Models of Inflammation: Carrageenan Air Pouch

The subcutaneous air pouch is an in vivo model that can be used to study the components of acute and chronic inflammation, the resolution of the inflammatory response, the oxidative stress response, and potential therapeutic targets for treating inflammation. Injection of irritants into an air pouch in rats or mice induces an inflammatory response that can be quantified by the volume of exudate produced, the infiltration of cells, and the release of inflammatory mediators. The model presented in this unit has been extensively used to identify potential anti-inflammatory drugs

Models of inflammation: Carrageenan- or complete Freund's Adjuvant (CFA)-induced edema and hypersensitivity in the rat

Animal models of inflammation are used to assess the production of inflammatory mediators at sites of inflammation, the anti-inflammatory properties of agents such as nonsteroidal anti-inflammatory drugs (NSAIDs), and the efficacy of putative analgesic compounds in reversing cutaneous hypersensitivity. This unit details methods to elicit and measure carrageenan- and complete Freund's adjuvant (CFA)-induced cutaneous inflammation. Due to possible differences between the dorsal root sensory system and the trigeminal sensory system, injections of either the footpad or vibrissal pad are described. In this manner, cutaneous inflammation can be assessed in tissue innervated by the lumbar dorsal root ganglion neurons (footpad) and by the trigeminal ganglion neurons (vibrissal pad)

Aromatase inhibitors augment nociceptive behaviors in rats and enhance the excitability of sensory neurons

Although aromatase inhibitors (AIs) are commonly used therapies for breast cancer, their use is limited because they produce arthralgia in a large number of patients. To determine whether AIs produce hypersensitivity in animal models of pain, we examined the effects of the AI, letrozole, on mechanical, thermal, and chemical sensitivity in rats. In ovariectomized (OVX) rats, administering a single dose of 1 or 5mg/kg letrozole significantly reduced mechanical paw withdrawal thresholds, without altering thermal sensitivity. Repeated injection of 5mg/kg letrozole in male rats produced mechanical, but not thermal, hypersensitivity that extinguished when drug dosing was stopped. A single dose of 5mg/kg letrozole or daily dosing of letrozole or exemestane in male rats also augmented flinching behavior induced by intraplantar injection of 1000nmol of adenosine 5'-triphosphate (ATP). To determine whether sensitization of sensory neurons contributed to AI-induced hypersensitivity, we evaluated the excitability of neurons isolated from dorsal root ganglia of male rats chronically treated with letrozole. Both small and medium-diameter sensory neurons isolated from letrozole-treated rats were more excitable, as reflected by increased action potential firing in response to a ramp of depolarizing current, a lower resting membrane potential, and a lower rheobase. However, systemic letrozole treatment did not augment the stimulus-evoked release of the neuropeptide calcitonin gene-related peptide (CGRP) from spinal cord slices, suggesting that the enhanced nociceptive responses were not secondary to an increase in peptide release from sensory endings in the spinal cord. These results provide the first evidence that AIs modulate the excitability of sensory neurons, which may be a primary mechanism for the effect of these drugs to augment pain behaviors in rats

Models of inflammation: carrageenan air pouch

The subcutaneous air pouch is an in vivo model that can be used to study acute and chronic inflammation, the resolution of the inflammatory response, and the oxidative stress response. Injection of irritants into an air pouch in rats or mice induces an inflammatory response that can be quantified by the volume of exudate produced, the infiltration of cells, and the release of inflammatory mediators. The model presented in this unit has been extensively used to identify potential anti-inflammatory drugs

Nerve growth factor mediates a switch in intracellular signaling for PGE2-induced sensitization of sensory neurons from protein kinase A to Epac.

We examined whether nerve growth factor (NGF), an inflammatory mediator that contributes to chronic hypersensitivity, alters the intracellular signaling that mediates the sensitizing actions of PGE2 from activation of protein kinase A (PKA) to exchange proteins directly activated by cAMP (Epacs). When isolated sensory neurons are grown in the absence of added NGF, but not in cultures grown with 30 ng/ml NGF, inhibiting protein kinase A (PKA) activity blocks the ability of PGE2 to augment capsaicin-evoked release of the neuropeptide CGRP and to increase the number of action potentials (APs) evoked by a ramp of current. Growing sensory neurons in culture in the presence of increasing concentrations of NGF increases the expression of Epac2, but not Epac1. An intradermal injection of complete Freund's adjuvant into the rat hindpaw also increases the expression of Epac2, but not Epac1 in the dorsal root ganglia and spinal cord: an effect blocked by intraplantar administration of NGF antibodies. Treating cultures grown in the presence of 30 ng/ml NGF with Epac1siRNA significantly reduced the expression of Epac1, but not Epac2, and did not block the ability of PGE2 to augment capsaicin-evoked release of CGRP from sensory neurons. Exposing neuronal cultures grown in NGF to Epac2siRNAreduced the expression of Epac2, but not Epac1 and prevented the PGE2-induced augmentation of capsaicin and potassium-evoked CGRP release in sensory neurons and the PGE2-induced increase in the number of APs generated by a ramp of current. In neurons grown with no added NGF, Epac siRNAs did not attenuate PGE2-induced sensitization. These results demonstrate that NGF, through increasing Epac2 expression, alters the signaling cascade that mediates PGE2-induced sensitization of sensory neurons, thus providing a novel mechanism for maintaining PGE2-induced hypersensitivity during inflammation

Reduced expression of Epac2 attenuates PGE₂-induced augmentation of capsaicin-evoked iCGRP release in sensory neurons grown in NGF, but has no effect in neurons grown without added NGF.

<p>A and B: Columns are the mean ± SEM of iCGRP release as % of total peptide content/well/10 min from sensory neurons grown without added NGF (A) or in the presence of 30 ng/ml NGF (B). Wells of cells (n = 9 from a minimum of 3 separate harvests) were exposed for 10 min to HEPES alone (Basal; open columns), or HEPES in the presence of 30 nM capsaicin (CAP; shaded columns) in the absence or presence of 1 µM PGE₂ as indicated by the horizontal bars. Release experiments were performed in cultures exposed to 200 nM scramble siRNA, siRNA to Epac1, or siRNA to Epac2 as indicated. An asterisk indicates a significant difference from basal release, whereas a cross indicates a significant difference in capsaicin-stimulated release in the presence of PGE₂ compared to vehicle control using analysis of variance with Tukey's post hoc test. C: The top portion shows a representative Western blots of Epac1 and actin from cultures grown without added NGF or with 30 ng/ml NGF as indicated and treated with 200 nM scramble siRNA (SCsiRNA), Epac1siRNA, or Epac2siRNA as indicated. In the lower portion, each column represents the mean ± SEM of the effects of exposure to SCsiRNA, Epac1siRNA or Epac2siRNA on Epac1 expression from 3 experiments. An asterisk indicates a statistically significant decrease in Epac expression using ANOVA and Tukey's post hoc test. D: The top portion shows a representative Western blots of Epac2 and actin from cultures grown without added NGF or with 30 ng/ml NGF as indicated and treated with 200 nM scramble siRNA (SCsiRNA), Epac1siRNA, or Epac2siRNA as indicated. In the lower portion, each column represents the mean ± SEM of the effects of exposure to SCsiRNA, Epac1siRNA or Epac2siRNA on Epac2 expression from 3 experiments. An asterisk indicates a statistically significant decrease in Epac expression using ANOVA and Tukey's post hoc test.</p

Inflammation-induced increase in Epac2 expression is attenuated by anti-NGF antibodies.

<p>The top figures show representative Western blots of Epacs and actin from L4-L5 dorsal root ganglia (left panel) and dorsal spinal cord (right panel) ipsilateral to the inflamed paw 5 days after the injection of CFA. In the middle and lower figures, each column represents the mean ± SEM of the effects of saline injection, injection of anti-NGF antibody alone, CFA, or CFA plus anti-NGF antibodies on Epac1 (middle panels) or Epac2 (lower panels) expression from 3 independent experiments normalized to the amount of actin detected using densitometry. An asterisk indicates a statistically significant increase in the expression of Epac2 compared to tissue from saline injected animals, whereas a cross indicates a significant difference in Epac2 expression compared to CFA-treatment alone using analysis of variance and Tukey's post hoc test.</p

Epac2 mediates PGE₂-induced increase in excitability in sensory neurons grown in NGF.

<p>The top panels show representative recordings from sensory neurons treated with scramble siRNA (SCsiRNA) or Epac2siRNA as indicated. Cells were exposed to the same ramp of depolarizing current prior to (control) and after a 10 min exposure to 1 µM PGE₂. The bottom panels are summary data from 4–9 capsaicin-sensitive sensory neurons. Each column is the mean ± SEM of number of APs at the various times and treatments as indicated. Ramp amplitudes ranged from 1000 to 8000 pA, but were the same for each individual cell prior to and after treatment. An asterisk indicates significant differences (p<0.05) compared to control values using analysis of variance with the Holm-Sidak post hoc test.</p

Nerve growth factor increases the expression of Epac2, but not Epac1 in adult sensory neurons in culture.

<p>A. The top portion shows a representative Western blot from neurons grown in culture for 8 days in various concentrations of NGF as indicated. In the lower portion, each column represents the mean ± SEM of the effects of NGF treatment on expression of Epac 1 (middle panel) or Epac2 (lower panel) from 3 experiments. B. Each column represents the mean ± SEM of the fold increase in mRNA (normalized to cells grown in the absence of added NGF) for Epac1 (open columns) or Epac2 (shaded columns) in cells grown for 8 days in various concentrations of NGF as indicated. In all panels, an asterisk indicates a statistically significant increase in Epac expression after NGF exposure using analysis of variance with Tukey's post hoc test.</p

The protein kinase inhibitor, H-89 attenuates PGE₂-induced sensitization of sensory neurons grown in the absence of NGF but not in cells grown in the presence of NGF.

<p>Each column represents the mean ± S.E.M. of iCGRP release in fmol/well/10 min in cultures for cells grown in the absence of added NGF (A) or in cultures grown in the presence of 30 ng/ml NGF (B). Wells of cells (n = 9–15 from a minimum of 3 separate harvests) were exposed for 10 min to HEPES alone (basal; open columns), or HEPES in the presence of 30 nM capsaicin (CAP; shaded columns) in the absence or presence of vehicle, 1 µM PGE₂, or 1 µM PGE₂ and 10 µM H89 as indicated by the horizontal bars. An asterisk indicates a significant difference from basal release, whereas a cross indicates a significant difference in capsaicin-stimulated release in the presence of PGE₂ compared to vehicle using analysis of variance with Tukey's post hoc test.</p