Methacholine induced airway contraction in porcine precision cut lung slices from indoor and outdoor reared pigs.

Repetitive exposure to bioaerosols in swine production facilities (SPF) promotes respiratory dysfunction in workers and animals. An adequate understanding of the impact of the SPF environment on pulmonary physiology is needed. However, there is currently no sufficient ex vivo model to investigate the cause for agriculture-related lung disease. The precision cut lung slices (PCLS) technique represents a practical and useful procedure for ex vivo studies. Our goal was to use the PCLS technique to develop a model of agriculture-related lung diseases using a physiologically relevant animal model, the domesticated pig. Freshly prepared pig lung tissue cores were sectioned into 300 µm slices and viability was measured by lactate dehydrogenase activity and live/dead staining. Airway contractility in response to a methacholine (MCh) dose gradient (10-7-10-4 M) was measured. After the last MCh dose, PCLS were incubated with 1 mM chloroquine to allow airways to relax. Time-lapse images were taken every minute for 35 minutes and used to determine airway lumen area changes. Porcine PCLS remained viable and demonstrated metabolic activity for three days. PCLS from indoor and outdoor pigs contracted in response to MCh exposure and relaxed when incubated with chloroquine. Notably, PCLS of indoor pigs showed greater airway constriction in response to 10-5 M MCh exposure compared to outdoor pig PCLS (P<0.05). These data suggest that exposure to the indoor pig production environment may be associated with hyperresponsiveness in swine airways, and support future studies to investigate lung response to inflammatory substances using the porcine PCLS model.

Sorrel Extract Reduces Oxidant Production in Airway Epithelial Cells Exposed to Swine Barn Dust Extract In Vitro.

Exposure to hog barn organic dust contributes to occupational lung diseases, which are mediated by inflammatory and oxidative stress pathways. Isoprostanes-a family of eicosanoids produced by oxidation of phospholipids by oxygen radicals-are biomarkers of pulmonary oxidative stress. Importantly, 8-isoprostane has been implicated as a key biomarker and mediator of oxidative stress because it is a potent pulmonary vasoconstrictor. Antioxidants found in fruits and vegetables hold promise for preventing or reducing effects of oxidative stress-related diseases including chronic bronchitis and chronic obstructive pulmonary disease (COPD). Here, we investigated 8-isoP and oxidant production by organic dust-exposed airway epithelial cells and the inhibitory effects of an extract from calyces of the sorrel plant, Hibiscus sabdariffa, on oxidant-producing pathways. Confluent cultures of normal human tracheobronchial epithelial cells were pretreated or not with 1% sorrel extract prior to 5% dust extract (DE) exposure. Following DE treatments, live cells, cell-free supernatants, or cell extracts were evaluated for the presence of 8-isoprostane, superoxide, hydrogen peroxide, nitric oxide, hydroxyl radical, peroxynitrite, and catalase activity to evaluate sorrel's inhibitory effect on oxidative stress. The well-known radical scavenging antioxidant, N-acetyl cysteine (NAC), was used for comparisons with sorrel. DE exposure augmented the production of all radicals measured including 8-isoprostane (p value < 0.001), which could be inhibited by NAC or sorrel. Among reactive oxygen and nitrogen species generated in response to DE exposure, sorrel had no effect on H2O2 production and NAC had no significant effect on NO· production. The observations reported here suggest a possible role for sorrel in preventing 8-isoprostane and oxidant-mediated stress responses in bronchial epithelial cells exposed to hog barn dust. These findings suggest a potential role for oxidative stress pathways in mediating occupational lung diseases and antioxidants within sorrel and NAC in reducing dust-mediated oxidative stress within the airways of exposed workers.

5-alk(en)ylresorcinols as the major active components in wheat bran inhibit human colon cancer cell growth.

We and others have found that wheat bran oil is the active constituent in wheat bran for colon cancer prevention. However, the active components in wheat bran oil are still unknown. Using human colon cancer cells (HCT-116 and HT-29) as the guiding assays, we further purified the active components from wheat bran using column chromatography. In this study, we identified that a fraction containing 5-n-alk(en)ylresorcinols had the strongest inhibitory effect on the proliferation of human HCT-116 and HT-29 colon cancer cells. Further purification led to the identification of 14 5-alk(en)ylresorcinols. Among them, 7, (10'Z,13'Z,16'Z)-5-(nonadeca-10',13',16'-trienyl)resorcinol, is a novel compound and 5, 6, 9, 10, and 13 were purified as individual compounds for the first time. The identification and structural elucidation of these compounds were based on 1D and 2D NMR and tandem mass spectral analyses. All these compounds (1-14) except 10 were evaluated for growth inhibition of human colon cancer cell lines (HCT-116 and HT-29). Our results indicate that increasing the length of the side chain will diminish the inhibitory activity, and the existence of a double bond and a carbonyl group will strengthen such an activity.

Assessment of behavior during labor in rats and effect of intrathecal morphine.

BACKGROUND: Efficacy of analgesics varies with the type of pain. Little is known in this regard concerning labor pain, given the ethical barriers to study in humans and the lack of surrogate animal models. To address this, the authors classified and quantified spontaneous behaviors during labor and delivery in rats and examined the effects of a known analgesic, intrathecal morphine. METHODS: Pregnant rats were video recorded for 72 h surrounding the time of anticipated labor and delivery. Specific behaviors were identified and classified into general activities, phasic stretching behaviors, and maternal attention activities. Rats received intrathecal infusion of saline or morphine, 0.035-3.5 microg/h, beginning approximately 1 day before delivery, and effects on behaviors and response to noxious heating of the paw were quantified. RESULTS: Phasic stretching behaviors occurred with high frequency before delivery of the first pup and were rare after delivery of the last pup. Intrathecal morphine at infusion rates greater than 0.035 microg/h abolished these behaviors without affecting general or maternal behaviors or the timing or duration of labor and delivery. Morphine was also antinociceptive to noxious heat, but only at infusion rates of 1.0 microg/h or higher. CONCLUSIONS: Phasic stretching behaviors are observed after distension or inflammation of pelvic viscera in rats, and similar behaviors occur during labor and delivery. Selective and dose-related blockade by intrathecal morphine of only these behaviors suggests that they reflect nociception and that this simple monitoring method can be used to study therapies for the pain of labor and delivery.

Preoperative inhibition of cyclooxygenase-1 in the spinal cord reduces postoperative pain.

Intrathecal administration of cyclooxygenase (COX)-1, but not COX-2, specific inhibitors given on postoperative day 1 has analgesic effects in an incisional model of postoperative pain. We investigated the effects of preoperative administration of intrathecal COX inhibitors in this model. Fifteen minutes before surgery, rats received intrathecally the COX-1 preferring inhibitor, ketorolac, the specific COX-1 inhibitor, SC-560, the COX-2 inhibitor, NS-398, or vehicle. A 1-cm longitudinal incision was then made through skin, fascia, and muscles of the plantar aspect of a left paw in male rats. Withdrawal threshold to von Frey filaments was measured at 2 h, 4 h, and at intervals up to 5 days later. Ketorolac and SC-560 increased withdrawal threshold to mechanical stimulation, but NS-398 had no significant effect. These results suggest that COX-1 plays an important role in spinal cord pain processing and sensitization after surgery and that preoperative intrathecal administration of specific COX-1 inhibitors may be useful to treat postoperative pain.

Intrathecal morphine reduces allodynia after peripheral nerve injury in rats via activation of a spinal A1 adenosine receptor.

BACKGROUND: The degree to which intrathecally administered morphine can alleviate hypersensitivity in animals after peripheral nerve injury is controversial, and the mechanisms by which morphine works in these circumstances are uncertain. In normal animals, morphine induces adenosine release, and in vitro data suggest that this link is disrupted after peripheral nerve injury. Therefore, using a controlled, blinded study design, the authors tested intrathecal morphine efficacy in rats with peripheral nerve injury and the role of spinal A1 adenosine receptors in the action of morphine. METHODS: Male rats underwent intrathecal catheter implantation and lumbar spinal nerve ligation, resulting in hypersensitivity to tactile stimulation of the paw. Intrathecal morphine alone or with naloxone or the specific A1 adenosine receptor antagonist, 1,3-dipropyl-8-cyclopentyxanthine (DPCPX), was administered, and withdrawal threshold to von Frey filament application to the hind paw was determined. RESULTS: Intrathecal morphine (0.25-30 microg) dose-dependently reversed mechanical hypersensitivity after spinal nerve ligation, with an ED50 of 0.79 microg. The effect of morphine was blocked by intrathecal naloxone. Intrathecal DPCPX alone had no effect on withdrawal threshold after spinal nerve ligation but completely reversed the effect of morphine, with an ID50 of 5.6 microg. CONCLUSIONS: This study is in accord with two recent reports that support short-term efficacy of intrathecal morphine to reverse hypersensitivity to mechanical stimuli in animal models of neuropathic pain. Despite previous studies demonstrating that morphine releases less adenosine after peripheral nerve injury, the current study suggests that the antihypersensitivity effect of morphine in these conditions is totally reliant on A1 adenosine receptor activation.

Intrathecal morphine and ketorolac analgesia after surgery: comparison of spontaneous and elicited responses in rats.

Pain after surgery results in significant morbidity, and systemic opioids often fail to provide adequate analgesia without marked sedation and respiratory depression. Intrathecal morphine provides better analgesia, but is limited by delayed respiratory depression. Intrathecal injection of the cyclooxygenase inhibitor, ketorolac, has recently entered clinical trials, and the current study examined the interaction between intrathecal morphine and ketorolac to treat postoperative pain. We also sought to compare these treatments on a commonly used assessment of withdrawal threshold and a new assessment of spontaneous behavior after surgery. Male Sprague Dawley rats and underwent hind paw incision or subcostal laparotomy surgery. Intrathecal morphine, ketorolac, or their combination were injected on the first postoperative day, with outcome measure being return to pre-surgery withdrawal threshold with von Frey filament testing of the paw after paw incision, or return to pre-surgery exploratory activity after laparotomy. Intrathecal morphine completely reversed the effects of surgery in both models, but intrathecal ketorolac only partially reversed them. Ketorolac enhanced the potency of morphine several fold in both models, and did so synergistically after paw incision. In all cases drug potency was greater for spontaneous than elicited responses. These data confirm that spinal opioid receptor and cyclooxygenase enzyme inhibition diminish elicited tactile hypersensitivity after surgery, and that they similarly return spontaneous behavior to normal. Differences in drug potency could reflect fundamental differences in outcome measures or in the surgical procedures themselves. These data support combination study of intrathecal morphine and ketorolac for postoperative pain.

Intrathecal ketorolac enhances antinociception from clonidine.

UNLABELLED: Although both alpha2-adrenergic agonists and cyclooxygenase inhibitors produce analgesia, their exact sites of action and interaction remain unclear. A previous report demonstrated a surprising inhibition of antinociception in rats from intrathecal clonidine by co-administered ketorolac. There are no other reports of interaction between these two classes of analgesics. We therefore reexamined this interaction, determining the effect of intrathecal clonidine and ketorolac alone and in combination in normal rats. Clonidine, but not ketorolac, produced antinociception to noxious hind paw thermal stimulation. The addition of ketorolac significantly enhanced the effect of clonidine, indicating a synergistic interaction for analgesia. Although the reasons for the discrepancy between this and the previous report are unclear, these results are consistent with previous studies that indicate an antinociceptive action of intrathecal alpha2-adrenergic agonists in the normal condition, a lack of such effect for cyclooxygenase inhibitors, and positive reinforcing effects of these two systems when co-stimulated. IMPLICATIONS: Spinal injection of the alpha2-adrenergic agonist clonidine and the cyclooxygenase inhibitor ketorolac results in a synergistic interaction for antinociception in normal animals, suggesting that the combination of these drugs will enhance rather than detract from the analgesia of either alone.