Ageing and gastrointestinal sensory function: Altered colonic mechanosensory and chemosensory function in the aged mouse

Ageing has a profound effect upon gastrointestinal function through mechanisms that are poorly understood. Here we investigated the effect of age upon gastrointestinal sensory signalling pathways in order to address the mechanisms underlying these changes. In vitro mouse colonic and jejunal preparations with attached splanchnic and mesenteric nerves were used to study mechanosensory and chemosensory afferent function in 3-, 12- and 24-month-old C57BL/6 animals. Quantitative RT-PCR was used to investigate mRNA expression in colonic tissue and dorsal root ganglion (DRG) cells isolated from 3- and 24-month animals, and immunohistochemistry was used to quantify the number of 5-HT-expressing enterochromaffin (EC) cells. Colonic and jejunal afferent mechanosensory function was attenuated with age and these effects appeared earlier in the colon compared to the jejunum. Colonic age-related loss of mechanosensory function was more pronounced in high-threshold afferents compared to low-threshold afferents. Chemosensory function was attenuated in the 24-month colon, affecting TRPV1 and serotonergic signalling pathways. High-threshold mechanosensory afferent fibres and small-diameter DRG neurons possessed lower functional TRPV1 receptor responses, which occurred without a change in TRPV1 mRNA expression. Serotonergic signalling was attenuated at 24 months, but TPH1 and TPH2 mRNA expression was elevated in colonic tissue. In conclusion, we saw an age-associated decrease in afferent mechanosensitivity in the mouse colon affecting HT units. These units have the capacity to sensitise in response to injurious events, and their loss in ageing may predispose the elderly to lower awareness of GI injury or disease

Critical role for iron accumulation in the pathogenesis of fibrotic lung disease

Increased iron levels and dysregulated iron homeostasis, or both, occur in several lung diseases. Here, the effects of iron accumulation on the pathogenesis of pulmonary fibrosis and associated lung function decline was investigated using a combination of murine models of iron overload and bleomycin-induced pulmonary fibrosis, primary human lung fibroblasts treated with iron, and histological samples from patients with or without idiopathic pulmonary fibrosis (IPF). Iron levels are significantly increased in iron overloaded transferrin receptor 2 (Tfr2) mutant mice and homeostatic iron regulator (Hfe) gene–deficient mice and this is associated with increases in airway fibrosis and reduced lung function. Furthermore, fibrosis and lung function decline are associated with pulmonary iron accumulation in bleomycin-induced pulmonary fibrosis. In addition, we show that iron accumulation is increased in lung sections from patients with IPF and that human lung fibroblasts show greater proliferation and cytokine and extracellular matrix responses when exposed to increased iron levels. Significantly, we show that intranasal treatment with the iron chelator, deferoxamine (DFO), from the time when pulmonary iron levels accumulate, prevents airway fibrosis and decline in lung function in experimental pulmonary fibrosis. Pulmonary fibrosis is associated with an increase in Tfr1+ macrophages that display altered phenotype in disease, and DFO treatment modified the abundance of these cells. These experimental and clinical data demonstrate that increased accumulation of pulmonary iron plays a key role in the pathogenesis of pulmonary fibrosis and lung function decline. Furthermore, these data highlight the potential for the therapeutic targeting of increased pulmonary iron in the treatment of fibrotic lung diseases such as IPF

The Mast Cell Degranulator Compound 48/80 Directly Activates Neurons

Background Compound 48/80 is widely used in animal and tissue models as a “selective” mast cell activator. With this study we demonstrate that compound 48/80 also directly activates enteric neurons and visceral afferents. Methodology/Principal Findings We used in vivo recordings from extrinsic intestinal afferents together with Ca++ imaging from primary cultures of DRG and nodose neurons. Enteric neuronal activation was examined by Ca++ and voltage sensitive dye imaging in isolated gut preparations and primary cultures of enteric neurons. Intraluminal application of compound 48/80 evoked marked afferent firing which desensitized on subsequent administration. In egg albumen-sensitized animals, intraluminal antigen evoked a similar pattern of afferent activation which also desensitized on subsequent exposure to antigen. In cross-desensitization experiments prior administration of compound 48/80 failed to influence the mast cell mediated response. Application of 1 and 10 µg/ml compound 48/80 evoked spike discharge and Ca++ transients in enteric neurons. The same nerve activating effect was observed in primary cultures of DRG and nodose ganglion cells. Enteric neuron cultures were devoid of mast cells confirmed by negative staining for c-kit or toluidine blue. In addition, in cultured enteric neurons the excitatory action of compound 48/80 was preserved in the presence of histamine H1 and H2 antagonists. The mast cell stabilizer cromolyn attenuated compound 48/80 and nicotine evoked Ca++ transients in mast cell-free enteric neuron cultures. Conclusions/Significance The results showed direct excitatory action of compound 48/80 on enteric neurons and visceral afferents. Therefore, functional changes measured in tissue or animal models may involve a mast cell independent effect of compound 48/80 and cromolyn

Compound 48/80 (c48/80) evoked activation of visceral afferents <i>in vivo</i>. A

<p>Jejunal afferent discharge stimulated by c48/80 administered intraluminally. A second administration 5 min later evoked a similar afferent response but subsequent administrations are completely desensitized, despite similar mechanosensitivity during the luminal perfusions. <b>B</b> In sensitized animals intraluminal application of the antigen egg albumin (EA) evoked a response similar to that observed with c48/80 and which too rapidly desensitized so that subsequent antigen failed to evoke a response. <b>C</b> Following desensitization to c48/80, intraluminal antigen still evoked a response similar to that in naïve control animals. When the order of administration was reversed and the response to antigen was desensitized, c48/80 was still able to evoke a response <b>D</b> illustrates mean data from these cross-desensitization experiments. The left panel shows the afferent response to saline or antigen in sensitized animals that had received pretreatment with vehicle (saline, N = 11) or c48/80 (N = 5). The panel on the right shows similar data for the response to vehicle (saline) or c48/80 in naïve control animals (N = 9) or sensitized animals following desensitization to antigen (N = 6). The response to c48/80 was significantly augmented after desensitization to antigen (P<0.01).</p

Compound 48/80 (c48/80) evoked Ca⁺⁺ transients in primary cultured enteric neurons. A

<p>shows two images of a Fluo-4 AM stained ganglion before c48/80 application and at the time of the maximal response. <b>B</b> illustrates the Ca⁺⁺ signal of the neuron marked by an arrow in A. Two consecutive c48/80 applications (marked by the bars below the traces) evoked comparable responses. In this ganglion all 8 neurons responded to c48/80. <b>C</b> confirms that responses to c48/80 application were not caused by activation of mechanosensors because spritz application of Krebs solution using the same application parameters had no effect. <b>D</b> illustrates that c48/80 evoked [Ca⁺⁺]_i increase is concentration dependent.</p

Compound 48/80 (c48/80) evoked spike discharge in primary cultured enteric neurons. A

<p>Di-8-ANEPPS stained ganglion; the dye incorporates into the outer membrane revealing the outline of individual neuronal cell bodies. <b>B</b> illustrates action potential discharge in response to two consecutive c48/80 applications (marked by bars below the traces) from the neuron marked by white arrow in A. The traces show responses during three recording periods with non-recording periods of 6 s in between. In this ganglion 17 of 19 neurons responded to c48/80. <b>C</b> PGP9.5-positive cultured enteric neurons. Lack of c-kit immunoreactivity demonstrated lack of mast cells in the culture. <b>D</b> demonstrates that c48/80 application still evokes spikes in cultures treated with a combination of the H₁ and H₂ blockers pyrilamine (1 µM) and ranitidine (10 µM), respectively. <b>E</b> shows green PGP9.5-positive neurons in a whole mount preparation of the guinea pig submucous plexus (left panel) and red c-kit-positive round and smooth mast cells (some marked by white triangles in center panel), which are morphologically distinct from the spindle shaped interstitial cells of Cajal. The right panel shows the merged image of the two stainings.</p

Neuronal activation by mucosal biopsy supernatants from irritable bowel syndrome patients is linked to visceral sensitivity

Based on the discomfort/pain threshold during rectal distension, irritable bowel syndrome (IBS) patients may be subtyped as normo- or hypersensitive. We previously showed that mucosal biopsy supernatants from IBS patients activated enteric and visceral afferent neurons. We tested the hypothesis that visceral sensitivity is linked to the degree of neuronal activation. Normo- and hypersensitive IBS patients were distinguished by their discomfort/pain threshold to rectal balloon distension with a barostat. Using potentiometric and Ca(2+) dye imaging, we recorded the response of guinea-pig enteric submucous and mouse dorsal root ganglion (DRG) neurons, respectively, to mucosal biopsy supernatants from normosensitive (n = 12 tested in enteric neurons, n = 9 tested in DRG) and hypersensitive IBS patients (n = 9, tested in both types of neurons). In addition, we analysed the association between neuronal activation and individual discomfort/pain pressure thresholds. The IBS supernatants evoked Ca(2+) transients in DRG neurons and spike discharge in submucous neurons. Submucous and DRG neurons showed significantly stronger responses to supernatants from hypersensitive IBS patients as reflected by higher spike frequency or stronger [Ca(2+)]i transients in a larger proportion of neurons. The neuroindex as a product of spike frequency or [Ca(2+)]i transients and proportion of responding neurons correlated significantly with the individual discomfort/pain thresholds of the IBS patients. Supernatants from hypersensitive IBS patients caused stronger activation of enteric and DRG neurons. The level of activation correlated with the individual discomfort/pain threshold pressure values. These findings support our hypothesis that visceral sensitivity is linked to activation of peripheral neurons by biopsy supernatant

Compound 48/80 (c48/80) evoked Ca⁺⁺ transients in isolated DRG and nodose ganglion cells in culture.

<p>In both cases c48/80 evoked a calcium signal that was about 40% of the maximum response to the calcium ionophore, ionomycin. The response of nodose and DRG neurons to c48/80 was different in two respects, first in the proportion of cells responding and in the time course of the onset of the response. More nodose neurons responded than DRG and the onset of the response was quicker.</p