Human BBB-on-a-chip reveals barrier disruption, endothelial inflammation, and T cell migration under neuroinflammatory conditions

The blood-brain barrier (BBB) is a highly selective barrier that ensures a homeostatic environment for the central nervous system (CNS). BBB dysfunction, inflammation, and immune cell infiltration are hallmarks of many CNS disorders, including multiple sclerosis and stroke. Physiologically relevant human in vitro models of the BBB are essential to improve our understanding of its function in health and disease, identify novel drug targets, and assess potential new therapies. We present a BBB-on-a-chip model comprising human brain microvascular endothelial cells (HBMECs) cultured in a microfluidic platform that allows parallel culture of 40 chips. In each chip, a perfused HBMEC vessel was grown against an extracellular matrix gel in a membrane-free manner. BBBs-on-chips were exposed to varying concentrations of pro-inflammatory cytokines tumor necrosis factor alpha (TNFα) and interleukin-1 beta (IL-1β) to mimic inflammation. The effect of the inflammatory conditions was studied by assessing the BBBs-on-chips’ barrier function, cell morphology, and expression of cell adhesion molecules. Primary human T cells were perfused through the lumen of the BBBs-on-chips to study T cell adhesion, extravasation, and migration. Under inflammatory conditions, the BBBs-on-chips showed decreased trans-endothelial electrical resistance (TEER), increased permeability to sodium fluorescein, and aberrant cell morphology in a concentration-dependent manner. Moreover, we observed increased expression of cell adhesion molecules and concomitant monocyte adhesion. T cells extravasated from the inflamed blood vessels and migrated towards a C-X-C Motif Chemokine Ligand 12 (CXCL12) gradient. T cell adhesion was significantly reduced and a trend towards decreased migration was observed in presence of Natalizumab, an antibody drug that blocks very late antigen-4 (VLA-4) and is used in the treatment of multiple sclerosis. In conclusion, we demonstrate a high-throughput microfluidic model of the human BBB that can be used to model neuroinflammation and assess anti-inflammatory and barrier-restoring interventions to fight neurological disorders

Connexin Hemichannel Mimetic Peptide Attenuates Cortical Interneuron Loss and Perineuronal Net Disruption Following Cerebral Ischemia in Near-Term Fetal Sheep

Perinatal hypoxia-ischemia is associated with disruption of cortical gamma-aminobutyric acid (GABA)ergic interneurons and their surrounding perineuronal nets, which may contribute to persisting neurological deficits. Blockade of connexin43 hemichannels using a mimetic peptide can alleviate seizures and injury after hypoxia-ischemia. In this study, we tested the hypothesis that connexin43 hemichannel blockade improves the integrity of cortical interneurons and perineuronal nets. Term-equivalent fetal sheep received 30 min of bilateral carotid artery occlusion, recovery for 90 min, followed by a 25-h intracerebroventricular infusion of vehicle or a mimetic peptide that blocks connexin hemichannels or by a sham ischemia + vehicle infusion. Brain tissues were stained for interneuronal markers or perineuronal nets. Cerebral ischemia was associated with loss of cortical interneurons and perineuronal nets. The mimetic peptide infusion reduced loss of glutamic acid decarboxylase-, calretinin-, and parvalbumin-expressing interneurons and perineuronal nets. The interneuron and perineuronal net densities were negatively correlated with total seizure burden after ischemia. These data suggest that the opening of connexin43 hemichannels after perinatal hypoxia-ischemia causes loss of cortical interneurons and perineuronal nets and that this exacerbates seizures. Connexin43 hemichannel blockade may be an effective strategy to attenuate seizures and may improve long-term neurological outcomes after perinatal hypoxia-ischemia

Modeling ischemic stroke in a triculture neurovascular unit on-a-chip

BACKGROUND: In ischemic stroke, the function of the cerebral vasculature is impaired. This vascular structure is formed by the so-called neurovascular unit (NVU). A better understanding of the mechanisms involved in NVU dysfunction and recovery may lead to new insights for the development of highly sought therapeutic approaches. To date, there remains an unmet need for complex human in vitro models of the NVU to study ischemic events seen in the human brain. METHODS: We here describe the development of a human NVU on-a-chip model using a platform that allows culture of 40 chips in parallel. The model comprises a perfused vessel of primary human brain endothelial cells in co-culture with induced pluripotent stem cell derived astrocytes and neurons. Ischemic stroke was mimicked using a threefold approach that combines chemical hypoxia, hypoglycemia, and halted perfusion. RESULTS: Immunofluorescent staining confirmed expression of endothelial adherens and tight junction proteins, as well as astrocytic and neuronal markers. In addition, the model expresses relevant brain endothelial transporters and shows spontaneous neuronal firing. The NVU on-a-chip model demonstrates tight barrier function, evidenced by retention of small molecule sodium fluorescein in its lumen. Exposure to the toxic compound staurosporine disrupted the endothelial barrier, causing reduced transepithelial electrical resistance and increased permeability to sodium fluorescein. Under stroke mimicking conditions, brain endothelial cells showed strongly reduced barrier function (35-fold higher apparent permeability) and 7.3-fold decreased mitochondrial potential. Furthermore, levels of adenosine triphosphate were significantly reduced on both the blood- and the brain side of the model (4.8-fold and 11.7-fold reduction, respectively). CONCLUSIONS: The NVU on-a-chip model presented here can be used for fundamental studies of NVU function in stroke and other neurological diseases and for investigation of potential restorative therapies to fight neurological disorders. Due to the platform's relatively high throughput and compatibility with automation, the model holds potential for drug compound screening

Role of Recurrent Hypoxia-Ischemia in Preterm White Matter Injury Severity

<div><p>Objective</p><p>Although the spectrum of white matter injury (WMI) in preterm infants is shifting from cystic necrotic lesions to milder forms, the factors that contribute to this changing spectrum are unclear. We hypothesized that recurrent hypoxia-ischemia (rHI) will exacerbate the spectrum of WMI defined by markers of inflammation and molecules related to the extracellular matrix (hyaluronan (HA) and the PH20 hyaluronidase) that regulate maturation of the oligodendrocyte (OL) lineage after WMI.</p><p>Methods</p><p>We employed a preterm fetal sheep model of <i>in utero</i> moderate hypoxemia and global severe but not complete cerebral ischemia that reproduces the spectrum of human WMI. The response to rHI was compared against corresponding early or later single episodes of HI. An ordinal rating scale of WMI was compared against an unbiased quantitative image analysis protocol that provided continuous histo-pathological outcome measures for astrogliosis and microglial activation. Late oligodendrocyte progenitors (preOLs) were quantified by stereology. Analysis of hyaluronan and the hyaluronidase PH20 defined the progressive response of the extracellular matrix to WMI.</p><p>Results</p><p>rHI resulted in a more severe spectrum of WMI with a greater burden of necrosis, but an expanded population of preOLs that displayed reduced susceptibility to cell death. WMI from single episodes of HI or rHI was accompanied by elevated HA levels and increased labeling for PH20. Expression of PH20 in fetal ovine WMI was confirmed by RT-PCR and RNA-sequencing.</p><p>Conclusions</p><p>rHI is associated with an increased risk for more severe WMI with necrosis, but reduced risk for preOL degeneration compared to single episodes of HI. Expansion of the preOL pool may be linked to elevated hyaluronan and PH20.</p></div

Spectrum of WMI in the four experimental conditions, as assessed by analysis of GFAP and Iba-1 staining using quantitation of area fractions stained for each marker.

<p>(A) Astrogliosis, measured by GFAP area fraction (AF), is increased following rHI (Kruskal-Wallis H = 13.96, p = 0.003, on 3df; mean ± SD Control: 0.20±0.06; Late HI: 0.25±0.05; Early HI: 0.33±0.05; rHI: 0.37±0.04; Bonferroni-corrected post-hoc Mann-Whitney U-tests: **rHI vs. Control, p = 0.002; *Early HI vs. Control, p = 0.018; *rHI vs. Late HI, p = 0.021). (B) Iba-1 AF revealed similar patterns to Iba-1 pathology in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112800#pone-0112800-g003" target="_blank">Fig. 3</a> (Kruskal-Wallis H = 11.7, p = 0.008; mean ± SD Control: 0.06±0.05; Late HI: 0.08±0.06; Early HI: 0.18±0.13; rHI: 0.33±0.170; Bonferroni-corrected post-hoc Mann-Whitney U-tests: **rHI vs. Control p = 0.01; *rHI vs. Late HI p = 0.020). (C) Iba-1 and GFAP AFs are significantly associated over a broad spectrum of WMI (***Spearman’s Rank Correlation: ρ = 0.91, p&lt;0.0001).</p

HA is present in fetal ovine white matter and displays a persistent increase for several weeks after HI.

<p>Representative confocal image of staining for HA with an HA binding protein (HABP) in frontal white matter (pseudocolors: green: HABP; red: Hoechst 33342-labeled nuclei). (A) Control: One-week post-insult. (B) Early HI. (C) Late HI. (D) rHI. (E) Control: 24-hours post-insult. (F) HI: 24-hours post-insult. (G) Control: Two weeks post-insult. (H) HI: Two weeks post-insult. (I) Control: Four weeks post-insult. (J) HI: Four weeks post-insult. Scale bars 20 µm.</p

The pool of premyelinating OL lineage cells is significantly increased following rHI; PreOLs are less susceptible to acute degeneration following rHI than following late HI.

<p>(A–B) Representative confocal images of premyelinating OL lineage cells in the PVWM labeled with the O4 monoclonal antibody (arrowheads) in controls (A) and after rHI (B). Scale bars 20 µm. (C) Unbiased stereological counts of O4-labeled cells show an expansion of the preOL pool in the rHI group relative to control and early HI (Kruskal-Wallis: H = 7.7, p = 0.053, on 3 df; mean ± SD Control: 16,854±4,752 cells/mm³; Early HI: 16,430±7,182 cells/mm³; Late HI: 22,855±3,210 cells/mm³; rHI: 29,917±11,254 cells/mm³; CE range: 0.048–0.154). (D) Stereology further shows a decreased rate of preOL death following rHI relative to late HI (Kruskal-Wallis: H = 7.5, p = 0.057, on 3 df; mean ± SD Control: 0.9±0.4%; Early HI: 1.2±0.9%; Late HI: 5.0±2.4%; rHI: 1.7±1.8%; Bonferroni-corrected post-hoc Mann-Whitney U-tests: *Late HI vs. Control, p = 0.037; CE range for pyknotic counts: 0.21–0.95). (E) There is a greater density of activated caspase 3-labeled cells following a single late HI episode (Kruskal-Wallis H = 10.5, p = 0.015, on 3df; mean ± SD, Control: 1.62±0.76 Cells/mm²; Late HI: 6.30±5.40; Early HI: 0.92±0.21 cells/mm²; rHI: 1.73±1.33 cells/mm²; Bonferroni-corrected post-hoc Mann-Whitney U-tests: **Early HI vs. Late HI, p = 0.001). (F) Representative confocal images of a degenerating O4-positive cell with a fragmented pyknotic nucleus in a late HI case. (F1) O4, (F2) Hoechst 33342 nuclear stain, (F3) Merge. Scale bar: 5 µm.</p

Spectrum of WMI in the four experimental conditions, as assessed by analysis of H &amp; E staining using ordinal rating scores.

<p>(A) Neuropathological scoring of H &amp; E staining in frontal white matter (Kruskal-Wallis H = 8.57, p = 0.036 on 3df; mean ± SD Control: 0.20±0.45; Late HI: 0.28±0.44; Early HI: 0.80±0.84; rHI: 2.0±1.3). (B) Neuropathological scoring of H &amp; E stained parietal white matter (Kruskal-Wallis H = 10.5, p = 0.015; mean ± SD Control: 0.60±0.89; Late HI: 1.00±0.00; Early HI: 1.4±0.89; rHI: 2.3±0.82; Bonferroni-corrected post-hoc Mann-Whitney U-test: *rHI vs. Late HI, p = 0.036). (C–F) Representative images of H &amp; E staining from (C) Control, (D) Early HI, (E) Late HI, (F) rHI frontal white matter. Panel scale bars: 200 µm; inset scale bars: 20 µm.</p

Schematic timeline of the ten day protocol for rHI studies showing the assignment of animals from twin pairs to the four experimental conditions.

<p>Animals recovered from surgery for 3 days before the initial exposure to hypoxemia with or without concurrent ischemia (black arrowheads indicate the timing of ischemia). The first gray shaded bar indicates that all groups sustained a 30-minute period of maternal hypoxemia at this time. Note that the twin control for the early HI group and the late HI twin of the rHI group did not sustain ischemia at this time. Thereafter, the early HI group and their twin controls survived for 7 days (i.e., 10 days after surgery). The rHI cases were exposed to a second episode of HI six days after the first HI episode. The corresponding twins for the rHI group were the late HI animals. The rHI group and the late HI cases were both exposed to an insult that involved maternal hypoxemia (gray shaded bar) and ischemia (arrowheads) at 24-hours before brain collection (i.e., 7 days after the initial insult and 10 days after surgery). Hence, the animals in all four groups survived for 10 days after surgery.</p