Gliotransmission modulates baseline mechanical nociception

Pain is a physiological and adaptive process which occurs to protect organisms from tissue damage and extended injury. Pain sensation beyond injury, however, is a pathological process which is poorly understood. Experimental models of neuropathic pain demonstrate that reactive astrocytes contribute to reduced nociceptive thresholds. Astrocytes release "gliotransmitters" such as D-serine, glutamate, and ATP, which is extracellularly hydrolyzed to adenosine. Adenosine 1 receptor activation in the spinal cord has anti-nociceptive effects on baseline pain threshold, but the source of the endogenous ligand (adenosine) in the spinal cord is unknown. In this study we used a transgenic mouse model in which SNARE-mediated gliotransmission was selectively attenuated (called dnSNARE mice) to investigate the role of astrocytes in mediating baseline nociception and the development of neuropathic pain. Under baseline conditions, immunostaining in the dorsal horn of the spinal cord showed astrocyte-specific transgene expression in dnSNARE mice, and no difference in expression levels of the astrocyte marker GFAP and the microglia marker Iba1 relative to wild-type mice. The Von Frey filament test was used to probe sensitivity to baseline mechanical pain thresholds and allodynia following the spared nerve injury model of neuropathic pain. DnSNARE mice exhibit a reduced nociceptive threshold in response to mechanical stimulation compared to wild-type mice under baseline conditions, but nociceptive thresholds following spared nerve injury were similar between dnSNARE and wild-types. This study is the first to provide evidence that gliotransmission contributes to basal mechanical nociception

Gliotransmission modulates baseline mechanical nociception

Abstract Pain is a physiological and adaptive process which occurs to protect organisms from tissue damage and extended injury. Pain sensation beyond injury, however, is a pathological process which is poorly understood. Experimental models of neuropathic pain demonstrate that reactive astrocytes contribute to reduced nociceptive thresholds. Astrocytes release "gliotransmitters" such as D-serine, glutamate, and ATP, which is extracellularly hydrolyzed to adenosine. Adenosine 1 receptor activation in the spinal cord has anti-nociceptive effects on baseline pain threshold, but the source of the endogenous ligand (adenosine) in the spinal cord is unknown. In this study we used a transgenic mouse model in which SNARE-mediated gliotransmission was selectively attenuated (called dnSNARE mice) to investigate the role of astrocytes in mediating baseline nociception and the development of neuropathic pain. Under baseline conditions, immunostaining in the dorsal horn of the spinal cord showed astrocyte-specific transgene expression in dnSNARE mice, and no difference in expression levels of the astrocyte marker GFAP and the microglia marker Iba1 relative to wild-type mice. The Von Frey filament test was used to probe sensitivity to baseline mechanical pain thresholds and allodynia following the spared nerve injury model of neuropathic pain. DnSNARE mice exhibit a reduced nociceptive threshold in response to mechanical stimulation compared to wild-type mice under baseline conditions, but nociceptive thresholds following spared nerve injury were similar between dnSNARE and wild-types. This study is the first to provide evidence that gliotransmission contributes to basal mechanical nociception. Keywords: Adenosine, Astrocyte, Gliotransmission, Pain Findings Pain sensation is an adaptive response to impending tissue damage that protects an organism from extended injury. Pain perception involves a series of cellular interactions and responses from immune cells, glia and neurons. Signals from glial cells trigger neuronal responses, and vice versa, initiating a complex cascade of cell-cell interactions and feedback mechanisms Acute pain stimuli excite primary nociceptive neurons, which synapse and release glutamate and substance-P onto postsynaptic neurons in the dorsal horn of the spinal cord. Under chronic pain conditions, this synapse exhibits an LTP-like state where increased responses from dorsal horn neurons are elicited by afferent stimulatio

Measures for assessing practice change in medical practitioners

BACKGROUND: There are increasing numbers of randomised trials and systematic reviews examining the efficacy of interventions designed to bring about a change in clinical practice. The findings of this research are being used to guide strategies to increase the uptake of evidence into clinical practice. Knowledge of the outcomes measured by these trials is vital not only for the interpretation and application of the work done to date, but also to inform future research in this expanding area of endeavour and to assist in collation of results in systematic reviews and meta-analyses. METHODS: The objective of this review was to identify methods used to measure change in the clinical practices of health professionals following an intervention aimed at increasing the uptake of evidence into practice. All published trials included in a recent, comprehensive Health Technology Assessment of interventions to implement clinical practice guidelines and change clinical practice (n = 228) formed the sample for this study. Using a standardised data extraction form, one reviewer (SH), extracted the relevant information from the methods and/or results sections of the trials. RESULTS: Measures of a change of health practitioner behaviour were the most common, with 88.8% of trials using these as outcome measures. Measures that assessed change at a patient level, either actual measures of change or surrogate measures of change, were used in 28.8% and 36.7% of studies (respectively). Health practitioners' knowledge and attitudes were assessed in 22.8% of the studies and changes at an organisational level were assessed in 17.6%. CONCLUSION: Most trials of interventions aimed at changing clinical practice measured the effect of the intervention at the level of the practitioner, i.e. did the practitioner change what they do, or has their knowledge of and/or attitude toward that practice changed? Less than one-third of the trials measured, whether or not any change in practice, resulted in a change in the ultimate end-point of patient health status

Extracellular Adenosine Protects against <i>Streptococcus pneumoniae</i> Lung Infection by Regulating Pulmonary Neutrophil Recruitment

<div><p>An important determinant of disease following <i>Streptococcus pneumoniae</i> (pneumococcus) lung infection is pulmonary inflammation mediated by polymorphonuclear leukocytes (PMNs). We found that upon intratracheal challenge of mice, recruitment of PMNs into the lungs within the first 3 hours coincided with decreased pulmonary pneumococci, whereas large numbers of pulmonary PMNs beyond 12 hours correlated with a greater bacterial burden. Indeed, mice that survived infection largely resolved inflammation by 72 hours, and PMN depletion at peak infiltration, i.e. 18 hours post-infection, lowered bacterial numbers and enhanced survival. We investigated host signaling pathways that influence both pneumococcus clearance and pulmonary inflammation. Pharmacologic inhibition and/or genetic ablation of enzymes that generate extracellular adenosine (EAD) (e.g. the ectoenzyme CD73) or degrade EAD (e.g. adenosine deaminase) revealed that EAD dramatically increases murine resistance to <i>S</i>. <i>pneumoniae</i> lung infection. Moreover, adenosine diminished PMN movement across endothelial monolayers <i>in vitro</i>, and although inhibition or deficiency of CD73 had no discernible impact on PMN recruitment within the first 6 hours after intratracheal inoculation of mice, these measures enhanced PMN numbers in the pulmonary interstitium after 18 hours of infection, culminating in dramatically elevated numbers of pulmonary PMNs at three days post-infection. When assessed at this time point, <i>CD73</i>^<i>-/-</i> mice displayed increased levels of cellular factors that promote leukocyte migration, such as CXCL2 chemokine in the murine lung, as well as CXCR2 and β-2 integrin on the surface of pulmonary PMNs. The enhanced pneumococcal susceptibility of <i>CD73</i>^<i>-/-</i> mice was significantly reversed by PMN depletion following infection, suggesting that EAD-mediated resistance is largely mediated by its effects on PMNs. Finally, CD73-inhibition diminished the ability of PMNs to kill pneumococci <i>in vitro</i>, suggesting that EAD alters both the recruitment and bacteriocidal function of PMNs. The EAD-pathway may provide a therapeutic target for regulating potentially harmful inflammatory host responses during Gram-positive bacterial pneumonia.</p></div

Inhibition of adenosine breakdown promotes resistance to <i>S</i>. <i>pneumoniae</i> lung challenge.

<p>C57BL/6J mice mock-treated or treated with EHNA-hydrochloride, an adenosine deaminase inhibitor, were inoculated I.T with 5x10⁵ CFU of <i>S</i>. <i>pneumoniae</i> TIGR4. Bacterial burdens in the lungs (A) or blood (B), as well as survival (C), was determined 3 days post-infection. Data pooled from 2 separate experiments (n = 6 mice per group) are shown. Data represent means +/- SEM. ** = <i>p</i>< 0.001 and * = <i>p</i><0.05 indicate means significantly different from mock-treated group by Student’s t-test. Below the graphs are indicated the fraction of surviving mice within each group.</p

PMNs promote pulmonary and systemic disease during later stages of <i>S</i>. <i>pneumoniae</i> lung infection.

<p>(A) C57BL/6J mice were inoculated I.T with 5x10⁵ CFU of <i>S</i>. <i>pneumoniae</i> TIGR4 and pulmonary (green) and bloodstream (red) bacterial loads, as well as pulmonary PMNs (blue) were monitored through 72 hours post-infection. Shown are representative data from one of two separate experiments (using 3 to 4 mice per time point). The numbers above the graph represent the fraction of surviving mice within that group at the corresponding time. (B-D) C57BL/6J mice were treated i.p with PMN depleting antibodies (anti-ly6G) or isotype control either 18 hours pre or post pulmonary challenge with 5x10⁵ CFU of <i>S</i>. <i>pneumoniae</i> TIGR4. Survival (B) and bacterial burdens in the blood (C) were monitored over time and shown are pooled data from two separate experiments. (D) Pneumococcal burdens in the lungs and blood were determined 3 days post-infection. The numbers below the graph represent the fraction of surviving mice within that group. Representative data from one of 4 separate experiments with 3 to 4 mice per group are shown. Means +/- SEM are given in Panels A, C and D, and values significantly (<i>p</i><0.05) different from isotype control-treated group by Student’s t-test are indicated by asterisk. In Panel B, asterisk indicates survival rate was significantly (<i>p</i><0.05) different from isotype control-treated controls by Log-rank (Mantel-Cox) test.</p

Inhibition of EAD production or signaling enhances susceptibility of mice to <i>S</i>. <i>pneumoniae</i> lung challenge.

<p>(A-D) Wild-type C57BL/6J mice were either treated with the CD73 inhibitor (α,β- methylene ADP), the pan adenosine receptor (AR) inhibitor (CGS 15943) or mock-treated with a vehicle control. (A) Bacterial loads in the lungs of a group of mice were determined 3 days after I.T. inoculation with 5x10³ CFU of <i>S</i>. <i>pneumoniae</i> TIGR4. (B-C) Bacteremia (B) and survival (C) were monitored over time for another group of mice. Pooled data from 3 separate experiments (n = 6–12 mice per group) are shown. (D) Mice were challenged I.T. with a high dose, ~1x10⁷ CFU of <i>S</i>. <i>pneumoniae</i> TIGR4 and bacterial burdens in the lungs and blood were assessed at two days post-infection. Pooled data from two separate experiments (n = 6 mice per group) are shown. Below figures A and D are indicated the fraction of surviving mice within each group. (E) <i>CD73</i>^<i>-/-</i> mice and wild-type B6 controls were inoculated I.T. with 5x10³ CFU of <i>S</i>. <i>pneumoniae</i> TIGR4 and bacterial burdens in the lungs and blood were measured at the indicated time points post-infection. Data pooled from 3 separate experiments (n = 6–9 mice per group) are shown. None of the mice died within the time frame of these (E) experiments. Data represent means +/- SEM. Means that are significantly different from mock-treated group (A-B) or wild-type control group (E) by student t-test are indicated by asterisks (*** = <i>p</i>< 0.0001; ** = <i>p</i>< 0.001; * = <i>p</i><0.05). In Panel C, a survival rate significantly (<i>p</i><0.05) different from mock-treated controls by Log-rank (Mantel-Cox) test is indicated by asterisk.</p

Inhibition of EAD production or signaling significantly increases PMN numbers in the pulmonary tissues.

<p>Wild-type untreated, mock-treated or CD73-inhibited C57BL/6 mice and CD73-ablated (<i>CD73</i>^<i>-/-</i>) mice were inoculated I.T. with 5x 10³ CFU of <i>S</i>. <i>pneumoniae</i> TIGR4. (A) H&E-stained lung sections examined by light microscopy at 3 days post-infection (10× magnification; inset at 40x magnification). (B) The mean +/- SEM of pulmonary PMNs (Ly6G⁺ cells) in mock-treated, CD73-inhibited, or adenosine receptor (AR)-inhibited mice, measured by flow cytometry at 72 hours post-infection. Uninfected mice of the different treatment groups had comparable low numbers (less than 10⁵) of PMNs in the lungs. (C) The number of PMNs in the bronchio-alveolar lavage fluid (BALF) was also determined by flow cytometry. (D) The mean +/- SEM of pulmonary PMNs of wild-type or <i>CD73</i>^<i>-/-</i> mice was determined over time at the indicated times post-infection. Pooled data from three separate experiments (Panels B and C n = 8–9 mice per group; Panel D n = 6–9 mice per group) are shown. Statistically significant differences determined by student’s t-test are indicated by asterisks (*** = <i>p</i>< 0.0001; ** = <i>p</i>< 0.001; * = <i>p</i><0.05).</p

EAD negatively regulates PMN migration across the endothelium but not the epithelium.

<p>(A) Media alone or <i>S</i>. <i>pneumoniae-</i>containing media was added to the lower chamber of HUVEC-seeded Transwell dishes for 3 hours. Transmigration of PMNs added to the upper chamber in media containing vehicle control or increasing concentrations of the CD73 inhibitor α,β- methylene ADP (left panel) or pan-adenosine receptor (AR) inhibitor CGS 15943 (right panel) was measured using a hemocytometer. The means +/- SEM from one representative of three experiments are shown and values significantly different from media control, determined by student’s t-test, are indicated by asterisk (*** = <i>p</i>< 0.0001; ** = <i>p</i>< 0.001; * = <i>p</i><0.05). (B) Polarized H292 epithelial cells, pre-treated with increasing concentrations CD73 inhibitor α,β- methylene ADP (left panel) or just adenosine (right panel), were left uninfected (“media”) or infected apically with pneumococcus (“+ <i>S</i>. <i>pneumoniae</i>”). The transmigration of PMNs, added to the basolateral side in media alone or the indicated concentrations of CD73 inhibitor or adenosine was measured by myeloperoxidase ELISA. Data represent means +/- SEM, and shown are one of three separate experiments.</p

CD73 modulates the induction of leukocyte recruitment signals upon I.T. challenge by <i>S</i>. <i>pneumoniae</i>.

<p>Wild-type C57BL/6 or <i>CD73</i>^<i>-/-</i> mice were mock-infected or I.T. challenged with 5 x 10³ CFU of <i>S</i>. <i>pneumoniae</i> TIGR4 (<i>+S</i>.<i>p</i>). Three days after challenge, levels of CXCL-2 in the lung homogenates were determined by ELISA (A) and the mean florescent intensities (MFI) of CXCR2 (B) or CD18 (C) on PMNs (Ly6G+) recruited into the lungs were determined by flow cytometry (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005126#sec014" target="_blank">Materials and Methods</a>). Pooled data from two separate experiments (n = 6 infected and n = 4 uninfected mice per group) are shown. Data represent means +/- SEM, and significant differences determined by Student’s t-test are indicated by asterisks (*** = <i>p</i>< 0.0001 and * = <i>p</i><0.05).</p