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Neuroinflammation and dysfunction of the blood–brain barrier (BBB) are two prominent mechanisms of secondary injury in neurotrauma. It has been suggested that Toll-like receptors (TLRs) play important roles in initiating and propagating neuroinflammation resulting from traumatic brain injury (TBI), but potential beneficial effects of targeting these receptors in TBI have not been broadly studied. Here, we investigated the effect of targeting TLRs with proteoglycan 4 (PRG4) on post-traumatic neuroinflammation and BBB function. PRG4 is a mucinous glycoprotein with strong anti-inflammatory properties, exerting its biological effects by interfering with TLR2/4 signaling. In addition, PRG4 has the ability to inhibit activation of cluster of differentiation 44 (CD44), a cell-surface glycoprotein playing an important role in inflammation. Using the controlled cortical impact model of TBI in rats, we showed a rapid and prolonged upregulation of message for TLR2/4 and CD44 in the injured cortex. In the in vitro model of the BBB, recombinant human PRG4 (rhPRG4) crossed the endothelial monolayers through a high-capacity, saturable transport system. In rats sustaining TBI, PRG4 delivery to the brain was enhanced by post-traumatic increase in BBB permeability. rhPRG4 injected intravenously at 1 h post-TBI potently inhibited post-traumatic activation of nuclear factor kappa B and extracellular signal-regulated kinases 1/2, the two major signal transduction pathways associated with TLR2/4 and CD44, and curtailed the post-traumatic influx of monocytes. In addition, PRG4 restored normal BBB function after TBI by preventing the post-traumatic loss of tight junction protein claudin 5 and reduced neuronal death. Our observations provide support for therapeutic strategies targeting TLRs in TBI.

This article highlights the scientific achievements, professional career, and personal interactions of Malcolm B. Segal who passed away in July this year. Born in 1937 in Goodmayes, Essex, UK, Segal rose to the Chairman position in the Division of Physiology at United Medical and Dental School of Guy’s and St. Thomas’ Hospitals, retiring in 2006 after his long professional career in biomedical science. Being trained in Hugh Davson’s laboratory, Segal became one of the pioneers in research on cerebrospinal fluid physiology and the choroid plexus. During the course of his career, Segal himself trained a number of young scientists and collaborated with many colleagues around the world, making long-lasting friendships along the way. In addition to his professional accomplishments as a researcher and educator, Segal was an avid sailor and wine connoisseur, and enjoyed teaching classes on navigation and wine tasting.

Background and objective Matrix metalloproteinases (MMPs) mediate blood–brain barrier dysfunction in inflammatory disease states. Our objective was to compare circulating MMPs in children with diabetic ketoacidosis (DKA) to children with type 1 diabetes mellitus without DKA. Research design and methods This was a prospective study performed at five tertiary-care pediatric hospitals. We measured plasma MMP-2, MMP-3, and MMP-9 early during DKA (time 1; within 2 h of beginning intravenous fluids) and during therapy (time 2; median 8 h; range: 4–16 h). The primary outcome was MMP levels in 34 children with DKA vs. 23 children with type 1 diabetes without DKA. Secondary outcomes included correlations between MMPs and measures of DKA severity. Results In children with DKA compared with diabetes controls, circulating MMP-2 levels were lower (mean 77 vs. 244 ng/mL, p

Brain-derived neurotrophic factor (BDNF), a key player in regulating synaptic strength and learning, is dysregulated following traumatic brain injury (TBI), suggesting that stimulation of BDNF signaling pathways may facilitate functional recovery. This study investigates whether CN2097, a peptidomimetic ligand which targets the synaptic scaffold protein, postsynaptic density protein 95, to enhance downstream signaling of tropomyosin-related kinase B, a receptor for BDNF, can improve neurological function after TBI. Moderate to severe TBI elicits neuroinflammation and c-Jun-N-terminal kinase (JNK) activation, which is associated with memory deficits. Here we demonstrate that CN2097 significantly reduces the post-traumatic synthesis of proinflammatory mediators and inhibits the post-traumatic activation of JNK in a rodent model of TBI. The recordings of field excitatory post-synaptic potentials in the hippocampal CA1 subfield demonstrate that TBI inhibits the expression of long-term potentiation (LTP) evoked by high-frequency stimulation of Schaffer collaterals, and that CN2097 attenuates this LTP impairment. Lastly, we demonstrate that CN2097 significantly improves the complex auditory processing deficits, which are impaired after injury. The multifunctionality of CN2097 strongly suggests that CN2097 could be highly efficacious in targeting complex secondary injury processes resulting from neurotrauma.

The blood-brain barrier (BBB) is formed by tightly connected cerebrovascular endothelial cells, but its normal function also depends on paracrine interactions between the brain endothelium and closely located glia. There is a growing consensus that brain injury, whether it is ischemic, hemorrhagic, or traumatic, leads to dysfunction of the BBB. Changes in BBB function observed after injury are thought to contribute to the loss of neural tissue and to affect the response to neuroprotective drugs. New discoveries suggest that considering the entire gliovascular unit, rather than the BBB alone, will expand our understanding of the cellular and molecular responses to traumatic brain injury (TBI). This review will address the BBB breakdown in TBI, the role of blood-borne factors in affecting the function of the gliovascular unit, changes in BBB permeability and post-traumatic edema formation, and the major pathophysiological factors associated with TBI that may contribute to post-traumatic dysfunction of the BBB. The key role of neuroinflammation and the possible effect of injury on transport mechanisms at the BBB will also be described. Finally, the potential role of the BBB as a target for therapeutic intervention through restoration of normal BBB function after injury and/or by harnessing the cerebrovascular endothelium to produce neurotrophic growth factors will be discussed.

The invasion of inflammatory cells occurring after ischemic or traumatic brain injury (TBI) has a detrimental effect on neuronal survival and functional recovery after injury. We have recently demonstrated that not only the blood-brain barrier, but also the blood-cerebrospinal fluid (CSF) barrier (BCSFB), has a role in posttraumatic recruitment of neutrophils. Here, we show that TBI results in a rapid increase in synthesis and release into the CSF of a major chemoattractant for monocytes, CCL2, by the choroid plexus epithelium, a site of the BCSFB. Using an in vitro model of the BCSFB, we also show that CCL2 is released across the apical and basolateral membranes of the choroidal epithelium, a pattern of chemokine secretion that promotes leukocyte migration across epithelial barriers. Immunohistochemical and electron microscopic analyses of choroidal tissue provide evidence for the movement of monocytes, sometimes in tandem with neutrophils, along the paracellular pathways between adjacent epithelial cells. These data further support the pathophysiological role of BCSFB in promoting the recruitment of inflammatory cells to the injured brain.

Previous studies have indicated that the primary targets for vasopressin actions on the injured brain are the cerebrovascular endothelium and astrocytes, and that vasopressin amplifies the posttraumatic production of proinflammatory mediators. Here, the controlled cortical impact model of traumatic brain injury in rats was used to identify the sources of vasopressin in the injured brain. Injury increased vasopressin synthesis in the hypothalamus and cerebral cortex adjacent to the posttraumatic lesion. In the cortex, vasopressin was predominantly produced by activated microglia/macrophages, and, to a lesser extent, by the cerebrovascular endothelium. These data further support the pathophysiological role of vasopressin in brain injury.

The choroid plexus (CP) epithelium is one of the extrahypothalamic sources of arginine vasopressin (AVP). However, it is unclear whether the regulation of choroidal AVP synthesis in response to pathophysiological stimuli, such as hyperosmotic stress, is similar to that observed in the hypothalamus. In the present study, rats chronically implanted with cisterna magna cannulas, enabling the collection of cerebrospinal fluid (CSF) in freely moving animals, were subjected to salt loading. CSF osmolality increased from the baseline normonatremic levels ranging between 292 ± 0.5 and 295 ± 2 to 309 ± 4 mosm/kg H2O at 2 days of hypernatremia. This elevated CSF osmolality was maintained at a relatively stable level until the end of a 10-day observation period. Changes in choroidal and hypothalamic AVP expression in response to hyperosmotic stress were assessed by semiquantitative reverse-transcriptase polymerase chain reaction. An increase in hypothalamic AVP expression was accompanied by augmented AVP synthesis in the CP. Compared to normonatremia, choroidal levels of AVP mRNA increased 5- and 10-fold at 2 and 5 days of salt loading, respectively. Salt loading also resulted in increased hypothalamic expression of the α-II, β1, and β2 subunits of voltage-gated Na+ channels. Similarly, the choroidal mRNA levels for the α-II and β1 subunits increased ∼2-fold after 5 days of salt loading; however, no changes in the β2 subunit expression were found in the CPs of hypernatremic rats. These experiments support the hypothesis that the regulation of choroidal AVP synthesis is similar to that observed in the hypothalamus. It is also suggested that the increased expression of voltage-gated Na+ channels found in the hypothalamus and CP after salt loading may play a role in the adaptation of AVP-producing cells to chronic hypernatremia.

In a number of stress conditions, the biological effects of tumor necrosis factor-alpha (TNF-alpha), such as the induction of neuronal apoptosis, are presumably attenuated by the soluble fragments of TNF receptors (sTNFRs). Within 1 h after spinal cord injury, increased synthesis and/or secretion of TNF-alpha is detectable at the injury site. However, the shedding of ectodomains of TNFRs in the traumatized spinal cord has not yet been reported. In the present study, adult Sprague-Dawley rats were subjected to acute spinal cord injury (ASCI) by applying a 25-g Walsh-Tator aneurysm clip at the C8-T1 level. Sham-injured animals underwent laminectomy and facetectomy only. A PE10 catheter was placed in the subarachnoid space to collect the samples of cerebrospinal fluid (CSF) from near the injury site. These CSF samples were analyzed by ELISA for the presence of TNF-alpha and soluble TNFR1 and TNFR2 (sTNFR1 and sTNFR2, respectively). The spinal cord tissue was analyzed by immunohistochemistry for the expression of TNF-alpha, TNFR1, and TNFR2, and by the TUNEL technique for the occurrence of neuronal death. The levels of TNFR1 and sTNFR1 in the injured tissue were determined by Western blotting. Immunohistochemistry demonstrated the increased neuronal expression of TNF-alpha and its receptors at 6 h post-ASCI. No changes in the intensity of staining were observed in the sham-injured rats. In addition, at 6 h after the injury, a significant increase in the number of TUNEL-positive neurons was observed. Numerous neurons in traumatized tissue were also immunoreactive for activated caspase-3, suggesting that the TUNEL-positive neurons were undergoing an apoptotic death. At 1 h after ASCI, TNF-alpha levels in the CSF were significantly higher than those found in the sham-injured animals, indicating the release of this cytokine into the interstitial fluid. This was followed by a significant increase, compared to the sham-injured controls, in sTNFR1 levels in the CSF at 3 and 6 h after the insult. Unlike sTNFR1, the levels of sTNFR2 in the CSF were unchanged at any time point post-ASCI. The increased shedding of TNFR1 was confirmed by Western blotting. It is concluded that the shedding of TNFR1 receptor may represent an important post-traumatic physiological response aimed at reducing the proapoptotic effect of TNF-alpha.

In addition to being the major site of cerebrospinal fluid formation, the choroid plexus epithelium emerges as an important source of polypeptides in the brain. Physiologically regulated release of some polypeptides synthesized by the choroid plexus has been shown. The molecular mechanisms underlying this polypeptide secretion have not been characterized, however. In the present study, synaptosome-associated protein of 25 kDa and vesicle-associated membrane protein, two membrane fusion proteins playing a critical role in exocytosis in neurons and endocrine cells, were found to be expressed in the choroid plexus epithelium. It was also shown that in choroidal epithelium, synaptosome-associated protein of 25 kDa and vesicle-associated membrane protein stably interact. Two members of the vesicle-associated membrane protein family, vesicle-associated membrane protein-1 and vesicle-associated membrane protein-2, were expressed in the rat choroid plexus at the messenger RNA and protein level. However, their newly discovered isoforms, vesicle-associated membrane protein-1b and vesicle-associated membrane protein-2b, produced by alternative RNA splicing, were not detected in choroidal tissue. Immunohistochemistry demonstrated that vesicle-associated membrane protein is confined to the cytoplasm of choroidal epithelium, whereas synaptosome-associated protein of 25 kDa is associated with plasma membranes, albeit with a varied cellular distribution among species studied. Specifically, in the rat choroid plexus, synaptosome-associated protein of 25 kDa was localized to the basolateral membrane domain of choroidal epithelium and was expressed in small groups of cells. In comparison, in ovine and human choroidal tissues, apical staining for synaptosome-associated protein of 25 kDa was found in the majority of epithelial cells. These species-related differences in cellular synaptosome-associated protein of 25 kDa distribution suggested that the synaptosome-associated protein of 25 kDa homologue, synaptosome-associated protein of 23 kDa, is also expressed in the rat choroid plexus, which was confirmed by reverse-transcriptase polymerase chain reaction. Our findings suggest that synaptosome-associated protein of 25 kDa and vesicle-associated membrane protein are involved in secretion of polypeptides from the choroid plexus epithelium. The presence of synaptosome-associated protein of 25 kDa and its homologue as well as multiple isoforms of vesicle-associated membrane protein in choroidal epithelium may play a role in the apical versus basolateral targeting of secretory vesicles.

International audience; In June 2014 Dr. Joseph D. Fenstermacher celebrated his 80th birthday, which was honored by the symposium held in New London, NH, USA. This review discusses Fenstermacher's contribution to the field of fluids and barriers of the CNS. Specifically, his fundamental work on diffusion of molecules within the brain extracellular space and the research on properties of the blood-brain barrier in health and disease are described. Fenstermacher's early research on cerebrospinal fluid dynamics and the regulation of cerebral blood flow is also reviewed, followed by the discussion of his more recent work involving the use of magnetic resonance imaging.

This article provides an overview of the Brown University Traumatic Brain Injury Research Consortium (TBIRC) and summarizes the multidisciplinary basic and clinical neuroscience work being conducted by investigators at Brown University and the affiliate hospitals in association with the Norman Prince Neurosciences Institute (NPNI).

Upregulation of certain growth factors in the central nervous system can alter brain fluid dynamics. Hydrocephalus was produced in adult Sprague-Dawley rats by infusing recombinant basic fibroblast growth factor (FGF-2) at 1 μg/day into a lateral ventricle for 2, 3, 5, or 10–12 days. Lateral and third ventricular enlargement progressively increased from 2 to 10 days. Ventriculomegaly was also induced by a 75% reduced dose of FGF-2. At 10–12 days, there was a 29% attenuation in cerebrospinal fluid (CSF) formation rate, from 2.5 to 1.8 μl/min ( P < 0.01). Choroid plexus, the main site of CSF secretion, had an augmented number of dark epithelial cells, which have previously been associated with decreased choroidal fluid formation. The twofold elevated resistance to CSF absorption, i.e., 0.8 to 1.7 mmHg ⋅ min−1 ⋅ μl−1, was attributable, at least in part, to enhanced fibrosis and collagen deposits in the arachnoid villi, a major site for CSF absorption. Normal CSF pressure (2–3 mmHg) was consistent with a patent cerebral aqueduct and reduced CSF formation rate. The FGF-2-induced ventriculomegaly is interpreted as an ex vacuuo hydrocephalus brought about by an altered neuropil and interstitium of the brain.

Despite significant advances in designing neuroprotective therapies, multiple clinical trials in head trauma and spinal cord injury have produced mixed results. A better understanding of the pathophysiology of central nervous system (CNS) barriers may improve clinical translation of therapies for injury of the CNS. The blood–brain barrier and blood-spinal cord barrier are formed by specialized CNS endothelial cells. These barriers play a fundamental role in restricting the entry of blood-borne factors into the CNS. However, they also function as the gateway for the delivery of neuroprotective drugs. Neurotrauma changes the properties of CNS barriers, which may significantly affect the efficacy of neuroprotective therapies. The endothelial barriers of the CNS together with the blood-cerebrospinal fluid barrier, which is predominantly formed by the epithelial cells of the choroid plexus, also restrict the influx of circulating leukocytes into the CNS. Dysfunction of these barriers resulting from injury plays a key role in the initiation and progression of inflammation that accompanies neurotrauma.

Several lines of evidence indicate that the blood-cerebrospinal fluid barrier (BCSFB), which primarily resides in the choroid plexus (CP), plays a significant pathophysiological role not only in neuroinflammatory diseases, such as multiple sclerosis, but also in traumatic brain injury (TBI). Here we investigated how arginine vasopressin (AVP) regulates function of the BCSFB in the context of post-traumatic neuroinflammation. It has previously been shown that AVP exacerbates various forms of brain injury, but the mechanisms underlying this AVP action are poorly understood. Type 1A AVP receptor is highly expressed on the CP epithelium and the CP synthesizes AVP. Using the controlled cortical impact model of TBI, we demonstrated decreased post-traumatic production of proinflammatory mediators by the CP and reduced influx of inflammatory cells across the BCSFB in AVP-deficient Brattleboro rats when compared with Long-Evans rats, a parental strain for Brattleboro rats. Arginine vasopressin was also found to play an important role in post-traumatic activation of c-Jun N-terminal kinase (JNK) in the CP. In the CP epithelial cell cultures, AVP augmented the tumor necrosis factor-α– and interleukin-1β–dependent increase in synthesis of proinflammatory mediators, including neutrophil chemoattractants, an action largely dependent on the JNK signaling pathway. Under in vivo conditions, a selective JNK inhibitor decreased the post-traumatic production of neutrophil chemoattractants by the CP and reduced the influx of neutrophils across the BCSFB. These results provide evidence for the synergistic interactions between proinflammatory cytokines and AVP, a ligand for G protein-coupled receptors, and support a pathophysiological role of AVP in post-traumatic neuroinflammation.

Nitric oxide (NO) has recently been shown to regulate blood flow to choroid plexus, a specialized brain structure responsible for production of most of cerebrospinal fluid. In the present study, we used a specific polyclonal rabbit antibody against the neuronal isoform of NO synthase (NOS), a synthetic enzyme for NO, to determine the localization of NOS in the choroid plexus of adult male Sprague-Dawley rats. NOS-containing nerve fibers were found in the anterior choroidal artery and its branches, and in stromal blood microvessels. Chronic denervation experiments indicated that these nerve fibers originate predominantly from the sphenopalatine ganglion. NOS-immunopositive staining was also detected in the cytoplasm of choroidal epithelial cells. NADPH-diaphorase, a histochemical marker for NOS, was found to colocalize with NOS-immunoreactive product in both nerve fibers and choroidal epithelium. Both neuronal and epithelium-derived NO may regulate secretory function and hemodynamics of choroidal tissue.

Choroid plexus is the major source of cerebrospinal fluid. The hemodynamics and secretory function of this tissue are controlled by multiple endocrine and neural mechanisms. Nitric oxide (NO) has been demonstrated to play an important role in regulating choroidal blood flow. In the present study, performed on adult male Sprague-Dawley rats, we employed a NADPH-diaphorase (NADPH-d) histochemical method to localize nitrergic innervation of choroidal blood vessels. This approach was based on previous observations that NADPH-d colocalizes with NO synthase, a synthetic enzyme for NO, in the central and peripheral nervous systems. NADPH-d-positive nerve fibers were found to accompany both large arteries and veins and blood microvessels (possibly arterioles) located in choroidal stroma. NADPH-d reaction product was also localized to the vascular endothelial lining and choroidal epithelial cells. All the above sources of NO may play important roles in the regulation of secretory and hemodynamic functions of the choroid plexus.

The effect of peripherally administered angiotensin II (AII) on blood flow to choroid plexuses was examined in pentobarbital-anesthetized rats. The indicator fractionation method with 123I- or 125I- N-isopropyl- p-iodoamphetamine as the marker was employed to measure blood flow. Basal blood flow to choroid plexus of the lateral cerebral ventricle (LVCP) (3.19 ± 0.23 ml g−1 min−1) was lower than that to choroid plexuses of the third (3VCP) and fourth (4VCP) ventricles (3.90 ± 0.38 and 3.95 ± 0.36 ml g−1 min−1, respectively). The effect of AII on choroidal blood flow varied depending on peptide dose and anatomical location of the choroidal tissue. AII infused intravenously at rates of 30 and 50 ng kg−1 min−1 decreased blood flow to both LVCP and 4VCP by 12–20%. Both lower (10 ng kg−1 min−1) and higher (100 and 300 ng kg−1 min−1) AII doses did not alter blood flow to LVCP and 4VCP. Blood flow to the 3VCP was not affected by any dose of the peptide used. In comparison, blood flow to cerebral cortex increased by 33% during intravenous AII infusion at a rate of 300 ng kg−1 min−1. The choroidal blood flow-lowering effect of moderate AII doses was abolished by both AT1 (losartan) and AT2 (PD 123319) receptor subtype antagonists (3 mg kg−1 i.v.). To determine whether the hemodynamic changes observed in choroid plexuses with moderate AII doses influence CSF formation, the ventriculocisternal perfusion was performed in rats (under the experimental conditions described) with Blue Dextran 2000 as the indicator. CSF production was not altered during intravenous infusion of AII at a rate of 30 ng kg−1 min−1. It is suggested that CSF formation is maintained in pathophysiological situations accompanied by increased plasma AII levels, which implicates a potential role for AII in regulating ion and water balance in the CNS.

Increasing evidence indicates that basic fibroblast growth factor (FGF2), a well-known mitogen, can regulate the synthesis and secretion of peptide hormones. FGF2 has also been recently shown to inhibit cerebrospinal fluid (CSF) formation and increase the number of dark choroid plexus epithelial cells. These latter FGF2 actions could be mediated by vasopressin (VP) synthesized by and released from choroidal epithelium. The present study was therefore designed to determine whether VP co-localizes with fibroblast growth factor receptors (FGFRs) in the choroid plexus epithelium. Using confocal laser-scanning microscopy, we demonstrated the apical (CSF-facing) distribution of FGFRs in epithelial cells. FGFRs were expressed on clusters of VP-positive cells, with the intensity of FGFR-immunopositive staining varying from one group of cells to the other. These observations are in line with the possible mediatory role of VP in the FGF2-dependent inhibition of CSF secretion and the induction of dark epithelial cells.

A complex set of molecular and functional reactions is set into motion by traumatic brain injury (TBI). New research that extends beyond pathological effects on neurons suggests a key role for the blood-brain barrier, neurovascular unit, arginine vasopressin, and neuroinflammation in the pathophysiology of TBI. The prevalence of molecular derangements in TBI holds promise for the identification and use of biomarkers to assess severity of injury, determine prognosis, and perhaps direct therapy. Hopefully, improved knowledge of these elements of pathophysiology will provide the mechanistic clues that lead to improved treatment of TBI. Language: en

Arginine vasopressin (AVP) has previously been shown to promote disruption of the blood-brain barrier, exacerbate edema, and augment the loss of neural tissue in various forms and models of brain injury. However, the mechanisms underlying these AVP actions are not well understood. These mechanisms were studied in AVP-deficient Brattleboro rats (Avp(di/di)), and their parental Long-Evans strain, using a controlled cortical impact model of traumatic brain injury (TBI). The increased influx of inflammatory cells into the injured cortex in wild-type versus Avp(di/di) rats was associated with higher levels of cortical synthesis of the CXC and CC chemokines found in wild-type versus Avp(di/di) rats. These chemokines were predominantly produced by the cerebrovascular endothelium and astrocytes. In astrocyte and brain endothelial cell cultures, AVP acted synergistically with tumor necrosis factor-alpha (TNF-alpha) to increase the TNF-alpha-dependent production of CXC and CC chemokines. These AVP actions were mediated by c-Jun N-terminal kinase (JNK), as shown by Western blotting and pharmacological inhibition of JNK activity. The activity of JNK was increased in response to injury, and the differences in the magnitude of its post-traumatic activation between Avp(di/di) and wild-type rats were observed. These data demonstrate that AVP plays an important role in exacerbating the brain inflammatory response to injury.

Traumatic brain injury (TBI) frequently results in neuroinflammation, which includes the invasion of neutrophils. After TBI, neutrophils infiltrate the choroid plexus (CP), a site of the blood—cerebrospinal fluid (CSF) barrier (BCSFB), and accumulate in the CSF space near the injury, from where these inflammatory cells may migrate to brain parenchyma. We have hypothesized that the CP functions as an entry point for neutrophils to invade the injured brain. Using the controlled cortical impact model of TBI in rats and an in vitro model of the BCSFB, we show that the CP produces CXC chemokines, such as cytokine-induced neutrophil chemoattractant (CINC)-1 or CXCL1, CINC-2α or CXCL3, and CINC-3 or CXCL2. These chemokines are secreted both apically and basolaterally from the choroidal epithelium, a prerequisite for neutrophil migration across epithelial barriers. Consistent with these findings, we also provide electron microscopic evidence that neutrophils infiltrate the choroidal stroma and subsequently reach the intercellular space between choroidal epithelial cells. This is the first detailed analysis of the BCSFB function related to neutrophil trafficking. Our observations support the role of this barrier in posttraumatic neutrophil invasion.

Background: GPR125 belongs to the family of Adhesion G protein-coupled receptors (GPCRs). A single copy of GPR125 was found in many vertebrate genomes. We also identified a Drosophila sequence, DmCG15744, which shares a common ancestor with the entire Group III of Adhesion GPCRs, and also contains Ig, LRR and HBD domains which were observed in mammalian GPR125.Results: We found specific expression of GPR125 in cells of the choroid plexus using in situ hybridization and protein-specific antibodies and combined in situ/immunohistochemistry co-localization using cytokeratin, a marker specific for epithelial cells. Induction of inflammation by LPS did not change GPR125 expression. However, GPR125 expression was transiently increased ( almost 2-fold) at 4 h after traumatic brain injury (TBI) followed by a decrease ( approximately 4-fold) from 2 days onwards in the choroid plexus as well as increased expression ( 2-fold) in the hippocampus that was delayed until 1 day after injury.Conclusion: These findings suggest that GPR125 plays a functional role in choroidal and hippocampal response to injury., The authors wish to thank Anders Hellström and Julie Sarri for technical assistance. ML was supported by Hjärnfonden (Brain Research Foundation). Studies were supported by grants from the Swedish Research Foundation and Novo Nordisk to HBS and TBI studies were supported by grant NS-49479 from the NIH to AC. JAP studies were supported by grant POCTI/SAU-NEU/56618/2004 from FEDER/Portuguese Foundation for Science and Technology (FCT). FM has a PhD fellowship from FCT., info:eu-repo/semantics/publishedVersion

To find out whether central arginine vasopressin (AVP) plays a role in regulation of cerebrospinal fluid (CSF) composition and of CSF bulk flow (CSFbf) in acute hyperosmolar state, the experiments were performed on 16 anesthetized cats in which the ventriculocisternal system was perfused either with artificial CSF (aCSF; 8 control experiments) or with aCSF containing AVP (10 microU/min, 8 experiments) before, during, and after intravenous infusion of 5% NaCl. In both series, hyperosmotic infusion significantly increased CSF osmolality (CSFosmol) and sodium (CSFNa) and potassium (CSFK) concentration and reduced CSFbf. Administration of AVP significantly influenced the dynamics of changes in CSFNa and slightly affected changes in CSFK concentration induced by infusion of hyperosmotic saline, while it did not modify changes in CSFosmol) and CSFbf. In control experiments, CSFbf was significantly related to plasma and CSF osmolalities and sodium concentrations, whereas in AVP experiments these relationships were markedly suppressed. The results suggest that during acute hyperosmolar state AVP significantly influences sodium handling in CSF and may play a role in adaptation of the brain to hyperosmolality.

The choroid plexus (CP) epithelium is one of the extrahypothalamic sources of arginine vasopressin (AVP). However, it is unclear whether the regulation of choroidal AVP synthesis in response to pathophysiological stimuli, such as hyperosmotic stress, is similar to that observed in the hypothalamus. In the present study, rats chronically implanted with cisterna magna cannulas, enabling the collection of cerebrospinal fluid (CSF) in freely moving animals, were subjected to salt loading. CSF osmolality increased from the baseline normonatremic levels ranging between 292 +/- 0.5 and 295 +/- 2 to 309 +/- 4 mosm/kg H(2)O at 2 days of hypernatremia. This elevated CSF osmolality was maintained at a relatively stable level until the end of a 10-day observation period. Changes in choroidal and hypothalamic AVP expression in response to hyperosmotic stress were assessed by semiquantitative reverse-transcriptase polymerase chain reaction. An increase in hypothalamic AVP expression was accompanied by augmented AVP synthesis in the CP. Compared to normonatremia, choroidal levels of AVP mRNA increased 5- and 10-fold at 2 and 5 days of salt loading, respectively. Salt loading also resulted in increased hypothalamic expression of the alpha-II, beta(1), and beta(2) subunits of voltage-gated Na(+) channels. Similarly, the choroidal mRNA levels for the alpha-II and beta(1) subunits increased approximately 2-fold after 5 days of salt loading; however, no changes in the beta(2) subunit expression were found in the CPs of hypernatremic rats. These experiments support the hypothesis that the regulation of choroidal AVP synthesis is similar to that observed in the hypothalamus. It is also suggested that the increased expression of voltage-gated Na(+) channels found in the hypothalamus and CP after salt loading may play a role in the adaptation of AVP-producing cells to chronic hypernatremia.

The function of the cerebrospinal fluid (CSF) and the tissue that secretes it, the choroid plexus (CP), has traditionally been thought of as both providing physical protection to the brain through buoyancy and facilitating the removal of brain metabolites through the bulk drainage of CSF. More recent studies suggest, however, that the CP-CSF system plays a much more active role in the development, homeostasis, and repair of the central nervous system (CNS). The highly specialized choroidal tissue synthesizes trophic and angiogenic factors, chemorepellents, and carrier proteins, and is strategically positioned within the ventricular cavities to supply the CNS with these biologically active substances. Through polarized transport systems and receptor-mediated transcytosis across the choroidal epithelium, the CP, a part of the blood-CSF barrier (BCSFB), controls the entry of nutrients, such as amino acids and nucleosides, and peptide hormones, such as leptin and prolactin, from the periphery into the brain. The CP also plays an important role in the clearance of toxins and drugs. During CNS development, CP-derived growth factors, such as members of the transforming growth factor-beta superfamily and retinoic acid, play an important role in controlling the patterning of neuronal differentiation in various brain regions. In the adult CNS, the CP appears to be critically involved in neuronal repair processes and the restoration of the brain microenvironment after traumatic and ischemic brain injury. Furthermore, recent studies suggest that the CP acts as a nursery for neuronal and astrocytic progenitor cells. The advancement of our knowledge of the neuroprotective capabilities of the CP may therefore facilitate the development of novel therapies for ischemic stroke and traumatic brain injury. In the later stages of life, the CP-CSF axis shows a decline in all aspects of its function, including CSF secretion and protein synthesis, which may in themselves increase the risk for development of late-life diseases, such as normal pressure hydrocephalus and Alzheimer's disease. The understanding of the mechanisms that underlie the dysfunction of the CP-CSF system in the elderly may help discover the treatments needed to reverse the negative effects of aging that lead to global CNS failure.

ONTOGENESIS AND MORPHOLOGY OF THE CHOROID PLEXUS Development of the Blood-Cerebrospinal Fluid Barrier, C. Joakim Ek, Katarzyna M. Dziegielewska, and Norman R. Saunders Normal and Pathologically Altered Structures of the Choroid Plexus, Lise Prescott, and Milton W. Brightman Junctional Proteins of the Blood-CSF Barrier, Susanne Angelow, and Hans-Joachim Galla PHYSIOLOGY AND MOLECULAR BIOLOGY OF THE BLOOD-CSF BARRIER Fluid Compartments of the Central Nervous System, Malcolm B. Segal Peptide-Mediated Regulation of CSF Formation and Blood Flow to the Choroid Plexus, Joanna Szmydynger-Chodobska and Adam Chodobski Ion Transporters and Channels Involved in CSF Formation. Peter D. Brown, Tracey Speake, Sarah L. Davies, and Ian D. Millar Molecular Basis of Xenobiotic Transport and Metabolism in Choroid Plexus, David S. Miller, Simon Lowes, and John B. Pritchard Homeostasis of Nucleosides and Nucleobases in the Brain: The Role of Flux Between the CSF and the Brain ISF, Transport Across the Choroid Plexus and the Blood-Brain Barrier, and Cellular Uptake, Zoran B. Redzic Regulation of Neuroactive Metals by the Choroid Plexus, G. Jane Li and Wei Zheng The Role of the Choroid Plexus in Transport and Production of Polypeptides, Adam Chodobski, Gerald D. Silverberg, and Joanna Szmydynger-Chodobska Expression of Transthyretin in the Choroid Plexus: Relation to the Brain Homeostasis of Thyroid Hormones, Samantha J. Richardson BLOOD-CSF BARRIER IN DISEASES Choroid Plexus and CSF in Alzheimer's Disease: Altered Expression and Transport of Proteins and Peptides, Conrad E. Johanson, Gerald D. Silverberg, John E. Donahue, John A. Duncan, and Edward G. Stopa The Blood-CSF Barrier and Cerebral Ischemia, Richard F. Keep, Steven R. Ennis, and Jianming Xiang Ageing of the Choroid Plexus and CSF System: Implications for Neurodegeneration, Jane E. Preston, Michael R. Wilson, and Ruo Li Chen The Role of the Choroid Plexus in HIV-1 Infection, Carol K. Petito Choroid Plexus and Drug Therapy for Neuro-AIDS, Julie E. Gibbs and Sarah A. Thomas Blood-CSF Barrier in Iron Regulation and Manganese-Induced Parkinsonism, Wei Zheng The Involvement of the Choroid Plexus and the Cerebrospinal Fluid in Immune Molecule Signaling in the CNS, Miles Herkenham Experimental Models of Hydrocephalus: Relation to Human Pathology, Charles E. Weaver and John A. Duncan, III Clinical Aspects of Disorders of the Choroid Plexus and the CSF Circulation, John D. Pickard, Marek Czosnyka, N. Higgins, B. Owler, S. Momjian, and Alonso Pena Grafting of the Choroid Plexus: Clinical Implications, Naoya Matsumoto, Yutaka Itokazu, Masaaki Kitada, Shushovan Chakrabortty, Kazushi Kimura, and Chizuka Ide CURRENT IN VIVO AND IN VITRO MODELS OF THE BLOOD-CSF BARRIER In situ Techniques Used in the Blood-CSF Barrier Research, Wei Zheng and Malcolm B. Segal In vitro Investigation of the Blood-Cerebrospinal Fluid Barrier Properties. Primary Cultures and Immortalized Cell Lines of the Choroidal Epithelium, Nathalie Strazielle and Jean-Francois Ghersi-Egea In vivo Techniques Used in the Blood-CSF Barrier Research: Measurement of CSF Formation, Adam Chodobski and Malcolm B. Segal

The function of the cerebrospinal fluid (CSF) and the tissue that secretes it, the choroid plexus (CP), has traditionally been thought of as both providing physical protection to the brain through buoyancy and facilitating the removal of brain metabolites through the bulk drainage of CSF. More recent studies suggest, however, that the CP-CSF system plays a much more active role in the development, homeostasis, and repair of the central nervous system (CNS). The highly specialized choroidal tissue synthesizes trophic and angiogenic factors, chemorepellents, and carrier proteins, and is strategically positioned within the ventricular cavities to supply the CNS with these biologically active substances. Through polarized transport systems and receptor-mediated transcytosis across the choroidal epithelium, the CP, a part of the blood-CSF barrier (BCSFB), controls the entry of nutrients, such as amino acids and nucleosides, and peptide hormones, such as leptin and prolactin, from the periphery into the brain. The CP also plays an important role in the clearance of toxins and drugs. During CNS development, CP-derived growth factors, such as members of the transforming growth factor-beta superfamily and retinoic acid, play an important role in controlling the patterning of neuronal differentiation in various brain regions. In the adult CNS, the CP appears to be critically involved in neuronal repair processes and the restoration of the brain microenvironment after traumatic and ischemic brain injury. Furthermore, recent studies suggest that the CP acts as a nursery for neuronal and astrocytic progenitor cells. The advancement of our knowledge of the neuroprotective capabilities of the CP may therefore facilitate the development of novel therapies for ischemic stroke and traumatic brain injury. In the later stages of life, the CP-CSF axis shows a decline in all aspects of its function, including CSF secretion and protein synthesis, which may in themselves increase the risk for development of late-life diseases, such as normal pressure hydrocephalus and Alzheimer's disease. The understanding of the mechanisms that underlie the dysfunction of the CP-CSF system in the elderly may help discover the treatments needed to reverse the negative effects of aging that lead to global CNS failure.

Experimental evidence obtained in various animal models of brain injury indicates that vasopressin promotes the formation of cerebral edema. However, the molecular and cellular mechanisms underlying this vasopressin action are not fully understood. In the present study, we analyzed the temporal changes in expression of vasopressin V1a receptors after traumatic brain injury (TBI) in rats. In the intact brain, the V1a receptor was expressed in neurons located in all layers of the frontoparietal cortex. The V1a receptor-immunoreactive product was predominantly localized to neuronal nuclei and had both a diffused and punctate staining pattern. The V1a receptors were also expressed in astrocytes, especially in layer 1 of the frontoparietal cortex. In these cells, two distinctive patterns of immunopositive staining for V1a receptors were observed: a diffused cytosolic staining of cell bodies and processes and a clearly punctate staining pattern that was predominantly localized to the astrocytic cell bodies. The real-time reverse-transcriptase polymerase chain reaction analysis of changes in mRNA for the V1a receptor demonstrated that after TBI, there is an early (4 h post-TBI) increase in the number of transcripts in the ipsilateral frontoparietal cortex, when compared to the contralateral hemisphere or the sham-injured rats. This increase in the message was followed by the up-regulation of expression of the V1a receptors at the protein level. This was most evident in cortical astrocytes in the areas surrounding the lesion. The number of the V1a receptor-immunopositive astrocytes in the traumatized parenchyma gradually increased, starting at 8 h and peaking at 4-6 days after TBI. Furthermore, a redistribution of V1a receptors from the astrocytic cell bodies to the astrocytic processes was observed. In addition to astrocytes, an increased expression of V1a receptors was found in the endothelium of both blood microvessels and the large-diameter blood vessels in the frontoparietal cortex ipsilateral to injury. This increase in the V1a receptor expression was apparent between 2 and 4 days after TBI. As early as 1-2 h following the impact, there was also a striking increase in the number of the V1a receptor-immunopositive beaded axonal processes, with greatly enlarged varicosities, that were localized to various areas of the injured parenchyma. It is suggested that the increased expression of V1a receptors plays an important role in the vasopressin-mediated formation of edema in the injured brain.

Choroid plexus (CP) is an important target organ for polypeptides. The fenestrated phenotype of choroidal endothelium facilitates the penetration of blood-borne polypeptides across the capillary walls. Thus, both circulating and cerebrospinal fluid (CSF)-borne polypeptides can reach their receptors on choroidal epithelium. Several polypeptides have been demonstrated to regulate CSF formation by controlling blood flow to choroid plexus and/or the activity of ion transport in choroidal epithelium. However, many ligand-receptor interactions occurring in the CP are not involved in the regulation of fluid secretion. Increasing evidence suggests that the choroidal epithelium plays an important role in hormonal signaling via a receptor-mediated transport into the brain (e.g., leptin) and helps to clear certain CSF-borne polypeptides (e.g., soluble amyloid β-protein). Thus, impaired choroidal transport or insufficient clearance of polypeptides may contribute to pathogenesis of systemic or central nervous system (CNS) disorders, such as obesity or Alzheimer's disease. CP epithelium is not only a target but is also a source of neuropeptides, growth factors, and cytokines in the CNS. These polypeptides following their release into the CSF may exert distal, endocrine-like effects on target cells in the brain due to bulk flow of this fluid. Distinct temporal patterns of choroidal expression of several polypeptides are observed during brain development and in various CNS disorders, including traumatic brain injury and ischemia. Therefore, it is proposed that the CP plays an integral role not only in normal brain functioning, but also in the recovery from the injury. This review attempts to critically analyze the available data to support the above hypothesis. Microsc. Res. Tech. 52:65–82, 2001. © 2001 Wiley-Liss, Inc.

The cerebrospinal fluid (CSF)-generating choroid plexus (CP) has many V1 binding sites for arginine vasopressin (AVP). AVP decreases CSF formation rate and choroidal blood flow, but little is known about how AVP alters ion transport across the blood-CSF barrier. Adult rat lateral ventricle CP was loaded with36Cl−, exposed to AVP for 20 min, and then placed in isotope-free artificial CSF to measure release of36Cl−. Effect of AVP at 10−12 to 10−7 M on the Cl− efflux rate coefficient (in s−1) was quantified. Maximal inhibition (by 20%) of Cl− extrusion at 10−9 M AVP was prevented by the V1 receptor antagonist [β-mercapto-β,β-cyclopentamethyleneproprionyl1, O-Me-Tyr2,Arg8]vasopressin. AVP also increased by more than twofold the number of dark and possibly dehydrated but otherwise morphologically normal choroid epithelial cells in adult CP. The V1 receptor antagonist prevented this AVP-induced increment in dark cell frequency. In infant rats (1 wk) with incomplete CSF secretory ability, 10−9 M AVP altered neither Cl− efflux nor dark cell frequency. The ability of AVP to elicit functional and structural changes in adult, but not infant, CP epithelium is discussed in regard to ion transport, CSF secretion, intracranial pressure, and hydrocephalus.

Cerebrospinal fluid (CSF) plays an important role in the brain’s adaptive response to acute osmotic disturbances. In the present experiments, the effect of 48-h dehydration on CSF formation and absorption rates was studied in conscious adult sheep. Animals had cannulas chronically implanted into the lateral cerebral ventricles and cisterna magna to enable the ventriculocisternal perfusion. A 48-h water deprivation altered neither CSF production nor resistance to CSF absorption. However, in the water-depleted sheep, intraventricular pressure tended to be lower than that found under control conditions. This likely resulted from decreased extracellular fluid volume and a subsequent drop in central venous pressure occurring in dehydrated animals. In conclusion, our findings provide evidence for the maintenance of CSF production during mild dehydration, which may play a role in the regulation of fluid balance in the brain during chronic hyperosmotic stress.

Angiotensin II infused at low doses into the cerebral ventricles decreases cerebrospinal fluid (CSF) production. Since central angiotensin II also activates the sympathetic nervous system and promotes vasopressin release, the roles of these two factors in mediating the inhibitory effect of angiotensin II on CSF formation were studied. CSF production was measured in rats by the ventriculocisternal perfusion method. During central angiotensin II infusion (5 pg min −1 ), the following adrenoceptor antagonists were administered intravenously (i.v.): phentolamine ( α 1 / α 2 , 2 mg/kg per h), prazosin ( α 1 , 1 mg/kg per h), and propranolol ( β , 1 mg/kg per h). None of these agents affected the inhibitory effect of angiotensin II on CSF formation. In comparison, in animals administered i.v., the vasopressin V 1 receptor antagonist, d(CH 2 ) 5 Tyr(Me)Arg-vasopressin (10 μ g/kg per h), the angiotensin II-induced decrease in CSF production was abolished. Our observations indicate, therefore, that vasopressin mediates the inhibitory effect of central angiotensin II on CSF formation.

Vasopressin (VP) levels in cerebrospinal fluid (CSF) change in response to physiological stimuli and under various pathological conditions. The sources of CSF VP have yet to be clarified, however. In the present study, we provide evidence indicating that VP is synthesized in the choroid plexus, the primary site of CSF formation. All experiments were performed on adult male Sprague-Dawley rats. The presence of VP mRNA in choroid plexus epithelium was demonstrated by in situ hybridization histochemistry using the 35S-labeled riboprobe that was complementary to cDNA fragment of rat VP encoding the C-terminus part of proVP. In situ hybridization findings were confirmed by reverse transcriptase-polymerase chain reaction analysis. Immunohistochemistry for VP-associated neurophysin (VP-NP), a polypeptide component of proVP, revealed subapical accumulation of VP-NP-immunopositive product in choroidal epithelial cells. Immunoprecipitation and immunoblotting of choroidal protein extracts with anti-VP-NP antibody demonstrated the presence of a ~10-kD polypeptide that was also detected in hypothalamus. We hypothesize that the choroid plexus-derived VP exerts autocrine and/or paracrine effects on tissues near the CSF system.

cerebral edema is a significant cause of mortality in patients with traumatic brain injury and ischemic or hemorrhagic stroke. Yet our understanding of the cellular and molecular mechanisms underlying this condition is rather limited, and new treatments showing promise in laboratory studies have

Arginine vasopressin (AVP) plays an important role in the regulation of secretory function and hemodynamics of choroid plexus, the primary site of cerebrospinal fluid (CSF) production. In the present study, localization of AVP and its transcripts in choroid plexus of adult male Sprague-Dawley rats was studied by immunohistochemistry and in situ hybridization histochemistry, respectively. For immunohistochemical analysis, AVP-specific polyclonal rabbit antibody was employed. Plasmid, pGrVP, containing a 232-bp fragment of rat AVP cDNA encoding the C-terminus of proAVP, was used as a probe to detect AVP mRNA. AVP-immunoreactive product was predominantly localized close to the apical (CSF-facing) membrane of choroidal epithelium while AVP transcripts were distributed throughout the cytoplasm of the cells. Our findings indicate that AVP is synthesized in choroid plexus epithelium, which suggests autocrine and/or paracrine actions of this peptide in choroidal tissue.

The effect of central administration of angiotensin II (AII) on cerebrospinal fluid (CSF) formation was studied in pentobarbital-anesthetized, artificially-ventilated rats. CSF production was measured by the ventriculocisternal perfusion method with Blue Dextran 2000 as the indicator. Baseline value of CSF production was 3.35 ± 0.08 μ l/min . Intracerebroventricular (i.c.v.) infusion of AII at rates of 0.5 and 5 pg/min significantly lowered ( P ) CSF formation by 23% and 16%, respectively. In comparison, high peptide doses (50 and 500 pg/min) did not alter this parameter. The inhibitory effect of low AII doses on CSF formation was blocked by the i.c.v. AT1 receptor subtype antagonists, losartan and SK&F 108566 (2.4 and 2.7 ng/min, respectively), but not by the AT2 receptor subtype-specific agent, PD 123319 (3.8 ng/min). Peptide AII antagonist, [Sar1,Ile8]AII (5 ng/min), which binds to both AT1 and AT2 receptors, had a similar effect to those of AT1-specific blockers. It is concluded that AII, by controlling CSF formation, may influence the water and electrolyte balance in the brain.

Postnatal developmental changes in blood flow to choroid plexuses of the lateral (LVCP) and fourth (4VCP) ventricles and cerebral cortex were studied in pentobarbital-anesthetized rats at 2, 3, 5, and 7-8 wk. Blood flow was measured by indicator fractionation with N-isopropyl-p-[125I]iodoamphetamine as the marker. Blood flow to the LVCP and 4VCP was 2.5 +/- 0.1 and 2.7 +/- 0.1 ml.g-1.min-1, respectively, and did not change between the 2nd and 3rd wk. However, it increased by 34% between the 3rd and 5th wk. From the age of 5 wk on, 4VCP was characterized by higher blood flow rates than LVCP. Cerebral cortical blood flow gradually increased between the 2nd and 5th wk. There was no difference in cortical blood flow between 5-wk-old and adult animals. The changes in choroidal blood flow likely represent a continuing adjustment of the choroidal vascular system to steadily increasing secretory capabilities of the maturing choroidal epithelium.