GluN2B inhibition rescues impaired potentiation and epileptogenicity at associational-commissural CA3 synapses in a model of anti-NMDAR encephalitis

Kupper M, Porath K, Sellmann T, Bien C, Kohling R, Kirschstein T. GluN2B inhibition rescues impaired potentiation and epileptogenicity at associational-commissural CA3 synapses in a model of anti-NMDAR encephalitis. Neuroscience Letters. 2022;795: 137031.Anti-N-methyl-D-aspartate receptor (anti-NMDAR) encephalitis is an autoimmune epilepsy associated with memory deficits. Research has demonstrated that anti-NMDAR inhibit long-term potentiation, and, at the same time, lead to disinhibition in the form of epileptiform afterpotentials in the potentiated state. While both effects may give rise to the key symptoms of the disease, the molecular basis of being simultaneously inhibitory and disinhibitory is difficult to explain. Here, we explored a possible involvement of the GluN2B subunit. To this aim, we injected cerebrospinal fluid from anti-NMDAR encephalitis patients into the rat hippocampus and prepared brain slices for in vitro field potential recordings. Associational-commissural-fiber-CA3 synapses from anti-NMDAR-treated animals showed increased field potential amplitudes with concomitantly enhanced paired-pulse ratios as compared to control tissue. GluN2B inhibition by Ro25-6981 mimicked these effects in controls but had no effect in anti-NMDAR tissues indicating a presynaptic and occluding effect of anti-NMDAR. We then induced potentiation of associational-commissural-fiber-CA3 synapses, and confirmed that slices from anti-NMDAR-treated animals showed reduced potentiation and pronounced epileptiform afterpotentials. Intriguingly, both effects were absent when Ro25-6981 was added in vitro before inducing potentiation. These results indicate that GluN2B-containing NMDARs, partially expressed presynaptically, show differential sensitivity to anti-NMDAR, and that altered GluN2B function is particularly apparent in the potentiated state rather than under baseline conditions. Since GluN2B inhibition rescued the effects of anti-NMDAR in the potentiated state, this opens the possibility that at least a subgroup of patients could benefit from a GluN2B antagonist. Copyright © 2022 Elsevier B.V. All rights reserved

Mycophenolate mofetil prevents the delayed T cell response after pilocarpine-induced status epilepticus in mice.

Growing clinical and laboratory evidence corroborates a role for the immune system in the pathophysiology of epilepsy. In order to delineate the immune response following pilocarpine-induced status epilepticus (SE) in the mouse, we monitored the kinetics of leukocyte presence in the hippocampus over the period of four weeks. SE was induced following a ramping protocol of pilocarpine injection into 4-5 weeks old C57BL/6 mice. Brains were removed at days 1-4, 14 or 28 after SE, and the hippocampi were analyzed via flow cytometry, via quantitative reverse transcriptase PCR (qRT-PCR) and via immunohistochemistry. Epileptogenesis was confirmed by Timm staining of mossy fiber sprouting in the inner molecular layer of the dentate gyrus. The flow cytometry data revealed a biphasic immune response following pilocarpine-induced SE with a transient increase in activated CD11b+ and F4/80+ macrophages within the first four days replaced by an increase in CD3+ T-lymphocytes around day 28. This delayed T cell response was confirmed via qRT-PCR and via immunohistochemistry. In addition, qRT-PCR data could show that the delayed T cell response was associated with an increased CD8/CD4 ratio indicating a cytotoxic T cell response after SE. Intriguingly, early intervention with mycophenolate mofetil administration on days 0-3 after SE prevented this delayed T cell response. These results show an orchestrated immunological sequela and provide evidence that the delayed T cell response is sensitive to early immunomodulatory intervention

DataSheet1_Cell-cell interactions and fluctuations in the direction of motility promote directed migration of osteoblasts in direct current electrotaxis.PDF

Under both physiological (development, regeneration) and pathological conditions (cancer metastasis), cells migrate while sensing environmental cues in the form of mechanical, chemical or electrical stimuli. In the case of bone tissue, osteoblast migration is essential in bone regeneration. Although it is known that osteoblasts respond to exogenous electric fields, the underlying mechanism of electrotactic collective movement of human osteoblasts is unclear. Here, we present a computational model that describes the osteoblast cell migration in a direct current electric field as the motion of a collection of active self-propelled particles and takes into account fluctuations in the direction of single-cell migration, finite-range cell-cell interactions, and the interaction of a cell with the external electric field. By comparing this model with in vitro experiments in which human primary osteoblasts are exposed to a direct current electric field of different field strengths, we show that cell-cell interactions and fluctuations in the migration direction promote anode-directed collective migration of osteoblasts.</p

Effects of Microbeam Irradiation on Rodent Esophageal Smooth Muscle Contraction

Background: High-dose-rate radiotherapy has shown promising results with respect to normal tissue preservation. We developed an ex vivo model to study the physiological effects of experimental radiotherapy in the rodent esophageal smooth muscle. Methods: We assessed the physiological parameters of the esophageal function in ex vivo preparations of the proximal, middle, and distal segments in the organ bath. High-dose-rate synchrotron irradiation was conducted using both the microbeam irradiation (MBI) technique with peak doses greater than 200 Gy and broadbeam irradiation (BBI) with doses ranging between 3.5&ndash;4 Gy. Results: Neither MBI nor BBI affected the function of the contractile apparatus. While peak latency and maximal force change were not affected in the BBI group, and no changes were seen in the proximal esophagus segments after MBI, a significant increase in peak latency and a decrease in maximal force change was observed in the middle and distal esophageal segments. Conclusion: No severe changes in physiological parameters of esophageal contraction were determined after high-dose-rate radiotherapy in our model, but our results indicate a delayed esophageal function. From the clinical perspective, the observed increase in peak latency and decreased maximal force change may indicate delayed esophageal transit

Effects of Microbeam Irradiation on Rodent Esophageal Smooth Muscle Contraction

Background: High-dose-rate radiotherapy has shown promising results with respect to normal tissue preservation. We developed an ex vivo model to study the physiological effects of experimental radiotherapy in the rodent esophageal smooth muscle. Methods: We assessed the physiological parameters of the esophageal function in ex vivo preparations of the proximal, middle, and distal segments in the organ bath. High-dose-rate synchrotron irradiation was conducted using both the microbeam irradiation (MBI) technique with peak doses greater than 200 Gy and broadbeam irradiation (BBI) with doses ranging between 3.5–4 Gy. Results: Neither MBI nor BBI affected the function of the contractile apparatus. While peak latency and maximal force change were not affected in the BBI group, and no changes were seen in the proximal esophagus segments after MBI, a significant increase in peak latency and a decrease in maximal force change was observed in the middle and distal esophageal segments. Conclusion: No severe changes in physiological parameters of esophageal contraction were determined after high-dose-rate radiotherapy in our model, but our results indicate a delayed esophageal function. From the clinical perspective, the observed increase in peak latency and decreased maximal force change may indicate delayed esophageal transit.Keywords:high-dose-rate radiotherapy; microbeam irradiation; broadbeam irradiation; organ bath; esophageal smooth muscle; carbachol-induced contraction; esophageal function and motilit

Quantitative PCR confirms the late response of CD3⁺ cells.

<p>Relative expressions of CD3g (normalized to Ptprc, 2^-ΔCT) in pilocarpine-treated and control animals on day 2 <b>(a)</b> and on day 28 <b>(b)</b> after SE (left panels). The right panels show the CD8/CD4 ratios in both experimental groups at the respective time-points. Each symbol represents one animal. Horizontal lines indicate means, and P-values result from unpaired t-tests or U-tests (see text).</p

Mycophenolate mofetil prevents the delayed response of CD3⁺ cells.

<p><b>(a)</b> Relative expression of CD3g (normalized to control, 2^-ΔCT) in control animals treated with mycophenolate mofetil (MMF) as well as in pilocarpine-treated animals on day 28 after SE with or without MMF treatment. The relative expression shown results from the comparison to mice neither treated with pilocarpine nor with MMF. Note that MMF given on day 0–3 after SE had no effect in controls, but significantly prevented the increased CD3g abundance in pilocarpine-treated animals. Each symbol represents one animal. Horizontal lines indicate means, and P-values result from unpaired t-tests. <b>(b)</b> CD3 immunoreactivity in the dentate gyrus of a control (left) and a pilocarpine-treated animal (right). Individual CD3⁺ cells (green) are indicated by white arrows. Nuclei are stained with DAPI (blue color). Magnification 600x.</p

The late phase of the hippocampal immune response following pilocarpine-induced SE correlates with an increase in CD3⁺ T cells.

<p><b>(a)</b> Sideward scatter versus CD3 analysis of control animals and animals on day 28 post SE. The gates indicate the CD3+ leukocytes. <b>(b)</b> Percentages of CD3⁺ leukocytes found in the hippocampi of either control or pilocarpine treated animals on days 14 and 28 post SE. Each symbol represents pooled hippocampal cells of 2–4 mice. Horizontal lines indicate means and P-values result from unpaired t-tests.</p

The early phase of the hippocampal immune response following pilocarpine-induced SE correlates with an increase in activated microglia.

<p><b>(a)</b> The left panel depicts a sideward scatter versus CD45 analysis of hippocampus cells on day 4 post SE. The panels on the right depict the CD11b versus F4/80 staining gating on either CD45⁺, CD45^- or ungated cells. Numbers indicate percentages in the respective quadrants and gates. <b>(b)</b> Summary of percentages of CD11b⁺ leukocytes found in the hippocampi of either control or pilocarpine treated animals on days 1–4, 14 and 28 post SE. Each symbol represents pooled hippocampal cells of 2–4 mice. Horizontal lines indicate means and P-values result from unpaired t-tests. <b>(c)</b> Sideward scatter versus CD11b analysis of control animals and animals on day 2 post SE. The gates indicate the CD11b⁺ leukocytes.</p

Biphasic immune response following pilocarpine-induced SE in the murine hippocampus.

<p><b>(a)</b> Sideward scatter (SSC) versus CD45 analysis of control animals and animals on day 2 post SE. The gates describe the CD45⁺ leukocytes. <b>(b)</b> Percentages of CD45⁺ leukocytes found in the hippocampi of either control or pilocarpine treated animals on days 1–4, 14 and 28 post SE. Each symbol represents pooled hippocampal cells of 2–4 mice. Horizontal lines indicate means and P-values result from unpaired t-tests.</p