SB366791, a TRPV1 antagonist, potentiates analgesic effects of systemic morphine in a murine model of bone cancer pain.

BACKGROUND: Bone cancer pain has a major impact on the quality of life of cancer patients but is difficult to treat. Therefore, development of a novel strategy for bone cancer pain is needed for improvement of the patient quality of life. In this study, we examined the analgesic effects of the combination of a transient receptor potential vanilloid subfamily 1 (TRPV1) antagonist and morphine on pain-related behaviours in a murine model of bone cancer pain. METHODS: C3H/HeJ mice underwent injection of osteolytic sarcoma cells into the intramedullary space of the femur. The analgesic effects of intraperitoneal morphine and the analgesic effect of a TRPV1 antagonist, SB366791 [N-(3-methoxyphenyl)-4-chlorocinnamide], on bone cancer pain-related behaviours were examined. The analgesic effects of the combination of SB366791 and morphine on bone cancer pain were also examined. RESULTS: Intraperitoneal morphine significantly reduced the number of spontaneous flinches and improved ambulation only at the highest dose of 10 mg kg(-1) whereas weight-bearing was not improved. Intraperitoneal SB366791 at doses of 0.3 and 1.0 mg kg(-1), but not at a dose of 0.1 mg kg(-1), reduced the number of spontaneous flinches, whereas neither weight-bearing nor ambulation was improved. Addition of a sub-analgesic dose of SB366791 (0.1 mg kg(-1)) to morphine significantly reduced the number of flinches and improved weight-bearing compared with the effects of morphine alone. CONCLUSIONS: Our findings showed that the combination of morphine and SB366791 has potent analgesic effects on bone cancer pain. The findings of this study may lead to novel strategies for the treatment of bone cancer pain.

Down-regulation of mu opioid receptor expression within distinct subpopulations of dorsal root ganglion neurons in a murine model of bone cancer pain.

Although micro opioid receptor (MOR) agonists are used for treatment of most types of pain, a recent study has suggested that the sensitivity of bone cancer pain to systemic morphine was lower than that of inflammatory pain. However, the reasons for this have remained unclear. In this study, MOR expression and the analgesic effects of morphine in a bone cancer model were compared with those in an inflammatory pain model. A bone cancer pain model and an inflammatory pain model were made by implantation of sarcoma cells into the intramedullary space of the femur and hind-paw injection of complete Freund's adjuvant (CFA), respectively. In a behavioral study, sarcoma-implanted mice showed flinching behavior of magnitude comparable to that induced by CFA injection. The flinching behavior of sarcoma-implanted mice was less sensitive to intrathecal morphine than that of CFA-injected mice. Western blot analysis showed that MOR expression in the dorsal root ganglion (DRG) ipsilateral to sarcoma implantation was significantly reduced, while that in the DRG ipsilateral to CFA injection was increased. In sarcoma-implanted mice, the percentage of MOR-positive DRG neuronal profiles was lower than that in control mice (30.3% vs. 45.2%). In particular, MOR expression was reduced among calcitonin gene-related peptide- and transient receptor potential vanilloid subfamily 1-positive DRG neuronal profiles, which are considered to be involved in the generation of bone cancer pain (from 61.5% to 41.5% and from 72.1% to 48.4%, respectively). These results suggest that down-regulation of MOR in the distinct populations of DRG neurons contributes to the fact that higher doses of morphine are needed to produce analgesia in bone cancer as compared with those used in non-malignant inflammatory situations.

Contribution of transient receptor potential vanilloid subfamily 1 to endothelin-1-induced thermal hyperalgesia.

Endothelin-1 (ET-1) plays an important role in peripheral pain processing. However, the mechanisms of the nociceptive action of ET-1 have not been fully elucidated. In this study, we investigated the contribution of transient receptor potential vanilloid subfamily 1 (TRPV1) to ET-1-induced thermal hyperalgesia. Intraplantar ET-1-induced thermal hyperalgesia was examined by assessing the paw withdrawal latency to noxious heat stimuli. In electrophysiological study, whole-cell patch-clamp recordings were performed to investigate the interaction of ET-1 and TRPV1 using human embryonic kidney 293 (HEK293) cells expressing endothelin type A receptor (ET(A)) and TRPV1. Intraplantar ET-1 (3, 10 and 30 pmol) produced thermal hyperalgesia in a dose-dependent manner. Thermal hyperalgesia was attenuated by the inhibition of ET(A) and protein kinase C (PKC) but not that of ET(B). ET-1-induced thermal hyperalgesia was significantly attenuated in TRPV1-deficient mice compared with that in wild-type mice. In voltage-clamp experiments, 10 nM capsaicin evoked small inward currents in HEK293 cells expressing TRPV1 and ET(A). In the presence of ET-1, capsaicin produced much larger current responses (P<0.05). Mutation at PKC-specific TRPV1 phosphorylation sites (S800A/S502A) and PKC inhibitors inhibited the potentiating effect of ET-1. In addition, ET-1 decreased the temperature threshold for TRPV1 activation in a PKC-dependent manner (from 41.0+/-0.4 degrees C to 32.6+/-0.6 degrees C). In addition, Western blot analysis was also performed to confirm ET-1-induced phosphorylation of TRPV1. Incubation of ET-1 and intraplantar ET-1 evoked phosphorylation of TRPV1 in HEK293 cells expressing TRPV1 and ET(A) and the skin, respectively. These results suggest that the sensitization of TRPV1 activity through an ET(A)-PKC pathway contributes to ET-1-induced thermal hyperalgesia.

Bone cancer increases transient receptor potential vanilloid subfamily 1 expression within distinct subpopulations of dorsal root ganglion neurons.

Bone cancer pain has a strong impact on the quality of life of patients but is difficult to treat. Therefore, the mechanisms of bone cancer pain require elucidation for the purpose of development of new therapeutics. A recent study showed that activation of transient receptor potential vanilloid subfamily 1 (TRPV1) was involved in bone cancer pain. In this study, we re-evaluated the analgesic effects of pharmacological blockade of TRPV1 using the potent TRPV1 antagonist 5-iodoresiniferatoxin (I-RTX) and examined whether bone cancer can change TRPV1 expression and distribution in the primary sensory neurons in a mouse model of bone cancer pain. Implantation of osteosarcoma into the femur induced ongoing and movement-evoked bone cancer-related pain behaviors. These behaviors were significantly reduced by i.p. administration of I-RTX, compared with vehicle. Western blot and reverse transcription-polymerase chain reaction (RT-PCR) analyses revealed that TRPV1 level was significantly increased in dorsal root ganglions (DRGs) ipsilateral to sarcoma implantation. Immunohistochemical analysis showed that implantation of osteosarcoma induced not only an increase in the percentage of TRPV1-positive neurons among DRG neurons (24.3+/-1.3% in sham mice and 31.2+/-1.3% in mice with osteosarcoma implantation, P<0.05) but also an overall shift in the distribution of area of profiles to the right. Colocalization study showed that the percentages of colocalization of TRPV1 with neurofilament 200 kD (NF200) and calcitonin gene-related peptide (CGRP) but not isolectin B4 (IB4) among DRG neurons in mice with osteosarcoma implantation were increased compared with those in sham mice (from 0.8+/-0.1% to 2.1+/-0.3% for TRPV1 and NF200 and from 21.1+/-1.3% to 26.5+/-0.2% for TRPV1 and CGRP). In conclusion, TRPV1 activation plays a critical role in the generation of bone cancer pain, and bone cancer increases TRPV1 expression within distinct subpopulation of DRG neurons. These findings may lead to novel strategies for the treatment of bone cancer pain.

Immunohistochemical analysis of acid-sensing ion channel 2 expression in rat dorsal root ganglion and effects of axotomy.

Several studies have suggested that acid-sensing ion channel 2 (ASIC2) plays a role in mechanoperception and acid sensing in the peripheral nervous system. We examined the expression and distribution of ASIC2 in the rat dorsal root ganglion, the co-localization of ASIC2 with tropomyosin-related kinase (trk) receptors, and the effects of axotomy on ASIC2 expression. ASIC2 immunoreactivity was observed in both neurons and satellite cells. ASIC2-positive neurons accounted for 16.5 +/- 2.4% of the total neurons in normal dorsal root ganglion. Most ASIC2-positive neurons were medium-to-large neurons and were labeled with neurofilament 200 kD (NF200). Within these neurons, ASIC2 was not evenly distributed throughout the cytoplasm, but rather was accumulated prominently in the cytoplasm adjacent to the axon hillock and axonal process. We next examined the co-localization of ASIC2 with trk receptors. trkA was expressed in few ASIC2-positive neurons, and trkB and trkC were observed in 85.2% and 53.4% of ASIC2-positive neurons, respectively, while only 6.9% of ASIC2-positive neurons were co-localized with trkC alone. Peripheral axotomy markedly reduced ASIC2 expression in the axotomized dorsal root ganglion neurons. On the other hand, intense ASIC2 staining was observed in satellite cells. These results show that ASIC2 is expressed in the distinct neurochemical population of sensory neurons as well as satellite cells, and that peripheral axotomy induced marked reductions in ASIC2 in neurons.

Marked suppression of cortical auditory evoked response shortly before the onset of REM sleep.

In 10 of 12 subjects examined, the amplitude of N300, a component of the cortical auditory evoked potential, was evidently smaller in rapid eye movement (REM) sleep than in non-REM sleep. The start of the reduction associated with the onset of the first episode of REM sleep was examined in these 10 subjects. In five of these, a marked reduction of N300 amplitude occurred 0.5-2.5 min before the appearance of muscle atonia of REM sleep. In two subjects, a similarly marked reduction of the N300 amplitude occurred 0.5-1.0 min before the disappearance of sleep spindles or K-complexes. This suggests that a suppression of the synchronizing mechanism in the cerebrum sometimes occurs briefly prior to the occurrence of other physiological phenomena associated with REM sleep.

Endogenous components of event-related potential appearing during NREM stage 1 and REM sleep in man.

Information processing in the brain during sleep was studied by analyzing the evoked cortical response to auditory stimulations presented in the odd-ball paradigm. Eight subjects were examined in different sleep stages. The subjects could provide the correct behavioral response to the auditory stimulation by pressing a key button in the light part of stage 1 of NREM sleep, just succeeding to the waking state, but none of the subjects could give the correct behavioral response in the other sleep stages. In the deep part of stage 1 of NREM sleep and REM sleep, a cortical potential corresponding to P300, the endogenous component of the event-related potential (ERP) recorded in the waking state, was recorded in 6 of the 8 subjects in spite of the absence of the behavioral response. In stages 2, 3 and 4 of NREM sleep, emergence of this endogenous component of ERP could not be confirmed. The present findings provide electrophysiological evidence indicating that selective information processing corresponding to sensory discrimination of auditory stimuli is actively performed in stage 1 of NREM sleep and REM sleep.

The effects of excess amino acids on maintenance of pregnancy and fetal growth in rats.

The effects of diets, each with an excess of one essential amino acid, on the maintenance of pregnancy and fetal growth were investigated in rats. Rats were fed on 6% casein diet containing 5% threonine, methionine, valine, isoleucine, leucine, tyrosine, phenylalanine, tryptophan, or lysine from day 1 to day 14 or 21 of pregnancy. Excess methionine and leucine diets resulted in complete and 80% loss of fetuses, respectively. This fetal wastage was prevented by daily injection of 0.5 microgram of estrone and 4 mg of progesterone. Judging from the total food consumptions and body weight gains during pregnancy, methionine had the most severe effects, followed in order by leucine, tryptophan, valine, lysine, isoleucine, threonine, phenylalanine, and tyrosine. The weights of fetuses in the excess amino acid groups were significantly lower than those in the respective pair-fed controls. Excess aromatic amino acids caused growth retardation of fetal brain, although the levels of free tyrosine and phenylalanine in fetal brain were not high. The concentrations of free methionine and threonine were markedly elevated in the maternal plasma when these amino acids were fed in excess, but those of other amino acids were not increased appreciably by excess amounts in the diet. Changes in the maternal plasma levels of individual amino acids other than those in excess in the diet were small. On the contrary, the levels of not only the excess amino acids but also of other amino acids in fetal brains were appreciably elevated by these diets. These findings suggest that the blood-brain barrier is immature and that the synthesis of proteins in fetal brain is impaired by excess amino acids in the mothers. The importance of experiments on diets with excess of single amino acids in pregnant animals is discussed in connection with studies on inborn errors of amino acid metabolism.

Effects of postnatal undernutrition on the catecholamine and serotonin contents of suckling rat brain.

The effect of postnatal undernutrition on the catecholamine and serotonin contents of various parts of the brain of suckling rats was examined. Undernourishment was induced by increasing the litter size to 18 pups from day 1 to 21 after birth. In control pups, the total amounts of norepinephrine and dopamine in the whole brain increased greatly during the suckling period (norepinephrine: 17.7 ng at birth, 154 ng on day 10, and 420 ng on day 21; dopamine: 12.6 ng at birth, 269 ng on day 10, and 1,022 ng on day 21). Similar, but less marked increases in the norepinephrine and dopamine contents of the brain were observed in malnourished pups. The norepinephrine contents of the forebrain, cerebellum, and brain stem of malnourished pups were comparable with those of normal pups on day 10 but the contents of the cerebellum and brain stem were significantly less than those of normal pups on day 21. Postnatal malnutrition also led to a significant decrease in the dopamine content of the forebrain. In contrast, the serotonin content of the brain of undernourished pups was significantly higher than that of controls. The control pups at the end of suckling period were significantly higher than those of undernourished pups (forebrain: 18.3 pmol in controls and 11.5 pmol in malnourished pups; brain stem: 12.3 pmol in controls and 9.8 pmol in malnourished pups). The tyrosine hydroxylase activity (pmol/g) correlated more closely with the norepinephrine content than with the dopamine or norepinephrine plus dopamine content. The tyrosine and phenylalanine contents of the brain were similar in the two groups. It is concluded from these findings that the catecholamine content of the brain is regulated by the enzyme activity rather than the levels of precursor amino acids.

Effects of dietary level of protein on the maintenance energy requirement and net energetic efficiency for growth of post-weaning rats.

The effects of dietary protein on the maintenance energy requirement (MEm) and net utilization efficiency of metabolizable energy for growth (MEg) were investigated by regression analysis of energy balance with various energy intakes. Weanling rats of the Wistar strain, weighing about 85 g, were given a diet containing 0 to 70% casein freely or in restricted amounts (equivalent to two-thirds or one-third of the intake of the ad libitum group) for 5 days. The MEm was fairly constant in rats given 10 to 50% casein diets, being about 29 kcal/100 g BW/day, but increased at higher or lower dietary protein levels, indicating inefficient energy utilization in protein-malnourished animals. From the slope of the regression line between energy balance and metabolizable energy intake, the net energetic efficiencies for growth were estimated as 68, 71, 74, 77, 82, 83, 80, 78, 77 and 74% with 0, 3, 6, 10, 20, 30, 40, 50, 60 and 70% casein diets, respectively. Weanling rats fed 20 to 30% casein diets utilized the dietary energy for growth most efficiently. At protein levels higher or lower than 20 to 30%, the efficiency was less, showing that MEg utilization depended on dietary protein. The energy necessary for 1 g body weight gain was 2.6 kcal in rats receiving 30% casein diet, but increased with an increase or decrease in the protein level. These data on the food efficiency, MEm, the net efficiency of MEg and the energy necessary for 1 g weight gain show that dietary protein affects energy utilization and that protein-malnourished animals use energy inefficiently.

Obligatory nitrogen losses in pregnant rats with special reference to estimation of net protein utilization.

For estimation of net protein utilization of dietary proteins during pregnancy, obligatory nitrogen losses were measured in protein-deficient rats in which pregnancy was maintained by administration of ovarian steroids. On shift from normal to protein-free diet, urinary nitrogen, expressed as mg/day or mg/100 g BW per day, decreased initially rapidly and then gradually during the first two weeks in both pregnant and nonpregnant rats. However, urinary endogenous nitrogen increased during the final week of pregnancy, whereas it continued to decrease in nonpregnant controls. The endogenous urinary nitrogen excretions during early-mid and late pregnancies were significantly higher in pregnant rats (666 mg/15 days and 234 mg/6 days, respectively) than in nonpregnant animals (585 mg/15 days and 153 mg/6 days, respectively), indicating pregnancy-induced protein hypercatabolism. The metabolic fecal nitrogen excretions in pregnant and nonpregnant rats were comparable. In pregnant rats, a protein-free diet resulted in decrease of basal energy expenditure, from 24 kcal/day on day 1 to about 15 kcal/day on days 16, 19 and 22 of pregnancy. Thus, the ratio of endogenous urinary nitrogen to basal energy expenditure increased in late pregnancy, indicating that "the law of a constant relationship of minimal nitrogen and energy output" is not applicable to the pregnant animals. We discuss which values for obligatory nitrogen loss should be used for estimating the net utilization efficiency of dietary proteins in pregnancy.

Net utilization of various levels of whole egg protein in pregnant rats.

Net protein utilization (NPU) was examined in pregnant rats fed various levels (1, 3, 6, 10 and 20%) of whole egg protein (WEP), based on their obligatory nitrogen losses. On increase in dietary protein, nitrogen balance improved curvilinearly and the NPU decreased exponentially in both pregnant and nonpregnant rats. The utilization efficiency was high in rats fed marginally low protein diets, mainly due to lower urinary nitrogen levels than the obligatory levels of nitrogen loss. The NPUs in pregnant rats fed 1, 3, 6, 10 and 20% WEP diets were 103, 99, 78, 66 and 46, respectively. These values were 15 to 20% higher than those in nonpregnant rats because in pregnant rats obligatory urinary nitrogen loss was higher and the animals took more energy. The problems in application of the NPUs in pregnant rats for estimating the protein allowance of pregnant humans are discussed.

Maintenance energy requirement in pregnant rats and net energetic efficiency for fetal growth during late pregnancy.

The maintenance energy requirement (MEm) of pregnant rats and net energetic efficiency for fetal growth during late pregnancy were examined by regression analysis. Pregnant rats, weighing about 180 g, were fed on 20% casein diet during early-mid pregnancy and then divided into three groups--ad libitum-fed, 50% food restricted, or starved. A linear relation between the energy balance (Y, kcal/100 g BW/day) and the metabolizable energy intake (X, kcal/100 g BW/day) was obtained as Y = 0.81 X-14.83 (n: 17, r = +0.99, p less than 0.001). The X-intercept, MEm, was calculated to be 18.31 kcal/100 g BW/day. Pregnant animals fed ad libitum retained 6.1 and 1.1 kcal of energy daily in their conceptuses and their own body, respectively, during late pregnancy. Assuming that the net efficiency of maternal energy deposition is equal to that for size- and age-matched nonpregnant rats, the net energetic efficiency for fetal growth was calculated to be 82%. This value was very close to the net efficiency for weanling rats reported previously, suggesting that the net energetic efficiency for maximum growth in well-nourished rats is about 82%.

Protein and energy metabolism in patients with progressive muscular dystrophy.

Studies were made on whether body weight loss in patients with muscular dystrophy is due to reduced intake and/or abnormal expenditure of energy. For this, food intakes and various physiological variables were surveyed in totals of 310 patients with Duchenne muscular dystrophy (DMD) of 11 to 29 years old and 28 patients with limb-girdle muscular dystrophy (LGMD) of 30 to 47 years old. Energy and protein intakes, expressed on a unit body weight basis, in DMD patients were comparable to, or higher than the allowances for age-matched healthy controls, whereas those in LGMD patients were 92 and 94% respectively of these allowances. The basal metabolic rate (BMR), expressed as kcal/kg/day, of DMD patients of all ages was higher than that of controls, the difference increasing with age, and being about 20 to 30% higher than that of controls in older patients with DMD. The BMR of LGMD patients was nearly normal. The maintenance requirements of conventional dietary protein in DMD and LGMD patients were 1.26 and 0.84 g/kg/day, respectively. These values were about 68 and 12% higher than the normal adult value (0.75 g/kg/day), indicating decreased protein utilization and increased protein catabolism. Daily excretion of urinary 3-methylhistidine (3MH) per unit muscle mass (micrograms/mg creatinine) by MD patients was significantly higher than that by controls, indicating increased degradation of muscle protein. The BMR, maintenance protein requirement and 3MH excretion of DMD patients suggest that DMD is a hypercatabolic disease. Comparison of the energy and protein intakes with the allowances estimated in consideration of increased requirements showed deficiencies of energy and protein in DMD patients. Thus, we conclude that the underweight of the DMD patients resulted from nutrient deficiencies due to hypercatabolism, despite their considerably high intakes of energy and protein, expressed as per kg body weight. These deficiencies were confirmed by demonstrating decreased concentrations of free essential amino acids, particularly branched chain amino acids, in their serum. The values of variables of LGMD patients were intermediate between those of DMD patients and control subjects.

Predictions of energy intake and energy allowance of patients with Duchenne muscular dystrophy and their validity.

Patients with Duchenne muscular dystrophy are so malnourished that energy supplementation is crucial. Their degree of energy deficiency was assessed as difference between their energy intake and their energy allowance, which were deduced from easily measured parameters. A significant, negative relationship was found between the basal metabolic rate (BMR) (Y, %, BMR/standard BMR) and body weight (X, %, body weight/standard body weight) in the patients, from which the formula for the BMR was deduced to be Y = -1.116X + 174.5 (n = 202, r = -0.72, p less than 0.001). Thus, it is possible to estimate the energy allowance for individual patients by a factorial procedure from the presumed BMR and a factor for physical activity. In addition, their energy intake was calculated from a constant protein-energy % (14.6%) in their diet and nitrogen intake which was deduced from a significant positive correlation between their nitrogen intake (Y, mg/kg/day) and their nitrogen excretion in 24 h urine samples (X, mg/kg/day). This correlation conformed to the equation Y = 1.053X + 32.4 (n = 267, r = +0.76, p less than 0.001). The validities of the above predictions for energy intake and allowance were examined by plotting the degree of energy deficiency (% ratio of presumed intake/presumed allowance) against the concentrations of retinal binding protein, prealbumin and transferrin in the serum, because rapid turnover proteins are sensitive to energy deficiency. Significant positive correlations were obtained with both variables, suggesting that these predictions were valid.

Effects of various levels of dietary calcium during pregnancy on maternal calcium utilization and fetal growth in rats.

The effects of calcium-free and normal (0.6%) and high (1.0%) calcium diets on the transfer of calcium from pregnant mothers to fetuses were investigated by balance experiments. Pregnant rats receiving calcium-free, normal and high calcium diets ate totals of 353, 324 and 280 g of the diet, respectively, during pregnancy, and the food consumption of the latter two groups decreased near term. The group on calcium-free diet was able to maintain pregnancy and produce normal fetuses by using calcium resorbed from the dam's bones. The calcium retentions due to pregnancy in rats on normal and high calcium diets were 116 and 128 mg, respectively, during the first 15 days, and -9 and -109 mg, respectively, during the last 6 days of pregnancy. Fetuses contained about 130 mg of calcium at term and most of this calcium was supplied from the dam's bones, in which extra calcium were retained during early-mid pregnancy. Unexpectedly, the true rate of calcium absorption was appreciably lower during late pregnancy than during early-mid pregnancy in both dietary groups. Thus, extra calcium retention during early-mid pregnancy seemed to be physiological adaptation to a decrease in either food consumption or calcium absorption during late pregnancy. Phosphorus absorption and its balance were examined in relation with the dietary calcium levels.

[The effect of continuous intra-articular and intra-bursal infusion of lidocaine on postoperative pain following shoulder arthroscopic surgery].

We evaluated the effects of continuous intra-articular and intra-bursal infusion of lidocaine on postoperative pain following shoulder arthroscopic surgery. Forty-one ASA I-II patients scheduled for shoulder arthroscopic surgery, were allocated into following four groups. The patients, after intra-articular arthroscopic surgery, either received intra-articular lidocaine (Group I, n = 10) or did not (Group III, n = 10). The patients after extra-articular arthroscopic surgery either received intra-bursal lidocaine (Group II, n = 11) or did not (Group IV, n = 10). Group I and Group II received 8 ml of 1% lidocaine intra-articularly and intra-bursally, respectively, at the end of surgery, followed by continuous infusion of 1% lidocaine at the rate of 2 ml.hr-1 for 24 hours. The intensities of postoperative pain were evaluated by Visual Analogue Scale (VAS), 2, 5, 8, 12, 18 and 24 hours after surgery, and by the number of patients' request for supplemental analgesic for 24 hours. The VAS scores and the number of analgesic requests were significantly lower (P < 0.05) in Group I than Group III, and in Group II than Group IV throughout the postoperative observation period. No adverse effects were observed during this study. We conclude that continuous intra-articular and intra-bursal infusion of lidocaine provides effective postoperative pain relief for shoulder arthroscopic surgery.