In silico-in vitro modeling to uncover cues involved in establishing microglia identity: TGF-β3 and laminin can drive microglia signature gene expression

Microglia are the resident macrophages of the central nervous system (CNS) and play a key role in CNS development, homeostasis, and disease. Good in vitro models are indispensable to study their cellular biology, and although much progress has been made, in vitro cultures of primary microglia still only partially recapitulate the transcriptome of in vivo microglia. In this study, we explored a combination of in silico and in vitro methodologies to gain insight into cues that are involved in the induction or maintenance of the ex vivo microglia reference transcriptome. First, we used the in silico tool NicheNet to investigate which (CNS-derived) cues could underlie the differences between the transcriptomes of ex vivo and in vitro microglia. Modeling on basis of gene products that were found to be upregulated in vitro, predicted that high mobility group box 2 (HMGB2)- and interleukin (IL)-1β-associated signaling pathways were driving their expression. Modeling on basis of gene products that were found to be downregulated in vitro, did not lead to predictions on the involvement of specific signaling pathways. This is consistent with the idea that in vivo microenvironmental cues that determine microglial identity are for most part of inhibitory nature. In a second approach, primary microglia were exposed to conditioned medium from different CNS cell types. Conditioned medium from spheres composed of microglia, oligodendrocytes, and radial glia, increased the mRNA expression levels of the microglia signature gene P2RY12. NicheNet analyses of ligands expressed by oligodendrocytes and radial glia predicted transforming growth factor beta 3 (TGF-β3) and LAMA2 as drivers of microglia signature gene expression. In a third approach, we exposed microglia to TGF-β3 and laminin. In vitro exposure to TGF-β3 increased the mRNA expression levels of the microglia signature gene TREM2. Microglia cultured on laminin-coated substrates were characterized by reduced mRNA expression levels of extracellular matrix-associated genes MMP3 and MMP7, and by increased mRNA expression levels of the microglia signature genes GPR34 and P2RY13. Together, our results suggest to explore inhibition of HMGB2- and IL-1β-associated pathways in in vitro microglia. In addition, exposure to TGF-β3 and cultivation on laminin-coated substrates are suggested as potential improvements to current in vitro microglia culture protocols

Somatosensory Profiles but Not Numbers of Somatosensory Abnormalities of Neuropathic Pain Patients Correspond with Neuropathic Pain Grading

Due to the lack of a specific diagnostic tool for neuropathic pain, a grading system to categorize pain as ‘definite’, ‘probable’, ‘possible’ and ‘unlikely’ neuropathic was proposed. Somatosensory abnormalities are common in neuropathic pain and it has been suggested that a greater number of abnormalities would be present in patients with ‘probable’ and ‘definite’ grades. To test this hypothesis, we investigated the presence of somatosensory abnormalities by means of Quantitative Sensory Testing (QST) in patients with a clinical diagnosis of neuropathic pain and correlated the number of sensory abnormalities and sensory profiles to the different grades. Of patients who were clinically diagnosed with neuropathic pain, only 60% were graded as ‘definite’ or ‘probable’, while 40% were graded as ‘possible’ or ‘unlikely’ neuropathic pain. Apparently, there is a mismatch between a clinical neuropathic pain diagnosis and neuropathic pain grading. Contrary to the expectation, patients with ‘probable’ and ‘definite’ grades did not have a greater number of abnormalities. Instead, similar numbers of somatosensory abnormalities were identified for each grade. The profiles of sensory signs in ‘definite’ and ‘probable’ neuropathic pain were not significantly different, but different from the ‘unlikely’ grade. This latter difference could be attributed to differences in the prevalence of patients with a mixture of sensory gain and loss and with sensory loss only. The grading system allows a separation of neuropathic and non-neuropathic pain based on profiles but not on the total number of sensory abnormalities. Our findings indicate that patient selection based on grading of neuropathic pain may provide advantages in selecting homogenous groups for clinical research

Bilateral Sensory Abnormalities in Patients with Unilateral Neuropathic Pain; A Quantitative Sensory Testing (QST) Study

In patients who experience unilateral chronic pain, abnormal sensory perception at the non-painful side has been reported. Contralateral sensory changes in these patients have been given little attention, possibly because they are regarded as clinically irrelevant. Still, bilateral sensory changes in these patients could become clinically relevant if they challenge the correct identification of their sensory dysfunction in terms of hyperalgesia and allodynia. Therefore, we have used the standardized quantitative sensory testing (QST) protocol of the German Research Network on Neuropathic Pain (DFNS) to investigate somatosensory function at the painful side and the corresponding non-painful side in unilateral neuropathic pain patients using gender- and age-matched healthy volunteers as a reference cohort. Sensory abnormalities were observed across all QST parameters at the painful side, but also, to a lesser extent, at the contralateral, non-painful side. Similar relative distributions regarding sensory loss/gain for non-nociceptive and nociceptive stimuli were found for both sides. Once a sensory abnormality for a QST parameter at the affected side was observed, the prevalence of an abnormality for the same parameter at the non-affected side was as high as 57% (for Pressure Pain Threshold). Our results show that bilateral sensory dysfunction in patients with unilateral neuropathic pain is more rule than exception. Therefore, this phenomenon should be taken into account for appropriate diagnostic evaluation in clinical practice. This is particularly true for mechanical stimuli where the 95% Confidence Interval for the prevalence of sensory abnormalities at the non-painful side ranges between 33% and 50%

Propionyl-IIGL tetrapeptide antagonizes β-amyloid excitotoxicity in rat nucleus basalis

A putative tetrapeptide β-amyloid (Aβ) antagonist (propionyl-Ile-Ile-Gly-Leu [Pr-IIGL]) based on the [31-34] sequence of Aβ was previously shown to rescue astrocytes from Aβ-induced membrane depolarization and subsequent long-term elevations of the intracellular Ca2+ concentration in vitro. Here we provide in vivo evidence that the Pr-IIGL tetrapeptide effectively attenuates the excitotoxic action of Aβ(1-42) on cholinergic neurons of the rat magnocellular nucleus basalis (MBN). We also demonstrate by means of microdialysis that administration of Pr-IIGL abolished Aβ(1-42)-induced increases in extracellular aspartate and glutamate concentrations in the MBN, which coincide with a significant preservation of cholinergic MBN neurons and their cortical projections. This neuroprotective effect was associated with preserved exploratory behavior in an open-field paradigm, and improved memory retention in a step-through passive avoidance task. Our data presented here indicate for the first time the efficacy of short, modified functional Aβ antagonists in ameliorating Aβ excitotoxicity in vivo.

Characterization of the effect of dopamine D₃ receptor stimulation on locomotion and striatal dopamine levels

By examining the effect of dopamine (DA) D3 receptor stimulation on locomotor activity and extracellular levels of DA in striatum we show that inhibition of locomotor activity induced by DA D3 receptor-selective agonists is mediated by two interacting mechanisms: (1) directly via the stimulation of DA D3 receptors that inhibit locomotor activity, and (2) indirectly via a decrease in extracellular levels of DA. Thus, the moderately DA D3 receptor-selective agonist R-(+)-7-OH- DPAT (R-(+)-7-hydroxy-2-(N,N-di-n-propylamino)tetralin) decreased locomotor activity after administration of 10 nmol/kg and extracellular DA levels in accumbens and striatum after administration of 30 nmol/kg. A decrease in locomotor activity that coincided with a decrease in extracellular DA levels in striatum was observed after administration of 100 nmol/kg of the DA D3 receptor-selective agonist PD128907 ((+)-trans-3,4,4a,10b-tetrahydro-4-propyl-2H,5H-[1]benzopyrano[4,3b]-1,4-oxasin-9-ol). In combination with the partial, DA D3 receptor-selective agonist PD151328 (2-[4[3-(4-phenyl)-1-piperazinyl)propoxy]phenyl]-benzamidazole), a reversal of the attenuating effect of PD128907 on locomotor activity was observed, without an effect on extracellular levels of DA. In combination with a low − 10 nmol/kg - dose of haloperidol, a reversal of the inhibitory effect of PD128907 on locomotor activity was observed that coincided with an increase in extracellular levels of DA. In the presence of 0.5 mg/kg amphetamine, PD128907 decreased amphetamine-induced locomotor activity. This effect could be reversed by PD151328

Overview of sensory abnormalities in QST.

<p>Patient numbers with sensory abnormalities at the affected (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037524#pone-0037524-t002" target="_blank">Table 2A</a>, top) and contralateral side (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037524#pone-0037524-t002" target="_blank">Table 2B</a>, bottom). Sensory abnormalities were defined as Z score <−.96 or >1.96 corresponding with 95% of values obtained from healthy volunteers. Shown are direction (n of sensory gain/n of sensory loss) and overall abnormalities in percent (% of sensory abnormality) for each Quantitative Sensory Testing (QST) parameters in 81 chronic pain patients. QST parameter: Cold Pain Threshold (CPT), Heat Pain Threshold (HPT), Warm Detection Threshold (WDT), Wind Up Ratio (WUR), Cold Detection Threshold (CDT), Thermal Sensory Limen (TSL), Paradoxical Heat Sensation (PHS), Mechanical Pain Threshold (MPT), Mechanical Pain Sensitivity (MPS), Mechanical Detection Threshold (MDT), Vibration Disappearance Threshold (VDT), Pressure Pain Threshold (PPT) and Dynamic Mechanical Allodynia (DMA). Wilson estimates with upper and lower bound of the 95% CI for each QST parameter (* p<0.05).</p

Quantitative Sensory Testing (QST) abnormalities at the affected and contralateral side in neuropathic pain patients.

<p>Quantitative Sensory Testing (QST) Z-score abnormalities in % at the affected (left) and contralateral side (right) in 81 neuropathic pain patients. Sensory abnormalities were defined as Z score < −1.96 or >1.96 corresponding with 95% of values obtained from healthy volunteers. QST parameter are ordered as sensory parameters: Cold Detection Threshold (CDT), Warm Detection Threshold (WDT), Thermal Sensory Limen (TSL), Mechanical Detection Threshold (MDT), Vibration Disappearance Threshold (VDT), Paradoxical Heat Sensation (PHS), Dynamic Mechanical Allodynia (DMA) and nociceptive parameters: Cold Pain Threshold (CPT), Heat Pain Threshold (HPT), Pressure Pain Threshold (PPT), Mechanical Pain Threshold (MPT), Mechanical Pain Sensitivity (MPS) and Wind Up Ratio (WUR). Z-scores with positive sensory signs (gain of sensory function) plotted rightwards and negative sensory signs (loss of sensory function) plotted leftwards. Absence of DMA is normal and therefore no negative sign possible.</p

Z-score profiles at the affected side and contralateral side in neuropathic pain patients.

<p>Sensory abnormalities (Z score <−1.96 or >1.96) for each Quantitative Sensory Testing (QST) parameter at the affected side (left) and contralateral side (right) in 81 neuropathic pain patients. Grey area indicates parameters within the normal range (−1.96</p

Overview of sensory abnormalities at the contralateral side given an abnormality at the affected side.

<p>Patient numbers with sensory abnormalities and in their direction (n of sensory gain/n of sensory loss). Sensory abnormalities were defined as Z score < −1.96 or >1.96 corresponding with 95% of values obtained from healthy volunteers. Percent (% of sensory abnormality) indicates overall occurrence of sensory abnormalities for each Quantitative Sensory Testing (QST) parameter at the contralateral side once there was already an abnormality for the same parameter detected at the affected side in 81 chronic pain patients. QST parameters: Cold Pain Threshold (CPT), Heat Pain Threshold (HPT), Warm Detection Threshold (WDT), Wind Up Ratio (WUR), Cold Detection Threshold (CDT), Thermal Sensory Limen (TSL), Paradoxical Heat Sensation (PHS), Mechanical Pain Threshold (MPT), Mechanical Pain Sensitivity (MPS), Mechanical Detection Threshold (MDT), Vibration Disappearance Threshold (VDT), Pressure Pain Threshold (PPT) and Dynamic Mechanical Allodynia (DMA). Wilson estimates with upper and lower bound of the 95% CI for each QST parameter (* p<0.05).</p