Proteoglycan 4 Reduces Neuroinflammation and Protects the Blood-Brain Barrier after Traumatic Brain Injury.

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.

Recombinant Human Proteoglycan-4 Mediates Interleukin-6 Response in Both Human and Mouse Endothelial Cells Induced Into a Sepsis Phenotype.

OBJECTIVES: Sepsis is a leading cause of death in the United States. Putative targets to prevent systemic inflammatory response syndrome include antagonism of toll-like receptors 2 and 4 and CD44 receptors in vascular endothelial cells. Proteoglycan-4 is a mucinous glycoprotein that interacts with CD44 and toll-like receptor 4 resulting in a blockade of the NOD-like receptor pyrin domain-containing-3 pathway. We hypothesized that endothelial cells induced into a sepsis phenotype would have less interleukin-6 expression after recombinant human proteoglycan 4 treatment in vitro. DESIGN: Enzyme-linked immunosorbent assay and reverse transcriptase-quantitative polymerase chain reaction to measure interleukin-6 protein and gene expression. SETTING: Research laboratory. SUBJECTS: Human umbilical vascular endothelial cells, human lung microvascular endothelial cells, and transgenic mouse (wild type) (Cd44 +/+/Prg4 +/+), Cd44 -/- (Cd44 tm1Hbg Prg4 +/+), Prg4 GT/GT (Cd44 +/+ Prg4 tm2Mawa/J), and double knockout (Cd44 tm1Hbg Prg4 tm2Mawa/J) lung microvascular endothelial cells. INTERVENTIONS: Cells were treated with 100 or 250 ng/mL lipopolysaccharide-Escherichia coli K12 and subsequently treated with recombinant human proteoglycan 4 after 30 minutes. Interleukin-6 levels in conditioned media were measured via enzyme-linked immunosorbent assay and gene expression was measured via reverse transcriptase-quantitative polymerase chain reaction with ΔΔ-Ct analysis. Additionally, human umbilical vascular endothelial cells and human lung microvascular endothelial cells were treated with 1:10 diluted plasma from 15 patients with sepsis in culture media. After 30 minutes, either 50 or 100 µg/mL recombinant human proteoglycan 4 was administered. Interleukin-6 protein and gene expression were assayed. Proteoglycan 4 levels were also compared between control and sepsis patient plasma. MEASUREMENTS AND MAIN RESULTS: Human umbilical vascular endothelial cell, human lung microvascular endothelial cell, and mouse lung microvascular endothelial cell treated with lipopolysaccharide had significantly increased interleukin-6 protein compared with controls. Recombinant human proteoglycan-4 significantly reduced interleukin-6 in human and mouse endothelial cells. Interleukin-6 gene expression was significantly increased after lipopolysaccharide treatment compared with controls. This response was reversed by 50 or 100 µg/mL recombinant human proteoglycan-4 in 80% of sepsis samples in human umbilical vascular endothelial cells and in 60-73% in human lung microvascular endothelial cells. In Cd44 -/- genotypes of the mouse lung microvascular endothelial cells, recombinant human proteoglycan-4 significantly reduced interleukin-6 protein levels after lipopolysaccharide treatment, indicating that Cd44 is not needed for recombinant human proteoglycan-4 to have an effect in a toll-like receptor 4 agonist inflammation model. Patient sepsis samples had higher plasma levels of native proteoglycan-4 than controls. INTERPRETATION AND CONCLUSIONS: Recombinant human proteoglycan-4 is a potential adjunct therapy for sepsis patients and warrants future in vivo model studies.

Effects of embryonic cyclosporine exposures on brain development and behavior.

Cyclosporine, a calcineurin inhibitor, is successfully used as an immunosuppressant in transplant medicine. However, the use of this pharmaceutical during pregnancy is concerning since calcineurin is thought to play a role in neural development. The risk for human brain development is difficult to evaluate because of a lack of basic information on the sensitive developmental times and the potentially pleiotropic effects on brain development and behavior. In the present study, we use zebrafish as a model system to examine the effects of embryonic cyclosporine exposures. Early embryonic exposures reduced the size of the eyes and brain. Late embryonic exposures did not affect the size of the eyes or brain, but did lead to substantial behavioral defects at the larval stages. The cyclosporine-exposed larvae displayed a reduced avoidance response to visual stimuli, low swim speeds, increased resting, an increase in thigmotaxis, and changes in the average distance between larvae. Similar results were obtained with the calcineurin inhibitor FK506, suggesting that most, but not all, effects on brain development and behavior are mediated by calcineurin inhibition. Overall, the results show that cyclosporine can induce either structural or functional brain defects, depending on the exposure window. The observed functional brain defects highlight the importance of quantitative behavioral assays when evaluating the risk of developmental exposures.

Exposure to female pheromones stimulates a specific type of neuronal population in the male but not female magnocellular division of the medial preoptic nucleus (MPN mag) of the Syrian hamster.

The magnocellular division of the medial preoptic area (MPN mag) integrates pheromonal and hormonal signals to play a critical role in the expression of male typical sex behavior. The MPN mag contains two morphologically distinct neuronal populations; the percentage of each type within the nucleus is sex specific. Males have more neurons with a single nucleolus whereas females have more with multiple nucleoli. To determine which neuronal subtype mediates pheromonal induction of copulation, tissue from male and female hamsters exposed to female pheromones was immunolabeled for the immediate early protein (EGR-1). Subsequently the tissue was counterstained and the number of ERG-1 neurons with one or two nuclei was determined. The results indicate that pheromones stimulate neurons with single nucleoli in males but fail to stimulate either neuronal subtype in females suggesting that synaptic input to the MPN mag is sexually differentiated.

A novel high-throughput imaging system for automated analyses of avoidance behavior in zebrafish larvae.

Early brain development can be influenced by numerous genetic and environmental factors, with long-lasting effects on brain function and behavior. The identification of these factors is facilitated by recent innovations in high-throughput screening. However, large-scale screening in whole organisms remains challenging, in particular when studying changes in brain function or behavior in vertebrate model systems. In this study, we present a novel imaging system for high-throughput analyses of behavior in zebrafish larvae. The three-camera system can image 12 multiwell plates simultaneously and is unique in its ability to provide local visual stimuli in the wells of a multiwell plate. The acquired images are converted into a series of coordinates, which characterize the location and orientation of the larvae. The developed imaging techniques were tested by measuring avoidance behaviors in seven-day-old zebrafish larvae. The system effectively quantified larval avoidance and revealed an increased edge preference in response to a blue or red 'bouncing ball' stimulus. Larvae also avoid a bouncing ball stimulus when it is counter-balanced with a stationary ball, but do not avoid blinking balls counter-balanced with a stationary ball. These results indicate that the seven-day-old larvae respond specifically to movement, rather than color, size, or local changes in light intensity. The imaging system and assays for measuring avoidance behavior may be used to screen for genetic and environmental factors that cause developmental brain disorders and for novel drugs that could prevent or treat these disorders.

Neuronal composition of the magnocellular division of the medial preoptic nucleus (MPN mag) is sex specific in the Syrian hamster (Mesocricetus auratus).

The magnocellular division of the medial Preoptic nucleus (MPN mag) plays a critical role in the regulation of male sexual behavior in the hamster. Results from previous studies indicated that the number of neurons in the MPN mag is greater in males than females but failed to find significant differences in the volume of the nucleus suggesting that other elements in the nucleus may be greater in the female. The results of the present study, using NeuN to identify neurons, are in line with this hypothesis. The data show that (1) neurons in the MPN mag display two distinct phenotypes, those with a single nucleolus and those with multiple nucleoli; (2) the percentage of each phenotype is sex specific, differing over the course of development and (3) there is no sex difference in the number of glial cells at any age. Sex differences in the numbers of each type are correlated with developmental milestones and suggest that morphological changes are influenced by changes in circulating gonadal steroids during development.

Temperature, peroxide concentration, and immunohistochemical staining method affects staining intensity, distribution, and background.

The techniques used to label neural tissue for specific antigens can vary significantly. Some immunostaining methods use free-floating tissue sections, whereas others use tissue sections mounted on slides. Mounting sections on glass slides before labeling the tissue with antigens is preferred method for neonatal tissue; processing young tissue by free-floating methods often destroy it. Surprisingly optimal temperature for storing tissue can vary with age. This study describes parameters developed to obtain robust staining of both young and old tissue. Our results show the most robust staining was found in tissue that was (1) stored at very low temperatures (-20 degrees C and -80 degrees C), (2) pretreated with 0.01% peroxide, and (3) entirely immersed in the staining solutions during immunohistochemistry.