 that reached fetal circulation 43 % transferred into brain and 38 % into CSF. The transfer of R123 from blood to brain and CSF was lower in postnatal pups and decreased with age (brain: 43 % at P4, 22 % at P14 and 9 % in adults; CSF: 8 % at P4, 8 % at P14 and 1 % in adults). Transfer from maternal blood across placental and brain barriers into fetal brain was approximately 9 %, similar to the transfer across adult blood-brain barriers (also 9 %). Following birth when placental protection was no longer present, transfer of R123 from blood into the newborn brain was significantly higher than into adult brain (3 fold, p < 0.05)., Conclusions: Administration of a PGP substrate to infant rats resulted in a higher transfer into the brain than equivalent doses at later stages of life or equivalent maternal doses during gestation. Toxicological testing of PGP substrate drugs should consider the possibility of these patient specific differences in safety analysis.

Drug entry into the adult brain is controlled by efflux mechanisms situated in various brain barrier interfaces. The effectiveness of these protective mechanisms in the embryo, fetus and newborn brain is less clear. The longstanding belief that "the" blood-brain barrier is absent or immature in the fetus and newborn has led to many misleading statements with potential clinical implications. Here we review the properties of brain barrier mechanisms in the context of drug entry into the developing brain and discuss the limited number of studies published on the subject. We noticed that most of available literature suffers from some experimental limitations, notably that drug levels in fetal blood and cerebrospinal fluid have not been measured. This means that the relative contribution to the overall brain protection provided by individual barriers such as the placenta (which contains similar efflux mechanisms) and the brain barriers cannot be separately ascertained. Finally, we propose that systematic studies in appropriate animal models of drug entry into the brain at different stages of development would provide a rational basis for use of medications in pregnancy and in newborns, especially prematurely born, where protection usually provided by the placenta is no longer present., (© 2020 John Wiley & Sons, Ltd.)

Background: Paracetamol (acetaminophen) is widely used in pregnancy and generally regarded as "safe" by regulatory authorities. Methods: Clinically relevant doses of paracetamol were administered intraperitoneally to pregnant rats twice daily from embryonic day E15 to 19 (chronic) or as a single dose at E19 (acute). Control samples were from un-treated age-matched animals. At E19, rats were anaesthetised, administered a final paracetamol dose, uteruses were opened and fetuses exposed for sample collection. For RNA sequencing, placentas and fetal brains were removed and flash frozen. Fetal and maternal plasma and cerebrospinal fluid were assayed for ⍺-fetoprotein and interleukin 1β (IL1β). Brains were fixed and examined (immunohistochemistry) for plasma protein distribution. Placental permeability to a small molecule ( 14 C-sucrose) was tested by injection into either mother or individual fetuses; fetal and maternal blood was sampled at regular intervals to 90 minutes. Results: RNA sequencing revealed a large number of genes up- or down-regulated in placentas from acutely or chronically treated animals compared to controls. Most notable was down-regulation of three acute phase plasma proteins (⍺-fetoprotein, transferrin, transthyretin) in acute and especially chronic experiments and marked up-regulation of immune-related genes, particularly cytokines, again especially in chronically treated dams. IL1β increased in plasma of most fetuses from treated dams but to variable levels and no IL1β was detectable in plasma of control fetuses or any of the dams. Increased placental permeability appeared to be only from fetus to mother for both 14 C-sucrose and ⍺-fetoprotein, but not in the reverse direction. In the fetal brain, gene regulatory changes were less prominent than in the placenta of treated fetuses and did not involve inflammatory-related genes; there was no evidence of increased blood-brain barrier permeability. Conclusion: Results suggest that paracetamol may induce an immune-inflammatory-like response in placenta and more caution should be exercised in use of paracetamol in pregnancy., Competing Interests: No competing interests were disclosed., (Copyright: © 2020 Koehn LM et al.)

Many pregnant women and prematurely born infants require medication for clinical conditions including cancer, cardiac defects and psychiatric disorders. In adults drug transfer from blood into brain is mostly restricted by efflux mechanisms (ATP-binding cassette, ABC transporters). These mechanisms have been little studied during brain development. Here expression of eight ABC transporters (abcb1a, abcb1b, abcg2, abcc1, abcc2, abcc3, abcc4, abcc5) and activity of conjugating enzyme glutathione-s-transferase (GST) were measured in livers, brain cortices (blood-brain-barrier) and choroid plexuses (blood-cerebrospinal fluid, CSF, barrier) during postnatal rat development. Controls were compared to animals chronically injected (4 days, 200 mg/kg/day) with known abcb1a inducer diallyl sulfide (DAS). Results reveal both tissue- and age-dependent regulation. In liver abcb1a and abcc3 were up-regulated at all ages. In cortex abcb1a/b, abcg2 and abcc4/abcc5 were up-regulated in adults only, while in choroid plexus abcb1a and abcc2 were up-regulated only at P14. DAS treatment increased GST activity in livers, but not in cortex or choroid plexuses. Immunocytochemistry of ABC transporters at the CSF-brain interface showed that PGP and BCRP predominated in neuroepithelium while MRP2/4/5 were prominent in adult ependyma. These results indicate an age-related capacity of brain barriers to dynamically regulate their defence mechanisms when chronically challenged by xenobiotic compounds.

Efflux mechanisms situated in various brain barrier interfaces control drug entry into the adult brain; this review considers the effectiveness of these protective mechanisms in the embryo, fetus, and newborn brain. The longstanding belief that the blood-brain barrier is absent or immature in the fetus and newborn has led to many misleading statements with potential clinical implications. The immature brain is undoubtedly more vulnerable to damage by drugs and toxins; as is reviewed here, some developmentally regulated normal brain barrier mechanisms probably contribute to this vulnerability. We propose that the functional status of brain barrier efflux mechanisms should be investigated at different stages of brain development to provide a rational basis for the use of drugs in pregnancy and in newborns, especially in those prematurely born, where protection usually provided by the placenta is no longer present.

Properties of the local internal environment of the adult brain are tightly controlled providing a stable milieu essential for its normal function. The mechanisms involved in this complex control are structural, molecular and physiological (influx and efflux transporters) frequently referred to as the 'blood-brain barrier'. These mechanisms include regulation of ion levels in brain interstitial fluid essential for normal neuronal function, supply of nutrients, removal of metabolic products, and prevention of entry or elimination of toxic agents. A key feature is cerebrospinal fluid secretion and turnover. This is much less during development, allowing greater accumulation of permeating molecules. The overall effect of these mechanisms is to tightly control the exchange of molecules into and out of the brain. This review presents experimental evidence currently available on the status of these mechanisms in developing brain. It has been frequently stated for over nearly a century that the blood-brain barrier is not present or at least is functionally deficient in the embryo, fetus and newborn. We suggest the alternative hypothesis that the barrier mechanisms in developing brain are likely to be appropriately matched to each stage of its development. The contributions of different barrier mechanisms, such as changes in constituents of cerebrospinal fluid in relation to specific features of brain development, for example neurogenesis, are only beginning to be studied. The evidence on this previously neglected aspect of brain barrier function is outlined. We also suggest future directions this field could follow with special emphasis on potential applications in a clinical setting., (© 2018 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.)

Adult brain is protected from entry of drugs and toxins by specific mechanisms such as ABC (ATP-binding Cassette) efflux transporters. Little is known when these appear in human brain during development. Cellular distribution of three main ABC transporters (ABCC1, ABCG2, ABCB1) was determined at blood-brain barriers and interfaces in human embryos and fetuses in first half of gestation. Antibodies against claudin-5 and -11 and antibodies to α-fetoprotein were used to describe morphological and functional aspects of brain barriers. First exchange interfaces to be established, probably at 4-5 weeks post conception, are between brain and embryonic cerebrospinal fluid (eCSF) and between outer surface of brain anlage and primary meninx. They already exclude α-fetoprotein and are immunopositive for both claudins, ABCC1 and ABCG2. ABCB1 is detectable within a week of blood vessels first penetrating into brain parenchyma (6-7 weeks post conception). ABCC1, ABCB1 and ABCG2 are present at blood-CSF barrier in all choroid plexuses from first appearance (7 weeks post conception). Outer CSF-brain interfaces are established between 9-11 weeks post conception exhibiting immunoreactivity for all three transporters. Results provide evidence for sequential establishment of brain exchange interfaces and spatial and temporal timetable for three main ABC transporters in early human brain.

Background : Most animal studies of spinal cord injury are conducted in quadrupeds, usually rodents. It is unclear to what extent functional results from such studies can be translated to bipedal species such as humans because bipedal and quadrupedal locomotion involve very different patterns of spinal control of muscle coordination. Bipedalism requires upright trunk stability and coordinated postural muscle control; it has been suggested that peripheral sensory input is less important in humans than quadrupeds for recovery of locomotion following spinal injury. Methods : We used an Australian macropod marsupial, the tammar wallaby (Macropus eugenii ), because tammars exhibit an upright trunk posture, human-like alternating hindlimb movement when swimming and bipedal over-ground locomotion. Regulation of their muscle movements is more similar to humans than quadrupeds. At different postnatal (P) days (P7-60) tammars received a complete mid-thoracic spinal cord transection. Morphological repair, as well as functional use of hind limbs, was studied up to the time of their pouch exit. Results: Growth of axons across the lesion restored supraspinal innervation in animals injured up to 3 weeks of age but not in animals injured after 6 weeks of age. At initial pouch exit (P180), the young injured at P7-21 were able to hop on their hind limbs similar to age-matched controls and to swim albeit with a different stroke. Those animals injured at P40-45 appeared to be incapable of normal use of hind limbs even while still in the pouch. Conclusions : Data indicate that the characteristic over-ground locomotion of tammars provides a model in which regrowth of supraspinal connections across the site of injury can be studied in a bipedal animal. Forelimb weight-bearing motion and peripheral sensory input appear not to compensate for lack of hindlimb control, as occurs in quadrupeds. Tammars may be a more appropriate model for studies of therapeutic interventions relevant to humans., Competing Interests: Competing interests: No competing interests were disclosed.

The brain functions within a specialized environment tightly controlled by brain barrier mechanisms. Understanding the regulation of barrier formation is important for understanding brain development and may also lead to finding new ways to deliver pharmacotherapies to the brain; access of many potentially promising drugs is severely hindered by these barrier mechanisms. The cellular composition of the neurovascular unit of the blood-brain barrier proper and their effects on regulation of its function are beginning to be understood. One hallmark of the neurovascular unit in the adult is the astroglial foot processes that tightly surround cerebral blood vessels. However their role in barrier formation is still unclear. In this study we examined barrier function in newborn, juvenile and adult mice lacking fibroblast growth factor-2 (FGF-2), which has been shown to result in altered astroglial differentiation during development. We show that during development of FGF-2 deficient mice the astroglial contacts with cerebral blood vessels are delayed compared with wild-type animals. However, this delay did not result in changes to the permeability properties of the blood brain barrier as assessed by exclusion of either small or larger sized molecules at this interface. In addition cerebral vessels were positive for tight-junction proteins and we observed no difference in the ultrastructure of the tight-junctions. The results indicate that the direct contact of astroglia processes to cerebral blood vessels is not necessary for either the formation of the tight-junctions or for basic permeability properties and function of the blood-brain barrier. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 76: 1201-1212, 2016., (© 2016 Wiley Periodicals, Inc.)

Tissue loss after spinal trauma is biphasic, with initial mechanical/haemorrhagic damage at the time of impact being followed by gradual secondary expansion into adjacent, previously unaffected tissue. Limiting the extent of this secondary expansion of tissue damage has the potential to preserve greater residual spinal cord function in patients. The acute tissue hypoxia resulting from spinal cord injury (SCI) activates acid-sensing ion channel 1a (ASIC1a). We surmised that antagonism of this channel should provide neuroprotection and functional preservation after SCI. We show that systemic administration of the spider-venom peptide PcTx1, a selective inhibitor of ASIC1a, improves locomotor function in adult Sprague Dawley rats after thoracic SCI. The degree of functional improvement correlated with the degree of tissue preservation in descending white matter tracts involved in hind limb locomotor function. Transcriptomic analysis suggests that PcTx1-induced preservation of spinal cord tissue does not result from a reduction in apoptosis, with no evidence of down-regulation of key genes involved in either the intrinsic or extrinsic apoptotic pathways. We also demonstrate that trauma-induced disruption of blood-spinal cord barrier function persists for at least 4 days post-injury for compounds up to 10 kDa in size, whereas barrier function is restored for larger molecules within a few hours. This temporary loss of barrier function provides a " treatment window " through which systemically administered drugs have unrestricted access to spinal tissue in and around the sites of trauma. Taken together, our data provide evidence to support the use of ASIC1a inhibitors as a therapeutic treatment for SCI. This study also emphasizes the importance of objectively grading the functional severity of initial injuries (even when using standardized impacts) and we describe a simple scoring system based on hind limb function that could be adopted in future studies., Competing Interests: The authors state that there are no conflicts of interest in the authorship of this study.

Barrier mechanisms in the brain are important for its normal functioning and development. Stability of the brain's internal environment, particularly with respect to its ionic composition, is a prerequisite for the fundamental basis of its function, namely transmission of nerve impulses. In addition, the appropriate and controlled supply of a wide range of nutrients such as glucose, amino acids, monocarboxylates, and vitamins is also essential for normal development and function. These are all cellular functions across the interfaces that separate the brain from the rest of the internal environment of the body. An essential morphological component of all but one of the barriers is the presence of specialized intercellular tight junctions between the cells comprising the interface: endothelial cells in the blood-brain barrier itself, cells of the arachnoid membrane, choroid plexus epithelial cells, and tanycytes (specialized glial cells) in the circumventricular organs. In the ependyma lining the cerebral ventricles in the adult brain, the cells are joined by gap junctions, which are not restrictive for intercellular movement of molecules. But in the developing brain, the forerunners of these cells form the neuroepithelium, which restricts exchange of all but the smallest molecules between cerebrospinal fluid and brain interstitial fluid because of the presence of strap junctions between the cells. The intercellular junctions in all these interfaces are the physical basis for their barrier properties. In the blood-brain barrier proper, this is combined with a paucity of vesicular transport that is a characteristic of other vascular beds. Without such a diffusional restrain, the cellular transport mechanisms in the barrier interfaces would be ineffective. Superimposed on these physical structures are physiological mechanisms as the cells of the interfaces contain various metabolic transporters and efflux pumps, often ATP-binding cassette (ABC) transporters, that provide an important component of the barrier functions by either preventing entry of or expelling numerous molecules including toxins, drugs, and other xenobiotics. In this review, we summarize these influx and efflux mechanisms in normal developing and adult brain, as well as indicating their likely involvement in a wide range of neuropathologies. There have been extensive attempts to overcome the barrier mechanisms that prevent the entry of many drugs of therapeutic potential into the brain. We outline those that have been tried and discuss why they may so far have been largely unsuccessful. Currently, a promising approach appears to be focal, reversible disruption of the blood-brain barrier using focused ultrasound, but more work is required to evaluate the method before it can be tried in patients. Overall, our view is that much more fundamental knowledge of barrier mechanisms and development of new experimental methods will be required before drug targeting to the brain is likely to be a successful endeavor. In addition, such studies, if applied to brain pathologies such as stroke, trauma, or multiple sclerosis, will aid in defining the contribution of brain barrier pathology to these conditions, either causative or secondary.

In recent years there has been a resurgence of interest in brain barriers and various roles their intrinsic mechanisms may play in neurological disorders. Such studies require suitable models and markers to demonstrate integrity and functional changes at the interfaces between blood, brain, and cerebrospinal fluid. Studies of brain barrier mechanisms and measurements of plasma volume using dyes have a long-standing history, dating back to the late nineteenth-century. Their use in blood-brain barrier studies continues in spite of their known serious limitations in in vivo applications. These were well known when first introduced, but seem to have been forgotten since. Understanding these limitations is important because Evans blue is still the most commonly used marker of brain barrier integrity and those using it seem oblivious to problems arising from its in vivo application. The introduction of HRP in the mid twentieth-century was an important advance because its reaction product can be visualized at the electron microscopical level, but it also has limitations. Advantages and disadvantages of these markers will be discussed together with a critical evaluation of alternative approaches. There is no single marker suitable for all purposes. A combination of different sized, visualizable dextrans and radiolabeled molecules currently seems to be the most appropriate approach for qualitative and quantitative assessment of barrier integrity.

The transcriptome of embryonic and adult rat lateral ventricular choroid plexus, using a combination of RNA-Sequencing and microarray data, was analyzed by functional groups of influx transporters, particularly solute carrier (SLC) transporters. RNA-Seq was performed at embryonic day (E) 15 and adult with additional data obtained at intermediate ages from microarray analysis. The largest represented functional group in the embryo was amino acid transporters (twelve) with expression levels 2-98 times greater than in the adult. In contrast, in the adult only six amino acid transporters were up-regulated compared to the embryo and at more modest enrichment levels (<5-fold enrichment above E15). In E15 plexus five glucose transporters, in particular Glut-1, and only one monocarboxylate transporter were enriched compared to the adult, whereas only two glucose transporters but six monocarboxylate transporters in the adult plexus were expressed at higher levels than in embryos. These results are compared with earlier published physiological studies of amino acid and monocarboxylate transport in developing rodents. This comparison shows correlation of high expression of some transporters in the developing brain with higher amino acid transport activity reported previously. Data for divalent metal transporters are also considered. Immunohistochemistry of several transporters (e.g., Slc16a10, a thyroid hormone transporter) gene products was carried out to confirm translational activity and to define cellular distribution of the proteins. Overall the results show that there is substantial expression of numerous influx transporters in the embryonic choroid plexus, many at higher levels than in the adult. This, together with immunohistochemical evidence and data from published physiological transport studies suggests that the choroid plexus in embryonic brain plays a major role in supplying the developing brain with essential nutrients.

Developmental studies of spinal cord injury in which regrowth of axons occurs across the site of transection rarely distinguish between the recovery of motor-controlling pathways and that of ascending axons carrying sensory information. We describe the morphological changes that occur in the dorsal column (DC) of the grey short-tailed opossum, Monodelphis domestica, following spinal cord injury at two early developmental ages. The spinal cords of opossums that had had their mid-thoracic spinal cords completely transected at postnatal day 7 (P7) or P28 were analysed. Profiles of neurofilament immunoreactivity in transected cords showing DC development were differentially affected by the injury compared with the rest of the cord and cytoarchitecture was modified in an age- and site-dependent manner. The ability of DC neurites to grow across the site of transection was confirmed by injection of fluorescent tracer below the injury. P7 transected cords showed labelling in the DC above the site of original transection indicating that neurites of this sensory tract were able to span the injury. No growth of any neuronal processes was seen after P28 transection. Thus, DC is affected by spinal injury in a differential manner depending on the age at which the transection occurs. This age-differential response, together with other facets of remodelling that occur after neonatal spinal injury, might explain the locomotor adaptations and recovery observed in these animals.

In the adult the interface between the cerebrospinal fluid and the brain is lined by the ependymal cells, which are joined by gap junctions. These intercellular connections do not provide a diffusional restrain between the two compartments. However, during development this interface, initially consisting of neuroepithelial cells and later radial glial cells, is characterized by "strap" junctions, which limit the exchange of different sized molecules between cerebrospinal fluid and the brain parenchyma. Here we provide a systematic study of permeability properties of this inner cerebrospinal fluid-brain barrier during mouse development from embryonic day, E17 until adult. Results show that at fetal stages exchange across this barrier is restricted to the smallest molecules (286Da) and the diffusional restraint is progressively removed as the brain develops. By postnatal day P20, molecules the size of plasma proteins (70 kDa) diffuse freely. Transcriptomic analysis of junctional proteins present in the cerebrospinal fluid-brain interface showed expression of adherens junctional proteins, actins, cadherins and catenins changing in a development manner consistent with the observed changes in the permeability studies. Gap junction proteins were only identified in the adult as was claudin-11. Immunohistochemistry was used to localize at the cellular level some of the adherens junctional proteins of genes identified from transcriptomic analysis. N-cadherin, β - and α-catenin immunoreactivity was detected outlining the inner CSF-brain interface from E16; most of these markers were not present in the adult ependyma. Claudin-5 was present in the apical-most part of radial glial cells and in endothelial cells in embryos, but only in endothelial cells including plexus endothelial cells in adults. Claudin-11 was only immunopositive in the adult, consistent with results obtained from transcriptomic analysis. These results provide information about physiological, molecular and morphological-related permeability changes occurring at the inner cerebrospinal fluid-brain barrier during brain development.

Careful examination of relevant literature shows that many of the most cherished concepts of the blood-brain barrier are incorrect. These include an almost mythological belief in its immaturity that is unfortunately often equated with absence or at least leakiness in the embryo and fetus. The original concept of a blood-brain barrier is often attributed to Ehrlich; however, he did not accept that permeability of cerebral vessels was different from other organs. Goldmann is often credited with the first experiments showing dye (trypan blue) exclusion from the brain when injected systemically, but not when injected directly into it. Rarely cited are earlier experiments of Bouffard and of Franke who showed methylene blue and trypan red stained all tissues except the brain. The term "blood-brain barrier" "Blut-Hirnschranke" is often attributed to Lewandowsky, but it does not appear in his papers. The first person to use this term seems to be Stern in the early 1920s. Studies in embryos by Stern and colleagues, Weed and Wislocki showed results similar to those in adult animals. These were well-conducted experiments made a century ago, thus the persistence of a belief in barrier immaturity is puzzling. As discussed in this review, evidence for this belief, is of poor experimental quality, often misinterpreted and often not properly cited. The functional state of blood-brain barrier mechanisms in the fetus is an important biological phenomenon with implications for normal brain development. It is also important for clinicians to have proper evidence on which to advise pregnant women who may need to take medications for serious medical conditions. Beliefs in immaturity of the blood-brain barrier have held the field back for decades. Their history illustrates the importance of taking account of all the evidence and assessing its quality, rather than selecting papers that supports a preconceived notion or intuitive belief. This review attempts to right the wrongs. Based on careful translation of original papers, some published a century ago, as well as providing discussion of studies claiming to show barrier immaturity, we hope that readers will have evidence on which to base their own conclusions.