or phosphorylated buffer solution (PBS) was injected into the bone cavity. Following injection, the entry site on the bone was sealed with bone wax. Doses of test reagents were given at 9, 11, 13 and 15 day respectively, including capsazepine (intravenous injection through the tail or intraperitoneal injection, i.p.) and resveratrol (i.p.). These regents were dissolved in DMSO (final concentration ,10%). Glutathione (i.p.) was dissolved in normal saline. All reagents were obtained from Sigma, unless otherwise indicated. The bone marrow and spinal cords of these rats were taken out for formaldehyde measurement with HPLC. To assess the bone destruction after inoculation, tibial bone radiographs from both hind limbs on 7 and 15 days were taken with a Digital Radiographer System (E-COM Technology Co. Ltd., Guangdong, China). Radiological scores were given based on careful, blind analysis of radiographs, taken from the ipsilateral and contralateral legs of MRMT-1-treated, heat-killed MRMT-1- treated, vehicle-treated and naive rats (n = 4 for each group). Scores were given as in previous report [44]. All scores related to the tibia (bone): 0, normal bone structure without any sign of deterioration; 1, small radiolucent lesions in the proximal epiphysis, close to the site of the injection; 2, increased number of radiolucent lesions, loss of medullary bone; 3, loss of medullary bone, plus erosion of the cortical bone; 4, full thickness unicortical bone loss; 5, full thickness bicortical bone loss and displaced fractures. Hot plate test for thermal hyperalgesia Male Sprague-Dawley rats (150,200 g) were provided by the Department of Animal Science of Peking University. Animals were raised under natural diurnal cycles and had free access to water and food. They were habituated to the testing paradigms for 3,5 days before experiment. Animal treatment was in compliance with the Guidelines of the International Association for the Study of Pain [68]. On days 1, 3, 7, 11 and 15 after injection of MRMT-1 cells, heat-killed cells or PBS injection, thermal hyperalgesia was tested with hot plate. Rats were habituated to the experimental environment for 30 min in their home cage. Rats were placed on the hot plate (5260.5uC) and the interval time until the rat jumped or licked either of its hind paws was recorded as hot plate latency. Following a response, the rat was immediately removed from the plate. Each test was repeated three times with a 15 min interval between tests [69]. Von Frey hair test for mechanical allodynia Each animal was placed in a clear Plexiglas compartment with a mesh floor and was allowed to habituate for 20 min. On days 1, 3, 7, 11 and 15 after injection of MRMT-1 cells, heat-killed cells or PBS, mechanical allodynia was evaluated with application of von Frey hair (Semmes-Weinstein Monofilaments, North Coast Medial Inc., San Jose, CA) in ascending order of force (0.41,15.1 g) to the plantar surface of the hind paw. The rat was placed in the test box and allowed to settle in for 5,10 min. An ascending series of von Frey hairs with logarithmically incremental stiffness (0.40, 0.60, 1.4, 2.0, 4.0, 6.0, 8.0, and 15.0 g) were applied perpendicular to the mid-plantar surface (avoiding the less sensitive tori) of each hind paw. Each von Frey hair was held about 1,2 s, with a 10-min interval between each application. A trial began with the application of the 2.0 g von Frey hair. The positive response was defined as a withdrawal of hind paw upon the stimulus. Whenever a positive response to a stimulus occurred, the next lower von Frey hair was applied, and whenever a negative response occurred, the next higher hair was applied. The testing consisted of five more stimuli after the first change in response occurred, and the pattern Formaldehyde and Cancer Pain of response was converted to a 50% von Frey threshold using the method as previously reported [70]. Classic formalin-induced spontaneous pain (formalin test) Following a 30-min habituation to the observation cage, male Sprague-Dawley rats (300,350 g) received an s.c. injection of 50 ml of a 5% formalin (1662 mM formaldehyde) solution into the dorsal aspect of the right hind paw. Nociceptive behaviors which were recorded included flinching, licking or biting the injected paw as previously described [22,51]. Test reagents with or without formalin was injected into paw plantar aspect of the paw. Capsazepine, melatonin, AP-18 and resveratrol were dissolved in DMSO (final concentration ,10%); formalin and glutathione were dissolved in normal saline. Formaldehyde test for acute nociception Following a 20 min adaptation, rats received a subcutaneous injection of formaldehyde (0.1,100 mM) at pH 5.0 or 6.0 (mimicking a moderate to severely acidic tumor microenviron- ment) into the plantar of right hind paw using a microsyringe with a 26-gauge needle. The length of time that the animals spent flinching, lifting, licking or biting the injected paw was recorded with a chronometer, and was considered as an indicator of pain response in the early phase (0,5 min) as previously described [46]. Agonists or antagonists (100 ml/paw) which were injected included capsazepine, AP-18 and melatonin which were dissolved in DMSO (final concentration ,10%). Formaldehyde was dissolved in normal saline. Cell culture Chinese hamster ovary (CHO) cells were cultured in Ham’s F- 12 medium. MRMT-1 rat mammary gland carcinoma cells (Novartis Oncology Research, Basel) and H1299 human lung cancer cells were cultured in RPMI 1640 (Gibco) medium, human SY5Y cell lines were cultured in Dulbecco’s modified Eagle’s medium (DMEM). All culture dishes were kept in an incubator under a humidified atmosphere (37uC, 95% air and 5% CO2). Dorsal root ganglion (DRG) neuron culture was carried out following a modification of a previously described procedure [71]. Briefly, DRG from 3-week-old SD rats were digested with trypsin/ EDTA solution for 40 min and dissociated in growth medium containing DMEM, 5% inactivated fetal bovine serum, 5 mM glutamine, B27 supplement (GIBCO), 100 ng/ml of nerve growth factor (Becton Dickinson) and 0.6% dextrose. Cells were seeded at a density of 56105 per well. After 2 days, cultures were treated with 10 mM cytosine arabinoside to control growth of dividing cells. Cells were used one week after culture. Cell viability assayed with MTT MTT solution was prepared in complete medium to a concentration of 1 mg/ml just before use. Cells were diluted in fresh complete medium and seeded in 96-well plates. After allowing overnight attachment, cells were treated with various concentrations of formaldehyde or reagents for 4 h. Optical density were determined by a spectrophotometer. Formaldehyde measurement with high performance liquid chromatography with fluorescence detection (Fluo-HPLC) Media from the cultured cancer cell lines, bone marrows, the homogenate of spinal cords of model rats and cancer patient tissue specimens were harvested for formaldehyde assay. An HP 1100 PLoS ONE | www.plosone.org 12 April 2010 | Volume 5 | Issue 4 | e10234