Retinal microglial cells increase expression and release of IL-1ß when exposed to ATP.

Cytokine IL-1ß is an early component of inflammatory cascades, with both priming and activation steps required before IL-1ß release. Here, the P2X7 receptor (P2X7R) for ATP was shown to both prime and release IL-1ß from retinal microglial cells. Isolated retinal microglial cells increased expression of Il1b when stimulated with endogenous receptor agonist extracellular ATP; ATP also rapidly downregulated expression of microglial markers Tmem119 and Cd206. Changes to all three genes were reduced by specific P2X7R antagonist A839977, implicating the P2X7R. Microglial cells expressed the P2X7R on ramifications and responded to receptor agonist BzATP with robust and rapid rises in intracellular Ca 2+ . BzATP increased expression of IL-1ß protein colocalizing with CX3CR1-GFP in retinal wholemounts consistent with microglial cells. ATP also triggered release of IL-1ß from isolated retinal microglia into the bath; release was inhibited by A839977 and induced by BzATP, supporting a role for the P2X7R in release as well as priming. The IL-1ß release triggered by ATP was substantially greater from microglial cells compared to astrocytes from the optic nerve head region. Il1b expression was increased by a transient rise in intraocular pressure and Il1b levels remained elevated 10 days after a single IOP elevation. In summary, this study suggests the P2X7 receptor can both prime IL-1ß levels in microglial cells and trigger its release. The P2Y12R was previously identified as a chemoattractant for retinal microglia, suggesting the recruitment of the cells towards the source of released extracellular ATP could position microglia for P2X7R receptor, enabling both priming and release of IL-1ß.

Fluorescent identification of axons, dendrites and soma of neuronal retinal ganglion cells with a genetic marker as a tool for facilitating the study of neurodegeneration.

This study characterizes a fluorescent Slc17a6 -tdTomato neuronal reporter mouse line offering strong labeling in axons throughout the optic nerve, dendrites and soma in 99% of retinal ganglion cells (RGCs). The model facilitates neuronal assessment ex vivo with wholemounts quantified to show neurodegeneration following optic nerve crush or elevated IOP as related to glaucoma, in vitro with robust Ca 2+ responses to P2X7 receptor stimulation in neuronal cultures, and in vivo using a confocal scanning laser ophthalmoscope (cSLO). While the tdTomato signal showed strong overlap with RGC markers, BRN3A and RBPMS, there was no cross-labeling of displaced amacrine cells in the ganglion cell layer. Controls indicated no impact of Slc17a6 -tdTomato expression on light-dependent neuronal function, as determined with a microelectrode array (MEA), or on structure, as measured with optical coherence tomography (OCT). In summary, this novel neuronal reporter mouse model offers an effective means to increase the efficiency for real-time, specific visualization of retinal ganglion cells. It holds substantial promise for enhancing our understanding of RGC pathology in glaucoma and other diseases of the optic nerve, and could facilitate the screening of targeted therapeutic interventions for neurodegeneration.

Piezo1 and Piezo2 channels in retinal ganglion cells and the impact of Piezo1 stimulation on light-dependent neural activity.

Piezo channels are associated with neuropathology in diseases like traumatic brain injury and glaucoma, but pathways linking tissue stretch to aberrant neural signaling remain unclear. The present study demonstrates that Piezo1 activation increases action potential frequency in response to light and the spontaneous dark signal from mouse retinal explants. Piezo1 stimulation was sufficient to increase cytoplasmic Ca 2+ in soma and neurites, while stretch increased spiking activity in current clamp recordings from of isolated retinal ganglion cells (RGCs). Axon-marker beta-tubulin III colocalized with both Piezo1 and Piezo2 protein in the mouse optic nerve head, while RGC nuclear marker BRN3A colocalized with Piezo channels in the soma. Piezo1 was also present on GFAP-positive regions in the optic nerve head and colocalized with glutamine synthetase in the nerve fiber layer, suggesting expression in optic nerve head astrocytes and Müller glia end feet, respectively. Human RGCs from induced pluripotent stem cells also expressed Piezo1 and Piezo2 in soma and axons, while staining patterns in rats resembled those in mice. mRNA message for Piezo1 was greatest in the RPE/choroid tissue, while Piezo2 levels were highest in the optic nerve, with both channels also expressed in the retina. Increased expression of Piezo1 and Piezo2 occurred both 1 and 10 days after a single stretch in vivo; this increase suggests a potential role in rising sensitivity to repeated nerve stretch. In summary, Piezo1 and Piezo2 were detected in the soma and axons of RGCs, and stimulation affected the light-dependent output of RGCs. The rise in RGCs excitability induced by Piezo stimulation may have parallels to the early disease progression in models of glaucoma and other retinal degenerations. Highlights: Activation of Piezo1 excites retinal ganglion cells, paralleling the early neurodegenerative progression in glaucoma mouse models and retinal degeneration.Piezo1 and Piezo2 were expressed in axons and soma of retinal ganglion cells in mice, rats, and human iPSC-RGCs.Functional assays confirmed Piezo1 in soma and neurites of neurons. Sustained elevation of Piezo1 and Piezo2 occurred after a single transient stretch may enhance damage from repeated traumatic nerve injury.

Increased Purinergic Signaling in Human Dental Pulps With Inflammatory Pain is Sex-Dependent.

An enhanced understanding of neurotransmitter systems contributing to pain transmission aids in drug development, while the identification of biological variables like age and sex helps in the development of personalized pain management and effective clinical trial design. This study identified enhanced expression of purinergic signaling components specifically in painful inflammation, with levels increased more in women as compared to men. Inflammatory dental pain is common and potentially debilitating; as inflammation of the dental pulp can occur with or without pain, it provides a powerful model to examine distinct pain pathways in humans. In control tissues, P2X3 and P2X2 receptors colocalized with PGP9.5-positive nerves. Expression of the ecto-nucleotidase NTPDase1 (CD39) increased with exposure to extracellular adenosine triphosphate (ATP), implying CD39 acted as a marker for sustained elevation of extracellular ATP. Both immunohistochemistry and immunoblots showed P2X2, P2X3, and CD39 increased in symptomatic pulpitis, suggesting receptors and the ATP agonist were elevated in patients with increased pain. The increased expression of P2X3 and CD39 was more frequently observed in women than men. In summary, this study identifies CD39 as a marker for chronic elevation of extracellular ATP in fixed human tissue. It supports a role for increased purinergic signaling in humans with inflammatory dental pain and suggests the contribution of purines shows sexual dimorphism. This highlights the potential for P2X antagonists to treat pain in humans and stresses the need to consider sex in clinical trials that target pain and purinergic pathways. PERSPECTIVE: This article demonstrates an elevation of ATP-marker CD39 and of ATP receptors P2X2 and P2X3 with inflammatory pain and suggests the rise is greater in women. This highlights the potential for P2X antagonists to treat pain and stresses the consideration of sexual dimorphism in studies of purines and pain.

Rapid morphologic changes to microglial cells and upregulation of mixed microglial activation state markers induced by P2X7 receptor stimulation and increased intraocular pressure.

BACKGROUND: The identification of endogenous signals that lead to microglial activation is a key step in understanding neuroinflammatory cascades. As ATP release accompanies mechanical strain to neural tissue, and as the P2X7 receptor for ATP is expressed on microglial cells, we examined the morphological and molecular consequences of P2X7 receptor stimulation in vivo and in vitro and investigated the contribution of the P2X7 receptor in a model of increased intraocular pressure (IOP). METHODS: In vivo experiments involved intravitreal injections and both transient and sustained elevation of IOP. In vitro experiments were performed on isolated mouse retinal and brain microglial cells. Morphological changes were quantified in vivo using Sholl analysis. Expression of mRNA for M1- and M2-like genes was determined with qPCR. The luciferin/luciferase assay quantified retinal ATP release while fura-2 indicated cytoplasmic calcium. Microglial migration was monitored with a Boyden chamber. RESULTS: Sholl analysis of Iba1-stained cells showed retraction of microglial ramifications 1 day after injection of P2X7 receptor agonist BzATP into mouse retinae. Mean branch length of ramifications also decreased, while cell body size and expression of Nos2, Tnfa, Arg1, and Chil3 mRNA increased. BzATP induced similar morphological changes in ex vivo tissue isolated from Cx3CR1+/GFP mice, suggesting recruitment of external cells was unnecessary. Immunohistochemistry suggested primary microglial cultures expressed the P2X7 receptor, while functional expression was demonstrated with Ca2+ elevation by BzATP and block by specific antagonist A839977. BzATP induced process retraction and cell body enlargement within minutes in isolated microglial cells and increased Nos2 and Arg1. While ATP increased microglial migration, this required the P2Y12 receptor and not P2X7 receptor. Transient elevation of IOP led to microglial process retraction, cell body enlargement, and gene upregulation paralleling changes observed with BzATP injection, in addition to retinal ATP release. Pressure-dependent changes were reduced in P2X7-/- mice. Death of retinal ganglion cells accompanied increased IOP in C57Bl/6J, but not P2X7-/- mice, and neuronal loss showed some association with microglial activation. CONCLUSIONS: P2X7 receptor stimulation induced rapid morphological activation of microglial cells, including process retraction and cell body enlargement, and upregulation of markers linked to both M1- and M2-type activation. Parallel responses accompanied IOP elevation, suggesting ATP release and P2X7 receptor stimulation influence the early microglial response to increased pressure.

Short-term and long-term effects of rapid maxillary expansion on the nasal soft and hard tissue.

OBJECTIVES: To evaluate nasal soft and hard tissue changes immediately post-rapid maxillary expansion (RME) and to assess the stability of these changes using cone beam computed tomography (CBCT). MATERIALS AND METHODS: A total of 35 treatment group (TG) patients (18 girls, 17 boys; 9.39 ± 1.4) had a pre-RME CBCT and a post-RME CBCT approximately 66 days after expansion, and 25 patients had a follow-up CBCT 2.84 years later. A total of 28 control group (CG; no RME) patients (16 girls, 12 boys; 8.81 ± 1.6) had an initial CBCT and a CBCT an average of 2.25 years later. Soft and hard tissue nasal landmarks were measured in transverse, sagittal, and coronal planes of space on CBCT scans. Differences within the same group were evaluated by paired t-tests or Wilcoxon signed-rank tests. Long-term comparisons between TG and CG were evaluated by independent-sample t-tests or Wilcoxon rank-sum tests. RESULTS: Immediately post-RME, there were statistically significant mean increases of 1.6 mm of alar base width, 1.77 mm of pyriform height, and 3.57 mm of pyriform width (P < .05). CG showed the significant increases over 2.25 years (P < .001). Compared with CG, the long-term evaluation of TG demonstrated only pyriform height and pyriform width showed a statistically significant difference (P < .01). CONCLUSIONS: Although RME produced some significant increase on the nasal soft tissue immediately after expansion, it regressed to the mean of normal growth and development over time. However, long-term evaluation of TG compared with CG showed only pyriform height and pyriform width to be affected by RME.