Manganese-enhanced MRI reveals changes within brain anxiety and aversion circuitry in rats with chronic neuropathic pain- and anxiety-like behaviors.

Chronic pain often predicts the onset of psychological distress. Symptoms including anxiety and depression after pain chronification reportedly are caused by brain remodeling/recruitment of the limbic and reward/aversion circuitries. Pain is the primary precipitating factor that has caused opioid overprescribing and continued overuse of opioids leading to the current opioid epidemic. Yet experimental pain therapies often fail in clinical trials. Better understanding of underlying pathologies contributing to pain chronification is needed to address these chronic pain related issues. In the present study, a chronic neuropathic pain model persisting 10 weeks was studied. The model develops both anxiety- and pain-related behavioral measures to mimic clinical pain. The manganese-enhanced magnetic resonance imaging (MEMRI) utilized improved MRI signal contrast in brain regions with higher neuronal activity in the rodent chronic constriction trigeminal nerve injury (CCI-ION) model. T1-weighted MEMRI signal intensity was increased compared to controls in supraspinal regions of the anxiety and aversion circuitry, including anterior cingulate gyrus (ACC), amygdala, habenula, caudate, ventrolateral and dorsomedial periaqueductal gray (PAG). Despite continuing mechanical hypersensitivity, MEMRI T1 signal intensity as the neuronal activity measure, was not significantly different in thalamus and decreased in somatosensory cortex (S1BF) of CCI-ION rats compared to naïve controls. This is consistent with decreased fMRI BOLD signal intensity in thalamus and cortex of patients with longstanding trigeminal neuropathic pain reportedly associated with gray matter volume decrease in these regions. Significant increase in MEMRI T2 signal intensity in thalamus of CCI-ION animals was indication of tissue water content, cell dysfunction and/or reactive astrogliosis. Decreased T2 signal intensity in S1BF cortex of rats with CCI-ION was similar to findings of reduced T2 signals in clinical patients with chronic orofacial pain indicating prolonged astrocyte activation. These findings support use of MEMRI and chronic rodent models for preclinical studies and therapeutic trials to reveal brain sites activated only after neuropathic pain has persisted in timeframes relevant to clinical pain and to observe treatment effects not possible in short-term models which do not have evidence of anxiety-like behaviors. Potential improvement is predicted in the success rate of preclinical drug trials in future studies with this model.

A Comparison of Cs-137 γ Rays and 320-kV X-Rays in a Mouse Bone Marrow Transplantation Model.

US homeland security concerns regarding the potential misuse of some radiation sources used in radiobiological research, for example, cesium-137 (137Cs), have resulted in recommendations by the National Research Council to conduct studies into replacing these sources with suitable X-ray instruments. The objective of this research is to compare the effectiveness of an X-RAD 320 irradiator (PXINC 2010) with a 137Cs irradiator (Gammacell-1000 Unit) using an established bone marrow chimeric model. Using measured radiation doses for each instrument, we characterized the dose-response relationships for bone marrow and splenocyte ablation, using a cytotoxicity-hazard model. Our results show that the X-RAD 320 photon energy spectrum was suitable for ablating bone marrow at the 3 exposure levels used, similar to that of 137Cs photons. However, the 320-kV X-rays were not as effective as the much higher energy γ rays at depleting mouse splenocytes. Furthermore, the 3 X-ray levels used were less effective than the higher energy γ rays in allowing the successful engraftment of donor bone marrow, potentially as a result of the incomplete depletion of the spleen cells. More defined studies are warranted for determining whether bone marrow transplantation in mice can be successfully achieved using 320-kV X-rays. A higher X-ray dose then used is likely needed for transplantation success.

Lipoxin Signaling in Murine Lung Host Responses to Cryptococcus neoformans Infection.

Lipoxins (LX) are proresolving mediators that augment host defense against bacterial infection. Here, we investigated roles for LX in lung clearance of the fungal pathogen Cryptococcus neoformans (Cne). After intranasal inoculation of 5,000 CFU Cne, C57BL/6 and C.B-17 mice exhibited strain-dependent differences in Cne clearance, immunologic responses, and lipoxin A4 (LXA4) formation and receptor (ALX/FPR2) expression. Compared with C.B-17 mice, C57BL/6 lungs had increased and persistent Cne infection 14 days after inoculation, increased eosinophils, and distinct profiles of inflammatory cytokines. Relative to C.B-17 mice, bronchoalveolar lavage fluid levels of LXA4 were increased before and after infection in C57BL/6. The kinetics for 15-epi-LXA4 production were similar in both strains. Lung basal expression of the LX biosynthetic enzyme Alox12/15 (12/15-lipoxygenase) was increased in C57BL/6 mice and further increased after Cne infection. In contrast, lung basal expression of the LXA4 receptor Alx/Fpr2 was higher in C.B-17 relative to C57BL/6 mice, and after Cne infection, Alx/Fpr2 expression was significantly increased in only C.B-17 mice. Heat-killed Cne initiated lung cell generation of IFN-γ and IL-17 and was further increased in C.B-17 mice by 15-epi-LXA4. A trend toward reduced Cne clearance and IFN-γ production was observed upon in vivo administration of an ALX/FPR2 antagonist. Together, these findings provide the first evidence that alterations in cellular immunity against Cne are associated with differences in LXA4 production and receptor expression, suggesting an important role for ALX/FPR2 signaling in the regulation of pathogen-mediated inflammation and antifungal lung host defense.

Role of nicotinic receptors and acetylcholine in mucous cell metaplasia, hyperplasia, and airway mucus formation in vitro and in vivo.

BACKGROUND: Airway mucus hypersecretion is a key pathophysiologic feature in a number of lung diseases. Cigarette smoke/nicotine and allergens are strong stimulators of airway mucus; however, the mechanism of mucus modulation is unclear. OBJECTIVES: We sought to characterize the pathway by which cigarette smoke/nicotine regulates airway mucus and identify agents that decrease airway mucus. METHODS: IL-13 and γ-aminobutyric acid type A receptors (GABA(A)Rs) are implicated in airway mucus. We examined the role of IL-13 and GABA(A)Rs in nicotine-induced mucus formation in normal human bronchial epithelial (NHBE) and A549 cells and secondhand cigarette smoke-induced, ovalbumin-induced, or both mucus formation in vivo. RESULTS: Nicotine promotes mucus formation in NHBE cells; however, the nicotine-induced mucus formation is independent of IL-13 but sensitive to the GABA(A)R antagonist picrotoxin. Airway epithelial cells express α7-, α9-, and α10-nicotinic acetylcholine receptors (nAChRs), and specific inhibition or knockdown of α7- but not α9/α10-nAChRs abrogates mucus formation in response to nicotine and IL-13. Moreover, addition of acetylcholine or inhibition of its degradation increases mucus in NHBE cells. Nicotinic but not muscarinic receptor antagonists block allergen- or nicotine/cigarette smoke-induced airway mucus formation in NHBE cells, murine airways, or both. CONCLUSIONS: Nicotine-induced airway mucus formation is independent of IL-13, and α7-nAChRs are critical in airway mucous cell metaplasia/hyperplasia and mucus production in response to various promucoid agents, including IL-13. In the absence of nicotine, acetylcholine might be the biological ligand for α7-nAChRs to trigger airway mucus formation. α7-nAChRs are downstream of IL-13 but upstream of GABA(A)Rα2 in the MUC5AC pathway. Acetylcholine and α7-nAChRs might serve as therapeutic targets to control airway mucus.

Low-dose gamma-irradiation inhibits IL-6 secretion from human lung fibroblasts that promotes bronchial epithelial cell transformation by cigarette-smoke carcinogen.

Despite decades of research in defining the health effects of low-dose (<100 mGy) ionizing photon radiation (LDR), the relationship between LDR and human cancer risk remains elusive. Because chemical carcinogens modify the tumor microenvironment, which is critical for cancer development, we investigated the role and mechanism of LDR in modulating the response of stromal cells to chemical carcinogen-induced lung cancer development. Secretion of proinflammatory cytokines such as interleukin-6 (IL-6), CXCL1 and CXCL5 from human lung fibroblasts was induced by cigarette-smoke carcinogen benzo[a]pyrene diol epoxide (BPDE), which was inhibited by a single dose of LDR. The activation of NF-κB, which is important for BPDE-induced IL-6 secretion, was also effectively suppressed by LDR. In addition, conditioned media from BPDE-treated fibroblasts activated STAT3 in the immortalized normal human bronchial epithelial cell line Beas-2B, which was blocked with an IL-6 neutralizing antibody. Conditioned medium from LDR-primed and BPDE-treated fibroblast showed diminished capacity in activating STAT3. Furthermore, IL-6 enhanced BPDE-induced Beas-2B cell transformation in vitro. These results suggest that LDR inhibits cigarette smoke-induced lung carcinogenesis by suppressing secretion of cytokines such as IL-6 from fibroblasts in lung tumor-prone microenvironment.

Low-dose gamma-radiation inhibits benzo[a]pyrene-induced lung adenoma development in a/j mice.

Low-dose ionizing radiation (LDR) may lead to suppression of smoking-related lung cancer. We examined the effects of a known cigarette smoke carcinogen Benzo[a]pyrene (B[a]P) alone or in combination with fractionated low-dose gamma radiation (60 - 600 mGy total dose) on the induction of lung neoplasms in the A/J mouse. Our results show that 600 mGy of gamma radiation delivered in six biweekly fractions of 100 mGy starting 1 month after B[a]P injection significantly inhibits the development of lung adenomas per animal induced by B[a]P. Our data also indicated that the six biweekly doses suppressed the occurrence of spontaneous hyperplastic foci in the lung, although this suppression failed to reach statistical significance when analyzed as average foci per lung possibly related to the small sample sizes used for the control and test groups.

Experimental hypersensitivity pneumonitis: role of MCP-1.

Inhalation of Saccharopolyspora rectivirgula causes "farmer's lung" disease, a classic example of hypersensitivity pneumonitis (HP). Monocyte chemoattractant protein-1 (MCP-1) is increased in the bronchoalveolar lavage fluid of mice challenged with S rectivirgula, and S rectivirgula induces MCP-1 secretion by alveolar macrophages. We tested the hypothesis that MCP-1 and its receptor CC chemokine receptor-2 (CCR2) are essential to the development of experimental HP by treating mice with MCP-1 antibody and using CCR2(-/-) mice. Administration of anti-MCP-1 did not change the response to intratracheally administered S rectivirgula. CCR2(-/-) animals responded in a fashion similar to that of wild-type animals to intratracheally administered.S rectivirgula. To determine the influence of the MCP-1-CCR2 interaction in vitro, we transferred S rectivirgula-cultured spleen cells from S rectivirgula-sensitized mice, to naïve recipients. Later, challenge of the recipients with intratracheal S rectivirgula and examination of both lung histology and bronchoalveolar lavage fluid characteristics were used to determine whether adoptive transfer had occurred. We found that cultured cells from CCR2(-/-) animals were fully capable of adoptive transfer. We conclude that interaction of MCP-1 with CCR2 is not necessary for the development of pulmonary inflammation in response to intratracheally administered S rectivirgula or cells able to adoptively transfer experimental HP.

LPS-induced neutrophilic inflammation and Bcl-2 expression in metaplastic mucous cells.

Our previous studies show that Bcl-2, a regulator of apoptosis, may be involved in the reduction of mucous cell metaplasia (MCM) during recovery from inflammatory responses. The present study was to determine whether neutrophilic inflammation mediates Bcl-2 expression in mucous cells. Rats were intratracheally instilled with 50-1000 microg of LPS. The number of neutrophils recovered by bronchoalveolar lavage (BAL) increased with the dose of LPS, and the percentage of Bcl-2-expressing cells increased with the numbers of neutrophils in the BAL. Depletion of neutrophils did not reduce MCM, but the percentage of Bcl-2-positive cells increased 1.8-fold in neutrophil-depleted compared with controls. Injection of rats with bezafibrate, an inducer of cytochrome P-450, doubled the number of neutrophils in the BAL, decreased MCM twofold compared with vehicle-injected controls, and reduced Bcl-2 expression. Bcl-2 mRNA levels decreased in a tracheal epithelial cell line treated with bezafibrate. These data demonstrate that Bcl-2 expression is independent of the number of neutrophils in the BAL and that bezafibrate may directly reduce Bcl-2 expression in epithelial cells.

Is IL12 necessary in experimental hypersensitivity pneumonitis?

Inhalation of Saccharopolyspora rectivirgula (SR) can cause the disease Farmer's Lung, a classic example of hypersensitivity pneumonitis. Th1, but not Th2, cell lines can adoptively transfer experimental hypersensitivity pneumonitis (EHP). Substantial amounts of IL12 appear in bronchoalveolar lavage fluid (BALF) after a single intratracheal (IT) injection of SR, and SR-induced IL12 secretion by both a macrophage cell line and alveolar macrophages. We tested the hypothesis that IL12 is essential for the development of EHP by addition of anti-IL12 to cultured cells, and adoptive transfer of EHP in IL12p40-/- animals. We transferred SR cultured spleen and lung associated lymph node cells from SR sensitized mice (both IL12p40-/- and wild type), to naïve recipients (both wild type and IL12p40-/-). The addition of anti-IL12 to cultures of sensitized cells could not ablate the ability of these cells to transfer EHP. Cultured cells from IL12p40-/- animals were fully capable of transferring EHP. In contrast, IL12p40-/- recipients of both wild type and IL12p40-/--cultured cells were less able to express EHP (lung histology and BALF characteristics) than wild type mice, and had more eosinophils in both lung tissue and BALF. We conclude that IL12 is not necessary for development of cells able to adoptively transfer EHP, but that it is required for full expression of EHP in recipient animals.