Complex chronic conditions among children born to women with schizophrenia.

PURPOSE: Maternal schizophrenia is linked to complications in offspring near the time of birth. Whether there is also a higher future risk of the child having a complex chronic condition (CCC) - a pediatric condition affecting any bodily system expected to last at least 12 months that is severe enough to require specialty care and/or a period of hospitalization - is not known. METHODS: In this population-based health administrative data cohort study (Ontario, Canada, 1995-2018), the risk for CCC was compared in 5066 children of women with schizophrenia (the exposed) vs. 2,939,320 unexposed children. Adjusted hazard ratios (aHR) were generated for occurrence of any CCC, by CCC category, and stratified by child sex, and child prematurity. RESULTS: CCC was more frequent in the exposed (7.7 per 1000 person-years [268 children]) than unexposed (4.2 per 100 person-years [124,452 children]) - an aHR of 1.25 (95% CI 1.10-1.41). aHRs were notably higher in 5 of 9 CCC categories: neuromuscular (1.73, 1.28-2.33), cardiovascular (1.94, 1.64-2.29), respiratory (1.83, 1.32-2.54), hematology/immunodeficiency (2.24, 1.24-4.05) and other congenital or genetic defect (1.59, 1.16-2.17). The aHR for CCC was more pronounced among boys (1.32, 1.13-1.55) than girls (1.16, 0.96-1.40), and of similar magnitude in term (1.22, 1.05-1.42) and preterm infants (1.18, 0.95-1.46). CONCLUSIONS: The risk for a CCC appears to be higher in children born to women with schizophrenia. This finding introduces opportunities for targeted preconception counselling, optimization of maternal risk factors, and intervention to support a vulnerable parent population who will experience unique challenges caring for a child with CCCs.

Modification of protein transfer across blood/cerebrospinal fluid barrier in response to altered plasma protein composition during development.

A developmentally regulated protein-specific transfer mechanism across choroid plexus epithelial cells has previously been proposed to contribute to the characteristically high concentration of protein in cerebrospinal fluid (CSF) in the immature brain. Here we demonstrate that this mechanism is sensitive to protein variations in plasma resulting in changed numbers of transferring cells for individual proteins and altered transfer into the CSF. Pups of Monodelphis domestica at postnatal day (P)9, P65 and P110 were injected intraperitoneally with either adult Monodelphis plasma or exogenous bovine fetuin. Samples of CSF, blood and brain were collected from terminally anaesthetized animals 3-48 h later. The concentration of total protein was measured and levels of albumin, hemopexin, α-fetoprotein and bovine fetuin were estimated by western blotting. Numbers of lateral ventricular choroid plexus cells positive for total and individual plasma proteins were counted in paraffin sections of brains stained with appropriate antibodies. Following intraperitoneal injections, the content of proteins in the CSF increased at all three ages, but the concentration increased only in the CSF of older animals. The total numbers of plexus cells positive for plasma protein did not change significantly, but cells positive for individual proteins did. Fetuin was detected in all protein-positive cells, but apparently displaced α-fetoprotein and, to a lesser degree, hemopexin. The results indicate that protein transfer across the blood/CSF barrier appears to be regulated by a molecular recognition mechanism that is probably saturable but may not be as specific for individual proteins as previously suggested.

SPARC/osteonectin, an endogenous mechanism for targeting albumin to the blood-cerebrospinal fluid interface during brain development.

Specialized populations of choroid plexus epithelial cells have previously been shown to be responsible for the transfer of individual plasma proteins from blood to the cerebrospinal fluid (CSF), contributing to their characteristically high concentrations in CSF of the developing brain. The mechanism of this protein transfer remains elusive. Using a marsupial, Monodelphis domestica, we demonstrate that the albumin-binding protein SPARC (osteonectin/BM-40/culture-shock protein) is present in a subset of choroid plexus epithelial cells from its first appearance, throughout development, and into adulthood. The synthesis of SPARC by the lateral ventricular plexus was confirmed with real-time PCR. The expression level of SPARC was higher in plexuses of younger than older animals. Western blot analysis of the gene product confirmed the quantitative PCR results. The co-localization of SPARC and albumin shown by immunocytochemistry and its cellular location indicate that this glycoprotein may act as a recognition site for albumin. In addition, the numbers of SPARC-immunopositive cells and its expression were responsive to experimental changes of albumin concentration in the blood. It is suggested that SPARC may be one of the molecules that govern the uptake and delivery of proteins from blood to the CSF. The results also confirm that protein transfer across the blood-CSF barrier is developmentally and physiologically regulated.

Stimulation of defecation in spinal cord-injured rats by a centrally acting ghrelin receptor agonist.

STUDY DESIGN: Animal proof of principle study. OBJECTIVES: To determine whether capromorelin, a compound that causes defecation by stimulating ghrelin receptors within the lumbosacral defecation centers, is effective after spinal cord injury (SCI), and whether SCI significantly alters sensitivity to the compound. SETTING: University of Melbourne and Austin Hospital, Melbourne, Australia. METHODS: Rats were subjected to spinal cord contusion injury or were sham-operated. At 6 weeks after surgery, effects of capromorelin on blood pressure, heart rate and propulsive contractions of the colorectum were investigated. RESULTS: Capromorelin caused robust propulsive activity in the colorectum soon after its application. The compound was similarly effective in naïve, sham-operated and spinal cord-injured rats. Blood pressure increases caused by capromorelin were not exaggerated after SCI, and there was no evidence of phasic blood pressure increases when the colon was contracted by the compound. CONCLUSION: Capromorelin is a therapeutic compound that could potentially be used to relieve constipation by triggering defecation in spinal cord-injured patients.

Efflux transporters in rat placenta and developing brain: transcriptomic and functional response to paracetamol.

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.

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.

Fetuin: the bovine homologue of human alpha 2HS glycoprotein.

The fetal protein fetuin has previously been considered to be confined to species of the order Artiodactyla (cattle, sheep, etc.) in spite of demonstrable biological in vitro effects in tissues of other species [(1983) Comp. Biochem. Physiol. 76A, 241-245]. We have determined the partial amino acid sequence of bovine fetuin and compared it with the published sequence of human alpha 2HS glycoprotein. The N-terminal 105 residues and a segment aligned with residues 170-225 of alpha 2HS glycoprotein revealed 109 of 161 residues to be identical between the two proteins (68% homology). Mouse polyclonal antibodies to fetuin, and trypsin digest fragments of this protein have been prepared and used for a comparison of native and digested proteins. Polyclonal antibodies to native protein showed little if any cross reactivity. However, antibodies to trypsin digest fragments of fetuin showed obvious cross reactivity with alpha 2HS.

Early cortical plate specific glycoprotein in a marsupial species belongs to the same family as fetuin and alpha 2HS glycoprotein.

Two related glycoproteins, fetuin in species of the order Artiodactyla (cattle, sheep, pig) and alpha 2HS glycoprotein in the human [(1987) Cell Tissue Res. 248, 33-41] have a very specific distribution in the developing brain. We have isolated and determined the first 15 N-terminal residues of a similarly distributed glycoprotein in the developing brain of the tammar wallaby (Macropus eugenii). The degree of homology is the same between wallaby glycoprotein and alpha 2HS glycoprotein as between fetuin and alpha 2HS glycoprotein (46%). Antibodies made to synthetic peptides of fetuin were used to identify the wallaby glycoprotein. A polyclonal antibody to the purified glycoprotein was used for immunocytochemical identification of brain cells positive for this protein.

Development of thalamocortical projections in the South American gray short-tailed opossum (Monodelphis domestica).

We determined the time-course and general pattern of thalamocortical development of Monodelphis domestica by tracing projections with carbocyanine dye in fixed postnatal brains between postnatal day 2 (P2) and P30. By P2, the first neurons have migrated to form the preplate of the lateral cortex and have sent out axons into the intermediate zone. By P3, fibers from the preplate of more dorsal cortex have entered the intermediate zone, and, by P5, they reach the primitive internal capsule. Crystal placements in the dorsal thalamus at P2-P3 reveal thalamic axons extending down through the diencephalon and growing out through the internal capsule among groups of back-labelled cells that already project into the thalamus. Thalamic axons arrive at the cortex after the arrival of cells of the true cortical plate has split the preplate into marginal zone and subplate. Axons from the ventral part of the dorsal thalamus reach the lateral cortex by P5: Dorsal thalamic fibers arrive at the extreme dorsal cortex by P9. The deeper layers of the cortex appear to mature relatively earlier in Monodelphis than in eutherian mammals, and the subplate becomes less distinct. Thalamic fibers and their side branches proceed into the cortex without an obvious period of waiting in the subplate, but they do not penetrate the dense cortical plate itself. Monodelphis could provide an excellent model species, because the development of its thalamocortical connections is entirely an extrauterine process: The period P0-P15 corresponds to that of E12-P0 in the rat.

Development of motoneurons and primary sensory afferents in the thoracic and lumbar spinal cord of the South American opossum Monodelphis domestica.

The postnatal development of the primary sensory afferent projection to the thoracic (T4) and lumbar (L4) spinal cord of the marsupial species Monodelphis domestica was studied by using anterograde and retrograde neuronal tracers. Large numbers of primary afferents and motoneurons were labelled by application of the carbocyanine dye DiI into individual dorsal root ganglia (DRG) afferents in short-term organ cultures. Dorsal root axons had entered the cord at birth, but most primary afferent innervation of the grey matter and the establishment of cytoarchitectural lamination occurs postnatally. In addition to ipsilateral projections, some primary afferents that projected to the dorsal horn extended across the midline into the equivalent contralateral regions of the grey matter. Similarly, motoneuron dendrites occasionally extended across midline and into the contralateral grey matter. The first fibres innervating the spinal cord project to the ventral horn and formed increasingly complex terminal arbours in the motor columns between P1 and P7. After P5 many afferents were seen projecting to the dorsal horn, with the superficial dorsal horn being the last region of the spinal grey to be innervated. Histochemical labelling with the lectin Griffonia simplicifolia indicated that C fibre primary afferents had arborised in the superficial dorsal horn by P14. The sequence of primary afferent innervation is thus similar to that described in the rat, but this sequence occurs over a period of several weeks in Monodelphis, compared with several days in the rat.

Fetuin in the developing neocortex of the rat: distribution and origin.

Immunocytochemical distribution of the fetal protein fetuin in the neocortex of developing rat brain and the presence of its mRNA, as detected by using reverse transcriptase-polymerase chain reaction analysis, was studied in fetuses at embryonic day 15 (E15) through E22, in neonates at postnatal day 0 (P0) through P20, and in adults. Quantitative estimates of fetuin in cerebrospinal fluid (CSF) and plasma were obtained over the same period. Exogenous (bovine) fetuin injected intraperitoneally into fetal and postnatal rats was used to study the uptake of fetuin into CSF and brain and its distribution compared with endogenous fetuin; bovine albumin was used as a control. Fetuin was identified immunocytochemically in the cortical plate and subplate cells of the developing neocortex. In the rat fetus, fetuin first was apparent at E17, mainly in cell processes, but a few subplate cells also were positive. By E18, there was strong staining in subplate neurons and in inner cells of the cortical plate. At E21, these inner cells of the cortical plate were beginning to differentiate into layer VI neurons, many of which were positive for fetuin. By P0-P1, more layer VI neurons and some layer V neurons had become positive for fetuin. Fetuin immunoreactivity generally was weaker at P1, and, by P2-P3, it had disappeared from all of the layers of the developing neocortex. Bovine fetuin (but not albumin), probably taken up through CSF over the neocortical dorsal surface, had a cytoplasmic distribution; endogenous rat fetuin was both cytoplasmic and membrane bound. Thus, much of this fetuin can be accounted for by uptake, although the presence of fetuin mRNA indicates that in situ synthesis may also contribute.

Regulation of GABAB receptors by histamine and neuronal activity in the isolated spinal cord of neonatal opossum in culture.

The aim of these experiments has been to analyse the properties of receptors for the transmitter gamma-aminobutyric acid (GABA) in developing mammalian nervous system. Changes in responses of GABAB receptors have been measured after alterations of the chemical environment and the level of electrical activity. We have previously shown that when the central nervous system (CNS) of the new-born opossum, Monodelphis domestica, is cultured for three to five days in the presence of histidine, inhibition by baclofen, a GABAB agonist, disappears (Stewart et al. 1991). We have now investigated whether histidine acts indirectly by way of conversion to histamine. As with histidine, culture with 150 microM histamine for five days virtually abolished the inhibition by baclofen. The effects of histidine, as well as histamine, were blocked by mepyramine, a histamine H1-receptor antagonist, and by ranitidine, an H2-antagonist. Tetrodotoxin (TTX), which blocks all electrical activity, protected preparations from the action of histidine but not histamine. Our results suggest that histidine is converted to histamine, which reduces the efficacy of GABAB agonists. We conclude that, in the developing mammalian CNS, transmitter levels and electrical activity can selectively influence the properties of receptors.

Growth of axons through a lesion in the intact CNS of fetal rat maintained in long-term culture.

The ability of neurons in the central nervous system (CNS) to grow through a lesion and restore conduction has been analysed in developing spinal cord in vitro. The preparation consists of the entire CNS of embryonic rat, isolated and maintained in culture. Conduction of electrical activity and normal morphological appearance (light microscopical and electron microscopical) were maintained in the spinal cord of such preparations for up to 7 d in culture. A complete transverse crush of the spinal cord abolished all conduction for 2 d. After 3-5 d, clear recovery had occurred: electrical conduction across the crush was comparable with that in uninjured preparations. Furthermore, the spinal cord had largely regained its gross normal appearance at the crush site. Axons stained in vivo by carbocyanine dyes had, by 5 d, grown in profusion through the lesion and several millimetres beyond it. These experiments, like those made in neonatal opossum (Treherne et al. 1992) demonstrate that central neurons of immature mammals, unlike those in adults, can respond to injury by rapid and extensive outgrowth of nerve fibres in the absence of peripheral nerve bridges or antibodies that neutralize inhibitory factors. However, unlike the opossum, in which outgrowth occurred at 24 degrees C, although there was prolonged survival of rat spinal cords at this temperature, outgrowth of axons across the lesion required a temperature of 29 degrees C. With rapid and reliable regeneration in vitro it becomes practicable to assay the effects of molecules that promote or inhibit restoration of functional connections.

Modification of macrophage response to lipopolysaccharide by fetuin.

The physiological role(s) of fetuin, a protein present in plasma and many tissues of developing animals at levels much higher than in the adult, is unknown. Here we show that fetuin can modify the responses of macrophages to lipopolysaccharide (LPS) stimulation. At concentrations of fetuin in the medium, corresponding to fetal levels of this protein in plasma, the production and secretion of interleukin 1beta (IL-1beta) and nitric oxide (NO) is almost abolished, tumour necrosis factor-alpha (TNF-alpha) reduced, while that of IL-6 is not affected. On the other hand, concentrations of fetuin corresponding to adult plasma levels (i.e. 40-60 mg/100 ml) were without much effect on macrophage synthesis and secretion of these cytokines.

Development of a clinical asthma score for use in hospitalized children between 1 and 5 years of age.

The objective of this study was to develop a clinical asthma score (CAS) for use in hospitalized children between 1 and 5 years of age. Formal approaches to item selection and reduction, reliability, discriminatory power, validity, and responsiveness were used. The final CAS consisted of five clinical characteristics: respiratory rate, wheezing, indrawing, observed dyspnea, and inspiratory-to-expiratory ratio. Interrater reliability was high (weighted kappa = 0.82), and the CAS was discriminatory (Ferguson's delta = 0.92). The CAS was valid, with a strong correlation with length of hospital stay (Spearman's correlation = 0.47, p < 0.05) and drug dosing interval (Spearman's correlation = -0.58, p < 0.01). The CAS was responsive, with a significant change in CAS from admission to discharge (Wilcoxon signed rank test, p < 0.01). This score, for use in hospitalized preschool children, is reliable, discriminatory, valid, and responsive.

Cyclosporin and bronchial healing in canine lung transplantation.

Long-term bronchial anastomotic healing has been assessed in the canine lung transplant model with cyclosporin as the primary immunosuppressant. Early bronchial revascularization was achieved by wrapping an omental pedicle around the bronchial anastomosis. Ten dogs underwent left lung transplantation and six survived 100 days or more before being put to death. No significant bronchial complications occurred. Late bronchostenosis was not seen, despite four biopsy-proved rejection episodes in three of the dogs surviving past 100 days. Histologically, all anastomoses were well healed at autopsy. Cyclosporin was shown to be an effective immunosuppressant in this model and was associated with prolonged survival and low morbidity. Transplant lung function was assessed at 100 days by contralateral pulmonary artery ligation in five dogs and was satisfactory in the three animals that had not had rejection episodes. The findings support our belief that bronchial anastomotic complications after human lung transplantation are mainly related to the effects of immunosuppression with steroids and to the ischemia resulting from division of the bronchial circulation at the time of transplantation.

Repair of connections in injured neonatal and embryonic spinal cord in vitro.

A remarkable preparation for studying development and repair is the CNS of the newborn opossum which, removed in its entirety, survives in culture for more than 1 week. In suitable medium, cells continue to divide, mature and reflex activity is maintained. Moreover, nerve fibers grow rapidly, reliably and extensively across lesions made in the spinal cord. Restoration of conduction has been demonstrated by recording electrically; labeled fibres have been observed directly by light and electron microscopy as they traverse the lesion. Similar experiments have also been made in embryonic (E15) rat CNS in culture. Open questions concern the identity of the fibers that traverse the lesion and the specificity of connections that they make with targets. We are now also analysing mechanisms that favor repair in younger opossums and that prevent it in their older siblings. Of particular interest are oligodendrocytes and myelin that start to appear at about 8-9 days after birth.

Development of the choroid plexus.

Mammalian choroid plexuses develop at four sites in the roof of the neural tube shortly after its closure, in the order IVth, lateral, and IIIrd ventricles. Bone morphogenetic proteins and tropomyosin are involved in early specification of these sites and in early plexus growth. Four stages of lateral ventricular plexus development have been defined, based on human and sheep fetuses; these depend mainly on the appearance of epithelial cells and presence or absence of glycogen. Other plexuses and other species are probably similar, although marsupials may lack glycogen. Choroid plexuses form one of the blood-brain barrier interfaces that control the brain's internal environment. The mechanisms involved combine a structural diffusion restraint (tight junctions between the plexus epithelial cells) and specific exchange mechanisms. In this review, it is argued that barrier mechanisms in the developing brain are different in important respects from those in the adult brain, but these differences do not necessarily reflect immaturity of the system. Absence of a barrier mechanism or presence of one not found in the adult may be a specialisation that is appropriate for that stage of brain development. Emphasis is placed on determining which mechanisms are present in the immature brain and relating them to brain development. One mechanism unique to the developing brain transfers specific proteins from blood to cerebrospinal fluid (CSF), via tubulocisternal endoplasmic reticulum in plexus epithelial cells. This results in a high concentration of proteins in early CSF. These proteins do not penetrate into brain extracellular space because of "strap" junctions between adjacent neuroependymal cells, which disappear later in development, when the protein concentration in CSF is much lower. Functions of the proteins in early CSF are discussed in terms of generation of a "colloid" osmotic pressure that expands the ventricular system as the brain grows; the proteins may also act as specific carriers and growth factors in their own right. The pathway for low molecular weight compounds, which is much more permeable in the developing choroid plexuses, appears also to be a transcellular one, rather than paracellular via tight junctions. There is thus good evidence to support a novel view of the state of development and functional significance of barrier mechanisms in the immature brain. It grows in an environment that is different from that of the rest of the fetus/neonate and that is also different in some respects from that of the adult. But these differences reflect developmental specialisation rather than immaturity.

Contribution of circulating formed elements to prostanoid production in complement-mediated lung injury in sheep.

Incubation of plasma with zymosan results in complement activation. Infusion of this "zymosan-activated plasma" (ZAP) into the superior vena cava in sheep results in pulmonary leukostasis, pulmonary hypertension with generation of thromboxane (TXB), and hypoxemia. To examine the contribution of circulating formed elements to TXB generated when ZAP is infused, we studied four pairs of sheep in a cross-circulation model. Acetylsalicylic acid (ASA) irreversibly acetylates cyclooxygenase and blocks TXB production. Four hours after an intravenous dose of ASA (10 mg/kg), no circulating ASA was detectable by high-pressure liquid chromatography. Infusion of ZAP in ASA-treated animals resulted in no pulmonary hypertension and no rise in serum TXB levels. Treated animals were then cross-circulated with untreated sheep through cannulas that attached a carotid artery of each animal to a jugular vein of the other, creating simultaneous arteriovenous fistulas between the animals. A roller pump maintained cross-circulation flow at 450 ml/min for 15 minutes. This resulted in greater than 80% mixing of the circulating blood volumes of each pair of animals, verified by measurement of an intravascular marker. After cross-circulation the ASA-treated animals were still unable to generate TXB in response to ZAP infusion. Untreated animals had a typical response when infused with ZAP after cross-circulation, with elaboration of large amounts of TXB. Since the treated animals with circulating, nonaspirinated formed elements showed no response and the untreated animals with similar blood composition had a normal response, we conclude that circulating formed elements do not contribute significantly to the TXB recovered from sheep infused with activated complement components.